Science.gov

Sample records for scfv antibody fragments

  1. Refolded scFv Antibody Fragment against Myoglobin Shows Rapid Reaction Kinetics

    PubMed Central

    Song, Hyung-Nam; Jang, Jun-Hyuck; Kim, Young-Wan; Kim, Dong-Hyung; Park, Sung-Goo; Lee, Myung Kyu; Paek, Se-Hwan; Woo, Eui-Jeon

    2014-01-01

    Myoglobin is one of the early biomarkers for acute myocardial infarction. Recently, we have screened an antibody with unique rapid reaction kinetics toward human myoglobin antigen. Antibodies with rapid reaction kinetics are thought to be an early IgG form produced during early stage of in vivo immunization. We produced a recombinant scFv fragment for the premature antibody from Escherichia coli using refolding technology. The scFv gene was constructed by connection of the VH–VL sequence with a (Gly4Ser)3 linker. The scFv fragment without the pelB leader sequence was expressed at a high level, but the solubility was extremely low. A high concentration of 8 M urea was used for denaturation. The dilution refolding process in the presence of arginine and the redox reagents GSH and GSSH successfully produced a soluble scFv protein. The resultant refolded scFv protein showed association and dissociation values of 9.32 × 10−4 M−1·s−1 and 6.29 × 10−3 s−1, respectively, with an affinity value exceeding 107 M−1 (kon/koff), maintaining the original rapid reaction kinetics of the premature antibody. The refolded scFv could provide a platform for protein engineering for the clinical application for diagnosis of heart disease and the development of a continuous biosensor. PMID:25530617

  2. Refolded scFv antibody fragment against myoglobin shows rapid reaction kinetics.

    PubMed

    Song, Hyung-Nam; Jang, Jun-Hyuck; Kim, Young-Wan; Kim, Dong-Hyung; Park, Sung-Goo; Lee, Myung Kyu; Paek, Se-Hwan; Woo, Eui-Jeon

    2014-01-01

    Myoglobin is one of the early biomarkers for acute myocardial infarction. Recently, we have screened an antibody with unique rapid reaction kinetics toward human myoglobin antigen. Antibodies with rapid reaction kinetics are thought to be an early IgG form produced during early stage of in vivo immunization. We produced a recombinant scFv fragment for the premature antibody from Escherichia coli using refolding technology. The scFv gene was constructed by connection of the V(H)-V(L) sequence with a (Gly4Ser)3 linker. The scFv fragment without the pelB leader sequence was expressed at a high level, but the solubility was extremely low. A high concentration of 8 M urea was used for denaturation. The dilution refolding process in the presence of arginine and the redox reagents GSH and GSSH successfully produced a soluble scFv protein. The resultant refolded scFv protein showed association and dissociation values of 9.32 × 10⁻⁴ M⁻¹·s⁻¹ and 6.29 × 10⁻³ s⁻¹, respectively, with an affinity value exceeding 10⁷ M⁻¹ (k(on)/k(off)), maintaining the original rapid reaction kinetics of the premature antibody. The refolded scFv could provide a platform for protein engineering for the clinical application for diagnosis of heart disease and the development of a continuous biosensor. PMID:25530617

  3. Production of stabilized scFv antibody fragments in the E. coli bacterial cytoplasm.

    PubMed

    Vaks, Lilach; Benhar, Itai

    2014-01-01

    Monoclonal antibodies (mAbs) are currently the fastest growing class of therapeutic proteins. Parallel to full-length IgG format the development of recombinant technologies provided the production of smaller recombinant antibody variants. The single-chain variable fragment (scFv) antibody is a minimal form of functional antibody comprised of the variable domains of immunoglobulin light and heavy chains connected by a flexible linker. In most cases, scFvs are expressed in the bacterium E. coli. The production of soluble scFvs under the reducing conditions of the E. coli bacterial cytoplasm is inefficient because of the inability of the disulfide bonds to form. Hence, scFvs are either secreted to the periplasm as soluble proteins or expressed in the cytoplasm as insoluble inclusion bodies and recovered by refolding. The cytoplasmic expression of scFvs as a C-terminal fusion to maltose-binding protein (MBP) provided the high-level production of stable, soluble, and functional fusion protein. The below protocol provides the detailed description of MBP-scFv production in E. coli utilizing two expression systems: pMalc-TNN and pMalc-NHNN. Although the MBP tag does not disrupt the most of antibody activities, the MBP-TNN-scFv product can be cleaved by TEV protease in order to obtain untagged scFv. PMID:24037842

  4. Development of single chain variable fragment (scFv) antibodies against surface proteins of ‘Ca. Liberibacter asiaticus’

    Technology Transfer Automated Retrieval System (TEKTRAN)

    ‘Ca. Liberibacter asiaticus’ is the causal agent of citrus huanglongbing, the most serious disease of citrus worldwide. We have developed and applied immunization and affinity screening methods to develop a primary library of recombinant single chain variable fragment (scFv) antibodies in an M13 vec...

  5. Construction of Recombinant Single Chain Variable Fragment (ScFv) Antibody Against Superantigen for Immunodetection Using Antibody Phage Display Technology.

    PubMed

    Singh, Pawan Kumar; Agrawal, Ranu; Kamboj, D V; Singh, Lokendra

    2016-01-01

    Superantigens are a class of antigens that bind to the major histocompatibility complex class (MHC) II and T-cell receptor (TCR) and cause the nonspecific activation of T cells, resulting in a massive release of pro-inflammatory mediators. They are produced by the gram-positive organisms Staphylococcus aureus and Streptococcus pyogenes, and by a variety of other microbes such as viruses and mycoplasma, and cause toxic shock syndrome (TSS) and even death in some cases. The immunodetection of superantigens is difficult due to the polyclonal activation of T-cells leading to nonspecific antibody production. The production of recombinant monoclonal antibodies against superantigens can solve this problem and are far better than polyclonal antibodies in terms of detection. Here, we describe the construction of recombinant single chain variable fragments (ScFv) antibodies against superantigens with specific reference to SEB (staphylococcal enterotoxin B) using antibody phage display technology. PMID:26676049

  6. NOVEL AMYLOID-BETA SPECIFIC scFv and VH ANTIBODY FRAGMENTS FROM HUMAN AND MOUSE PHAGE DISPLAY ANTIBODY LIBRARIES

    PubMed Central

    Medecigo, M.; Manoutcharian, K.; Vasilevko, V.; Govezensky, T.; Munguia, M. E.; Becerril, B.; Luz-Madrigal, A.; Vaca, L.; Cribbs, D. H.; Gevorkian, G.

    2010-01-01

    Anti-amyloid immunotherapy has been proposed as an appropriate therapeutic approach for Alzheimer’s disease (AD). Significant efforts have been made towards the generation and assessment of antibody-based reagents capable of preventing and clearing amyloid aggregates as well as preventing their synaptotoxic effects. In this study, we selected a novel set of human anti-amyloid-beta peptide 1-42 (Aβ1-42) recombinant monoclonal antibodies in a single chain fragment variable (scFv) and a single domain (VH) formats. We demonstrated that these antibody fragments recognize in a specific manner amyloid beta deposits in APP/Tg mouse brains, inhibit toxicity of oligomeric Aβ1-42 in neuroblastoma cell cultures in a concentration-dependently manner and reduced amyloid deposits in APP/Tg2576 mice after intracranial administration. These antibody fragments recognize epitopes in the middle/C-terminus region of Aβ, which makes them strong therapeutic candidates due to the fact that most of the Aβ species found in the brains of AD patients display extensive N-terminus truncations/modifications. PMID:20451261

  7. Targeted Multiplex Imaging Mass Spectrometry with Single Chain Fragment Variable (scfv) Recombinant Antibodies

    NASA Astrophysics Data System (ADS)

    Thiery, Gwendoline; Mernaugh, Ray L.; Yan, Heping; Spraggins, Jeffrey M.; Yang, Junhai; Parl, Fritz F.; Caprioli, Richard M.

    2012-10-01

    Recombinant scfv antibodies specific for CYP1A1 and CYP1B1 P450 enzymes were combined with targeted imaging mass spectrometry to simultaneously detect the P450 enzymes present in archived, paraffin-embedded, human breast cancer tissue sections. By using CYP1A1 and CYP1B1 specific scfv, each coupled to a unique reporter molecule (i.e., a mass tag) it was possible to simultaneously detect multiple antigens within a single tissue sample with high sensitivity and specificity using mass spectrometry. The capability of imaging multiple antigens at the same time is a significant advance that overcomes technical barriers encountered when using present day approaches to develop assays that can simultaneously detect more than a single antigen in the same tissue sample.

  8. Isolation of a human-like antibody fragment (scFv) that neutralizes ricin biological activity

    PubMed Central

    Pelat, Thibaut; Hust, Michael; Hale, Martha; Lefranc, Marie-Paule; Dübel, Stefan; Thullier, Philippe

    2009-01-01

    Background Ricin is a lethal toxin that inhibits protein synthesis. It is easily extracted from a ubiquitously grown plant, Ricinus communis, and thus readily available for use as a bioweapon (BW). Anti-ricin antibodies provide the only known therapeutic against ricin intoxication. Results In this study, after immunizing a non-human primate (Macaca fascicularis) with the ricin chain A (RTA), a phage-displayed immune library was built (2 × 108 clones), that included the λ light chain fragment. The library was screened against ricin, and specific binders were sequenced and further analyzed. The best clone, 43RCA, was isolated using a new, stringent neutralization test. 43RCA had a high, picomolar affinity (41 pM) and neutralized ricin efficiently (IC50 = 23 ± 3 ng/ml, corresponding to a [scFv]/[ricin] molar ratio of 4). The neutralization capacity of 43RCA compared favourably with that of polyclonal anti-deglycosylated A chain (anti-dgRCA) IgGs, obtained from hyperimmune mouse serum, which were more efficient than any monoclonal at our disposal. The 43RCA sequence is very similar to that for human IgG germline genes, with 162 of 180 identical amino acids for the VH and VL (90% sequence identity). Conclusion Results of the characterization studies, and the high degree of identity with human germline genes, altogether make this anti-ricin scFv, or an IgG derived from it, a likely candidate for use in humans to minimize effects caused by ricin intoxication. PMID:19563687

  9. Development of single chain variable fragment (scFv) antibodies against Xylella fastidiosa subsp. pauca by phage display.

    PubMed

    Yuan, Qing; Jordan, Ramon; Brlansky, Ronald H; Istomina, Olga; Hartung, John

    2015-10-01

    Xylella fastidiosa is a member of the gamma proteobacteria. It is fastidious, insect-vectored and xylem-limited and causes a variety of diseases, some severe, on a wide range of economically important perennial crops, including grape and citrus. Antibody based detection assays are commercially available for X. fastidiosa, and are effective at the species, but not at the subspecies level. We have made a library of scFv antibody fragments directed against X. fastidiosa subsp. pauca strain 9a5c (citrus) by using phage display technology. Antibody gene repertoires were PCR-amplified using 23 primers for the heavy chain variable region (V(H)) and 21 primers for the light chain variable region (V(L)). The V(H) and V(L) were joined by overlap extension PCR, and then the genes of the scFv library were ligated into the phage vector pKM19. The library contained 1.2×10(7) independent clones with full-length scFv inserts. In each of 3cycles of affinity-selection with 9a5c, about 1.0×10(12) phage were used for panning with 4.1×10(6), 7.1×10(6), 2.1×10(7) phage recovered after the first, second and third cycles, respectively. Sixty-six percent of clones from the final library bound X. fastidiosa 9a5c in an ELISA. Some of these scFv antibodies recognized strain 9a5c and did not recognize X. fastidiosa strains that cause Pierce's disease of grapevine. PMID:26232710

  10. Cloning single-chain antibody fragments (ScFv) from hyrbidoma cells.

    PubMed

    Toleikis, Lars; Frenzel, André

    2012-01-01

    Despite the rising impact of the generation of antibodies by phage display and other technologies, hybridoma technology still provides a valuable tool for the generation of high-affinity binders against different targets. But there exist several limitations of using hybridoma-derived antibodies. The source of the hybridoma clones are mostly rat or mouse B-lymphocytes. Therefore a human-anti-mouse or human-anti-rat antibody response may result in immunogenicity of these antibodies. This leads to the necessity of humanization of these antibodies where the knowledge of the amino acid sequence of the proteins is inalienable. Furthermore, additional in vitro modifications, e.g., affinity maturation or fusion to other proteins, are dependent on cloning of the antigen-binding domains.Here we describe the isolation of RNA from hybridoma cells and the primers that can be used for the amplification of VL and VH as well as the cloning of the antibody in scFv format and its expression in Escherichia coli. PMID:22907345

  11. High throughput ranking of recombinant avian scFv antibody fragments from crude lysates using the Biacore A100.

    PubMed

    Leonard, Paul; Säfsten, Pär; Hearty, Stephen; McDonnell, Barry; Finlay, William; O'Kennedy, Richard

    2007-06-30

    Advances in molecular evolution strategies have made it possible to identify antibodies with exquisite specificities and also to fine-tune their biophysical properties for practically any specified application. Depending on the desired function, antibody/antigen interactions can be long-lived or short-lived and, therefore, particular attention is needed when seeking to identify antibodies with specific reaction-rate and affinity properties. Surface plasmon resonance (SPR) biosensors routinely generate sensitive and reliable kinetic data from antibody/antigen interactions for both therapeutic and diagnostic applications. However, many kinetic-based screening assays require rigorous sample preparation and purification prior to analysis. To ameliorate this problem, we developed a rapid and reliable assay for characterising recombinant scFv antibody fragments, directly from crude bacterial lysates. Ninety-six scFv antibodies derived from chickens immunised with C-reactive protein (CRP) were selected by phage display and evaluated using the Biacore A100 protein interaction array system. Antibodies were captured from crude bacterial extracts on the sensor chip surface and ranked based on the percentage of the complex left (% left) after dissociation in buffer. Kinetic rate constants (k(a) and k(d)) and affinity (K(D)) data were obtained for six clones that bound monomeric CRP across a broad affinity range (2.54 x 10(-8) to 3.53 x 10(-10) M). Using this assay format the A100 biosensor yielded high quality kinetic data, permitting the screening of nearly 400 antibody clones per day. PMID:17532001

  12. Production of biologically active scFv and VHH antibody fragments in Bifidobacterium longum.

    PubMed

    Shkoporov, A N; Khokhlova, E V; Savochkin, K A; Kafarskaia, L I; Efimov, B A

    2015-06-01

    Bifidobacteria constitute a significant part of healthy intestinal microbiota in adults and infants and present a promising platform for construction of genetically modified probiotic agents for treatment of gastrointestinal disorders. In this study, three strains of Bifidobacterium longum were constructed that express and secrete biologically active single-chain antibodies against human TNF-α and Clostridium difficile exotoxin A. Anti-TNF-α scFv antibody D2E7 was produced at the level of 25 μg L(-1) in broth culture and was mostly retained in the cytoplasm, while VHH-type antibodies A20.1 and A26.8 against C. difficile exotoxin A were produced at the levels of 0.3-1 mg L(-1) and secreted very efficiently. The biological activity of both antibody types was demonstrated in the mammalian cell-based assays. Expression of A20.1 and A26.8 was also observed in vivo after intragastric administration of transformed B. longum strains to (C57/BL6 × DBA/2)F1 mice. The obtained B. longum strains may serve as prototypes for construction of novel probiotic medications against inflammatory bowel disease and C. difficile-associated disease. PMID:25994292

  13. Selection of scFv Antibody Fragments Binding to Human Blood versus Lymphatic Endothelial Surface Antigens by Direct Cell Phage Display

    PubMed Central

    Keller, Thomas; Kalt, Romana; Raab, Ingrid; Schachner, Helga; Mayrhofer, Corina; Kerjaschki, Dontscho; Hantusch, Brigitte

    2015-01-01

    The identification of marker molecules specific for blood and lymphatic endothelium may provide new diagnostic tools and identify new targets for therapy of immune, microvascular and cancerous diseases. Here, we used a phage display library expressing human randomized single-chain Fv (scFv) antibodies for direct panning against live cultures of blood (BECs) and lymphatic (LECs) endothelial cells in solution. After six panning rounds, out of 944 sequenced antibody clones, we retrieved 166 unique/diverse scFv fragments, as indicated by the V-region sequences. Specificities of these phage clone antibodies for respective compartments were individually tested by direct cell ELISA, indicating that mainly pan-endothelial cell (EC) binders had been selected, but also revealing a subset of BEC-specific scFv antibodies. The specific staining pattern was recapitulated by twelve phage-independently expressed scFv antibodies. Binding capacity to BECs and LECs and differential staining of BEC versus LEC by a subset of eight scFv antibodies was confirmed by immunofluorescence staining. As one antigen, CD146 was identified by immunoprecipitation with phage-independent scFv fragment. This antibody, B6-11, specifically bound to recombinant CD146, and to native CD146 expressed by BECs, melanoma cells and blood vessels. Further, binding capacity of B6-11 to CD146 was fully retained after fusion to a mouse Fc portion, which enabled eukaryotic cell expression. Beyond visualization and diagnosis, this antibody might be used as a functional tool. Overall, our approach provided a method to select antibodies specific for endothelial surface determinants in their native configuration. We successfully selected antibodies that bind to antigens expressed on the human endothelial cell surfaces in situ, showing that BECs and LECs share a majority of surface antigens, which is complemented by cell-type specific, unique markers. PMID:25993332

  14. Isolation of soluble scFv antibody fragments specific for small biomarker molecule, L-Carnitine, using phage display.

    PubMed

    Abou El-Magd, Rabab M; Vozza, Nicolas F; Tuszynski, Jack A; Wishart, David S

    2016-01-01

    Isolation of single chain antibody fragment (scFv) clones from naïve Tomlinson I+J phage display libraries that specifically bind a small biomarker molecule, L-Carnitine, was performed using iterative affinity selection procedures. L-Carnitine has been described as a conditionally essential nutrient for humans. Abnormally high concentrations of L-Carnitine in urine are related to many health disorders including diabetes mellitus type 2 and lung cancer. ELISA-based affinity characterization results indicate that selectants preferentially bind to L-Carnitine in the presence of key bioselecting component materials and closely related L-Carnitine derivatives. In addition, the affinity results were confirmed using biophysical fluorescence quenching for tyrosine residues in the V segment. Small-scale production of the soluble fragment yielded 1.3mg/L using immunopure-immobilized protein A affinity column. Circular Dichroism data revealed that the antibody fragment (Ab) represents a folded protein that mainly consists of β-sheets. These novel antibody fragments may find utility as molecular affinity interface receptors in various electrochemical biosensor platforms to provide specific L-Carnitine binding capability with potential applications in metabolomic devices for companion diagnostics and personalized medicine applications. It may also be used in any other biomedical application where detection of the L-Carnitine level is important. PMID:26608419

  15. Aptamers, antibody scFv, and antibody Fab' fragments: An overview and comparison of three of the most versatile biosensor biorecognition elements.

    PubMed

    Crivianu-Gaita, Victor; Thompson, Michael

    2016-11-15

    The choice of biosensing elements is crucial for the development of the optimal biosensor. Three of the most versatile biosensing elements are antibody single-chain Fv fragments (scFv), antibody fragment-antigen binding (Fab') units, and aptamers. This article provides an overview of these three biorecognition elements with respects to their synthesis/engineering, various immobilization techniques, and examples of their use in biosensors. Furthermore, the final section of the review compares and contrasts their characteristics (time/cost of development, ease and variability of immobilization, affinity, stability) illustrating their advantages and disadvantages. Overall, scFv fragments are found to display the highest customizability (i.e. addition of functional groups, immobilizing peptides, etc.) due to recombinant synthesis techniques. If time and cost are an issue in the development of the biosensor, Fab' fragments should be chosen as they are relatively cheap and can be developed quickly from whole antibodies (several days). However, if there are sufficient funds and time is not a factor, aptamers should be utilized as they display the greatest affinity towards their target analytes and are extremely stable (excellent biosensor regenerability). PMID:27155114

  16. High efficient expression of a functional humanized single-chain variable fragment (scFv) antibody against CD22 in Pichia pastoris.

    PubMed

    Zarei, Najmeh; Vaziri, Behrouz; Shokrgozar, Mohammad Ali; Mahdian, Reza; Fazel, Ramin; Khalaj, Vahid

    2014-12-01

    Single-chain variable fragments (scFvs) have recently emerged as attractive candidates in targeted immunotherapy of various malignancies. The anti-CD22 scFv is able to target CD22, on B cell surface and is being considered as a promising molecule in targeted immunotherapy of B cell malignancies. The recombinant anti-CD22 scFv has been successfully expressed in Escherichia coli; however, the insufficient production yield has been a major bottleneck for its therapeutic application. The methylotrophic yeast Pichia pastoris has become a highly popular expression host for the production of a wide variety of recombinant proteins such as antibody fragments. In this study, we used the Pichia expression system to express a humanized scFv antibody against CD22. The full-length humanized scFv gene was codon optimized, cloned into the pPICZαA and expressed in GS115 strain. The maximum production level of the scFv (25 mg/L) were achieved at methanol concentration, 1 %; pH 6.0; inoculum density, OD600 = 3 and the induction time of 72 h. The correlation between scFv gene dosage and expression level was also investigated by real-time PCR, and the results confirmed the presence of such correlation up to five gene copies. Immunofluorescence and flow cytometry studies and Biacore analysis demonstrated binding to CD22 on the surface of human lymphoid cell line Raji and recombinant soluble CD22, respectively. Taken together, the presented data suggest that the Pichia pastoris can be considered as an efficient host for the large-scale production of anti-CD22 scFv as a promising carrier for targeted drug delivery in treatment of CD22(+) B cell malignancies. PMID:25239038

  17. Development of single chain variable fragment (scFv) antibodies against surface proteins of 'Ca. Liberibacter asiaticus'.

    PubMed

    Yuan, Qing; Jordan, Ramon; Brlansky, Ronald H; Minenkova, Olga; Hartung, John

    2016-03-01

    'Candidatus Liberibacter asiaticus' is the causal agent of citrus huanglongbing, the most serious disease of citrus worldwide. We have developed and applied immunization and affinity screening methods to develop a primary library of recombinant single chain variable fragment (scFv) antibodies in an M13 vector, pKM19. The antibody population is enriched for antibodies that bind antigens of 'Ca. Liberibacter asiaticus'. The primary library has more than 10(7) unique antibodies and the genes that encode them. We have screened this library for antibodies that bind to specifically-chosen proteins that are present on the surface of 'Ca. Liberibacter asiaticus'. These proteins were used as targets for affinity-based selection of scFvs that bind to the major outer membrane protein, OmpA; the polysaccharide capsule protein KpsF; a protein component of the type IV pilus (CapF); and, two flagellar proteins FlhA and FlgI. These scFvs have been used in ELISA and dot blot assays against purified protein antigens and 'Ca. Liberibacter asiaticus' infected plant extracts. We have also recloned many of these scFvs into a plasmid expression vector designed for the production of scFvs. Screening of these scFvs was more efficient when phage-bound, rather than soluble scFvs, were used. We have demonstrated a technology to produce antibodies at will and against any protein target encoded by 'Ca. Liberibacter asiaticus'. Applications could include advanced diagnostic methods for huanglongbing and the development of immune labeling reagents for in planta applications. PMID:26744234

  18. Construction of a Single Chain Variable Fragment Antibody (scFv) against Carbaryl and Its Interaction with Carbaryl.

    PubMed

    Xiuyuan, Zhang; Zhihong, Huang; Lixia, Wang; Xiaonan, Liu

    2015-05-01

    Carbaryl is a low molecular weight insecticide that inhibits cholinesterase. Residues of carbaryl in food and the environment have damaged human health. A high-specificity scFv that can identify carbaryl is still lacking. In the present study, an anti-carbaryl scFv gene was prepared by cloning VL and VH genes from hybridoma cells secreting monoclonal antibody, then VH and VL were fused together using splicing by overlap extension (SOE) PCR with a flexible polypeptide linker connector (Gly4Ser)3, and then the scFv-pET-26b recombinant plasmid was constructed and transformed into E. coli BL21 for expression using IPTG as an inducer. The expressed recombinant protein was identified by SDS-PAGE and ELISA. The three-dimensional structure of the anti-carbaryl scFv was constructed by computer modeling, and carbaryl was docked to the scFv model to obtain the structure of the binding complex. The binding site was composed of Ala51, Ser52, Ile51, Gly54, Ser56, Arg98, and Gly100. This helps to understand the mechanism of interaction between anti-carbaryl antibody and antigen. Furthermore, it provides guidance for in vitro affinity maturation of anti-carbaryl antibody. PMID:26071785

  19. Successful engineering of a highly potent single-chain variable-fragment (scFv) bispecific antibody to target disialoganglioside (GD2) positive tumors

    PubMed Central

    Cheng, Ming; Santich, Brian H.; Xu, Hong; Ahmed, Mahiuddin; Huse, Morgan; Cheung, Nai-Kong V.

    2016-01-01

    ABSTRACT Engineering potent bispecific antibodies from single-chain variable fragments (scFv) remains difficult due to the inherent instability and insufficient binding of scFv's compared to their parental immunoglobulin format. Previously, we described a scFv-based bispecific antibody (scBA) against disialoganglioside (GD2) based on the anti-GD2 murine 5F11-scFv and the anti-CD3 huOKT3-scFv (5F11-scBA). In this study, we substituted the 5F11-scFv with the higher affinity (13-fold) hu3F8-scFv to form hu3F8-scBA. With this modification, hu3F8-scBA redirected T cells to kill GD2(+) cancer cell lines with up to 5,000-fold higher potency (femtomolar EC50) compared with 5F11-scBA (picomolar EC50) in cytotoxicity assays, even against target cells with low GD2 densities. Furthermore, hu3F8-scBA induced stronger T-cell activation than 5F11-scBA, as measured by Ca2+ flux and cytokine release. Additionally, in vivo, hu3F8-scBA suppressed tumor growth and prolonged mice survival much more effectively than 5F11-scBA, in both neuroblastoma and melanoma xenograft models. We conclude that the functional properties of scBA's can be increased substantially by relatively modest increases in antigen affinity. PMID:27471647

  20. A new expression vector facilitating production and functional analysis of scFv antibody fragments selected from Tomlinson I+J phagemid libraries.

    PubMed

    Ossysek, Karolina; Uchański, Tomasz; Kulesza, Małgorzata; Bzowska, Monika; Klaus, Tomasz; Woś, Klaudia; Madej, Mariusz; Bereta, Joanna

    2015-10-01

    Tomlinson I+J are synthetic phagemid human scFv libraries widely employed to obtain specific antibody fragments via a phage display method. The pIT2/HB2151 expression system proposed by the designers of the libraries has certain drawbacks which result in the lack of expression or low expression levels of numerous soluble scFvs. At the stage of scFv screening, this may lead to losing some excellent antibodies, which can be avoided but requires laborious and expensive work. Here we present a new, pET-30-based vector, which is compatible with Tomlinson libraries, retains all virtues of pIT2 used as a plasmid and eliminates all its flaws. We demonstrate that pET-scFv-T is frequently superior to pIT2 in terms of efficient scFv expression. Moreover, an amber suppressor bacterial strain, RosettaBlue(DE3)pLysS, transformed with the new vector, pET-scFv-T, coding for a number of scFvs, produces substantial amounts of functional, easy to purify recombinant antibody fragments, regardless of whether their coding sequences contain amber codons. Thus, pET-scFv-T/RosettaBlue(DE3)pLysS expression system seems to be a perfect tool for screening for the finest soluble scFvs selected from Tomlinson I+J, as well as from many other phagemid libraries. PMID:26219832

  1. Application of adaptive DO-stat feeding control to Pichia pastoris X33 cultures expressing a single chain antibody fragment (scFv).

    PubMed

    Ferreira, A R; Ataíde, F; von Stosch, M; Dias, J M L; Clemente, J J; Cunha, A E; Oliveira, R

    2012-11-01

    In this study, fed-batch cultures of a Pichia pastoris strain constitutively expressing a single chain antibody fragment (scFv) under the control of the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter were performed in a pilot 50 L bioreactor. Due to the very high cell density achieved within the first 75 h, typically between 140 and 160 g-DCW/L of dry cell weight (DCW), most of the scFv is produced under hard oxygen transfer limitation. To improve scFv productivity, a direct adaptive dissolved oxygen (DO)-stat feeding controller that maximizes glycerol feeding under the constraint of available oxygen transfer capacity was developed and applied to this process. The developed adaptive controller enabled to maximize glycerol feeding through the regulation of DO concentration between 3 and 5 % of saturation, thereby improving process productivity. Set-point convergence dynamics are characterized by a fast response upon large perturbations to DO, followed by a slower but very robust convergence in the vicinity of the boundary with almost imperceptible overshoot. Such control performance enabled operating closer to the 0 % boundary for longer periods of time when compared to a traditional proportional-integral-derivative algorithm, which tends to destabilize with increasing cell density. PMID:22610694

  2. Production and characterization of a single chain variable fragment (scFv) for the mycotoxin deoxynivalenol

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Deoxynivalenol (DON)is a mycotoxin produced by certain fungi that infest cereal grains worldwide. A hybridoma cell line producing a monoclonal antibody (Mab) recognizing DON was used as the starting point in the development of a recombinant single chain variable fragment (scFv) antibody. The scFv wa...

  3. Blocking monocyte transmigration in in vitro system by an anti-CD99 human antibody in single chain fragment variable (scFv) format. Efficient large scale purification of biological active scFv from inclusion bodies in E. coli expression system

    PubMed Central

    Moricoli, Diego; Muller, William A.; Carbonella, Damiano Cosimo; Balducci, Maria Cristina; Dominici, Sabrina; Fiori, Valentina; Watson, Richard; Weber, Evan; Cianfriglia, Maurizio; Scotlandi, Katia; Magnani, Mauro

    2015-01-01

    Migration of leukocytes into a site of inflammation involves several steps mediated by various families of adhesion molecules. CD99 play a significant role in transendothelial migration (TEM) of leukocytes. Inhibition of TEM by specific monoclonal antibody (mAb) can provide a potent therapeutic approach to treating inflammatory conditions. However, the therapeutic utilization of whole IgG can lead to an inappropriate activation of Fc receptor-expressing cells inducing serious adverse side effects due to cytokine release. In this regard, specific recombinant antibody in single chain variable fragments (scFvs) originated by phage library may offer a solution by affecting TEM function in a safe clinical context. However, this consideration requires large scale production of functional scFv antibodies under GMP conditions and hence, the absence of toxic reagents utilized for the solubilization and refolding steps of inclusion bodies that may discourage industrial application of these antibody fragments. In order to apply the scFv anti-CD99 named C7A in a clinical setting we herein describe an efficient and large scale production of the antibody fragments expressed in E.coli as insoluble protein avoiding gel filtration chromatography approach, and laborious refolding step pre- and post-purification. Using differential salt elution which is a simple, reproducible and effective procedure we are able to separate scFv in monomer format from aggregates. The purified scFv antibody C7A exhibits inhibitory activity comparable to an antagonistic conventional mAb, thus providing an excellent agent for blocking CD99 signalling. Thanks to the original purification protocol that can be extended to other scFvs that are expressed as inclusion bodies in bacterial systems, the scFv anti-CD99 C7A herein described represents the first step towards the construction of new antibody therapeutic. PMID:24798881

  4. Expression of functional single-chain variable domain fragment antibody (scFv) against mycotoxin zearalenone in Pichia pastoris.

    PubMed

    Chang, Hyun-Joo; Choi, Sung-Wook; Chun, Hyang Sook

    2008-10-01

    A synthetic gene coding for single-chain variable domain fragment antibody against mycotoxin zearalenone (scFv-ZEN) has been designed, constructed and expressed in Pichia pastoris. The native scFv-ZEN sequence was optimized to Pichia preference codon usage. The expression level of codon-optimized scFv-ZEN was slightly higher than that of native scFv-ZEN, and its maximum yield reached 328 mg total protein/l in flask culture. The binding activities of two selected clones to ZEN using surface plasmon resonance analysis were comparable or better than that of monoclonal antibody. Our results demonstrate the potential of soluble scFv-ZEN for developing a rapid and affordable immunoassay for detection of ZEN in food and feedstuff. PMID:18575809

  5. Isolation and characterisation of a human-like antibody fragment (scFv) that inactivates VEEV in vitro and in vivo.

    PubMed

    Rülker, Torsten; Voß, Luzie; Thullier, Philippe; O' Brien, Lyn M; Pelat, Thibaut; Perkins, Stuart D; Langermann, Claudia; Schirrmann, Thomas; Dübel, Stefan; Marschall, Hans-Jürgen; Hust, Michael; Hülseweh, Birgit

    2012-01-01

    Venezuelan equine encephalitis virus (VEEV) belongs to the Alphavirus genus and several species of this family are pathogenic to humans. The viruses are classified as potential agents of biological warfare and terrorism and sensitive detection as well as effective prophylaxis and antiviral therapies are required.In this work, we describe the isolation of the anti-VEEV single chain Fragment variable (scFv), ToR67-3B4, from a non-human primate (NHP) antibody gene library. We report its recloning into the bivalent scFv-Fc format and further immunological and biochemical characterisation.The scFv-Fc ToR67-3B4 recognised viable as well as formalin and ß-propionolactone (ß-Pl) inactivated virus particles and could be applied for immunoblot analysis of VEEV proteins and immuno-histochemistry of VEEV infected cells. It detected specifically the viral E1 envelope protein of VEEV but did not react with reduced viral glycoprotein preparations suggesting that recognition depends upon conformational epitopes. The recombinant antibody was able to detect multiple VEEV subtypes and displayed only marginal cross-reactivity to other Alphavirus species except for EEEV. In addition, the scFv-Fc fusion described here might be of therapeutic use since it successfully inactivated VEEV in a murine disease model. When the recombinant antibody was administered 6 hours post challenge, 80% to 100% of mice survived lethal VEEV IA/B or IE infection. Forty to sixty percent of mice survived when scFv-Fc ToR67-3B4 was applied 6 hours post challenge with VEEV subtypes II and former IIIA. In combination with E2-neutralising antibodies the NHP antibody isolated here could significantly improve passive protection as well as generic therapy of VEE. PMID:22666347

  6. Characterization of the Native and Denatured Herceptin by ELISA and QCM using a High-Affinity Single Chain Fragment Variable (scFv) Recombinant Antibody

    PubMed Central

    Shang, Yuqin; Mernaugh, Ray

    2012-01-01

    Herceptin/Trastuzumab is a humanized IgG1κ light chain antibody used to treat some forms of breast cancer. A phage-displayed recombinant antibody library was used to obtain an scFv (designated 2B4) to a linear synthetic peptide representing Herceptin’s heavy chain CDR3. ELISAs and piezoimmunosensor/quartz crystal microbalance (QCM) assays were used to characterize 2B4-binding activity to both native and heat denatured Herceptin. The 2B4 scFv specifically bound to heat denatured Herceptin in a concentration dependent manner over a wide (35–220.5 nM) dynamic range. Herceptin denatures and forms significant amount of aggregates when heated. UV-Vis characterization confirms that Herceptin forms aggregates as the temperature used to heat Herceptin increases. QCM affinity assay shows that binding stoichiometry between 2B4 scFv and Herceptin follows a 1:2 relationship proving that 2B4 scFv binds strongly to the dimers of heat denatured Herceptin aggregates and exhibits an affinity constant of 7.17 × 1013 M−2. The 2B4-based QCM assay was more sensitive than the corresponding ELISA. Combining QCM with ELISA can be used to more fully characterize non-specific binding events in assays. The potential theoretical and clinical implications of these results and the advantages of using QCM to characterize human therapeutic antibodies in samples are also discussed. PMID:22934911

  7. Enhancement of hERG channel activity by scFv antibody fragments targeted to the PAS domain.

    PubMed

    Harley, Carol A; Starek, Greg; Jones, David K; Fernandes, Andreia S; Robertson, Gail A; Morais-Cabral, João H

    2016-08-30

    The human human ether-à-go-go-related gene (hERG) potassium channel plays a critical role in the repolarization of the cardiac action potential. Changes in hERG channel function underlie long QT syndrome (LQTS) and are associated with cardiac arrhythmias and sudden death. A striking feature of this channel and KCNH channels in general is the presence of an N-terminal Per-Arnt-Sim (PAS) domain. In other proteins, PAS domains bind ligands and modulate effector domains. However, the PAS domains of KCNH channels are orphan receptors. We have uncovered a family of positive modulators of hERG that specifically bind to the PAS domain. We generated two single-chain variable fragments (scFvs) that recognize different epitopes on the PAS domain. Both antibodies increase the rate of deactivation but have different effects on channel activation and inactivation. Importantly, we show that both antibodies, on binding to the PAS domain, increase the total amount of current that permeates the channel during a ventricular action potential and significantly reduce the action potential duration recorded in human cardiomyocytes. Overall, these molecules constitute a previously unidentified class of positive modulators and establish that allosteric modulation of hERG channel function through ligand binding to the PAS domain can be attained. PMID:27516548

  8. Generation of a Novel Bacteriophage Library Displaying scFv Antibody Fragments from the Natural Buffalo Host to Identify Antigens from Adult Schistosoma japonicum for Diagnostic Development.

    PubMed

    Hosking, Christopher G; McWilliam, Hamish E G; Driguez, Patrick; Piedrafita, David; Li, Yuesheng; McManus, Donald P; Ilag, Leodevico L; Meeusen, Els N T; Veer, Michael J de

    2015-12-01

    The development of effective diagnostic tools will be essential in the continuing fight to reduce schistosome infection; however, the diagnostic tests available to date are generally laborious and difficult to implement in current parasite control strategies. We generated a series of single-chain antibody Fv domain (scFv) phage display libraries from the portal lymph node of field exposed water buffaloes, Bubalus bubalis, 11-12 days post challenge with Schistosoma japonicum cercariae. The selected scFv-phages showed clear enrichment towards adult schistosomes and excretory-secretory (ES) proteins by immunofluorescence, ELISA and western blot analysis. The enriched libraries were used to probe a schistosome specific protein microarray resulting in the recognition of a number of proteins, five of which were specific to schistosomes, with RNA expression predominantly in the adult life-stage based on interrogation of schistosome expressed sequence tags (EST). As the libraries were enriched by panning against ES products, these antigens may be excreted or secreted into the host vasculature and hence may make good targets for a diagnostic assay. Further selection of the scFv library against infected mouse sera identified five soluble scFv clones that could selectively recognise soluble whole adult preparations (SWAP) relative to an irrelevant protein control (ovalbumin). Furthermore, two of the identified scFv clones also selectively recognised SWAP proteins when spiked into naïve mouse sera. These host B-cell derived scFvs that specifically bind to schistosome protein preparations will be valuable reagents for further development of a cost effective point-of-care diagnostic test. PMID:26684756

  9. Generation of a Novel Bacteriophage Library Displaying scFv Antibody Fragments from the Natural Buffalo Host to Identify Antigens from Adult Schistosoma japonicum for Diagnostic Development

    PubMed Central

    Hosking, Christopher G.; McWilliam, Hamish E. G.; Driguez, Patrick; Piedrafita, David; Li, Yuesheng; McManus, Donald P.; Ilag, Leodevico L.; Meeusen, Els N. T.; de Veer, Michael J.

    2015-01-01

    The development of effective diagnostic tools will be essential in the continuing fight to reduce schistosome infection; however, the diagnostic tests available to date are generally laborious and difficult to implement in current parasite control strategies. We generated a series of single-chain antibody Fv domain (scFv) phage display libraries from the portal lymph node of field exposed water buffaloes, Bubalus bubalis, 11–12 days post challenge with Schistosoma japonicum cercariae. The selected scFv-phages showed clear enrichment towards adult schistosomes and excretory-secretory (ES) proteins by immunofluorescence, ELISA and western blot analysis. The enriched libraries were used to probe a schistosome specific protein microarray resulting in the recognition of a number of proteins, five of which were specific to schistosomes, with RNA expression predominantly in the adult life-stage based on interrogation of schistosome expressed sequence tags (EST). As the libraries were enriched by panning against ES products, these antigens may be excreted or secreted into the host vasculature and hence may make good targets for a diagnostic assay. Further selection of the scFv library against infected mouse sera identified five soluble scFv clones that could selectively recognise soluble whole adult preparations (SWAP) relative to an irrelevant protein control (ovalbumin). Furthermore, two of the identified scFv clones also selectively recognised SWAP proteins when spiked into naïve mouse sera. These host B-cell derived scFvs that specifically bind to schistosome protein preparations will be valuable reagents for further development of a cost effective point-of-care diagnostic test. PMID:26684756

  10. High throughput screening of scFv antibodies against viral hemorrhagic septicaemia virus by flow cytometry.

    PubMed

    Zhou, Yao; Xie, Zhi-Gang

    2015-07-01

    Viral hemorrhagic septicaemia (VHS) is an economically important disease that affects salmon and trout worldwide. In this study, a recombinant single chain variable fragment (scFv) antibody library derived from rainbow trout immunized with purified viral hemorrhagic septicaemia virus (VHSV) was constructed. The library was subjected to three rounds of screening by flow cytometry (FCM) against VHSV through a bacteria display technology, resulting in the enrichment of scFv. Four scFv clones with different fluorescence intensity were obtained by colony pick up at random following three rounds of screening. The isolated scFv antibodies were expressed and purified. Relative affinity assay showed the four clones had different sensitivity to VHSV, in accordance with FCM. The potential use of the selected VHSV-specific scFv antibodies was demonstrated by the successful application in Western blotting assay, ELISA and immunofluorescence antibody test (IFAT), and one of the isolated scFv molecular showed excellent in vitro and in vivo blocking activities against VHSV. scFv isolated in this study can be promising diagnostic and/or therapeutic reagents for VHS. This study provides powerful strategies for screening antibodies against new diseases. PMID:25813596

  11. Synergistic capture of Clostridium botulinum Type A neurotoxin by scFv antibodies to novel epitopes

    SciTech Connect

    Gray, Sean A.; Barr, John R.; Kalb, Suzanne R.; Marks, James D.; Baird, Cheryl L.; Cangelosi, Gerard A.; Miller, Keith D.; Feldhaus, Michael J.

    2011-10-01

    A non-immune library of human single chain fragment variable (scFv) antibodies displayed on Saccharomyces cerevisiae was screened for binding to the Clostridium botulinum neurotoxin serotype A binding domain [BoNT/A (Hc)] with the goal of identifying scFv to novel epitopes. To do this, an antibody-mediated labeling strategy was used in which antigen-binding yeast clones were selected after labeling with previously characterized monoclonal antibodies (MAbs) specific to the Hc. Twenty unique scFv clones were isolated that bound Hc. Of these, three also bound to full-length BoNT/A toxin complex with affinities ranging from 5 nM to 170 nM. Epitope binning showed that the three unique clones recognized at least two epitopes that were distinct from one another and from the detection MAbs. After production in E. coli, the scFv were coupled to magnetic particles and tested for their ability to capture BoNT/A holotoxin using an Endopep-MS assay. In this assay, toxin captured by scFv coated magnetic particles was detected by incubation of the complex with a peptide containing a BoNT/A-specific cleavage sequence. Mass spectrometry was used to detect the ratio of intact peptide to cleavage products as evidence for toxin capture. When tested individually, each of the scFv showed a weak positive Endopep-MS result. However, when the particles were coated with all three scFv simultaneously, they exhibited significantly higher Endopep-MS activity, consistent with synergistic binding. These results demonstrate novel approaches toward the isolation and characterization of scFv antibodies specific to unlabeled antigen. They also provide evidence that distinct scFv antibodies can work synergistically to increase the efficiency of antigen capture onto a solid support.

  12. Bacterial expression and characterization of a novel human anti-IgE scFv fragment.

    PubMed

    Wang, MingRong; Zhang, YongXia; Du, TianFei; Dai, YunJian; He, YongZhi; Yu, Xin; Cong, Cong; He, MingYue

    2011-01-01

    Antibodies highly specific to human immunoglobulin (Ig) E are capable of selectively blocking the IgE interaction or eliminating IgE-producing cells, thus providing valuable agents for diagnostics and treatment of various allergic illness. An example is omalizumab, a humanized monoclonal anti-IgE antibody that is approved for the treatment of patients with moderate-to-severe allergic diseases in the United States, European Union and other countries. Here, we describe the generation and characterization of a novel human anti-IgE as a single-chain antibody fragment (scFv). The bacterially-synthesized scFv showed high affinity (86 nM) and specificity to the Fc region of human IgE. To our knowledge, this is the first report of the production of a human anti-IgE scFv in E. coli. Its further development as a potential candidate for medical applications is discussed. PMID:21785278

  13. A neutralizing scFv antibody against infectious bursal disease virus screened by flow cytometry.

    PubMed

    Zhou, Yao; Xie, Zhi-Gang

    2015-09-01

    Infectious bursal disease (IBD) is considered a vital viral disease that threatens the poultry industry worldwide. In this study, a recombinant single chain variable fragment (scFv) antibody library derived from chickens immunized with VP2 protein of infectious bursal disease virus (IBDV) was constructed. The library was subjected to three rounds of screening by flow cytometry (FCM) against VP2/IBDV through a bacteria display technology, resulting in the enrichment of scFvs. Three scFv clones with different fluorescence intensity were obtained by colony pick up at random. The obtained scFv antibodies were expressed and purified. Relative affinity assay showed the three clones had different sensitivity to VP2, in accordance with fluorescence activity cell sorting analysis (FACS). The potential use of the isolated IBDV-specific scFv antibodies was demonstrated by the successful application of these antibodies in Western blotting and ELISA assay. What's more, in vitro neutralization measurement showed that one of the three isolated antibodies possessed the neutralization function against IBDV. This study provides new strategies for screening of antibody library, and scFv antibodies isolated in this study may be utilized as lead candidates for further development of diagnostic or therapeutic antibodies for detection and treatment of IBDV infection. PMID:26003676

  14. Fast conversion of scFv to Fab antibodies using type IIs restriction enzymes.

    PubMed

    Sanmark, Hanna; Huovinen, Tuomas; Matikka, Tero; Pettersson, Tiina; Lahti, Maria; Lamminmäki, Urpo

    2015-11-01

    Single chain variable fragment (scFv) antibody libraries are widely used for developing novel bioaffinity reagents, although Fab or IgG molecules are the preferred antibody formats in many final applications. Therefore, rapid conversion methods for combining multiple DNA fragments are needed to attach constant domains to the scFv derived variable domains. In this study we describe a fast and easy cloning method for the conversion of single framework scFv fragments to Fab fragments using type IIS restriction enzymes. All cloning steps excluding plating of the Fab transformants can be done in 96 well plates and the procedure can be completed in one working day. The concept was tested by converting 69 scFv clones into Fab format on 96 well plates, which resulted in 93% success rate. The method is particularly useful as a high-throughput tool for the conversion of the chosen scFv clones into Fab molecules in order to analyze them as early as possible, as the conversion can significantly affect the binding properties of the chosen clones. PMID:26271437

  15. Engineering production of functional scFv antibody in E. coli by co-expressing the molecule chaperone Skp.

    PubMed

    Wang, Rongzhi; Xiang, Shuangshuang; Feng, Youjun; Srinivas, Swaminath; Zhang, Yonghui; Lin, Mingshen; Wang, Shihua

    2013-01-01

    Single-chain variable fragment (scFv) is a class of engineered antibodies generated by the fusion of the heavy (VH) and light chains (VL) of immunoglobulins through a short polypeptide linker. ScFv play a critical role in therapy and diagnosis of human diseases, and may in fact also be developed into a potential diagnostic and/or therapeutic agent. However, the fact that current scFv antibodies have poor stability, low solubility, and affinity, seriously limits their diagnostic and clinical implication. Here we have developed four different expression vectors, and evaluated their abilities to express a soluble scFv protein. The solubility and binding activity of the purified proteins were determined using both SDS-PAGE and ELISA. Amongst the four purified proteins, the Skp co-expressed scFv showed the highest solubility, and the binding activity to antigen TLH was 3-4 fold higher than the other three purified scFv. In fact, this scFv is specific for TLH and does not cross-react with other TLH-associated proteins and could be used to detect TLH directly in real samples. These results suggest that the pACYC-Duet-skp co-expression vector might be a useful tool for the production of soluble and functional scFv antibody. PMID:24224158

  16. Insilico analysis of three different tag polypeptides with dual roles in scFv antibodies.

    PubMed

    Mohammadi, Mozafar; Nejatollahi, Foroogh; Sakhteman, Amirhossein; Zarei, Neda

    2016-08-01

    Single chain fragment variable (scFv) antibodies are composed of variable heavy (VH) and variable light (VL) domains that are joined by a polypeptide linker. Typically, [(Gly4Ser) n] sequence is used as a linker to retain the integrity of the antigen-binding domain. Due to its low immunogenicity, this sequence cannot be used as a tag for scFv detection and purification. Several evidences have shown that the addition of an N or C-terminal tag for scFv detection and purification will result in the decreased expression and binding capacity of this antibody fragment. In this study, we substituted the traditional linker (GGGGS) with His-tag, C-myc or E-tag sequences through molecular modeling. Stability and integrity of all models were assessed by molecular dynamic (MD) simulation. Based on MD simulation analysis, the model containing E-tag sequence as a linker indicated more stability compared to other molecules. The results suggest that E-tag not only can be substituted for the traditional linker, also eliminates the necessity of using additional tag for scFv detection and purification. PMID:27113782

  17. Expression and characterization of a functional single-chain variable fragment (scFv) protein recognizing MCF7 breast cancer cells in E. coli cytoplasm.

    PubMed

    Mahgoub, Ilham Omer

    2012-08-01

    Single-chain variable fragment (scFv) is one of the most common antibody forms. This report describes the expression of the scFv gene as a soluble protein in Origami DE3 cytoplasm. The purified scFv recognized the epidermal growth factor receptor (EGFRvIII) on the surface of MCF-7 cells. The scFv protein was purified in soluble form at a concentration of 10 mg/l, and the scFv protein activity and specificity were characterized using several immunological assays. The purified scFv protein showed specific binding to MCF-7 cells, evidenced by a band of 68 kDa in Western blot analysis, and immunofluorescence clearly proved that the scFv antibody recognized the EGFRvIII antigen epitopes. Furthermore, 53 % of the MCF-7 cells were bound to scFv protein, as measured by flow cytometry analysis. This study demonstrated that the Origami DE3 expression system can produce single-chain antibodies in active form for later use in gene therapy and vaccine production. PMID:22552770

  18. Anti-ABCG2 scFv antibody of lung adenocarcinoma increases chemosensitivity and induces apoptosis through the activation of mitochondrial pathway

    PubMed Central

    Zhao, Wen-Si; Luo, Yi; Li, Bo-Yi; Zhou, Han-Jing; Zhang, Tao

    2016-01-01

    ABCG2 is a multidrug resistance efflux pump expressed in many diverse tumors. The overexpression of ABCG2 is associated with resistance to a wide variety of anticancer agents, providing a noticeable setback to successful cancer therapy. Therapies targeting ABCG2 may therefore be a promising candidate for reversal of chemoresistance. The anti-ABCG2 single-chain variable fragment (scFv) antibody was constructed by phage display peptide library technology. Immunoblotting, ELISA and immunocytochemistry were used to evaluate the soluble expression and immunoreactivity of the scFv. The effects of scFv on cell function and chemosensitization were confirmed by colony formation, cell migration and CCK-8 assays. Flow cytometry was used to analyse the cell cycle and apoptosis. Radioimmunoimaging and nude mouse tumorigenicity assays were taken to determine the biodistribution and antitumor capacity of the scFv antibody. We have successfully screened out the candidate scFv antibody with an apparent molecular weight of 34 kDa. The scFv demonstrated favourable binding ability to lung adenocarcinoma cells and ABCG2 antigen, and the radioactivity was specifically aggregated at the tumor location. Furthermore, the internalized scFv resulted in antibody-mediated downregulation of ABCG2, proliferation inhibition, apoptosis and cisplatin (DDP) sensitivity. The anti-ABCG2 scFv antibody possesses good tumoraffin and antitumor activity and may therefore be an effective therapeutic agent for lung adenocarcinoma that is dependent on ABCG2 for drug resistance and survival. PMID:27293996

  19. Primary structure and functional scFv antibody expression of an antibody against the human protooncogen c-myc.

    PubMed

    Fuchs, P; Breitling, F; Little, M; Dübel, S

    1997-06-01

    The immunoglobulin heavy- and light-chain variable region (Vh and Vl) genes were isolated from Myc1-9E10 hybridoma cells, which secreted monoclonal antibody against human oncogen c-myc. The expression vector pOPE52-c-myc was constructed for the recombinant production in E. coli. A 30 kDa single chain fragment (scFv) expression product was found in the periplasmic space by SDS-PAGE and immunoblotting. A significant fraction was processed correctly as demonstrated with an antiserum recognizing the processed aminoterminus only. The specific binding of the scFv fragment to the peptide epitope of the maternal monoclonal antibody was demonstrated and the primary sequence of the variable regions was determined. Sequence comparison with previously published partial Vh and Vl sequences from this hybridoma cell line revealed a genetic heterogeneity for the light chain variable region. The potential use of this scFv as a new tool for detection and purification of tagged proteins, for adding costimulatory signals to the surface of cancer cells as well as for analyzing c-myc function in the living cell by cytoplasmic expression is discussed. PMID:9219032

  20. Functional Characteristics and Molecular Mechanism of a New scFv Antibody Against Aβ42 Oligomers and Immature Protofibrils.

    PubMed

    Zhang, Yuan; Sun, Yuanhong; Huai, Yangyang; Zhang, Ying-Jiu

    2015-12-01

    Amyloid β peptide (Aβ42) is a major determinant of Alzheimer's disease (AD). In this study, we studied a novel single-chain variable fragment (scFv), AS, generated from an antibody library of AD patients, which recognized and bound specifically to medium-size amyloid β peptide (Aβ42) oligomers and immature protofibrils (25-55 kDa) and, more importantly, reduced their level by blocking their formation or inducing their disassembly. Consequently, scFv AS ameliorated or prevented their cytotoxicity and protected SH-SY5Y cells and primary cultured neurons in vitro from their damage in a concentration-dependent manner. Comparison of its cytotoxicity-inhibiting and cytotoxicity-neutralizing activities indicated that scFv AS displayed its protective effect on target cells mainly due to its cytotoxicity-inhibitory activity though it could also neutralize the cytotoxicity. We also found that scFv AS could efficiently cross the in vitro BBB model with a delivery efficiency of over 70% after a 60-min post-administration. The scFv AS was a monovalent antibody with an affinity constant (KD) of 5.5 × 10(-6) M and a binding threshold of 6.25 × 10(-4) μM for Aβ42 oligomers. The molecular docking simulations of Aβ42 to scFv AS revealed that scFv AS tends to approached Aβ42 oligomers and immature protofibrils mainly by their hydrophobic interaction and then drew Aβ42 molecule into the gap between VL and VH domains of scFv AS by hydrophilic interaction between scFv AS and the N-terminal region (residues 1-15) of Aβ42 and the hydrophobic interactions between scFv AS and the middle region (residues 20-33) of Aβ42. The combination of scFv AS with Aβ42 was realized likely through an induced-fit process. PMID:25330935

  1. A simple and robust approach to immobilization of antibody fragments.

    PubMed

    Ikonomova, Svetlana P; He, Ziming; Karlsson, Amy J

    2016-08-01

    Antibody fragments, such as the single-chain variable fragment (scFv), have much potential in research and diagnostics because of their antigen-binding ability similar to a full-sized antibody and their ease of production in microorganisms. Some applications of antibody fragments require immobilization on a surface, and we have established a simple immobilization method that is based on the biotin-streptavidin interaction and does not require a separate purification step. We genetically fused two biotinylation tags-the biotin carboxyl carrier protein (BCCP) or the AviTag minimal sequence-to six different scFvs (scFv13R4, scFvD10, scFv26-10, scFv3, scFv5, and scFv12) for site-specific biotinylation in vivo by endogenous biotin ligases produced by Escherichia coli. The biotinylated scFvs were immobilized onto streptavidin-coated plates directly from cell lysates, and immobilization was detected through enzyme-linked immunosorbent assays. All scFvs fusions were successfully immobilized, and scFvs biotinylated via the BCCP tag tended to immobilize better than those biotinylated via the AviTag, even when biotinylation efficiency was improved with the biotin ligase BirA. The ability of immobilized scFvs to bind antigens was confirmed using scFv13R4 and scFvD10 with their respective targets β-galactosidase and bacteriophage lambda head protein D (gpD). The immobilized scFv13R4 bound to β-galactosidase at the same level for both biotinylation tags when the surface was saturated with the scFv, and immobilized scFvs retained their functionality for at least 100days after immobilization. The simplicity and robustness of our method make it a promising approach for future applications that require antibody fragment immobilization. PMID:27142477

  2. Tetanus Neurotoxin Neutralizing Antibodies Screened from a Human Immune scFv Antibody Phage Display Library.

    PubMed

    Wang, Han; Yu, Rui; Fang, Ting; Yu, Ting; Chi, Xiangyang; Zhang, Xiaopeng; Liu, Shuling; Fu, Ling; Yu, Changming; Chen, Wei

    2016-01-01

    Tetanus neurotoxin (TeNT) produced by Clostridium tetani is one of the most poisonous protein substances. Neutralizing antibodies against TeNT can effectively prevent and cure toxicosis. Using purified Hc fragments of TeNT (TeNT-Hc) as an antigen, three specific neutralizing antibody clones recognizing different epitopes were selected from a human immune scFv antibody phage display library. The three antibodies (2-7G, 2-2D, and S-4-7H) can effectively inhibit the binding between TeNT-Hc and differentiated PC-12 cells in vitro. Moreover, 2-7G inhibited TeNT-Hc binding to the receptor via carbohydrate-binding sites of the W pocket while 2-2D and S-4-7H inhibited binding of the R pocket. Although no single mAb completely protected mice from the toxin, they could both prolong survival when challenged with 20 LD50s (50% of the lethal dose) of TeNT. When used together, the mAbs completely neutralized 1000 LD50s/mg Ab, indicating their high neutralizing potency in vivo. Antibodies recognizing different carbohydrate-binding pockets could have higher synergistic toxin neutralization activities than those that recognize the same pockets. These results could lead to further production of neutralizing antibody drugs against TeNT and indicate that using TeNT-Hc as an antigen for screening human antibodies for TeNT intoxication therapy from human immune antibody library was convenient and effective. PMID:27626445

  3. Selection of single chain variable fragment (scFv) antibodies from a hyperimmunized phage display library for the detection of the antibiotic monensin.

    PubMed

    Makvandi-Nejad, Shokouh; Sheedy, Claudia; Veldhuis, Linda; Richard, Gabrielle; Hall, J Christopher

    2010-08-31

    Concerns over the occurrence of the veterinary antibiotic monensin (MW 671Da) in animal food products and water have given rise to the need for a sensitive and rapid detection method. In this study, four monensin-specific single chain variable fragments (scFvs) were isolated from a hyperimmunized phage-displayed library originating from splenocytes of a mouse immunized with monensin conjugated to bovine serum albumin (BSA). The coding sequences of the scFvs were engineered in the order 5'-V(L)-linker-V(H)-3', where the linker encodes for Gly(10)Ser(7)Arg. Three rounds of selection were performed against monensin conjugated to chicken ovalbumin (OVA) and keyhole limpet hemocyanin (KLH), alternately. In the third round of selection, two different strategies, which differed in the number of washes and the concentration of the coating conjugates, were used to select for specific binders to monensin. A total of 376 clones from round two and three were screened for their specific binding to monensin conjugates and positive clones were sequenced. It was found that 80% of clones from round three contained a stop codon. After removing the stop codon by site-directed mutagenesis, ten binders with different amino acid sequences were subcloned into the vector pMED2 for soluble expression in Escherichia coli HB2151. Four of these scFvs bound to free monensin as determined using competitive fluorescence polarization assays (C-FPs). IC(50) values ranged from 0.031 and 231 microM. A cross-reactivity assay against salinomycin, lasalocid A, kanamycin and ampicillin revealed that the two best binders were highly specific to monensin. PMID:20600077

  4. Construction and Periplasmic Expression of the Anti-EGFRvIII ScFv Antibody Gene in Escherichia coli.

    PubMed

    Dewi, Kartika Sari; Retnoningrum, Debbie Sofie; Riani, Catur; Fuad, Asrul Muhamad

    2016-01-01

    In the previous study, we constructed an expression vector carrying the anti-EGFRvIII scFv antibody gene with VH-linker-VL orientation. The proteins were successfully produced in the periplasmic space of Escherichia coli. In this study, we substituted the inserted DNA with VL-linker-VH orientation of the anti-EGFRvIII scFv gene and analyzed its expression in E. coli. The DNA fragment was amplified from its cloning vector (pTz-rscFv), subsequently cloned into a previous expression vector containing the pelB signal sequence and his-tag, and then transformed into E. coli TOP10. The recombinant plasmids were characterized by restriction, PCR, and DNA sequencing analyses. The new anti-EGFRvIII scFv antibody proteins have been successfully expressed in the periplasmic compartment of E. coli Nico21(DE3) using 0.1 mM final concentration of IPTG induction. Total proteins, soluble periplasmic and cytoplasmic proteins, solubilized inclusion bodies, and extracellular proteins were analyzed by SDS-PAGE and Western Blot analyses. The results showed that soluble scFv proteins were found in all fractions except from the cytoplasmic space. PMID:27110505

  5. Construction and Periplasmic Expression of the Anti-EGFRvIII ScFv Antibody Gene in Escherichia coli

    PubMed Central

    Dewi, Kartika Sari; Retnoningrum, Debbie Sofie; Riani, Catur; Fuad, Asrul Muhamad

    2016-01-01

    In the previous study, we constructed an expression vector carrying the anti-EGFRvIII scFv antibody gene with VH-linker-VL orientation. The proteins were successfully produced in the periplasmic space of Escherichia coli. In this study, we substituted the inserted DNA with VL-linker-VH orientation of the anti-EGFRvIII scFv gene and analyzed its expression in E. coli. The DNA fragment was amplified from its cloning vector (pTz-rscFv), subsequently cloned into a previous expression vector containing the pelB signal sequence and his-tag, and then transformed into E. coli TOP10. The recombinant plasmids were characterized by restriction, PCR, and DNA sequencing analyses. The new anti-EGFRvIII scFv antibody proteins have been successfully expressed in the periplasmic compartment of E. coli Nico21(DE3) using 0.1 mM final concentration of IPTG induction. Total proteins, soluble periplasmic and cytoplasmic proteins, solubilized inclusion bodies, and extracellular proteins were analyzed by SDS-PAGE and Western Blot analyses. The results showed that soluble scFv proteins were found in all fractions except from the cytoplasmic space. PMID:27110505

  6. Production of recombinant antibody fragments in Bacillus megaterium

    PubMed Central

    Jordan, Eva; Hust, Michael; Roth, Andreas; Biedendieck, Rebekka; Schirrmann, Thomas; Jahn, Dieter; Dübel, Stefan

    2007-01-01

    Background Recombinant antibodies are essential reagents for research, diagnostics and therapy. The well established production host Escherichia coli relies on the secretion into the periplasmic space for antibody synthesis. Due to the outer membrane of Gram-negative bacteria, only a fraction of this material reaches the medium. Recently, the Gram-positive bacterium Bacillus megaterium was shown to efficiently secrete recombinant proteins into the growth medium. Here we evaluated B. megaterium for the recombinant production of antibody fragments. Results The lysozyme specific single chain Fv (scFv) fragment D1.3 was succesfully produced using B. megaterium. The impact of culture medium composition, gene expression time and culture temperatures on the production of functional scFv protein was systematically analyzed. A production and secretion at 41°C for 24 h using TB medium was optimal for this individual scFv. Interestingly, these parameters were very different to the optimal conditions for the expression of other proteins in B. megaterium. Per L culture supernatant, more than 400 μg of recombinant His6-tagged antibody fragment were purified by one step affinity chromatography. The material produced by B. megaterium showed an increased specific activity compared to material produced in E. coli. Conclusion High yields of functional scFv antibody fragments can be produced and secreted into the culture medium by B. megaterium, making this production system a reasonable alternative to E. coli. PMID:17224052

  7. Baculovirus display of single chain antibody (scFv) using a novel signal peptide

    PubMed Central

    2010-01-01

    Background Cells permissive to virus can become refractory to viral replication upon intracellular expression of single chain fragment variable (scFv) antibodies directed towards viral structural or regulatory proteins, or virus-coded enzymes. For example, an intrabody derived from MH-SVM33, a monoclonal antibody against a conserved C-terminal epitope of the HIV-1 matrix protein (MAp17), was found to exert an inhibitory effect on HIV-1 replication. Results Two versions of MH-SVM33-derived scFv were constructed in recombinant baculoviruses (BVs) and expressed in BV-infected Sf9 cells, N-myristoylation-competent scFvG2/p17 and N-myristoylation-incompetent scFvE2/p17 protein, both carrying a C-terminal HA tag. ScFvG2/p17 expression resulted in an insoluble, membrane-associated protein, whereas scFvE2/p17 was recovered in both soluble and membrane-incorporated forms. When coexpressed with the HIV-1 Pr55Gag precursor, scFvG2/p17 and scFvE2/p17 did not show any detectable negative effect on virus-like particle (VLP) assembly and egress, and both failed to be encapsidated in VLP. However, soluble scFvE2/p17 isolated from Sf9 cell lysates was capable of binding to its specific antigen, in the form of a synthetic p17 peptide or as Gag polyprotein-embedded epitope. Significant amounts of scFvE2/p17 were released in the extracellular medium of BV-infected cells in high-molecular weight, pelletable form. This particulate form corresponded to BV particles displaying scFvE2/p17 molecules, inserted into the BV envelope via the scFv N-terminal region. The BV-displayed scFvE2/p17 molecules were found to be immunologically functional, as they reacted with the C-terminal epitope of MAp17. Fusion of the N-terminal 18 amino acid residues from the scFvE2/p17 sequence (N18E2) to another scFv recognizing CD147 (scFv-M6-1B9) conferred the property of BV-display to the resulting chimeric scFv-N18E2/M6. Conclusion Expression of scFvE2/p17 in insect cells using a BV vector resulted in

  8. In vivo imaging of prostate cancer using an anti-PSMA scFv fragment as a probe.

    PubMed

    Mazzocco, Claire; Fracasso, Giulio; Germain-Genevois, Coralie; Dugot-Senant, Nathalie; Figini, Mariangela; Colombatti, Marco; Grenier, Nicolas; Couillaud, Franck

    2016-01-01

    We aimed to evaluate a fluorescent-labeled single chain variable fragment (scFv) of the anti-PSMA antibody as a specific probe for the detection of prostate cancer by in vivo fluorescence imaging. An orthotopic model of prostate cancer was generated by injecting LNCaP cells into the prostate lobe. ScFvD2B, a high affinity anti-PSMA antibody fragment, was labeled using a near-infrared fluorophore to generate a specific imaging probe (X770-scFvD2B). PSMA-unrelated scFv-X770 was used as a control. Probes were injected intravenously into mice with prostate tumors and fluorescence was monitored in vivo by fluorescence molecular tomography (FMT). In vitro assays showed that X770-scFvD2B specifically bound to PSMA and was internalized in PSMA-expressing LNCaP cells. After intravenous injection, X770-scFvD2B was detected in vivo by FMT in the prostate region. On excised prostates the scFv probe co-localized with the cancer cells and was found in PSMA-expressing cells. The PSMA-unrelated scFv used as a control did not label the prostate cancer cells. Our data demonstrate that scFvD2B is a high affinity contrast agent for in vivo detection of PSMA-expressing cells in the prostate. NIR-labeled scFvD2B could thus be further developed as a clinical probe for imaging-guided targeted biopsies. PMID:26996325

  9. In vivo imaging of prostate cancer using an anti-PSMA scFv fragment as a probe

    PubMed Central

    Mazzocco, Claire; Fracasso, Giulio; Germain-Genevois, Coralie; Dugot-Senant, Nathalie; Figini, Mariangela; Colombatti, Marco; Grenier, Nicolas; Couillaud, Franck

    2016-01-01

    We aimed to evaluate a fluorescent-labeled single chain variable fragment (scFv) of the anti-PSMA antibody as a specific probe for the detection of prostate cancer by in vivo fluorescence imaging. An orthotopic model of prostate cancer was generated by injecting LNCaP cells into the prostate lobe. ScFvD2B, a high affinity anti-PSMA antibody fragment, was labeled using a near-infrared fluorophore to generate a specific imaging probe (X770-scFvD2B). PSMA-unrelated scFv-X770 was used as a control. Probes were injected intravenously into mice with prostate tumors and fluorescence was monitored in vivo by fluorescence molecular tomography (FMT). In vitro assays showed that X770-scFvD2B specifically bound to PSMA and was internalized in PSMA-expressing LNCaP cells. After intravenous injection, X770-scFvD2B was detected in vivo by FMT in the prostate region. On excised prostates the scFv probe co-localized with the cancer cells and was found in PSMA-expressing cells. The PSMA-unrelated scFv used as a control did not label the prostate cancer cells. Our data demonstrate that scFvD2B is a high affinity contrast agent for in vivo detection of PSMA-expressing cells in the prostate. NIR-labeled scFvD2B could thus be further developed as a clinical probe for imaging-guided targeted biopsies. PMID:26996325

  10. Fluorescent labeling of antibody fragments using split GFP.

    PubMed

    Ferrara, Fortunato; Listwan, Pawel; Waldo, Geoffrey S; Bradbury, Andrew R M

    2011-01-01

    Antibody fragments are easily isolated from in vitro selection systems, such as phage and yeast display. Lacking the Fc portion of the antibody, they are usually labeled using small peptide tags recognized by antibodies. In this paper we present an efficient method to fluorescently label single chain Fvs (scFvs) using the split green fluorescent protein (GFP) system. A 13 amino acid tag, derived from the last beta strand of GFP (termed GFP11), is fused to the C terminus of the scFv. This tag has been engineered to be non-perturbing, and we were able to show that it exerted no effect on scFv expression or functionality when compared to a scFv without the GFP11 tag. Effective functional fluorescent labeling is demonstrated in a number of different assays, including fluorescence linked immunosorbant assays, flow cytometry and yeast display. Furthermore, we were able to show that this split GFP system can be used to determine the concentration of scFv in crude samples, as well an estimate of antibody affinity, without the need for antibody purification. We anticipate this system will be of widespread interest in antibody engineering and in vitro display systems. PMID:21998685

  11. Human scFv antibodies (Afribumabs) against Africanized bee venom: Advances in melittin recognition.

    PubMed

    Pessenda, Gabriela; Silva, Luciano C; Campos, Lucas B; Pacello, Elenice M; Pucca, Manuela B; Martinez, Edson Z; Barbosa, José E

    2016-03-15

    Africanized Apis mellifera bees, also known as killer bees, have an exceptional defensive instinct, characterized by mass attacks that may cause envenomation or death. From the years 2000-2013, 77,066 bee accidents occurred in Brazil. Bee venom comprises several substances, including melittin and phospholipase A2 (PLA2). Due to the lack of antivenom for bee envenomation, this study aimed to produce human monoclonal antibody fragments (single chain fragment variable; scFv), by using phage display technology. These fragments targeted melittin and PLA2, the two major components of bee venom, to minimize their toxic effects in cases of mass envenomation. Two phage antibody selections were performed using purified melittin. As the commercial melittin is contaminated with PLA2, phages specific to PLA2 were also obtained during one of the selections. Specific clones for melittin and PLA2 were selected for the production of soluble scFvs, named here Afribumabs: prefix: afrib- (from Africanized bee); stem/suffix: -umab (fully human antibody). Afribumabs 1 and 2 were tested in in vitro and in vivo assays to assess their ability to inhibit the toxic actions of purified melittin, PLA2, and crude bee venom. Afribumabs reduced hemolysis caused by purified melittin and PLA2 and by crude venom in vitro and reduced edema formation in the paws of mice and prolonged the survival of venom-injected animals in vivo. These results demonstrate that Afribumabs may contribute to the production of the first non-heterologous antivenom treatment against bee envenomation. Such a treatment may overcome some of the difficulties associated with conventional immunotherapy techniques. PMID:26829652

  12. Isolation of a high affinity scFv from a monoclonal antibody recognising the oncofoetal antigen 5T4.

    PubMed

    Shaw, D M; Embleton, M J; Westwater, C; Ryan, M G; Myers, K A; Kingsman, S M; Carroll, M W; Stern, P L

    2000-12-15

    The oncofoetal antigen 5T4 is a 72 kDa glycoprotein expressed at the cell surface. It is defined by a monoclonal antibody, mAb5T4, that recognises a conformational extracellular epitope in the molecule. Overexpression of 5T4 antigen by tumours of several types has been linked with disease progression and poor clinical outcome. Its restricted expression in non-malignant tissue makes 5T4 antigen a suitable target for the development of antibody directed therapies. The use of murine monoclonal antibodies for targeted therapy allows the tumour specific delivery of therapeutic agents. However, their use has several drawbacks, including a strong human anti-mouse immune (HAMA) response and limited tumour penetration due to the size of the molecules. The use of antibody fragments leads to improved targeting, pharmacokinetics and a reduced HAMA. A single chain antibody (scFv) comprising the variable regions of the mAb5T4 heavy and light chains has been expressed in Escherichia coli. The addition of a eukaryotic leader sequence allowed production in mammalian cells. The two 5T4 single chain antibodies, scFv5T4WT19 and LscFv5T4, described the same pattern of 5T4 antigen expression as mAb5T4 in normal human placenta and by FACS. Construction of a 5T4 extracellular domain-IgGFc fusion protein and its expression in COS-7 cells allowed the relative affinities of the antibodies to be compared by ELISA and measured in real time using a biosensor based assay. MAb5T4 has a high affinity, K(D)=1.8x10(-11) M, as did both single chain antibodies, scFv5T4WT19 K(D)=2.3x10(-9) M and LscFv5T4 K(D)=7.9x10(-10) M. The small size of this 5T4 specific scFv should allow construction of fusion proteins with a range of biological response modifiers to be prepared whilst retaining the improved pharmacokinetic properties of scFvs. PMID:11113573

  13. The production of antibody fragments and antibody fusion proteins by yeasts and filamentous fungi

    PubMed Central

    Joosten, Vivi; Lokman, Christien; van den Hondel, Cees AMJJ; Punt, Peter J

    2003-01-01

    In this review we will focus on the current status and views concerning the production of antibody fragments and antibody fusion proteins by yeasts and filamentous fungi. We will focus on single-chain antibody fragment production (scFv and VHH) by these lower eukaryotes and the possible applications of these proteins. Also the coupling of fragments to relevant enzymes or other components will be discussed. As an example of the fusion protein strategy, the 'magic bullet' approach for industrial applications, will be highlighted. PMID:12605725

  14. Peptide docking of HIV-1 p24 with single chain fragment variable (scFv) by CDOCKER algorithm

    NASA Astrophysics Data System (ADS)

    Karim, Hana Atiqah Abdul; Tayapiwatana, Chatchai; Nimmanpipug, Piyarat; Zain, Sharifuddin M.; Rahman, Noorsaadah Abdul; Lee, Vannajan Sanghiran

    2014-10-01

    In search for the important residues that might have involve in the binding interaction between the p24 caspid protein of HIV-1 fragment (MET68 - PRO90) with the single chain fragment variable (scFv) of FAB23.5, modern computational chemistry approach has been conducted and applied. The p24 fragment was initially taken out from the 1AFV protein molecule consisting of both light (VL) and heavy (VH) chains of FAB23.5 as well as the HIV-1 caspid protein. From there, the p24 (antigen) fragment was made to dock back into the protein pocket receptor (antibody) by using the CDOCKER algorithm to conduct the molecular docking process. The score calculated from the CDOCKER gave 15 possible docked poses with various docked ligand's positions, the interaction energy as well as the binding energy. The best docked pose that imitates the original antigen's position was determined and further processed to the In Situ minimization to obtain the residues interaction energy as well as to observe the hydrogen bonds interaction in the protein-peptide complex. Based on the results demonstrated, the specific residues in the complex that have shown immense lower interaction energies in the 5Å vicinity region from the peptide are from the heavy chain (VH:TYR105) and light chain (VL: ASN31, TYR32, and GLU97). Those residues play vital roles in the binding mechanism of Antibody-Antigen (Ab-Ag) complex of p24 with FAB23.5.

  15. Thermodynamic stability and flexibility characteristics of antibody fragment complexes

    PubMed Central

    Li, Tong; Verma, Deeptak; Tracka, Malgorzata B.; Casas-Finet, Jose; Livesay, Dennis R.; Jacobs, Donald J.

    2015-01-01

    Free energy landscapes, backbone flexibility and residue-residue couplings for being co-rigid or co-flexible are calculated from the minimal Distance Constraint Model (mDCM) on an exploratory dataset consisting of VL, scFv and Fab antibody fragments. Experimental heat capacity curves are reproduced markedly well, and an analysis of quantitative stability/flexibility relationships (QSFR) is applied to a representative VL domain and several complexes in the scFv and Fab forms. Global flexibility in the denatured ensemble typically decreases in the larger complexes due to domain-domain interfaces. Slight decreases in global flexibility also occur in the native state of the larger fragments, but with a concurrent large increase in correlated flexibility. Typically, a VL fragment has more co-rigid residue pairs when isolated compared to the scFv and Fab forms, where correlated flexibility appears upon complex formation. This context dependence on residue-residue couplings in the VL domain across length scales of a complex is consistent with the evolutionary hypothesis of antibody maturation. In comparing two scFv mutants with similar thermodynamic stability, local and long-ranged changes in backbone flexibility are observed. In the case of anti-p24 HIV-1 Fab, a variety of QSFR metrics were found to be atypical, which includes comparatively greater co-flexibility in the VH domain and less co-flexibility in the VL domain. Interestingly, this fragment is the only example of a polyspecific antibody in our dataset. Finally, the mDCM method is extended to cases where thermodynamic data is incomplete, enabling high throughput QSFR studies on large numbers of antibody fragments and their complexes. PMID:23855672

  16. Production, purification, and characterization of human scFv antibodies expressed in Saccharomyces cerevisiae, Pichia pastoris, and Escherichia coli.

    SciTech Connect

    Miller, Keith D.; Feldhaus, Jane M.; Gray, Sean A.; Siegel, Robert W.; Feldhaus, Michael J.

    2005-08-01

    Single chain (scFv) antibodies are used as affinity reagents for diagnostics, therapeutics, and proteomic analyses. The antibody discovery platform we use to identify novel antigen binders involves discovery, characterization, and production. The discovery and characterization components have previously been characterized but in order to fully utilize the capabilities of affinity reagents from our yeast surface display library, efforts were focused on developing a production component to obtain purified, soluble, and active scFvs. Instead of optimizing conditions to achieve maximum yield, efforts were focused on using a system that could quickly and easily produce and process hundreds of scFv antibodies. Heterologous protein expression in Saccharomyces cerevisiae, Pichia pastoris, and Escherichia coli were evaluated for their ability to rapidly, efficaciously, and consistently produce scFv antibodies for use in downstream proteomic applications. Following purification, the binding activity of several scFv antibodies were quantified using a novel Biacore assay. All three systems produced soluble scFv antibodies which ranged in activity from 0-99%. scFv antibody yields from Saccharomyces, Pichia, and E. coli were 1.5-4.2, 0.4-7.3, and 0.63-16.4 mg L-1 culture, respectively. For our purposes, expression in E. coli proved to be the quickest and most consistent way to obtain and characterize purified scFv for downstream applications. The E. coli expression system was also used to compare scFv production levels from the periplasm, inclusion bodies, and culture media. The E. coli production system was then used to produce variants of several scFv to determine structure function relationships.

  17. Effects of Environmental Factors on Soluble Expression of a Humanized Anti-TNF-α scFv Antibody in Escherichia coli

    PubMed Central

    Sina, Mohammad; Farajzadeh, Davoud; Dastmalchi, Siavoush

    2015-01-01

    Purpose: The bacterial cultivation conditions for obtaining anti-TNF-α single chain variable fragment (scFv) antibody as the soluble product in E. coli was investigated. Methods: To avoid the production of inclusion bodies, the effects of lactose, IPTG, incubation time, temperature, shaking protocol, medium additives (Mg+2, sucrose), pH, osmotic and heat shocks were examined. Samples from bacterial growth conditions with promising results of soluble expression of GST-hD2 scFv were affinity purified and quantified by SDS-PAGE and image processing for further evaluation. Results: The results showed that cultivation in LB medium under induction by low concentrations of lactose and incubation at 10 °C led to partial solubilization of the expressed anti-TNF-α scFv (GST-hD2). Other variables which showed promising increase in soluble expression of GST-hD2 were osmotic shock and addition of magnesium chloride. Furthermore, addition of sucrose to medium suppressed the expression of scFv completely. The other finding was that the addition of sorbitol decreased the growth rate of bacteria. Conclusion: It can be concluded that low cultivation temperature in the presence of low amount of inducer under a long incubation time or addition of magnesium chloride are the most effective environmental factors studied for obtaining the maximum solubilization of GST-hD2 recombinant protein. PMID:26819916

  18. A neutralizing recombinant single chain antibody, scFv, against BaP1, A P-I hemorrhagic metalloproteinase from Bothrops asper snake venom.

    PubMed

    Castro, J M A; Oliveira, T S; Silveira, C R F; Caporrino, M C; Rodriguez, D; Moura-da-Silva, A M; Ramos, O H P; Rucavado, A; Gutiérrez, J M; Magalhães, G S; Faquim-Mauro, E L; Fernandes, I

    2014-09-01

    BaP1 is a P-I class snake venom metalloproteinase (SVMP) relevant in the local tissue damage associated with envenomings by Bothrops asper, a medically important snake species in Central America and parts of South and North America. The main treatment for these accidents is the passive immunotherapy using antibodies raised in horses. In order to obtain more specific and batch-to-batch consistent antivenons, recombinant antibodies are considered a good option compared to animal immunization. We constructed a recombinant single chain variable fragment (scFv) from a monoclonal antibody against BaP1 (MABaP1) formerly secreted by a hybridoma clone. This recombinant antibody was cloned into pMST3 vector in fusion with SUMO protein and contains VH and VL domains linked by a flexible (G4S)3 polypeptide (scFvBaP1). The aim of this work was to produce scFvBaP1 and to evaluate its potential concerning the neutralization of biologically important activities of BaP1. The cytoplasmic expression of this construct was successfully achieved in C43 (DE3) bacteria. Our results showed that scFvBaP1-SUMO fusion protein presented an electrophoretic band of around 43 kDa from which SUMO alone corresponded to 13.6 kDa, and only the scFv was able to recognize BaP1 as well as the whole venom by ELISA. In contrast, neither an irrelevant scFv anti-LDL nor its MoAb partner recognized it. BaP1-induced fibrinolysis was significantly neutralized by scFvBaP1, but not by SUMO, in a concentration-dependent manner. In addition, scFvBaP1, as well as MaBaP1, completely neutralized in vivo hemorrhage, muscle necrosis, and inflammation induced by the toxin. Docking analyses revealed possible modes of interaction of the recombinant antibody with BaP1. Our data showed that scFv recognized BaP1 and whole B. asper venom, and neutralized biological effects of this SVMP. This scFv antibody can be used for understanding the molecular mechanisms of neutralization of SVMPs, and for exploring the potential of

  19. Multi-channeled single chain variable fragment (scFv) based microfluidic device for explosives detection.

    PubMed

    Charles, Paul T; Davis, Jasmine; Adams, André A; Anderson, George P; Liu, Jinny L; Deschamps, Jeffrey R; Kusterbeck, Anne W

    2015-11-01

    The development of explosives detection technologies has increased significantly over the years as environmental and national security agencies implement tighter pollution control measures and methods for improving homeland security. 2, 4, 6-Trinitrotoluene (TNT), known primarily as a component in munitions, has been targeted for both its toxicity and carcinogenic properties that if present at high concentrations can be a detriment to both humans, marine and plant ecosystems. Enabling end users with environmental detection and monitoring systems capable of providing real-time, qualitative and quantitative chemical analysis of these toxic compounds would be extremely beneficial. Reported herein is the development of a multi-channeled microfluidic device immobilized with single chain fragment variable (scFv) recombinant proteins specific for the explosive, TNT. Fluorescence displacement immunoassays performed under constant flow demonstrated trace level sensitivity and specificity for TNT. The utility of three multi-channeled devices immobilized with either (1) scFv recombinant protein, (2) biotinylated-scFv (bt-scFv) and (3) monoclonal anti-TNT (whole IgG molecule) were investigated and compared. Fluorescence dose response curves, crossreactivity measurements and limits of detection (LOD) for TNT were determined. Fluorescence displacement immunoassays for TNT in natural seawater demonstrated detection limits at sub-parts-per-billion levels (0.5 ppb) utilizing the microfluidic device with immobilized bt-scFv. PMID:26452845

  20. Expression and Purification of the scFv from Hybridoma Cells Secreting a Monoclonal Antibody Against S Protein of PEDV

    PubMed Central

    Zhu, Qinghe; Guo, Donghua; Feng, Li

    2013-01-01

    The variable regions of the heavy chain (VH) and light chain (VL) were amplified by RT-PCR from the hybridoma 6E6, which secretes the monoclonal antibody against PEDV S protein. The VL and VH amplicons were combined using SOE-PCR by a 12 amino acid flexible linker (SSGGGGSGGGGS), which produced the scFv gene (named scFv/6E6). After sequence analysis, the scFv/6E6 gene was cloned into the prokaryotic expression vector pGEX-6p-1 with a GST-tag. The recombinant scFv/6E6 protein was successfully expressed in recombinant Escherichia coli by IPTG induction. Moreover, the recombinant scFv/6E6 protein was purified from the inclusion body form by the gel-cutting measure followed by electroelution and dialysis. The recombinant scFv/6E6 protein reported here will provide some basis for further antiviral drug research based on the scFv molecule. PMID:23600505

  1. Expression of anti-tumor necrosis factor alpha (TNFα) single-chain variable fragment (scFv) in Spirodela punctata plants transformed with Agrobacterium tumefaciens.

    PubMed

    Balaji, Parthasarathy; Satheeshkumar, P K; Venkataraman, Krishnan; Vijayalakshmi, M A

    2016-05-01

    Therapeutic antibodies against tumor necrosis factor alpha (TNFα) have been considered effective for some of the autoimmune diseases such as rheumatoid arthritis, Crohn's diseases, and so on. But associated limitations of the current therapeutics in terms of cost, availability, and immunogenicity have necessitated the need for alternative candidates. Single-chain variable fragment (scFv) can negate the limitations tagged with the anti-TNFα therapeutics to a greater extent. In the present study, Spirodela punctata plants were transformed with anti-TNFα through in planta transformation using Agrobacterium tumefaciens strain, EHA105. Instead of cefotaxime, garlic extract (1 mg/mL) was used to remove the agrobacterial cells after cocultivation. To the best of our knowledge, this report shows for the first time the application of plant extracts in transgenic plant development. 95% of the plants survived screening under hygromycin. ScFv cDNA integration in the plant genomic DNA was confirmed at the molecular level by PCR. The transgenic protein expression was followed up to 10 months. Expression of scFv was confirmed by immunodot blot. Protein expression levels of up to 6.3% of total soluble protein were observed. β-Glucuronidase and green fluorescent protein expressions were also detected in the antibiotic resistant plants. The paper shows the generation of transgenic Spirodela punctuata plants through in planta transformation. PMID:25786575

  2. Engineered single-chain variable fragment antibody for immunodiagnosis of groundnut bud necrosis virus infection.

    PubMed

    Maheshwari, Yogita; Vijayanandraj, S; Jain, R K; Mandal, Bikash

    2015-05-01

    Few studies have been done on engineered antibodies for diagnosis of tospovirus infections. The present study was undertaken to develop a single-chain variable fragment (scFv) for specific diagnosis of infection by groundnut bud necrosis virus (GBNV), the most prevalent serogroup IV tospovirus in India. Heavy chain (372 nucleotide [nt]) and light chain (363 nt) variable region clones obtained from a hybridoma were used to make an scFv construct that expressed a ~29-kDa protein in E. coli. The scFv specifically detected GBNV in field samples of cowpea, groundnut, mung bean, and tomato, and it did not recognize watermelon bud necrosis virus, a close relative of GBNV belonging to tospovirus serogroup IV. This study for the first time demonstrated the application of a functional scFv against a serogroup-IV tospovirus. PMID:25698103

  3. Isolation of a new human scFv antibody recognizing a cell surface binding site to CEACAM1. Large yield production, purification and characterization in E. coli expression system.

    PubMed

    Moricoli, Diego; Laguardia, Maria Elena; Carbonella, Damiano Cosimo; Balducci, Maria Cristina; Dominici, Sabrina; Fiori, Valentina; Serafini, Giordano; Flego, Michela; Cianfriglia, Maurizio; Magnani, Mauro

    2014-01-01

    The CEACAM1 cell adhesion molecule has recently received considerable interest as a tumour target antigen since its re-expression often occurs in the advanced stages of multiple malignancies including malignant melanoma, non-small cell lung cancer and other types of solid tumors. In this study, we describe the expression-purification and characterization of the new single chain variable fragment (scFv) antibody named DIATHIS1, that recognizes the N-terminal IgV-like domain present in CEACAM1. Three validation batches show that the production process is robust and reproducible. The scFv DIATHIS1 is formulated as a naturally occurring mixture of monomer and dimer. The antibody is biophysically stable at low temperature (-80°C), different concentrations and remains biologically active for at least 24months. The thermal stability of scFv DIATHIS1 at 37°C shows important features for its activity in vivo. The dimer behaves as a reservoir converting slowly into monomer. The monomer and dimer forms of scFv DIATHIS1 were isolated and characterized, showing high reactivity for CEACAM1. This new composition of antibody could have advantageous pharmacokinetics parameters over conventional scFv for in vivo applications. PMID:24184403

  4. The Development of a Recombinant scFv Monoclonal Antibody Targeting Canine CD20 for Use in Comparative Medicine

    PubMed Central

    Jain, Saurabh; Aresu, Luca; Comazzi, Stefano; Shi, Jianguo; Worrall, Erin; Clayton, John; Humphries, William; Hemmington, Sandra; Davis, Paul; Murray, Euan; Limeneh, Asmare A.; Ball, Kathryn; Ruckova, Eva; Muller, Petr; Vojtesek, Borek; Fahraeus, Robin; Argyle, David; Hupp, Ted R.

    2016-01-01

    Monoclonal antibodies are leading agents for therapeutic treatment of human diseases, but are limited in use by the paucity of clinically relevant models for validation. Sporadic canine tumours mimic the features of some human equivalents. Developing canine immunotherapeutics can be an approach for modeling human disease responses. Rituximab is a pioneering agent used to treat human hematological malignancies. Biologic mimics that target canine CD20 are just being developed by the biotechnology industry. Towards a comparative canine-human model system, we have developed a novel anti-CD20 monoclonal antibody (NCD1.2) that binds both human and canine CD20. NCD1.2 has a sub-nanomolar Kd as defined by an octet red binding assay. Using FACS, NCD1.2 binds to clinically derived canine cells including B-cells in peripheral blood and in different histotypes of B-cell lymphoma. Immunohistochemical staining of canine tissues indicates that the NCD1.2 binds to membrane localized cells in Diffuse Large B-cell lymphoma, Marginal Zone Lymphoma, and other canine B-cell lymphomas. We cloned the heavy and light chains of NCD1.2 from hybridomas to determine whether active scaffolds can be acquired as future biologics tools. The VH and VL genes from the hybridomas were cloned using degenerate primers and packaged as single chains (scFv) into a phage-display library. Surprisingly, we identified two scFv (scFv-3 and scFv-7) isolated from the hybridoma with bioactivity towards CD20. The two scFv had identical VH genes but different VL genes and identical CDR3s, indicating that at least two light chain mRNAs are encoded by NCD1.2 hybridoma cells. Both scFv-3 and scFv-7 were cloned into mammalian vectors for secretion in CHO cells and the antibodies were bioactive towards recombinant CD20 protein or peptide. The scFv-3 and scFv-7 were cloned into an ADEPT-CPG2 bioconjugate vector where bioactivity was retained when expressed in bacterial systems. These data identify a recombinant anti-CD20

  5. The Development of a Recombinant scFv Monoclonal Antibody Targeting Canine CD20 for Use in Comparative Medicine.

    PubMed

    Jain, Saurabh; Aresu, Luca; Comazzi, Stefano; Shi, Jianguo; Worrall, Erin; Clayton, John; Humphries, William; Hemmington, Sandra; Davis, Paul; Murray, Euan; Limeneh, Asmare A; Ball, Kathryn; Ruckova, Eva; Muller, Petr; Vojtesek, Borek; Fahraeus, Robin; Argyle, David; Hupp, Ted R

    2016-01-01

    Monoclonal antibodies are leading agents for therapeutic treatment of human diseases, but are limited in use by the paucity of clinically relevant models for validation. Sporadic canine tumours mimic the features of some human equivalents. Developing canine immunotherapeutics can be an approach for modeling human disease responses. Rituximab is a pioneering agent used to treat human hematological malignancies. Biologic mimics that target canine CD20 are just being developed by the biotechnology industry. Towards a comparative canine-human model system, we have developed a novel anti-CD20 monoclonal antibody (NCD1.2) that binds both human and canine CD20. NCD1.2 has a sub-nanomolar Kd as defined by an octet red binding assay. Using FACS, NCD1.2 binds to clinically derived canine cells including B-cells in peripheral blood and in different histotypes of B-cell lymphoma. Immunohistochemical staining of canine tissues indicates that the NCD1.2 binds to membrane localized cells in Diffuse Large B-cell lymphoma, Marginal Zone Lymphoma, and other canine B-cell lymphomas. We cloned the heavy and light chains of NCD1.2 from hybridomas to determine whether active scaffolds can be acquired as future biologics tools. The VH and VL genes from the hybridomas were cloned using degenerate primers and packaged as single chains (scFv) into a phage-display library. Surprisingly, we identified two scFv (scFv-3 and scFv-7) isolated from the hybridoma with bioactivity towards CD20. The two scFv had identical VH genes but different VL genes and identical CDR3s, indicating that at least two light chain mRNAs are encoded by NCD1.2 hybridoma cells. Both scFv-3 and scFv-7 were cloned into mammalian vectors for secretion in CHO cells and the antibodies were bioactive towards recombinant CD20 protein or peptide. The scFv-3 and scFv-7 were cloned into an ADEPT-CPG2 bioconjugate vector where bioactivity was retained when expressed in bacterial systems. These data identify a recombinant anti-CD20

  6. Latex agglutination test based on single-chain Fv recombinant antibody fragment.

    PubMed

    Golchin, M; Khalili-Yazdi, A; Karamouzian, M; Abareghi, A

    2012-01-01

    Recombinant antibodies have been proposed as invaluable tools for various therapeutic and diagnostic purposes. Here, we describe the development of a novel latex agglutination test (LAT) using single-chain Fv recombinant antibody fragment for the detection of K99(+) enterotoxigenic Escherichia coli strains. For the production of a single-chain Fv antibody fragment (scFv) against the major colonization factor (FanC) of K99 antigen, the scFv gene was integrated into a bacterial expression vector under the control of T7 promoter. After high-level expression of soluble scFv (approximately 50 mg/l) in flask cultivation of E. coli DE3 and purification, scFv was immobilized on different latex particles, and then, these sensitized beads were used in LAT. Results obtained with our latex reagents revealed that the recombinant antibody-coated particles were able to give a good agglutination signal with purified antigen, intact cells displaying this protein and clinical specimens. The strength of agglutination of scFv-coated beads for antigen was comparable to that of polyclonal anti-K99-coated particles. However, the assay proved to be simple and rapid, similar to conventional LATs, and owing to more convenient and economical production of recombinant antibodies, they can be considered as a useful reagent for replacing monoclonal antibodies in LATs. PMID:21916915

  7. Generation of a naïve human single chain variable fragment (scFv) library for the identification of monoclonal scFv against Salmonella Typhi Hemolysin E antigen.

    PubMed

    Lim, Bee Nar; Chin, Chai Fung; Choong, Yee Siew; Ismail, Asma; Lim, Theam Soon

    2016-07-01

    Antibody phage display is a useful tool for the isolation and identification of monoclonal antibodies. Naive antibody libraries are able to overcome the limitations associated with the traditional hybridoma method for monoclonal antibody generation. Antibody phage display is also a preferred method for antibody generation against toxins as it does not suffer from toxicity mediated complications. Here, we describe a naïve multi ethnic scFv antibody library generated via two-step cloning with an estimated diversity of 2 × 10(9). The antibody library was used to screen for monoclonal antibodies against Hemolysin E antigen, a pore forming toxin produced by Salmonella enterica serovar Typhi. A soluble monoclonal scFv antibody against the HlyE toxin (IgM scFv D7 anti-hlyE) was isolated from the library. This shows the value of the naïve library to generate antibodies against toxin targets in addition to the potential use of the library to isolate antibodies against other immunogenic targets. PMID:27090555

  8. Possible therapeutic potential of a recombinant group 2 grass pollen allergen-specific antibody fragment.

    PubMed

    Gadermaier, E; Flicker, S; Blatt, K; Valent, P; Valenta, R

    2014-02-01

    The induction of blocking IgG antibodies that compete with IgE for allergen binding is one important mechanism of allergen-specific immunotherapy. The application of blocking antibodies may be an alternative treatment strategy. A synthetic gene coding for a single-chain fragment (ScFv) specific for the major timothy grass pollen allergen Phl p 2 was inserted into plasmid pCANTAB 5 E, and the recombinant ScFv was expressed in Escherichia coli and purified by affinity chromatography. The ScFv was tested for allergen binding by ELISA, and its association and dissociation were measured by surface plasmon resonance (Biacore) technology. The ability of the ScFv to inhibit allergic patients' IgE binding to Phl p 2 and Phl p 2-induced basophil degranulation was studied by ELISA competition and basophil activation (CD203c) assays. We report the expression, purification, biochemical and immunological characterization of a monomeric single-chain fragment (ScFv) of human origin specific for the major timothy grass pollen allergen, Phl p 2. The Phl p 2-ScFv showed high affinity binding to the allergen and blocked the binding of allergic patients' polyclonal IgE to Phl p 2 up to 98%. Furthermore, it inhibited allergen-induced basophil activation. The Phl p 2-ScFv inhibited allergic patients' IgE binding to Phl p 2 as well as Phl p 2-induced basophil activation and might be useful for passive immunotherapy of grass pollen allergy. PMID:24251384

  9. Blocking monocyte transmigration in in vitro system by a human antibody scFv anti-CD99. Efficient large scale purification from periplasmic inclusion bodies in E. coli expression system.

    PubMed

    Moricoli, Diego; Muller, William Anthony; Carbonella, Damiano Cosimo; Balducci, Maria Cristina; Dominici, Sabrina; Watson, Richard; Fiori, Valentina; Weber, Evan; Cianfriglia, Maurizio; Scotlandi, Katia; Magnani, Mauro

    2014-06-01

    Migration of leukocytes into site of inflammation involves several steps mediated by various families of adhesion molecules. CD99 play a significant role in transendothelial migration (TEM) of leukocytes. Inhibition of TEM by specific monoclonal antibody (mAb) can provide a potent therapeutic approach to treating inflammatory conditions. However, the therapeutic utilization of whole IgG can lead to an inappropriate activation of Fc receptor-expressing cells, inducing serious adverse side effects due to cytokine release. In this regard, specific recombinant antibody in single chain variable fragments (scFvs) originated by phage library may offer a solution by affecting TEM function in a safe clinical context. However, this consideration requires large scale production of functional scFv antibodies and the absence of toxic reagents utilized for solubilization and refolding step of inclusion bodies that may discourage industrial application of these antibody fragments. In order to apply the scFv anti-CD99 named C7A in a clinical setting, we herein describe an efficient and large scale production of the antibody fragments expressed in E. coli as periplasmic insoluble protein avoiding gel filtration chromatography approach, and laborious refolding step pre- and post-purification. Using differential salt elution which is a simple, reproducible and effective procedure we are able to separate scFv in monomer format from aggregates. The purified scFv antibody C7A exhibits inhibitory activity comparable to an antagonistic conventional mAb, thus providing an excellent agent for blocking CD99 signaling. This protocol can be useful for the successful purification of other monomeric scFvs which are expressed as periplasmic inclusion bodies in bacterial systems. PMID:24798881

  10. Optimization of a single-chain antibody fragment overexpression in Escherichia coli using response surface methodology.

    PubMed

    Akbari, V; Sadeghi, H Mir Mohammad; Jafarian-Dehkordi, A; Chou, C Perry; Abedi, D

    2015-01-01

    Human epidermal growth factor receptor (HER) family plays an important role in various types of cancers. As a result, antibodies against HER and the mechanism of antigen-antibody binding action are under active investigation. We previously constructed a single-chain variable fragment (ScFv) against HER2, i.e. anti-Her2 ScFv, for expressing in the Escherichia coli. In the present study, we report the optimization of anti-Her2 ScFv expression in an E. coli host of BL21 (DE3) pLysS using response surface methodology based on tuning of three cultivation variables, including isopropyl-beta-D-thiogalactopyranoside (IPTG) concentration, temperature and post-induction time. A model for protein expression according to the Box-Behnken design predicted a maximal anti-Her2 ScFv expression at 37 °C, a post-induction time of 10.45 h and 0.75 mM IPTG. In addition, strategies based on inclusion body isolation and affinity chromatography were applied to purify anti-Her2 ScFv. The purity of the final product for inclusion bodies isolation and purification by Ni-NTA resin were 70 % and 95 %, respectively. The solubilization of the inclusion bodies was carried out using two denaturant agents, guanidine hydrochloride and urea. The present study showed that guanidine hydrochloride was more effective than urea in solubilizing the inclusion bodies. PMID:26430460

  11. Production of a recombinant anti-human CD4 single-chain variable-fragment antibody using phage display technology and its expression in Escherichia coli.

    PubMed

    Babaei, Arash; Zarkesh-Esfahani, Sayyed Hamid; Gharagozloo, Marjan

    2011-05-01

    Single-chain variable fragment (scFv) is a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of immunoglobulin, connected with a short linker peptide of 10 to about 20 amino acids. In this study, the scFv of a monoclonal antibody against the third domain of human CD4 was cloned from OKT4 hybridoma cells using the phage display technique and produced in E. coli. The expression, production, and purification of anti-CD4 scFv were tested using SDS-PAGE and Western blot, and the specificity of anti-CD4 scFv was examined using ELISA. A 31 kDa recombinant anti-CD4 scFv was expressed and produced in bacteria, which was confirmed by SDS-PAGE and Western blot assays. Sequence analysis proved the ScFv structure of the construct. It was able to bind to CD4 in quality ELISA assay. The canonical structure of anti-CD4 scFv antibody was obtained using the SWISS_MODEL bioinformatics tool for comparing with the scFv general structure. To the best of our knowledge, this is the first report for generating scFv against human CD4 antigen. Engineered anti-CD4 scFv could be used in immunological studies, including fluorochrome conjugation, bispecific antibody production, bifunctional protein synthesis, and other genetic engineering manipulations. Since the binding site of our product is domain 3 (D3) of the CD4 molecule and different from the CD4 immunological main domain, including D1 and D2, further studies are needed to evaluate the anti-CD4 scFv potential for diagnostic and therapeutic applications. PMID:21617352

  12. Single chain antibody fragment with serine protease inhibitory property capable of neutralizing toxicity of Trimeresurus mucrosquamatus venom.

    PubMed

    Lee, Yu-Ching; Chen, Wang-Chuan; Liang, Meng-Huei; Lee, Chi-Hsin; Tsai, Keng-Chang; Chiang, Jen-Ron; Chiang, Liao-Chun; Chen, Chi-Ching; Chang, Chang-Yu; Lee, Ching-Hsiao; Leu, Sy-Jye; Yang, Yi-Yuan

    2015-05-01

    Trimeresurus mucrosquamatus (TM) is one of majorities of snake envenomation with necrotic and hemorrhagic toxin in Taiwan. In this study, chickens were used as an alternative animal model for immunization with TM venom. Using phage display technology to process four rounds of panning, selected single chain variable fragments (scFv) could specifically recognize TM venom proteins, which were later identified as a group of homogeneous venom serine protease. The specific scFv antibodies showed various inhibitory effects on sheep RBC lysis induced by TM venom using an indirect hemolytic assay in vitro. In addition, the survival times of mice were extended to certain degrees when treated with these scFv antibodies individually or in a combination. To elucidate the inhibitory mechanism, we used molecular modeling to build up the serine protease structure to simulate the possible interactions with scFv antibodies. The results suggested that the CDR-loop of the scFv antibodies (3S10 or 4S1) might bind at the 99-loop of venom serine protease so as to affect substrate access due to the partial collapse of the subsite S2 and the partial movement of the subsite S4. It is hoped these chicken-derived antibodies could be applied to develop diagnostic and therapeutic agents against snakebites. PMID:25769957

  13. Flow cytometry-based methods for assessing soluble scFv activities and detecting pathogen antigens in solution

    SciTech Connect

    Gray, Sean; Weigel, Kris M.; Miller, Keith D.; Ndung'u, Joseph; Buscher, Philippe; Tran, Thao N.; Baird, Cheryl L.; Cangelosi, Gerard A.

    2010-04-01

    Novel methods are reported for evaluating and utilizing single chain fragment variable (scFv) antibodies derived from yeast-display libraries. Yeast-display was used to select scFv specific to invariant surface glycoproteins (ISG) of Trypanosoma brucei. A limiting step in the isolation of scFv from nonimmune libraries is the conversion of highly active yeast-displayed scFv into soluble antibodies that can be used in standard immunoassays. Challenges include limited solubility or activity following secretion and purification of scFv. For this reason, few scFv derived from yeast-display platforms have moved into development and implementation as diagnostic reagents. To address this problem, assays were developed that employ both yeastdisplayed and secreted scFv as analytical reagents. The first is a competitive inhibition flow cytometry (CIFC) assay that detects secreted scFv by virtue of its ability to competitively inhibit the binding of biotinylated antigen to yeast-displayed scFv. The second is an epitope binning assay that uses secreted scFv toidentify additional yeast-displayed scFv that bind nonoverlapping or noncompeting epitopes on an antigen. The epitope binning assay was used not only to identify sandwich assay pairs with yeast-displayed scFv, but also to identify active soluble scFv present in low concentration in a crude expression extract. Finally, a CIFC assay was developed that bypasses entirely the need for soluble scFv expression, by using yeast displayed scFv to detect unlabeled antigen in samples. These methods will facilitate the continued development and practical implementation of scFv derived from yeast-display libraries.

  14. Expression and purification of a novel therapeutic single-chain variable fragment antibody against BNP from inclusion bodies of Escherichia coli.

    PubMed

    Bu, Dawei; Zhou, Yuwei; Tang, Jian; Jing, Fang; Zhang, Wei

    2013-12-01

    Abnormal brain natriuretic peptide (BNP) secretion is regarded as the dominating mechanism of cerebral salt wasting syndrome (CSW), which results from a renal loss of sodium and water during intracranial disease leading to hyponatremia. Scale preparation of therapeutic single-chain variable fragment (scFv) that can neutralize elevated circulating BNP may have potential value for clinical use. In this report, we used a recently isolated humanized anti-BNP scFv fragment (3C1) as model antibody (Ab) to evaluate the potential of scale production of this therapeutic protein. The truncated gene encoding for scFv fragment cloned in pET22b (+) was mainly overexpressed as inclusion bodies in Escherichia coli (E. coli) Rosetta (DE3) pLysS cells. The insoluble fragment was solubilized and purified by Ni-NTA agarose resin under denaturation conditions, and recovered via an effective refolding buffer containing 50 mM Tris-HCl, pH 8.0, 0.15 M NaCl, 1 mM EDTA, 0.5 M arginine, 2 mM GSH, 1 mM GSSG, and 5% glycerol. The refolded scFv fragment was concentrated by PEG20000, and dialyzed in PBS (containing 5% glycerol, pH 7.4). The final yield was approximately 10.2 mg active scFv fragment per liter of culture (3.4 g wet weight cells). The scFv fragment was more than 95% pure assessed by SDS-PAGE assay. Recombinant scFv fragment with His tag displayed its immunoreactivity with anti-His tag Ab by western blotting. ELISA showed the scFv fragment specifically bound to BNP, and it displayed similar activity as the traditional anti-BNP monoclonal Ab (mAb). Thus, the current strategy allows convenient small-scale production of this therapeutic protein. PMID:24128692

  15. Anti-Aβ single-chain variable fragment antibodies exert synergistic neuroprotective activities in Drosophila models of Alzheimer's disease.

    PubMed

    Fernandez-Funez, Pedro; Zhang, Yan; Sanchez-Garcia, Jonatan; de Mena, Lorena; Khare, Swati; Golde, Todd E; Levites, Yona; Rincon-Limas, Diego E

    2015-11-01

    Both active and passive immunotherapy protocols decrease insoluble amyloid-ß42 (Aß42) peptide in animal models, suggesting potential therapeutic applications against the main pathological trigger in Alzheimer's disease (AD). However, recent clinical trials have reported no significant benefits from humanized anti-Aß42 antibodies. Engineered single-chain variable fragment antibodies (scFv) are much smaller and can easily penetrate the brain, but identifying the most effective scFvs in murine AD models is slow and costly. We show here that scFvs against the N- and C-terminus of Aß42 (scFv9 and scFV42.2, respectively) that decrease insoluble Aß42 in CRND mice are neuroprotective in Drosophila models of Aß42 and amyloid precursor protein neurotoxicity. Both scFv9 and scFv42.2 suppress eye toxicity, reduce cell death in brain neurons, protect the structural integrity of dendritic terminals in brain neurons and delay locomotor dysfunction. Additionally, we show for the first time that co-expression of both anti-Aß scFvs display synergistic neuroprotective activities, suggesting that combined therapies targeting distinct Aß42 epitopes can be more effective than targeting a single epitope. Overall, we demonstrate the feasibility of using Drosophila as a first step for characterizing neuroprotective anti-Aß scFvs in vivo and identifying scFv combinations with synergistic neuroprotective activities. PMID:26253732

  16. Generation of Anti-Idiotype scFv for Pharmacokinetic Measurement in Lymphoma Patients Treated with Chimera Anti-CD22 Antibody SM03

    PubMed Central

    Zhao, Qi; Wong, Pui-Fan; Lee, Susanna S. T.; Leung, Shui-On; Cheung, Wing-Tai; Wang, Jun-Zhi

    2014-01-01

    Pre-clinical and clinical studies of therapeutic antibodies require highly specific reagents to examine their immune responses, bio-distributions, immunogenicity, and pharmacodynamics in patients. Selective antigen-mimicking anti-idiotype antibody facilitates the assessment of therapeutic antibody in the detection, quantitation and characterization of antibody immune responses. Using mouse specific degenerate primer pairs and splenocytic RNA, we generated an idiotype antibody-immunized phage-displayed scFv library in which an anti-idiotype antibody against the therapeutic chimera anti-CD22 antibody SM03 was isolated. The anti-idiotype scFv recognized the idiotype of anti-CD22 antibody and inhibited binding of SM03 to CD22 on Raji cell surface. The anti-idiotype scFv was subsequently classified as Ab2γ type. Moreover, our results also demonstrated firstly that the anti-idiotype scFv could be used for pharmacokinetic measurement of circulating residual antibody in lymphoma patients treated with chimera anti-CD22 monoclonal antibody SM03. Of important, the present approach could be easily adopted to generate anti-idiotype antibodies for therapeutic antibodies targeting membrane proteins, saving the cost and time for producing a soluble antigen. PMID:24816427

  17. Construction and bacterial expression of a recombinant single-chain antibody fragment against Wuchereria bancrofti SXP-1 antigen for the diagnosis of lymphatic filariasis.

    PubMed

    Kamatchi, R; Charumathi, J; Ravishankaran, R; Kaliraj, P; Meenakshisundaram, S

    2016-01-01

    Global programmes to eliminate lymphatic filariasis (GPELF) require mapping, monitoring and evaluation using filarial antigen diagnostic kits. To meet this objective, a functional single-chain fragment variable (ScFv) specific for filarial Wuchereria bancrofti SXP-1 (Wb-SXP-1) antigen was constructed for the diagnosis of active filarial infection, an alternative to the production of complete antibodies using hybridomas. The variable heavy chain (VH) and the variable light chain (kappa) (Vκ) genes were amplified from the mouse hybridoma cell line and were linked together with a flexible linker by overlap extension polymerase chain reaction (PCR). The ScFv construct (Vκ-Linker-VH) was expressed as a fusion protein with N-terminal His tag in Escherichia coli and purified using immobilized metal affinity chromatography (IMAC) without the addition of reducing agents. Immunoblotting and sandwich enzyme-linked immunosorbent assay (ELISA) were used to analyse the antigen binding affinity of purified ScFv. The purified ScFv was found to recognize recombinant and native Wb-SXP-1 antigen in microfilariae (Mf)-positive patient sera. The affinity of ScFv was comparable with that of the monoclonal antibody. The development of recombinant ScFv to replace monoclonal antibody for detection of filarial antigen was achieved. The recombinant ScFv was purified, on-column refolded and its detection ability validated using field samples. PMID:26693887

  18. Development of an Immunoassay for Chloramphenicol Based on the Preparation of a Specific Single-Chain Variable Fragment Antibody.

    PubMed

    Du, Xin-Jun; Zhou, Xiao-Nan; Li, Ping; Sheng, Wei; Ducancel, Frédéric; Wang, Shuo

    2016-04-13

    Specific antibodies are essential for the immune detection of small molecule contaminants. In the present study, the heavy and light variable regions (VH and VL) of the immunoglobulin genes from a hybridoma secreting a chloramphenicol (CAP)-specific monoclonal antibody (mAb) were cloned and sequenced. In addition, the light and heavy chains obtained from the monoclonal antibody were separated using SDS-PAGE and analyzed using Orbitrap mass spectrometry. The results of DNA sequencing and mass spectrometry analysis were compared, and the VH and VL chains specific for CAP were determined and used to construct a single-chain variable fragment (scFv). This fragment was recombinantly expressed as a soluble scFv-alkaline phosphatase fusion protein and used to develop a direct competitive ELISA. Compared with the parent mAb, scFv exhibits lower sensitivity but better food matrix resistance. This work highlights the application of engineered antibodies for CAP detection. PMID:27003441

  19. Cell-free production of Gaussia princeps luciferase – antibody fragment bioconjugates for ex vivo detection of tumor cells

    PubMed Central

    Patel, Kedar G.; Ng, Patrick P.; Kuo, Chiung-Chi; Levy, Shoshana; Levy, Ronald; Swartz, James R.

    2016-01-01

    Antibody fragments (scFvs) fused to luciferase reporter proteins have been used as highly sensitive optical imaging probes. Gaussia princeps luciferase (GLuc) is an attractive choice for a reporter protein because it is small and bright and does not require ATP to stimulate bioluminescence-producing reactions. Both GLuc and scFv proteins contain multiple disulfide bonds, and consequently the production of active and properly folded GLuc–scFv fusions is challenging. We therefore produced both proteins individually in active form, followed by covalent coupling to produce the intended conjugate. We used an Escherichia coli-based cell-free protein synthesis (CFPS) platform to produce GLuc and scFv proteins containing non-natural amino acids (nnAAs) for subsequent conjugation by azide–alkyne click chemistry. GLuc mutants with exposed alkyne reactive groups were produced by global replacement of methionine residues in CFPS. Antibody fragment scFvs contained a single exposed azide group using a scheme for site-specific incorporation of tyrosine analogs. Incorporation of tyrosine analogs at specific sites in proteins was performed using an engineered orthogonal tRNA–tRNA synthetase pair from an archaebacterium. The unique azide and alkyne side chains in GLuc and the antibody fragment scFv facilitated conjugation by click chemistry. GLuc–scFv conjugates were shown to differentiate between cells expressing a surface target of the scFv and cells that did not carry this marker. PMID:19852937

  20. A human scFv antibody generation pipeline for proteome research.

    PubMed

    Hust, Michael; Meyer, Torsten; Voedisch, Bernd; Rülker, Torsten; Thie, Holger; El-Ghezal, Aymen; Kirsch, Martina Inga; Schütte, Mark; Helmsing, Saskia; Meier, Doris; Schirrmann, Thomas; Dübel, Stefan

    2011-04-10

    The functional decryption of the human proteome is the challenge which follows the sequencing of the human genome. Specific binders to every human protein are key reagents for this purpose. In vitro antibody selection using phage display offers one possible solution that can meet the demand for 25,000 or more antibodies, but needs substantial standardisation and minimalisation. To evaluate this potential, three human, naive antibody gene libraries (HAL4/7/8) were constructed and a standardised antibody selection pipeline was set up. The quality of the libraries and the selection pipeline was validated with 110 antigens, including human, other mammalian, fungal or bacterial proteins, viruses or haptens. Furthermore, the abundance of VH, kappa and lambda subfamilies during library cloning and the E. coli based phage display system on library packaging and the selection of scFvs was evaluated from the analysis of 435 individual antibodies, resulting in the first comprehensive comparison of V gene subfamily use for all steps of an antibody phage display pipeline. Further, a compatible cassette vector set for E. coli and mammalian expression of antibody fragments is described, allowing in vivo biotinylation, enzyme fusion and Fc fusion. PMID:20883731

  1. Site-specific scFv labelling with invertase via Sortase A mechanism as a platform for antibody-antigen detection using the personal glucose meter

    PubMed Central

    Ismail, Nur Faezee; Lim, Theam Soon

    2016-01-01

    Antibody labelling to reporter molecules is gaining popularity due to its many potential applications for diagnostics and therapeutics. However, non-directional bioconjugation methods which are commonly used often results in the loss of target binding capabilities. Therefore, a site-specific enzymatic based bioconjugation such as sortase-mediated transpeptidation allows for a more rapid and efficient method of antibody conjugation for diagnostic applications. Here we describe the utilization of sortase A bioconjugation to conjugate a single chain fragment variable (scFv) to the extracellular invertase (invB) from Zymomonas mobilis with the aim of developing an invertase based immunoassay. In addition, conjugation to enhanced green fluorescent protein (eGFP) was also validated to show the flexibility of the method. The invertase conjugated complex was successfully applied for the detection of antibody-antigen interaction using a personal glucose meter (PGM) for assay readout. The setup was used in both a direct and competitive assay highlighting the robustness of the conjugate for assay development. The method provides an alternative conjugation process to allow easy exchange of antibodies to facilitate rapid development of diagnostic assays for various diseases on the PGM platform. PMID:26782912

  2. Site-specific scFv labelling with invertase via Sortase A mechanism as a platform for antibody-antigen detection using the personal glucose meter.

    PubMed

    Ismail, Nur Faezee; Lim, Theam Soon

    2016-01-01

    Antibody labelling to reporter molecules is gaining popularity due to its many potential applications for diagnostics and therapeutics. However, non-directional bioconjugation methods which are commonly used often results in the loss of target binding capabilities. Therefore, a site-specific enzymatic based bioconjugation such as sortase-mediated transpeptidation allows for a more rapid and efficient method of antibody conjugation for diagnostic applications. Here we describe the utilization of sortase A bioconjugation to conjugate a single chain fragment variable (scFv) to the extracellular invertase (invB) from Zymomonas mobilis with the aim of developing an invertase based immunoassay. In addition, conjugation to enhanced green fluorescent protein (eGFP) was also validated to show the flexibility of the method. The invertase conjugated complex was successfully applied for the detection of antibody-antigen interaction using a personal glucose meter (PGM) for assay readout. The setup was used in both a direct and competitive assay highlighting the robustness of the conjugate for assay development. The method provides an alternative conjugation process to allow easy exchange of antibodies to facilitate rapid development of diagnostic assays for various diseases on the PGM platform. PMID:26782912

  3. Femtosecond spectroscopy probes the folding quality of antibody fragments expressed as GFP fusions in the cytoplasm

    SciTech Connect

    Didier, P.; Weiss, E.; Sibler, A.-P.; Philibert, P.; Martineau, P.; Bigot, J.-Y.; Guidoni, L.

    2008-02-22

    Time-resolved femtosecond spectroscopy can improve the application of green fluorescent proteins (GFPs) as protein-folding reporters. The study of ultrafast excited-state dynamics (ESD) of GFP fused to single chain variable fragment (scFv) antibody fragments, allowed us to define and measure an empirical parameter that only depends on the folding quality (FQ) of the fusion. This method has been applied to the analysis of genetic fusions expressed in the bacterial cytoplasm and allowed us to distinguish folded and thus functional antibody fragments (high FQ) with respect to misfolded antibody fragments. Moreover, these findings were strongly correlated to the behavior of the same scFvs expressed in animal cells. This method is based on the sensitivity of the ESD to the modifications in the tertiary structure of the GFP induced by the aggregation state of the fusion partner. This approach may be applicable to the study of the FQ of polypeptides over-expressed under reducing conditions.

  4. Stainless steel surface functionalization for immobilization of antibody fragments for cardiovascular applications.

    PubMed

    Foerster, A; Hołowacz, I; Sunil Kumar, G B; Anandakumar, S; Wall, J G; Wawrzyńska, M; Paprocka, M; Kantor, A; Kraskiewicz, H; Olsztyńska-Janus, S; Hinder, S J; Bialy, D; Podbielska, H; Kopaczyńska, M

    2016-04-01

    Stainless steel 316 L material is commonly used for the production of coronary and peripheral vessel stents. Effective biofunctionalization is a key to improving the performance and safety of the stents after implantation. This paper reports the method for the immobilization of recombinant antibody fragments (scFv) on stainless steel 316 L to facilitate human endothelial progenitor cell (EPC) growth and thus improve cell viability of the implanted stents for cardiovascular applications. The modification of stent surface was conducted in three steps. First the stent surface was coated with titania based coating to increase the density of hydroxyl groups for successful silanization. Then silanization with 3 aminopropyltriethoxysilane (APTS) was performed to provide the surface with amine groups which presence was verified using FTIR, XPS, and fluorescence microscopy. The maximum density of amine groups (4.8*10(-5) mol/cm(2)) on the surface was reached after reaction taking place in ethanol for 1 h at 60 °C and 0.04M APTS. On such prepared surface the glycosylated scFv were subsequently successfully immobilized. The influence of oxidation of scFv glycan moieties and the temperature on scFv coating were investigated. The fluorescence and confocal microscopy study indicated that the densest and most uniformly coated surface with scFv was obtained at 37 °C after oxidation of glycan chain. The results demonstrate that the scFv cannot be efficiently immobilized without prior aminosilanization of the surface. The effect of the chemical modification on the cell viability of EPC line 55.1 (HucPEC-55.1) was performed indicating that the modifications to the 316 L stainless steel are non-toxic to EPCs. PMID:26566715

  5. Immunodiagnosis of platelet activation in immune thrombocytopenia through scFv antibodies cognate to activated IIb3 integrins

    PubMed Central

    Bhoria, Preeti; Varma, Neelam; Malhotra, Pankaj; Varma, Subhash; Luthra-Guptasarma, Manni

    2015-01-01

    Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder characterized by low platelet count and presence of IgG autoantibodies to platelet surface glycoproteins, such as αIIbβ3 and GPIb/IX. Our previous work has shown that platelets in ITP patients exist in an activated state. Two different marker-based approaches are used to study the course of platelet activation: (1) binding of PAC-1 antibody, signifying a change in αIIbβ3 conformation, and (2) expression of P-selectin, signifying alpha granule content release from platelets. Here, we describe the development of a new scFv antibody (R38) that, compared with PAC-1, appears to better distinguish between platelets of ITP patients and healthy controls. Notably, R38 was generated using commercially sourced resting-state integrin that was coated on a microtiter plate. Its ability to distinguish between ITP patients and healthy controls thus suggests that inadvertent integrin activation caused by coating involves a conformational change and exposure of a cryptic epitope. This report also describes for the first time the potential use of an scFv antibody in the immunodiagnosis of platelet activation in ITP patients. PMID:26301697

  6. In Vivo Tumor Targeting and Imaging with Engineered Trivalent Antibody Fragments Containing Collagen-Derived Sequences

    PubMed Central

    Cuesta, Ángel M.; Sánchez-Martín, David; Sanz, Laura; Bonet, Jaume; Compte, Marta; Kremer, Leonor; Blanco, Francisco J.; Oliva, Baldomero; Álvarez-Vallina, Luis

    2009-01-01

    There is an urgent need to develop new and effective agents for cancer targeting. In this work, a multivalent antibody is characterized in vivo in living animals. The antibody, termed “trimerbody”, comprises a single-chain antibody (scFv) fragment connected to the N-terminal trimerization subdomain of collagen XVIII NC1 by a flexible linker. As indicated by computer graphic modeling, the trimerbody has a tripod-shaped structure with three highly flexible scFv heads radially outward oriented. Trimerbodies are trimeric in solution and exhibited multivalent binding, which provides them with at least a 100-fold increase in functional affinity than the monovalent scFv. Our results also demonstrate the feasibility of producing functional bispecific trimerbodies, which concurrently bind two different ligands. A trimerbody specific for the carcinoembryonic antigen (CEA), a classic tumor-associated antigen, showed efficient tumor targeting after systemic administration in mice bearing CEA-positive tumors. Importantly, a trimerbody that recognizes an angiogenesis-associated laminin epitope, showed excellent tumor localization in several cancer types, including fibrosarcomas and carcinomas. These results illustrate the potential of this new antibody format for imaging and therapeutic applications, and suggest that some laminin epitopes might be universal targets for cancer targeting. PMID:19401768

  7. Production and characterization of a biotinylated single-chain variable fragment antibody for detection of parathion-methyl.

    PubMed

    Wang, Huimin; Zhao, Fengchun; Han, Xiao; Yang, Zhengyou

    2016-10-01

    In this article, we reported the development of a biotinylated single-chain variable fragment (scFv) antibody based indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) for parathion-methyl (PM) detection. Firstly, a phage display library was generated using a pre-immunized BALB/C mouse against a specific hapten of PM. After four rounds of panning, the scFv gene fragments were transferred into a secreted expression vector. Then, the scFv antibodies were secreted expressed and screened by IC-ELISA against PM. The selected scFv antibody was fused with a biotin acceptor domain (BAD) and inserted into pET-28a(+) vector for high-level expression in Escherichia coli BL2 (DE3). After optimizing expression conditions, the scFv-BAD antibody was expressed as a soluble protein and biotinylated in vitro by the E. coli biotin ligase (BirA). Subsequently, the biotinylated scFv-BAD antibody was purified with a high yield of 59.2 ± 3.7 mg/L of culture, and was characterized by SDS-PAGE and western blotting. Finally, based on the biotinylated scFv-BAD, a sensitive IC-ELISA for detection of PM was developed, and the 50% inhibition value (IC50) of PM was determined as 14.5 ng/mL, with a limit of detection (LOD, IC10) of 0.9 ng/mL. Cross-reactivity (CR) studies revealed that the scFv antibody showed desirable specificity for PM. PMID:27181246

  8. CD176 single-chain variable antibody fragment inhibits the adhesion of cancer cells to endothelial cells and hepatocytes.

    PubMed

    Liu, Jiangnan; Yi, Bin; Zhang, Zhe; Cao, Yi

    2016-06-01

    CD176 (Thomsen-Friedenreich antigen) is a tumor-associated carbohydrate epitope (glycotope) functionally involved in blood spread and liver metastasis of cancer cells by mediating the adhesion of cancer cells to endothelial cells and hepatocytes, respectively. CD176 could be a promising target for antitumor immunotherapy. We applied B lymphocytes obtained from mice immunized with CD176 antigen and constructed a phage display library. A positive clone of CD176 single-chain variable antibody fragment (scFv) was successfully screened from this library. The CD176 scFv was expressed in Escherichia coli and purified. The purified scFv can bind to the natural CD176 expressed on the surface of cancer cells. Furthermore, the CD176 scFv inhibits the adhesion of CD176(+) cancer cells to endothelial cells and hepatocytes. This CD176 scFv provides a basis for future development of recombinant CD176-specific antibodies that can be used in therapeutic application. PMID:27090911

  9. Expression of a single-chain variable-fragment antibody against a Fusarium virguliforme toxin peptide enhances tolerance to sudden death syndrome in transgenic soybean plants.

    PubMed

    Brar, Hargeet K; Bhattacharyya, Madan K

    2012-06-01

    Plants do not produce antibodies. However, plants can correctly assemble functional antibody molecules encoded by mammalian antibody genes. Many plant diseases are caused by pathogen toxins. One such disease is the soybean sudden death syndrome (SDS). SDS is a serious disease caused by the fungal pathogen Fusarium virguliforme. The pathogen, however, has never been isolated from diseased foliar tissues. Thus, one or more toxins produced by the pathogen have been considered to cause foliar SDS. One of these possible toxins, FvTox1, was recently identified. We investigated whether expression of anti-FvTox1 single-chain variable-fragment (scFv) antibody in transgenic soybean can confer resistance to foliar SDS. We have created two scFv antibody genes, Anti-FvTox1-1 and Anti-FvTox1-2, encoding anti-FvTox1 scFv antibodies from RNAs of a hybridoma cell line that expresses mouse monoclonal anti-FvTox1 7E8 antibody. Both anti-FvTox1 scFv antibodies interacted with an antigenic site of FvTox1 that binds to mouse monoclonal anti-FvTox1 7E8 antibody. Binding of FvTox1 by the anti-FvTox1 scFv antibodies, expressed in either Escherichia coli or transgenic soybean roots, was initially verified on nitrocellulose membranes. Expression of anti-FvTox1-1 in stable transgenic soybean plants resulted in enhanced foliar SDS resistance compared with that in nontransgenic control plants. Our results suggest that i) FvTox1 is an important pathogenicity factor for foliar SDS development and ii) expression of scFv antibodies against pathogen toxins could be a suitable biotechnology approach for protecting crop plants from toxin-induced diseases. PMID:22397408

  10. In situ gastrointestinal protection against anthrax edema toxin by single-chain antibody fragment producing lactobacilli

    PubMed Central

    2011-01-01

    Background Anthrax is caused by the bacterium Bacillus anthracis and is regarded as one of the most prominent bioterrorism threats. Anthrax toxicity is induced by the tripartite toxin complex, composed of the receptor-binding anthrax protective antigen and the two enzymatic subunits, lethal factor and edema factor. Recombinant lactobacilli have previously been used to deliver antibody fragments directed against surface epitopes of a variety of pathogens, including Streptococcus mutans, Porphyromonas gingivalis, and rotavirus. Here, we addressed whether or not anthrax toxins could be targeted and neutralised in the gastrointestinal tract by lactobacilli producing recombinant antibody fragments as a model system for toxin neutralisation in the gastrointestinal lumen. Results The neutralising anti-PA scFv, 1H, was expressed in L. paracasei as a secreted protein, a cell wall-anchored protein or both secreted and wall-anchored protein. Cell wall display on lactobacilli and PA binding of the anchored constructs was confirmed by flow cytometry analysis. Binding of secreted or attached scFv produced by lactobacilli to PA were verified by ELISA. Both construct were able to protect macrophages in an in vitro cytotoxicity assay. Finally, lactobacilli producing the cell wall attached scFv were able to neutralise the activity of anthrax edema toxin in the GI tract of mice, in vivo. Conclusion We have developed lactobacilli expressing a neutralising scFv fragment against the PA antigen of the anthrax toxin, which can provide protection against anthrax toxins both in vitro and in vivo. Utilising engineered lactobacilli therapeutically for neutralising toxins in the gastrointestinal tract can potential be expanded to provide protection against a range of additional gastrointestinal pathogens. The ability of lactobacilli to colonise the gastrointestinal tract may allow the system to be used both prophylactically and therapeutically. PMID:22185669

  11. [Construction and panning of scFv phage display library against recombinant interleukin 4 receptor].

    PubMed

    Yang, Guangyong; Guo, Haitao; Liu, Ximing; He, Guangzhi; Tian, Weiyi; Cai, Kun; Wang, Ping; Wang, Wenjia

    2016-06-01

    Objective To construct the recombinant human interleukin 4 receptor (rhIL-4R) single-chain Fv (scFv) antibody library by phage display technique to obtain the anti-IL-4R scFv clones selected from the library. Methods Total RNA was extracted from splenocytes of the BALB/c mice immunized with rhIL-4R. Complementary DNA fragments of variable heavy (VH) and variable light (VL) chains of the antibodies were prepared by reverse transcription PCR and assembled into scFv by splice overlap extension PCR (SOE-PCR). Both scFv and the pCANTAB5E vector were respectively double-digested with restriction endonuclease Sfi I and Not I, connected with T4 ligase, and then transformed into the competent cells E.coli TG1; it was cultured in medium to obtain the phage scFv antibody library; after three rounds of enrichment and panning, the specific antigen scFv with high affinity was selected for the sequencing. Results After three rounds of panning, we obtained a diversity of approximately 2×10(8) anti-rhIL-4R scFv antibody library. Sequencing analysis of one positive clone showed that the anti-rhIL-4R scFv was 741 bp and coded 247 amino acids. The analysis of VBASE2 database indicated that VH and VL gene sequences of anti-rhIL-4R protein all had three complementarity determining regions and four backbone areas.Conclusion The anti-rhIL-4R scFv was obtained from the scFv antibody library. PMID:27371853

  12. Optimal Neutralization of Centruroides noxius Venom Is Understood through a Structural Complex between Two Antibody Fragments and the Cn2 Toxin.

    PubMed

    Riaño-Umbarila, Lidia; Ledezma-Candanoza, Luis M; Serrano-Posada, Hugo; Fernández-Taboada, Guillermo; Olamendi-Portugal, Timoteo; Rojas-Trejo, Sonia; Gómez-Ramírez, Ilse V; Rudiño-Piñera, Enrique; Possani, Lourival D; Becerril, Baltazar

    2016-01-22

    The current trend of using recombinant antibody fragments in research to develop novel antidotes against scorpion stings has achieved excellent results. The polyclonal character of commercial antivenoms, obtained through the immunization of animals and which contain several neutralizing antibodies that recognize different epitopes on the toxins, guarantees the neutralization of the venoms. To avoid the use of animals, we aimed to develop an equivalent recombinant antivenom composed of a few neutralizing single chain antibody fragments (scFvs) that bind to two different epitopes on the scorpion toxins. In this study, we obtained scFv RU1 derived from scFv C1. RU1 showed a good capacity to neutralize the Cn2 toxin and whole venom of the scorpion Centruroides noxius. Previously, we had produced scFv LR, obtained from a different parental fragment (scFv 3F). LR also showed a similar neutralizing capacity. The simultaneous administration of both scFvs resulted in improved protection, which was translated as a rapid recovery of previously poisoned animals. The crystallographic structure of the ternary complex scFv LR-Cn2-scFv RU1 allowed us to identify the areas of interaction of both scFvs with the toxin, which correspond to non-overlapping sites. The epitope recognized by scFv RU1 seems to be related to a greater efficiency in the neutralization of the whole venom. In addition, the structural analysis of the complex helped us to explain the cross-reactivity of these scFvs and how they neutralize the venom. PMID:26589800

  13. Improved soluble expression of a single-chain antibody fragment in E. coli for targeting CA125 in epithelial ovarian cancer.

    PubMed

    Sharma, Sai Kiran; Suresh, Mavanur R; Wuest, Frank R

    2014-10-01

    Production of antibody fragments in heterologous hosts such as Escherichiacoli provides a unique and cost-effective method to develop engineered vectors for tumor targeting. A single-chain Fragment variable (scFv) of the murine monoclonal antibody MAb-B43.13 targeting CA125 in epithelial ovarian cancer was previously developed, expressed, purified and proposed as a functional targeting entity for biomedical applications. However, the yields from its soluble expression in heterologous systems were very low for any practical use in preclinical translational research; leave alone the defeated objective of convenient and cost-effective production. In the present work, the anti-CA125 scFv gene was re-organized and sub-cloned into pET-22b(+) vector to be in frame with the pelB leader peptide for periplasmic localization and C-terminal hexa-histidine tag to facilitate downstream purification. Six variants of the scFv were constructed to investigate the impact of variable domain orientations, inter-domain peptide linker sequences and codon optimization on the soluble expression of the scFv using Rosetta 2(DE3) as the E. coli host supplemented with tRNAs for rare codons. Expression in shake flask cultures under the control of an inducible T7 promoter and subsequent purification by cobalt based immobilized metal affinity chromatography yielded differential amounts of high purity scFv for all constructs. Here, we report up to 14-fold increase in the soluble expression of the scFv primarily as a result of codon optimization with minor effects from inter-domain peptide linkers and variable domain orientation in the anti-CA125 scFv molecule. All the scFv constructs expressed and purified were found to be immunoreactive for in vitro targeting of CA125 antigen. PMID:25079010

  14. Delineation of BmSXP antibody V-gene usage from a lymphatic filariasis based immune scFv antibody library.

    PubMed

    Rahumatullah, Anizah; Ahmad, Azimah; Noordin, Rahmah; Lim, Theam Soon

    2015-10-01

    Phage display technology is an important tool for antibody generation or selection. This study describes the development of a scFv library and the subsequent analysis of identified monoclonal antibodies against BmSXP, a recombinant antigen for lymphatic filariasis. The immune library was generated from blood of lymphatic filariasis infected individuals. A TA based intermediary cloning approach was used to increase cloning efficiency for the library construction process. A diverse immune scFv library of 10(8) was generated. Six unique monoclonal antibodies were identified from the 50 isolated clones against BmSXP. Analysis of the clones showed a bias for the IgHV3 and Vκ1 (45.5%) and IgHV2 and Vκ3 (27.3%) gene family. The most favored J segment for light chain is IgKJ1 (45.5%). The most favored D and J segment for heavy chain are IgHD6-13 (75%) and IgHJ3 (47.7%). The information may suggest a predisposition of certain V genes in antibody responses against lymphatic filariasis. PMID:26277276

  15. Expression and characterization of single-chain variable fragment antibody against staphylococcal enterotoxin A in Escherichia coli.

    PubMed

    Chen, Weifeng; Hu, Li; Liu, Aiping; Li, Jinquan; Chen, Fusheng; Wang, Xiaohong

    2014-11-01

    The staphylococcal enterotoxins (SEs) are potent gastrointestinal exotoxins synthesized by Staphylococcus aureus, which is responsible for various diseases including septicemia, food poisoning, and toxic shock syndrome, as well as bovine mastitis. Among them, staphylococcal enterotoxin A (SEA) is one of the most commonly present serotypes in staphylococcal food poisoning cases. In this study, the stable hybridoma 3C12 producing anti-SEA monoclonal antibody was established with an equilibrium dissociation constant (KD) of 1.48 × 10(-8) mol·L(-1), its ScFv-coding genes were obtained and then the anti-SEA single chain variable fragment (ScFv) protein was expressed in Escherichia coli. Characterization of the expressed target ScFv protein was analyzed by sodium dodecyl sulfate - polyacrylamide gel electrophoresis, Western blot, and enzyme-linked immunosorbent assay. The results demonstrated that the recombinant anti-SEA ScFv protein retained a specific binding activity for SEA, and the KD value of the soluble ScFv was about 3.75 × 10(-7) mol·L(-1). The overall yield of bioactive anti-SEA ScFv in E. coli flask culture was more than 10 mg·L(-1). PMID:25322256

  16. A method to confer Protein L binding ability to any antibody fragment

    PubMed Central

    Lakhrif, Zineb; Pugnière, Martine; Henriquet, Corinne; di Tommaso, Anne; Dimier-Poisson, Isabelle; Billiald, Philippe; Juste, Matthieu O.; Aubrey, Nicolas

    2016-01-01

    abstract Recombinant antibody single-chain variable fragments (scFv) are difficult to purify homogeneously from a protein complex mixture. The most effective, specific and fastest method of purification is an affinity chromatography on Protein L (PpL) matrix. This protein is a multi-domain bacterial surface protein that is able to interact with conformational patterns on kappa light chains. It mainly recognizes amino acid residues located at the VL FR1 and some residues in the variable and constant (CL) domain. Not all kappa chains are recognized, however, and the lack of CL can reduce the interaction. From a scFv composed of IGKV10-94 according to IMGT®, it is possible, with several mutations, to transfer the motif from the IGKV12-46 naturally recognized by the PpL, and, with the single mutation T8P, to confer PpL recognition with a higher affinity. A second mutation S24R greatly improves the affinity, in particular by modifying the dissociation rate (kd). The equilibrium dissociation constant (KD) was measured at 7.2 10-11 M by surface plasmon resonance. It was possible to confer PpL recognition to all kappa chains. This protein interaction can be modulated according to the characteristics of scFv (e.g., stability) and their use with conjugated PpL. This work could be extrapolated to recombinant monoclonal antibodies, and offers an alternative for protein A purification and detection. PMID:26683650

  17. Gladiolus plants transformed with single-chain variable fragment antibodies to Cucumber mosaic virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transgenic plants of Gladiolus ‘Peter Pears’ or ‘Jenny Lee’ were developed that contain single-chain variable fragments (scFv) to Cucumber mosaic virus (CMV) subgroup I or II. The CMV subgroup I heavy and light chain scFv fragments were placed under control of either the duplicated CaMV 35S or suga...

  18. Antitumor effects of an engineered and energized fusion protein consisting of an anti-CD20 scFv fragment and lidamycin.

    PubMed

    Fang, Hong; Miao, Qingfang; Zhang, Shenghua; Cheng, Xin; Xiong, Dongsheng; Zhen, Yongsu

    2011-03-01

    Antibody-based fusion proteins are the next generation of antibody therapies for cancer and other diseases. CD20 antigen, which is overexpressed on cell membranes in nearly 95% of cases of B-cell Non-Hodgkin's Lymphoma, is an attractive target for the therapy of B-lymphoid malignancies. Lidamycin (LDM) is a potent enediyne-containing antitumor antibiotic that now has entered phase II clinical trials. In this study, we prepared an engineered fusion protein, scFv-LDP, consisting of an anti-CD20 scFv fragment and the apoprotein LDP of LDM using DNA recombination. After purification and refolding, scFv-LDP was found to bind specifically to CD20-positive lymphoma cells using ELISA and indirect immunofluorescent cytochemical staining assays. The energized fusion protein scFv-LDP-AE was obtained using molecular reconstitution of the active chromophore AE of LDM and scFv-LDP. MTT assay revealed potent cytotoxicity of scFv-LDP-AE to CD20-positive Raji and Daudi cells, with IC(50) values of 1.21×10(-11) and 6.24×10(-11) mol L(-1), respectively. An in vivo subcutaneous xenograft model of CD20-positive B cell lymphoma in BALB/c (nu/nu) mice was also utilized. Drugs were given intravenously on day 14 and 21 after tumor transplantation. In terms of maximal tolerated doses, scFv-LDP-AE at 0.3 mg kg(-1) suppressed tumor growth by 79.3%, and LDM at 0.05 mg kg(-1) by 68.6% (P<0.05). Results suggested scFv-LDP-AE could be a potential candidate for tumor-targeting therapy. PMID:21416325

  19. A novel variable antibody fragment dimerized by leucine zippers with enhanced neutralizing potency against rabies virus G protein compared to its corresponding single-chain variable antibody fragment.

    PubMed

    Li, Zhuang; Cheng, Yue; Xi, Hualong; Gu, Tiejun; Yuan, Ruosen; Chen, Xiaoxu; Jiang, Chunlai; Kong, Wei; Wu, Yongge

    2015-12-01

    Fatal rabies can be prevented effectively by post-exposure prophylactic (PEP) with rabies immunoglobulin (RIG). Single-chain variable fragments (scFv), which are composed of a variable heavy chain (VH) and a variable light chain (VL) connected by a peptide linker, can potentially be used to replace RIG. However, in our previous study, a scFv (scFV57S) specific for the rabies virus (RV) G protein showed a lower neutralizing potency than that of its parent IgG due to lower stability and altered peptide assembly pattern. In monoclonal antibodies, the VH and VL interact non-covalently, while in scFvs the VH is connected covalently with the VL by the artificial linker. In this study, we constructed and expressed two peptides 57VL-JUN-HIS and 57VH-FOS-HA in Escherichia coli. The well-known Fos and Jun leucine zippers were utilized to dimerize VH and VL similarly to the IgG counterpart. The two peptides assembled to form zipFv57S in vitro. Due to the greater similarity in structure with IgG, the zipFv57S protein showed a higher binding ability and affinity resulting in notable improvement of in vitro neutralizing activity over its corresponding scFv. The zipFv57S protein was also found to be more stable and showed similar protective rate as RIG in mice challenged with a lethal dose of RV. Our results not only indicated zipFv57S as an ideal alternative for RIG in PEP but also offered a novel and efficient hetero-dimerization pattern of VH and VL leading to enhanced neutralizing potency. PMID:26325475

  20. Targeting Prostate Cancer Cells In Vivo Using a Rapidly Internalizing Novel Human Single-Chain Antibody Fragment

    PubMed Central

    He, Jiang; Wang, Yong; Feng, Jinjin; Zhu, Xiaodong; Lan, Xiaoli; Iyer, Arun K.; Zhang, Niu; Seo, Youngho; VanBrocklin, Henry F.; Liu, Bin

    2010-01-01

    Human antibodies targeting prostate cancer cell surface epitopes may be useful for imaging and therapy. The objective of this study was to evaluate the tumor targeting of an internalizing human antibody fragment, a small-size platform, to provide high contrast in a mouse model of human prostate carcinoma. Methods A prostate tumor-targeting single-chain antibody fragment (scFv), UA20, along with a nonbinding control scFv, N3M2, were labeled with 99mTc and evaluated for binding and rapid internalization into human prostate tumor cells in vitro and tumor homing in vivo using xenograft models. For the in vitro studies, the labeled UA20 scFv was incubated at 37°C for 1 h with metastatic prostate cancer cells (DU145) to assess the total cellular uptake versus intracellular uptake. For the animal studies, labeled UA20 and N3M2 scFvs were administered to athymic mice implanted subcutaneously with DU145 cells. Mice were imaged with small-animal SPECT/CT with concomitant biodistribution at 1 and 3 h after injection. Results The UA20 scFv was labeled in 55%–65% yield and remained stable in phosphate buffer within 24 h. The labeled UA20 scFv was taken up specifically by prostate tumor cells. Internalization was rapid, because incubation at 37°C for less than 1 h resulted in 93% internalization of total cell-associated scFvs. In animal studies, SPECT/CT showed significant tumor uptake as early as 1 h after injection. At 3 h after injection, tumor uptake was 4.4 percentage injected dose per gram (%ID/g), significantly greater than all organs or tissues studied (liver, 2.7 %ID/g; other organs or tissues, <1 %ID/g), except the kidneys (81.4 %ID/g), giving tumor-to-blood and tumor-to-muscle ratios of 12:1 and 70:1, respectively. In contrast, the control antibody exhibited a tumor uptake of only 0.26 %ID/g, similar to that of muscle and fat. Tumor-specific targeting was evidenced by reduced tumor uptake of nearly 70% on administration of a 10-fold excess of unlabeled UA20 scFv

  1. Diagnostic Potential of Recombinant scFv Antibodies Generated Against Hemagglutinin Protein of Influenza A Virus

    PubMed Central

    Rajput, Roopali; Sharma, Gaurav; Rawat, Varsha; Gautam, Anju; Kumar, Binod; Pattnaik, B.; Pradhan, H. K.; Khanna, Madhu

    2015-01-01

    Human influenza A viruses have been the cause of enormous socio-economic losses worldwide. In order to combat such a notorious pathogen, hemagglutinin protein (HA) has been a preferred target for generation of neutralizing-antibodies as potent therapeutic/diagnostic agents. In the present study, recombinant anti-HA single chain variable fragment antibodies were constructed using the phage-display technology to aid in diagnosis and treatment of human influenza A virus infections. Spleen cells of mice hyper-immunized with A/New Caledonia/20/99 (H1N1) virus were used as the source for recombinant antibody (rAb) production. The antigen-binding phages were quantified after six rounds of bio-panning against A/New Caledonia/20/99 (H1N1), A/California/07/2009 (H1N1)-like, or A/Udorn/307/72(H3N2) viruses. The maximum phage yield was for the A/New Caledonia/20/99 (H1N1), however, considerable cross-reactivity was observed for the other virus strains as well. The HA-specific polyclonal rAb preparation was subjected to selection of single clones for identification of high reactive relatively conserved epitopes. The high-affinity rAbs were tested against certain known conserved HA epitopes by peptide ELISA. Three recombinant mAbs showed reactivity with both the H1N1 strains and one (C5) showed binding with all the three viral strains. The C5 antibody was thus used for development of an ELISA test for diagnosis of influenza virus infection. Based on the sample size in the current analysis, the ELISA test demonstrated 83.9% sensitivity and 100% specificity. Thus, the ELISA, developed in our study, may prove as a cheaper alternative to the presently used real time RT–PCR test for detection of human influenza A viruses in clinical specimens, which will be beneficial, especially in the developing countries. PMID:26388868

  2. Preparation and identification of a single-chain variable fragment antibody against Newcastle diseases virus F48E9.

    PubMed

    Li, Benqiang; Ye, Jiaxin; Lin, Yuan; Wang, Man; Zhu, Jianguo

    2014-10-15

    This article describes a proposed method for convenient and efficient detection of Newcastle diseases virus (NDV) that uses the fusion of single-chain variable fragment (scFv) and pOPE101 vector. In order to select the single chain variable fragment (scFv) against NDV F48E9, the total RNA was extracted from the spleen of immunized chicken, and then was converted into cDNA via the reverse transcription. The scFv was spliced by using splice-overlap extension polymerase chain reaction (SOE-PCR). The scFv gene was cloned into a pOPE101 vector and expressed in E. coli. Under the optimized conditions, antibody affinity was studied by indirect ELISA. One positive clone was selected by ELISA screening, named ZL.6. Based on the positive clone and the germline sequence, the results of sequence analysis showed that there are more variation in CDR of VH and VL. In addition, BHK21 cell culture was conducted to examine the potential antiviral activity of ZL.6. The experimental result demonstrated that ZL.6 was able to neutralize NDV F48E9 which infected BHK21 cells. So ZL.6 will be proved useful for further characterization of NDV as potential diagnostic tool and therapeutic agent. PMID:25183016

  3. "Diabodies": small bivalent and bispecific antibody fragments.

    PubMed Central

    Holliger, P; Prospero, T; Winter, G

    1993-01-01

    Bivalent and bispecific antibodies and their fragments have immense potential for practical application. Here we describe the design of small antibody fragments with two antigen-binding sites. The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) on the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. As indicated by a computer graphic model of the dimers, the two pairs of domains can pack together with the antigen-binding sites pointing in opposite directions. The dimeric antibody fragments, or "diabodies," can be designed for bivalent or bispecific interactions. Starting from the monoclonal antibodies NQ11.7.22 (NQ11) and D1.3 directed against the hapten phenyloxazolone and hen egg lysozyme, respectively, we built bivalent fragments (VHNQ11-VLNQ11)2 and (VHD1.3-VLD1.3)2 and bispecific fragments VHNQ11-VLD1.3 and VHD1.3-VLNQ11. The fragments were expressed by secretion from bacteria and shown to bind specifically to the hapten and/or antigen. Those with 5- and 15-residue linkers had similar binding affinities to the parent antibodies, but a fragment with the VH domain joined directly to the VL domain was found to have slower dissociation kinetics and an improved affinity for hapten. Diabodies offer a ready means of constructing small bivalent and bispecific antibody fragments in bacteria. Images Fig. 1 Fig. 3 PMID:8341653

  4. Selection of affinity-improved neutralizing human scFv against HBV PreS1 from CDR3 VH/VL mutant library.

    PubMed

    Chen, YanMin; Bai, Yin; Guo, XiaoChen; Wang, WenFei; Zheng, Qi; Wang, FuXiang; Sun, Dejun; Li, DeShan; Ren, GuiPing; Yin, JieChao

    2016-07-01

    A CDR3 mutant library was constructed from a previously isolated anti-HBV neutralizing Homo sapiens scFv-31 template by random mutant primers PCR. Then the library was displayed on the inner membrane surface in Escherichia coli periplasmic space. Seven scFv clones were isolated from the mutant library through three rounds of screening by flow cytometry. Competition ELISA assay indicates that isolated scFv fragments show more efficient binding ability to HBV PreS1 compared with parental scFv-31. HBV neutralization assay indicated that two clones (scFv-3 and 59) show higher neutralizing activity by blocking the HBV infection to Chang liver cells. Our method provides a new strategy for rapid screening of mutant antibody library for affinity-enhanced scFv clones and the neutralizing scFvs obtained from this study provide a potential alternative of Hepatitis B immune globulin. PMID:27255707

  5. Expression and Functional Properties of an Anti-Triazophos High-Affinity Single-Chain Variable Fragment Antibody with Specific Lambda Light Chain

    PubMed Central

    Liu, Rui; Liang, Xiao; Xiang, Dandan; Guo, Yirong; Liu, Yihua; Zhu, Guonian

    2016-01-01

    Triazophos is a widely used organophosphorous insecticide that has potentially adverse effects to organisms. In the present study, a high-affinity single-chain variable fragment (scFv) antibody with specific lambda light chain was developed for residue monitoring. First, the specific variable regions were correctly amplified from a hybridoma cell line 8C10 that secreted monoclonal antibody (mAb) against triazophos. The regions were then assembled as scFv via splicing by overlap extension polymerase chain reaction. Subsequently, the recombinant anti-triazophos scFv-8C10 was successfully expressed in Escherichia coli strain HB2151 in soluble form, purified through immobilized metal ion affinity chromatography, and verified via Western blot and peptide mass fingerprinting analyses. Afterward, an indirect competitive enzyme-linked immunosorbent assay was established based on the purified anti-triazophos scFv-8C10 antibody. The assay exhibited properties similar to those based on the parent mAb, with a high sensitivity (IC50 of 1.73 ng/mL) to triazophos and no cross reaction for other organophosphorus pesticides; it was reliable in detecting triazophos residues in spiked water samples. Moreover, kinetic measurement using a surface plasmon resonance biosensor indicated that the purified scFv-8C10 antibody had a high affinity of 1.8 × 10−10 M and exhibited good binding stability. Results indicated that the recombinant high-affinity scFv-8C10 antibody was an effective detection material that would be promising for monitoring triazophos residues in environment samples. PMID:27338340

  6. Expression and Functional Properties of an Anti-Triazophos High-Affinity Single-Chain Variable Fragment Antibody with Specific Lambda Light Chain.

    PubMed

    Liu, Rui; Liang, Xiao; Xiang, Dandan; Guo, Yirong; Liu, Yihua; Zhu, Guonian

    2016-01-01

    Triazophos is a widely used organophosphorous insecticide that has potentially adverse effects to organisms. In the present study, a high-affinity single-chain variable fragment (scFv) antibody with specific lambda light chain was developed for residue monitoring. First, the specific variable regions were correctly amplified from a hybridoma cell line 8C10 that secreted monoclonal antibody (mAb) against triazophos. The regions were then assembled as scFv via splicing by overlap extension polymerase chain reaction. Subsequently, the recombinant anti-triazophos scFv-8C10 was successfully expressed in Escherichia coli strain HB2151 in soluble form, purified through immobilized metal ion affinity chromatography, and verified via Western blot and peptide mass fingerprinting analyses. Afterward, an indirect competitive enzyme-linked immunosorbent assay was established based on the purified anti-triazophos scFv-8C10 antibody. The assay exhibited properties similar to those based on the parent mAb, with a high sensitivity (IC50 of 1.73 ng/mL) to triazophos and no cross reaction for other organophosphorus pesticides; it was reliable in detecting triazophos residues in spiked water samples. Moreover, kinetic measurement using a surface plasmon resonance biosensor indicated that the purified scFv-8C10 antibody had a high affinity of 1.8 × 10(-10) M and exhibited good binding stability. Results indicated that the recombinant high-affinity scFv-8C10 antibody was an effective detection material that would be promising for monitoring triazophos residues in environment samples. PMID:27338340

  7. Structural Characterization of a Therapeutic Anti-Methamphetamine Antibody Fragment: Oligomerization and Binding of Active Metabolites

    PubMed Central

    Gokulan, Kuppan; Varughese, Kottayil I.

    2013-01-01

    Vaccines and monoclonal antibodies (mAb) for treatment of (+)-methamphetamine (METH) abuse are in late stage preclinical and early clinical trial phases, respectively. These immunotherapies work as pharmacokinetic antagonists, sequestering METH and its metabolites away from sites of action in the brain and reduce the rewarding and toxic effects of the drug. A key aspect of these immunotherapy strategies is the understanding of the subtle molecular interactions important for generating antibodies with high affinity and specificity for METH. We previously determined crystal structures of a high affinity anti-METH therapeutic single chain antibody fragment (scFv6H4, KD = 10 nM) in complex with METH and the (+) stereoisomer of 3,4-methylenedioxymethamphetamine (MDMA, or “ecstasy”). Here we report the crystal structure of scFv6H4 in homo-trimeric unbound (apo) form (2.60Å), as well as monomeric forms in complex with two active metabolites; (+)-amphetamine (AMP, 2.38Å) and (+)-4-hydroxy methamphetamine (p-OH-METH, 2.33Å). The apo structure forms a trimer in the crystal lattice and it results in the formation of an intermolecular composite beta-sheet with a three-fold symmetry. We were also able to structurally characterize the coordination of the His-tags with Ni2+. Two of the histidine residues of each C-terminal His-tag interact with Ni2+ in an octahedral geometry. In the apo state the CDR loops of scFv6H4 form an open conformation of the binding pocket. Upon ligand binding, the CDR loops adopt a closed formation, encasing the drug almost completely. The structural information reported here elucidates key molecular interactions important in anti-methamphetamine abuse immunotherapy. PMID:24349338

  8. High throughput cytotoxicity screening of anti-HER2 immunotoxins conjugated with antibody fragments from phage-displayed synthetic antibody libraries

    PubMed Central

    Hou, Shin-Chen; Chen, Hong-Sen; Lin, Hung-Wei; Chao, Wei-Ting; Chen, Yao-Sheng; Fu, Chi-Yu; Yu, Chung-Ming; Huang, Kai-Fa; Wang, Andrew H.-J.; Yang, An-Suei

    2016-01-01

    Immunotoxins are an important class of antibody-based therapeutics. The potency of the immunotoxins depends on the antibody fragments as the guiding modules targeting designated molecules on cell surfaces. Phage-displayed synthetic antibody scFv libraries provide abundant antibody fragment candidates as targeting modules for the immunoconjugates, but the discovery of optimally functional immunoconjugates is limited by the scFv-payload conjugation procedure. In this work, cytotoxicity screening of non-covalently assembled immunotoxins was developed in high throughput format to discover highly functional synthetic antibody fragments for delivering toxin payloads. The principles governing the efficiency of the antibodies as targeting modules have been elucidated from large volume of cytotoxicity data: (a) epitope and paratope of the antibody-based targeting module are major determinants for the potency of the immunotoxins; (b) immunotoxins with bivalent antibody-based targeting modules are generally superior in cytotoxic potency to those with corresponding monovalent targeting module; and (c) the potency of the immunotoxins is positively correlated with the densities of the cell surface antigen. These findings suggest that screening against the target cells with a large pool of antibodies from synthetic antibody libraries without the limitations of natural antibody responses can lead to optimal potency and minimal off-target toxicity of the immunoconjugates. PMID:27550798

  9. High throughput cytotoxicity screening of anti-HER2 immunotoxins conjugated with antibody fragments from phage-displayed synthetic antibody libraries.

    PubMed

    Hou, Shin-Chen; Chen, Hong-Sen; Lin, Hung-Wei; Chao, Wei-Ting; Chen, Yao-Sheng; Fu, Chi-Yu; Yu, Chung-Ming; Huang, Kai-Fa; Wang, Andrew H-J; Yang, An-Suei

    2016-01-01

    Immunotoxins are an important class of antibody-based therapeutics. The potency of the immunotoxins depends on the antibody fragments as the guiding modules targeting designated molecules on cell surfaces. Phage-displayed synthetic antibody scFv libraries provide abundant antibody fragment candidates as targeting modules for the immunoconjugates, but the discovery of optimally functional immunoconjugates is limited by the scFv-payload conjugation procedure. In this work, cytotoxicity screening of non-covalently assembled immunotoxins was developed in high throughput format to discover highly functional synthetic antibody fragments for delivering toxin payloads. The principles governing the efficiency of the antibodies as targeting modules have been elucidated from large volume of cytotoxicity data: (a) epitope and paratope of the antibody-based targeting module are major determinants for the potency of the immunotoxins; (b) immunotoxins with bivalent antibody-based targeting modules are generally superior in cytotoxic potency to those with corresponding monovalent targeting module; and (c) the potency of the immunotoxins is positively correlated with the densities of the cell surface antigen. These findings suggest that screening against the target cells with a large pool of antibodies from synthetic antibody libraries without the limitations of natural antibody responses can lead to optimal potency and minimal off-target toxicity of the immunoconjugates. PMID:27550798

  10. Purification and on-column refolding of a single-chain antibody fragment against rabies virus glycoprotein expressed in Escherichia coli.

    PubMed

    Xi, Hualong; Yuan, Ruosen; Chen, Xiaoxu; Gu, Tiejun; Cheng, Yue; Li, Zhuang; Jiang, Chunlai; Kong, Wei; Wu, Yongge

    2016-10-01

    An anti-rabies virus single-chain antibody fragment of an anti-glycoprotein with the VL-linker-VH orientation, designated scFv57RN, was successfully and conveniently prepared in this study. The scFv57RN protein was mainly expressed in inclusion bodies in Escherichia coli. After washing and purification, the inclusion bodies were finally obtained with an on-column refolding procedure. Further purification by gel exclusion chromatography was performed to remove inactive multimers. About 360 mg of final product was recovered from 1 L of bacterial culture. The final product showed a high neutralizing titer of 950 IU/mg to the CVS-11 strain as measured using the rapid fluorescent focus inhibition test. Our study demonstrated a highly efficient method to mass produce scFV57RN with activity from inclusion bodies, which may be applied in the purification of other insoluble proteins. PMID:27157441

  11. Generation of “LYmph Node Derived Antibody Libraries” (LYNDAL) for selecting fully human antibody fragments with therapeutic potential

    PubMed Central

    Diebolder, Philipp; Keller, Armin; Haase, Stephanie; Schlegelmilch, Anne; Kiefer, Jonathan D; Karimi, Tamana; Weber, Tobias; Moldenhauer, Gerhard; Kehm, Roland; Eis-Hübinger, Anna M; Jäger, Dirk; Federspil, Philippe A; Herold-Mende, Christel; Dyckhoff, Gerhard; Kontermann, Roland E; Arndt, Michaela AE; Krauss, Jürgen

    2014-01-01

    The development of efficient strategies for generating fully human monoclonal antibodies with unique functional properties that are exploitable for tailored therapeutic interventions remains a major challenge in the antibody technology field. Here, we present a methodology for recovering such antibodies from antigen-encountered human B cell repertoires. As the source for variable antibody genes, we cloned immunoglobulin G (IgG)-derived B cell repertoires from lymph nodes of 20 individuals undergoing surgery for head and neck cancer. Sequence analysis of unselected “LYmph Node Derived Antibody Libraries” (LYNDAL) revealed a naturally occurring distribution pattern of rearranged antibody sequences, representing all known variable gene families and most functional germline sequences. To demonstrate the feasibility for selecting antibodies with therapeutic potential from these repertoires, seven LYNDAL from donors with high serum titers against herpes simplex virus (HSV) were panned on recombinant glycoprotein B of HSV-1. Screening for specific binders delivered 34 single-chain variable fragments (scFvs) with unique sequences. Sequence analysis revealed extensive somatic hypermutation of enriched clones as a result of affinity maturation. Binding of scFvs to common glycoprotein B variants from HSV-1 and HSV-2 strains was highly specific, and the majority of analyzed antibody fragments bound to the target antigen with nanomolar affinity. From eight scFvs with HSV-neutralizing capacity in vitro, the most potent antibody neutralized 50% HSV-2 at 4.5 nM as a dimeric (scFv)2. We anticipate our approach to be useful for recovering fully human antibodies with therapeutic potential. PMID:24256717

  12. SNAP-Tag Technology: A Useful Tool To Determine Affinity Constants and Other Functional Parameters of Novel Antibody Fragments.

    PubMed

    Niesen, Judith; Sack, Markus; Seidel, Melanie; Fendel, Rolf; Barth, Stefan; Fischer, Rainer; Stein, Christoph

    2016-08-17

    Antibody derivatives, such as the single chain fragment variable (scFv), can be developed as diagnostic and therapeutic tools in cancer research, especially in the form of fusion proteins. Such derivatives are easier to produce and modify than monoclonal antibodies (mAbs) and achieve better tissue/tumor penetration. The genetic modification of scFvs is also much more straightforward than the challenging chemical modification of mAbs. Therefore, we constructed two scFvs derived from the approved monoclonal antibodies cetuximab (scFv2112) and panitumumab (scFv1711), both of which are specific for the epidermal growth factor receptor (EGFR), a well-characterized solid tumor antigen. Both scFvs were genetically fused to the SNAP-tag, an engineered version of the human DNA repair enzyme O(6)-alkylguanine DNA alkyltransferase that allows the covalent coupling of benzylguanine (BG)-modified substrates such as fluorescent dyes. The SNAP-tag achieves controllable and irreversible protein modification and is an important tool for experimental studies in vitro and in vivo. The affinity constant of a scFv is a key functional parameter, especially in the context of a fusion protein. Therefore, we developed a method to define the affinity constants of scFv-SNAP fusion proteins by surface plasmon resonance (SPR) spectroscopy. We could confirm that both scFvs retained their functionality after fusion to the SNAP-tag in a variety of procedures and assays, including ELISA, flow cytometry, and confocal microscopy. The experimental procedures described herein, and the new protocol for affinity determination by SPR spectroscopy, are suitable for the preclinical evaluation of diverse antibody formats and derivatives. PMID:27391930

  13. Broadening the neutralizing capacity of a family of antibody fragments against different toxins from Mexican scorpions.

    PubMed

    Rodríguez-Rodríguez, Everardo Remi; Olamendi-Portugal, Timoteo; Serrano-Posada, Hugo; Arredondo-López, Jonathan Noé; Gómez-Ramírez, Ilse; Fernández-Taboada, Guillermo; Possani, Lourival D; Anguiano-Vega, Gerardo Alfonso; Riaño-Umbarila, Lidia; Becerril, Baltazar

    2016-09-01

    New approaches aimed at neutralizing the primary toxic components present in scorpion venoms, represent a promising alternative to the use of antivenoms of equine origin in humans. New potential therapeutics developed by these approaches correspond to neutralizing antibody fragments obtained by selection and maturation processes from libraries of human origin. The high sequence identity shared among scorpion toxins is associated with an important level of cross reactivity exhibited by these antibody fragments. We have exploited the cross reactivity showed by single chain variable antibody fragments (scFvs) of human origin to re-direct the neutralizing capacity toward various other scorpion toxins. As expected, during these evolving processes several variants derived from a parental scFv exhibited the capacity to simultaneously recognize and neutralize different toxins from Centruroides scorpion venoms. A sequence analyses of the cross reacting scFvs revealed that specific mutations are responsible for broadening their neutralizing capacity. In this work, we generated a set of new scFvs that resulted from the combinatorial insertion of these point mutations. These scFvs are potential candidates to be part of a novel recombinant antivenom of human origin that could confer protection against scorpion stings. A remarkable property of one of these new scFvs (ER-5) is its capacity to neutralize at least three different toxins and its complementary capacity to neutralize the whole venom from Centruroides suffusus in combination with a second scFv (LR), which binds to a different epitope shared by Centruroides scorpion toxins. PMID:27212628

  14. Production of antibody fragments in Escherichia coli.

    PubMed

    Katsuda, Tomohisa; Sonoda, Hiroyuki; Kumada, Yoichi; Yamaji, Hideki

    2012-01-01

    Escherichia coli is a host widely used in the industrial production of recombinant proteins. However, the expression of heterologous proteins in E. coli often encounters the formation of inclusion bodies, which are insoluble and nonfunctional protein aggregates. For the successful production of antibody fragments, which includes single-chain variable fragments (scFvs), we describe here the modification of linker, signal, and Shine-Dalgarno (SD) sequences, the coexpression of cytoplasmic and periplasmic chaperones, and a method for fed-batch cultivation with exponential feed. PMID:22907360

  15. In vitro folding and thermodynamic stability of an antibody fragment selected in vivo for high expression levels in Escherichia coli cytoplasm.

    PubMed

    Martineau, P; Betton, J M

    1999-10-01

    We recently isolated a mutant of a human anti-beta-galactosidase single chain antibody fragment (scFv) able to fold at high levels in Escherichia coli cytoplasm. When targeted to the periplasm, this mutant and the wild-type scFv are both expressed at comparable levels in a soluble, active and oxidized form. If a reducing agent is added to the growth medium, only the mutant scFv is still able to fold, showing that in vivo aggregation is a direct consequence of the lack of disulphide bond formation and not of the cellular localization. In vitro denaturation/renaturation experiments show that the mutant protein is more stable than the wild-type scFv. Furthermore, refolding kinetics under reducing conditions show that the mutant folds faster than the wild-type protein. Aggregation does not proceed from the native or unfolded conformation of the protein, but from a species only present during the unfolding/refolding transition. In conclusion, the in vivo properties of the mutant scFv can be explained by, first, an increase in the stability of the protein in order to tolerate the removal of the two disulphide bonds and, second, a modification of its folding properties that reduces the kinetic competition between folding and aggregation of a reduced folding intermediate. PMID:10525415

  16. Production of a soluble single-chain variable fragment antibody against okadaic acid and exploration of its specific binding.

    PubMed

    He, Kuo; Zhang, Xiuyuan; Wang, Lixia; Du, Xinjun; Wei, Dong

    2016-06-15

    Okadaic acid is a lipophilic marine algal toxin commonly responsible for diarrhetic shellfish poisoning (DSP). Outbreaks of DSP have been increasing and are of worldwide public health concern; therefore, there is a growing demand for more rapid, reliable, and economical analytical methods for the detection of this toxin. In this study, anti-okadaic acid single-chain variable fragment (scFv) genes were prepared by cloning heavy and light chain genes from hybridoma cells, followed by fusion of the chains via a linker peptide. An scFv-pLIP6/GN recombinant plasmid was constructed and transformed into Escherichia coli for expression, and the target scFv was identified with IC-CLEIA (chemiluminescent enzyme immunoassay). The IC15 was 0.012 ± 0.02 μg/L, and the IC50 was 0.25 ± 0.03 μg/L. The three-dimensional structure of the scFv was simulated with computer modeling, and okadaic acid was docked to the scFv model to obtain a putative structure of the binding complex. Two predicted critical amino acids, Ser32 and Thr187, were then mutated to verify this theoretical model. Both mutants exhibited significant loss of binding activity. These results help us to understand this specific scFv-antigen binding mechanism and provide guidance for affinity maturation of the antibody in vitro. The high-affinity scFv developed here also has potential for okadaic acid toxin detection. PMID:26772159

  17. In silico experiments of single-chain antibody fragment against drugs of abuse

    PubMed Central

    Hu, Guodong; Chen, L. Y.

    2010-01-01

    SUMMARY Three sets of in silico experiments have been conducted to elucidate the binding mechanics of two drugs, (+)-methamphetamine (METH) and amphetamine (AMP) to the single-chain variable fragment (scFv) recently engineered from anti-METH monoclonal antibody mAb6H4 (IgG, κ light chain, Kd = 11nM). The first set of in silico experiments are long time equilibration runs of scFv:drug complexes and of drug-free scFv both in solution. They demonstrate how the solution structures of scFv deviate from its crystallographic form with or without drug molecules bound to it. And they lead to the prediction that the Arrhenius activation barrier is nearly zero for transitions from the dissociated state to the bound state. The second set of in silico experiments are nonequilibrium dynamics of pulling the drug molecules out of the binding pocket of scFv and the equilibration runs for drugs to fall back into binding pocket. They demonstrate that extra water molecules (in addition to the two crystallographic waters) exist inside the binding pocket, underneath the drug molecules. These extra waters must have been evaporated from the binding pockets during the crystallization process of the in vitro experiments of structural determination. The third set of in silico experiments are nonequilibrium steered molecular dynamics simulations to determine the absolute binding free energies of METH and AMP to scFv. The center-of-mass of a drug molecule (METH or AMP) is steered (pulled) towards (forward) and away from (reverse) the binding site, sampling forward and reverse pulling paths. Mechanic work is measured along the pulling paths. The work measurements are averaged through the Brownian dynamics fluctuation dissipation theorem to produce the free-energy profiles of the scFv:drug complexes as a function of the drug-scFv separation. These experiments lead to the theoretical prediction of absolute binding energies of METH and AMP that are in agreement with the in vitro experimental

  18. High-Affinity Recombinant Antibody Fragments (Fabs) Can Be Applied in Peptide Enrichment Immuno-MRM Assays

    PubMed Central

    2015-01-01

    High-affinity antibodies binding to linear peptides in solution are a prerequisite for performing immuno-MRM, an emerging technology for protein quantitation with high precision and specificity using peptide immunoaffinity enrichment coupled to stable isotope dilution and targeted mass spectrometry. Recombinant antibodies can be generated from appropriate libraries in high-throughput in an automated laboratory and thus may offer advantages over conventional monoclonal antibodies. However, recombinant antibodies are typically obtained as fragments (Fab or scFv) expressed from E. coli, and it is not known whether these antibody formats are compatible with the established protocols and whether the affinities necessary for immunocapture of small linear peptides can be achieved with this technology. Hence, we performed a feasibility study to ask: (a) whether it is feasible to isolate high-affinity Fabs to small linear antigens and (b) whether it is feasible to incorporate antibody fragments into robust, quantitative immuno-MRM assays. We describe successful isolation of high-affinity Fab fragments against short (tryptic) peptides from a human combinatorial Fab library. We analytically characterize three immuno-MRM assays using recombinant Fabs, full-length IgGs constructed from these Fabs, or traditional monoclonals. We show that the antibody fragments show similar performance compared with traditional mouse- or rabbit-derived monoclonal antibodies. The data establish feasibility of isolating and incorporating high-affinity Fabs into peptide immuno-MRM assays. PMID:24568200

  19. Probing the soybean Bowman-Birk inhibitor using recombinant antibody fragments.

    PubMed

    Muzard, Julien; Fields, Conor; O'Mahony, James John; Lee, Gil U

    2012-06-20

    The nutritional and health benefits of soy protein have been extensively studied over recent decades. The Bowman-Birk inhibitor (BBI), derived from soybeans, is a double-headed inhibitor of chymotrypsin and trypsin with anticarcinogenic and anti-inflammatory properties, which have been demonstrated in vitro and in vivo. However, the lack of analytical and purification methodologies complicates its potential for further functional and clinical investigations. This paper reports the construction of anti-BBI antibody fragments based on the principle of protein design. Recombinant antibody (scFv and diabody) molecules targeting soybean BBI were produced and characterized in vitro (K(D)~1.10(-9) M), and the antibody-binding site (epitope) was identified as part of the trypsin-specific reactive loop. Finally, an extremely fast purification strategy for BBI from soybean extracts, based on superparamagnetic particles coated with antibody fragments, was developed. To the best of the authors' knowledge, this is the first report on the design and characterization of recombinant anti-BBI antibodies and their potential application in soybean processing. PMID:22642722

  20. Production and characterization of a recombinant anti-MUC1 scFv reactive with human carcinomas.

    PubMed Central

    Denton, G.; Sekowski, M.; Spencer, D. I.; Hughes, O. D.; Murray, A.; Denley, H.; Tendler, S. J.; Price, M. R.

    1997-01-01

    Recombinant single-chain fragments (scFv) of the murine anti-MUC1 monoclonal antibody C595 have been produced using the original hybridoma cells as a source of variable heavy (V(H))- and variable light (V(L))-chain-encoding antibody genes. The use of the polymerase chain reaction (PCR), bacteriophage (phage) display technology and gene expression systems in E. coli has led to the production of soluble C595 scFv. The scFv has been purified from the bacterial supernatant by peptide epitope affinity chromatography, leading to the recovery of immunoreactive C595 scFv, which was similar in activity to the C595 parent antibody. Analysis by DNA sequencing, SDS-PAGE and Western blotting has demonstrated the integrity of the scFv, while ELISA, FACScan analysis, fluorescence quenching, quantitative immunoreactivity experiments and immunohistochemistry confirm that the activity of the scFv compares favourably with that of the parent antibody. The retention of binding activity to MUC1 antigen on human bladder and breast carcinoma tissue specimens illustrates the potential application of this novel product as an immunodiagnostic and immunotherapeutic reagent. Images Figure 1 Figure 2 Figure 3 Figure 7 PMID:9303360

  1. Human antibody fragments specific for the epidermal growth factor receptor selected from large non-immunised phage display libraries.

    PubMed

    Souriau, Christelle; Rothacker, Julie; Hoogenboom, Hennie R; Nice, Edouard

    2004-09-01

    Antibodies to EGFR have been shown to display anti-tumour effects mediated in part by inhibition of cellular proliferation and angiogenesis, and by enhancement of apoptosis. Humanised antibodies are preferred for clinical use to reduce complications with HAMA and HAHA responses frequently seen with murine and chimaeric antibodies. We have used depletion and subtractive selection strategies on cells expressing the EGFR to sample two large antibody fragment phage display libraries for the presence of human antibodies which are specific for the EGFR. Four Fab fragments and six scFv fragments were identified, with affinities of up to 2.2nM as determined by BIAcore analysis using global fitting of the binding curves to obtain the individual rate constants (ka and kd). This overall approach offers a generic screening method for the identification of growth factor specific antibodies and antibody fragments from large expression libraries and has potential for the rapid development of new therapeutic and diagnostic reagents. PMID:15518242

  2. Yeast display of antibody fragments: a discovery and characterization platform

    SciTech Connect

    Feldhaus, Michael; Siegel, Robert W.

    2004-07-01

    This review will focus on some of the novel attributes of the yeast surface display platform for the discovery and characterization of novel affinity reagents, optimization of those reagents, and novel uses of the platform. This is not intended to serve as an exhaustive review on the broader topic of general scFv technologies (see Winter et al., 1994; Smith and Petrenko, 1997; Bradbury et al., 2003) Furthermore, the scFv format of antibodies are easily manipulated through molecular cloning into a number of other formats such IgG, Fab, diabodies and such, for use in down steam applications and the reader is encouraged to read ?IgG?, ?Fab?, or your favorite format whenever scFv is seen in this review. This review is presented in 5 parts; (1) description of yeast display and its components, (2) library types and construction methods, (3) screening approaches for non-immune libraries and benefits, (4) screening approaches for directed evolution, kinetic on and off rates and (5) epitope complementation binning of clones.

  3. Selection and characterization of human antibody fragments specific for psoriasin - a cancer associated protein.

    PubMed

    Cyranka-Czaja, Anna; Wulhfard, Sarah; Neri, Dario; Otlewski, Jacek

    2012-03-01

    S100A7 (psoriasin) is a calcium-binding protein that is upregulated in many types of cancer and often associated with poor prognosis. Its role in carcinogenesis has been associated with the stimulation of VEGF and EGF activity. The recent research showed that psoriasin directly interacts with αvβ6 integrin, a protein related to the invasive phenotype of cancer. Moreover, this interaction promotes the αvβ6-dependent invasive activity. The important function of S100A7 in carcinoma development determines a great need for valuable tools enabling its detection, quantification and also activity inhibition. Here, we show the selection of S100A7 specific antibody fragments from the human scFv phage library ETH-2 Gold. We have selected antibody fragments specific for psoriasin, purified them and analyzed by BIAcore affinity measurements. The best clone was subjected to affinity maturation procedure yielding molecule with a subnanomolar affinity towards human S100A7 protein. Selected clone was expressed in a bivalent format and applied for immunostaining analysis, which confirmed the ability of the antigen recognition in physiological conditions. We therefore propose that obtained antibody, that is the first phage display-derived human antibody against psoriasin, can serve as a useful psoriasin binding platform in research, diagnostics and therapy of cancer. PMID:22342672

  4. Rigidity Emerges during Antibody Evolution in Three Distinct Antibody Systems: Evidence from QSFR Analysis of Fab Fragments

    PubMed Central

    Li, Tong; Tracka, Malgorzata B.; Uddin, Shahid; Casas-Finet, Jose; Jacobs, Donald J.; Livesay, Dennis R.

    2015-01-01

    The effects of somatic mutations that transform polyspecific germline (GL) antibodies to affinity mature (AM) antibodies with monospecificity are compared among three GL-AM Fab pairs. In particular, changes in conformational flexibility are assessed using a Distance Constraint Model (DCM). We have previously established that the DCM can be robustly applied across a series of antibody fragments (VL to Fab), and subsequently, the DCM was combined with molecular dynamics (MD) simulations to similarly characterize five thermostabilizing scFv mutants. The DCM is an ensemble based statistical mechanical approach that accounts for enthalpy/entropy compensation due to network rigidity, which has been quite successful in elucidating conformational flexibility and Quantitative Stability/Flexibility Relationships (QSFR) in proteins. Applied to three disparate antibody systems changes in QSFR quantities indicate that the VH domain is typically rigidified, whereas the VL domain and CDR L2 loop become more flexible during affinity maturation. The increase in CDR H3 loop rigidity is consistent with other studies in the literature. The redistribution of conformational flexibility is largely controlled by nonspecific changes in the H-bond network, although certain Arg to Asp salt bridges create highly localized rigidity increases. Taken together, these results reveal an intricate flexibility/rigidity response that accompanies affinity maturation. PMID:26132144

  5. Alternative downstream processes for production of antibodies and antibody fragments.

    PubMed

    Arakawa, Tsutomu; Tsumoto, Kouhei; Ejima, Daisuke

    2014-11-01

    Protein-A or Protein-L affinity chromatography and virus inactivation are key processes for the manufacturing of therapeutic antibodies and antibody fragments. These two processes often involve exposure of therapeutic proteins to denaturing low pH conditions. Antibodies have been shown to undergo conformational changes at low pH, which can lead to irreversible damages on the final product. Here, we review alternative downstream approaches that can reduce the degree of low pH exposure and consequently damaged product. We and others have been developing technologies that minimize or eliminate such low pH processes. We here cover facilitated elution of antibodies using arginine in Protein-A and Protein-G affinity chromatography, a more positively charged amidated Protein-A, two Protein-A mimetics (MEP and Mabsorbent), mixed-mode and steric exclusion chromatography, and finally enhanced virus inactivation by solvents containing arginine. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody. PMID:24859179

  6. Identification of Novel Single Chain Fragment Variable Antibodies Against TNF-α Using Phage Display Technology

    PubMed Central

    Alizadeh, Ali Akbar; Hamzeh-Mivehroud, Maryam; Dastmalchi, Siavoush

    2015-01-01

    Purpose: Tumor necrosis factor alpha (TNF-α) is an inflammatory cytokine, involved in both physiological and pathological pathways. Because of central role of TNF-α in pathogenesis of inflammatory diseases, in the current study, we aimed to identify novel scFv antibodies against TNF-α using phage display technology. Methods: Using libraries composed of phagemid displaying scFv antibodies, four rounds of biopanning against TNF-α were carried out, which led to identification of scFvs capable of binding to TNF-α. The scFv antibody with appropriate binding affinity towards TNF-α, was amplified and used in ELISA experiment. Results: Titration of phage achieved from different rounds of biopanning showed an enrichment of specific anti-TNF-α phages during biopanning process. Using ELISA experiment, a binding constant (Kd) of 1.11 ± 0.32 nM was determined for the phage displaying J48 scFv antibody. Conclusion: The findings in the current work revealed that the identified novel scFv antibody displayed at the N-terminal of minor coat proteins of phagemid binds TNF-α with suitable affinity. However, the soluble form of the antibody is needed to be produced and evaluated in more details regarding its binding properties to TNF-α. PMID:26793613

  7. Preparation and Identification of a Single-chain Variable Fragment Antibody Against Canine Distemper Virus.

    PubMed

    Yi, Li; Cheng, Shipeng

    2015-08-01

    The variable regions of the heavy chain (VH) and light chain (VL) were amplified by RT-PCR from the hybridoma 1N8, which secretes the monoclonal antibody against CDV N protein (aa 277-471). The VL and VH amplicons were combined using SOE-PCR by a 12 amino acid flexible linker (SSGGGGSGGGGS), which produced the scFv gene (named scFv/1N8). After sequence analysis, the scFv/1N8 gene was cloned into the prokaryotic expression vector PET32a with a His-tag. The recombinant scFv/1N8 protein was successfully expressed in recombinant Escherichia coli by IPTG induction. Moreover, the binding activity and specificity of the scFv were determined by indirect ELISA (His-tag) and competitive ELISA. The recombinant scFv/1N8 protein reported here will provide some basis for further antiviral drug research based on the scFv molecule. PMID:26301925

  8. A strategy for high-level expression of a single-chain variable fragment against TNFα by subcloning antibody variable regions from the phage display vector pCANTAB 5E into pBV220.

    PubMed

    Yang, Tao; Yang, Lijun; Chai, Weiran; Li, Renke; Xie, Jun; Niu, Bo

    2011-03-01

    A phage display single-chain variable fragment (scFv) library against TNFα was constructed using a recombinant phage antibody system (RPAS). The cloned scFv gene was introduced into the phage display vector pCANTAB 5E and expressed in Escherichia coli (E. coli) with a yield of up to 0.15 mg/l of total protein. With the attempt to improve the expression level of TNF-scFv, a strategy was established for subcloning the scFv gene from pCANTAB 5E into the plasmid pBV220. Under the control of a highly efficient tandem P(R)P(L) promoter system, scFv production was increased to 30% of total protein as inclusion bodies. After extraction from the cell pellet by sonication, the inclusion bodies were solubilized and denatured in the presence of 8M urea. Purification of denatured scFv was performed using nickel column chromatography followed by renaturation. The purity and activity of the refolded scFv were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting and by an enzyme-linked immunoabsorbent assay (ELISA). The results reveal that the overall yield of bioactive TNF-scFv from E. coli flask cultures was more than 45 mg/l culture medium and 15 mg/g wet weight cells. The renatured scFv exhibited binding activity similarly to soluble scFv. In conclusion we developed a method to over-express TNF-scFv, which have biological function after purification and renaturation. PMID:20951213

  9. Therapeutic Strategy for the Prevention of Pseudorabies Virus Infection in C57BL/6 Mice by 3D8 scFv with Intrinsic Nuclease Activity

    PubMed Central

    Lee, Gunsup; Cho, SeungChan; Hoang, Phuong Mai; Kim, Dongjun; Lee, Yongjun; Kil, Eui-Joon; Byun, Sung-June; Lee, Taek-Kyun; Kim, Dae-Hyun; Kim, Sunghan; Lee, Sukchan

    2015-01-01

    3D8 single chain variable fragment (scFv) is a recombinant monoclonal antibody with nuclease activity that was originally isolated from autoimmune-prone MRL mice. In a previous study, we analyzed the nuclease activity of 3D8 scFv and determined that a HeLa cell line expressing 3D8 scFv conferred resistance to herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PRV). In this study, we demonstrate that 3D8 scFv could be delivered to target tissues and cells where it exerted a therapeutic effect against PRV. PRV was inoculated via intramuscular injection, and 3D8 scFv was injected intraperitoneally. The observed therapeutic effect of 3D8 scFv against PRV was also supported by results from quantitative reverse transcription polymerase chain reaction, southern hybridization, and immunohistochemical assays. Intraperitoneal injection of 5 and 10 μg 3D8 scFv resulted in no detectable toxicity. The survival rate in C57BL/6 mice was 9% after intramuscular injection of 10 LD50 PRV. In contrast, the 3D8 scFv-injected C57BL/6 mice showed survival rates of 57% (5 μg) and 47% (10 μg). The results indicate that 3D8 scFv could be utilized as an effective antiviral agent in several animal models. PMID:26255831

  10. Purification of single-chain antibody fragments exploiting pH-gradients in simulated moving bed chromatography.

    PubMed

    Martínez Cristancho, Carlos Andrés; Seidel-Morgenstern, Andreas

    2016-02-19

    This paper deals with the theoretical design and experimental validation of an affinity-based continuous multi-column chromatography process for the purification of single-chain Fragment variable (scFv) antibodies. An open-loop 3-zone pH-gradient simulated moving bed (SMB) process was investigated exploiting the highly specific affinity of metal ions toward histidine-tagged recombinant proteins. The separation problem was simplified by considering the cell culture supernatant as a pseudo-binary mixture. The influence of mobile phase pH on the adsorption isotherm parameters was estimated by the inverse method using recorded pH-gradient batch elution profiles. Suitable operating parameters for the SMB process were identified using an equilibrium stage model and subsequently validated in a lab-scale SMB unit. Finally, the performance of the pH-gradient SMB process was compared against a non-optimized batch process. Biologically active single-chain Fragment variable antibody formats were purified continuously with 9% more recovery, 11 times more productivity (576 mg of purified scFv per day and liter stationary phase in SMB) and enriched by a factor of 2.5 compared to those obtained in the non-optimized batch process. PMID:26810806

  11. A chimera of green fluorescent protein with single chain variable fragment antibody against ginsenosides for fluorescence-linked immunosorbent assay.

    PubMed

    Sakamoto, Seiichi; Tanizaki, Yusuke; Pongkitwitoon, Benyakan; Tanaka, Hiroyuki; Morimoto, Satoshi

    2011-05-01

    A chimera of green fluorescent protein extracted from Aequorea coerulescens (AcGFP), a mutant that has been codon optimized for mammalian expression, with single-chain variable fragment (scFv) antibody against ginsenoside Re (GRe-scFv), named fluobody, has been successfully expressed in Escherichia coli (E. coli) to develop simple, speedy, and sensitive fluorescence-linked immunosorbent assay (FLISA). Two chimera proteins were constructed to contain GRe-scFv at the C-terminus of AcGFP (C-fluobody) and at the N-terminus of AcGFP (N-fluobody). These fluobodies were then purified by ion metal affinity chromatography and refolded by stepwise dialysis. The characterization of both fluobodies revealed that C-fluobody was found to be appropriate probe for FLISA as compare with N-fluobody. Furthermore, improvement of limit of detection (LOD) was observed in FLISA using C-fluobody (10 ng/mL) due to its strong fluorescence intensity of AcGFP compared with conventional enzyme-linked immunosorbent assay (ELISA) using parental monoclonal antibody against ginsenoside Re (G-Re), MAb-4G10 (100 ng/mL). Since some steps required in ELISA can be avoided in this present FLISA, speedy and sensitive immunoassay also could be performed using fluobody instead of monoclonal antibody and scFv. PMID:21277981

  12. Selection and Characterization of Single Chain Antibody Fragments Specific for Hsp90 as a Potential Cancer Targeting Molecule

    PubMed Central

    Petters, Edyta; Sokolowska-Wedzina, Aleksandra; Otlewski, Jacek

    2015-01-01

    Heat shock proteins play an essential role in facilitating malignant transformation and they have been recognized as important factors in human cancers. One of the key elements of the molecular chaperones machinery is Hsp90 and it has recently become a target for anticancer therapeutic approaches. The potential and importance of Hsp90-directed agents becomes apparent when one realizes that disruption of Hsp90 function may influence over 200 oncogenic client proteins. Here, we described the selection and characterization of Hsp90-specific antibody fragments from commercially available Tomlinson I and J phage display libraries. The affinities of Hsp90-binding scFv variants were measured using SPR method. Then, based on the best clone selected, we performed the affinity maturation procedure and obtained valuable Hsp90-specific clones. The selected binders were expressed and applied for immunostaining, ELISA and SPR analysis using model cancer cell lines. All performed experiments confirmed the ability of selected antibodies to interact with the Hsp90. Therefore, the presented Hsp90-specific scFv, might be a starting point for the development of a novel antibody-based strategy targeting cancer. PMID:26307975

  13. Selection and Characterization of Single Chain Antibody Fragments Specific for Hsp90 as a Potential Cancer Targeting Molecule.

    PubMed

    Petters, Edyta; Sokolowska-Wedzina, Aleksandra; Otlewski, Jacek

    2015-01-01

    Heat shock proteins play an essential role in facilitating malignant transformation and they have been recognized as important factors in human cancers. One of the key elements of the molecular chaperones machinery is Hsp90 and it has recently become a target for anticancer therapeutic approaches. The potential and importance of Hsp90-directed agents becomes apparent when one realizes that disruption of Hsp90 function may influence over 200 oncogenic client proteins. Here, we described the selection and characterization of Hsp90-specific antibody fragments from commercially available Tomlinson I and J phage display libraries. The affinities of Hsp90-binding scFv variants were measured using SPR method. Then, based on the best clone selected, we performed the affinity maturation procedure and obtained valuable Hsp90-specific clones. The selected binders were expressed and applied for immunostaining, ELISA and SPR analysis using model cancer cell lines. All performed experiments confirmed the ability of selected antibodies to interact with the Hsp90. Therefore, the presented Hsp90-specific scFv, might be a starting point for the development of a novel antibody-based strategy targeting cancer. PMID:26307975

  14. Production and Purification of a Novel Anti-TNF-α Single Chain Fragment Variable Antibody

    PubMed Central

    Alizadeh, Ali Akbar; Hamzeh-Mivehroud, Maryam; Dastmalchi, Siavoush

    2015-01-01

    Purpose: TNF-α is an inflammatory cytokine with a key role in initiation of inflammatory responses. Anti-TNF-α antibodies are being used in clinic for the purpose of diagnosis and treatment due to their high specificity. The objective of the current study was to express and purify an anti-TNF-α scFv antibody identified by phage display technology. Methods: The DNA coding sequence of the identified scFv was cloned into pET28a vector and the corresponding protein was expressed as 6×His tagged using E.coli BL21 (DE3) pLysS expression system followed by affinity purification on Ni-Sepharose affinity column. Results: The J44 scFv antibody was cloned into the expression vector and successfully expressed and purified. The purity of the scFv fraction was confirmed using SDS-PAGE analysis. Western blotting technique was used to detect expression of 6×His tagged protein. Conclusion: In the current study an anti-TNF-α scFv antibody was successfully expressed in bacterial expression system and purified on affinity column. The purified protein can be used in different in vitro and in vivo experiments in order to elucidate its functionality. PMID:26793614

  15. Improved biological activity of a single chain antibody fragment against human epidermal growth factor receptor 2 (HER2) expressed in the periplasm of Escherichia coli.

    PubMed

    Akbari, Vajihe; Sadeghi, Hamid Mir Mohammad; Jafarian-Dehkordi, Abbas; Abedi, Daryoush; Chou, C Perry

    2015-12-01

    A novel monoclonal antibody against human epidermal growth factor receptor 2 (HER2), i.e., pertuzumab (Perjeta®) developed by Genentech, has been verified to be effective in treating metastatic HER2-overexpressing breast cancer. The fact that the presence of the Fc region of the anti-HER2 is uncritical for growth inhibition of tumor cells suggests the potential biological activity of the associated antibody fragments. In the present study, we report functional expression of anti-HER2his-scFv, a single-chain variable fragment (scFv) derived from pertuzumab, in the periplasm of Escherichia coli and its purification. Biological activity of the soluble scFv produced in this manner was characterized using immunofluorescent staining, immunocytochemistry, flow cytometry and cytotoxicity assay. The effect of anti-HER2his-scFv on HER2 dimerization was also assessed by tyrosine kinase assay. It was observed that the purified scFv had a high specificity and affinity to HER2 receptors expressed on the surface of tumor cells with a selective cytotoxic effect on HER2-overexpressing SK-OV-3 cells. In addition, anti-HER2his-scFv was able to suppress phosphorylation of HER2 in the presence of heregulin. The results suggest that anti-HER2his-scFv can be a potential candidate for various therapeutic and diagnosis applications. PMID:26166178

  16. Considerations in producing preferentially reduced half-antibody fragments.

    PubMed

    Makaraviciute, Asta; Jackson, Carolyn D; Millner, Paul A; Ramanaviciene, Almira

    2016-02-01

    Half-antibody fragments are a promising reagent for biosensing, drug-delivery and labeling applications, since exposure of the free thiol group in the Fc hinge region allows oriented reaction. Despite the structural variations among the molecules of different IgG subclasses and those obtained from different hosts, only generalized preferential antibody reduction protocols are currently available. Preferential reduction of polyclonal sheep anti-digoxin, rabbit anti-Escherichia coli and anti-myoglobin class IgG antibodies to half-antibody fragments has been investigated. A mild reductant 2-mercaptoethylamine (2-MEA) and a slightly stronger reductant tris(2-carboxyethyl)phosphine (TCEP) were used and the fragments obtained were quantitatively determined by SDS-PAGE analysis. It has been shown that the yields of half-antibody fragments could be increased by lowering the pH of the reduction mixtures. However, antibody susceptibility to the reductants varied. At pH4.5 the highest yield of sheep anti-digoxin IgG half-antibody fragments was obtained with 1M 2-MEA. Conversely, rabbit IgG half-antibody fragments could only be obtained with the stronger reductant TCEP. Preferential reduction of rabbit anti-myoglobin IgG antibodies was optimized and the highest half-antibody yield was obtained with 35 mM TCEP. Finally, it has been demonstrated that produced anti-myoglobin half-IgG fragments retained their binding activity. PMID:26779832

  17. Novel human single chain antibody fragments that are rapidly interalizing effectively target epithelioid and sarcomatoid mesotheliomas

    PubMed Central

    Iyer, Arun K.; Lan, Xiaoli; Zhu, Xiaodong; Su, Yang; Feng, Jinjin; Zhang, Xiaoju; Gao, Dongwei; Seo, Youngho; VanBrocklin, Henry F.; Broaddus, V. Courtney; Liu, Bin; He, Jiang

    2011-01-01

    Human antibodies targeting all subtypes of mesothelioma could be useful to image and treat this deadly disease. Here we report tumor targeting of a novel internalizing human single chain antibody fragment (scFv) labeled with 99mTc (99mTc-M40) in murine models of mesothelioma of both epithelioid (M28) and sarcomatoid (VAMT-1) origins. 99mTc-M40 was taken up rapidly and specifically by both subtype tumor cells in vitro, with 68–92% internalized within 1h. The specificity of binding was evidenced by blocking (up to 95%) with 10-fold excess of unlabeled M40. In animal studies, tumors of both subtypes were clearly visualized by SPECT/CT as early as 1h post-injection of 99mTc-M40. Tumor uptake measured as percent of injected dose per gram tissue (%ID/g) at 3h was 4.38 and 5.84 for M28 and VAMT-1 tumors respectively, significantly greater than all organs or tissues studied (liver, 2.62%ID/g; other organs or tissues <1.7%ID/g), except the kidneys (130.7%ID/g), giving tumor-to-blood ratios of 5:1 and 7:1 and tumor-to-muscle ratios of 45:1 and 60:1, for M28 and VAMT-1 respectively. The target-mediated uptake was confirmed by a nearly 70% reduction in tumor activity following administration of 10-fold excess of unlabeled scFv. Taken together, these results indicate that M40 can rapidly and specifically target epithelioid and sarcomatoid tumor cells, demonstrating the potential of this agent as a versatile targeting ligand for imaging and therapy of all subtypes of mesothelioma. PMID:21447742

  18. Efficient construct of a large and functional scFv yeast display library derived from the ascites B cells of ovarian cancer patients by three-fragment transformation-associated recombination.

    PubMed

    Yuan, Xiaopeng; Chen, Xiang; Yang, Mingjuan; Hu, Jia; Yang, Wei; Chen, Tingtao; Wang, Qirui; Zhang, Xuhua; Lin, Ruihe; Zhao, Aizhi

    2016-05-01

    Over the past decade, yeast display technology has emerged as a powerful tool for the isolation of high-affinity immunoglobulin fragments with potential utility as clinical diagnostic and therapeutic reagents. Despite significant refinement of the various methodologies underpinning library construction and selections, certain aspects remain challenging and process limiting. We have sought to significantly improve the robustness of the single-chain Fv (scFv) library construction step by overcoming the technical inefficiencies frequently encountered during the PCR-mediated assembly of scFvs from the discrete heavy and light V-domain repertoires. Using a novel primer set designed to provide maximum amplification coverage of the known germ-line V-domain repertoire, we have exploited the potential of the in vivo homologous gap-repair apparatus of Saccharomyces cerevisiae to assemble intact scFvs directly from co-transformed PBMC-derived VH, VL, and linearized vector component fragments. We have successfully applied this three-fragment assembly strategy to construct a large (>10(9)) scFv yeast display library from the ascites immune repertoire of ovarian cancer patients and validated the approach by applying FACS-based sorting to readily isolate scFvs that recognize various tumor marker antigens (TMAs). It is expected that this simplified construction method may find general utility, both for de novo scFv library construction and for subsequent combinatorial affinity maturation manipulations that require more than two fragments. PMID:26782745

  19. Expression and purification of a human anti-cyclin D1 single-chain variable fragment antibody AD5 and its characterization.

    PubMed

    Wu, Yan; Zou, Desheng; Cao, Yuhua; Yao, Nannan; Wang, Junye; Wang, Wenhan; Jiang, Hongyu; Li, Guiying

    2013-12-01

    Cyclin D1 plays an important role in cell cycle progression. Increasing evidence indicates that cyclin D1 is overexpressed in the majority of tumor cells and has become a potential target for tumor therapy. However, little research has been done on the specific inhibition of cyclin D1 for cancer therapy. With the rapid development of the phage display antibody library technique, single-chain variable fragment (scFv) antibodies have emerged, which have tremendous application prospects in cancer therapy and diagonosis. In this study, a human scFv binding specifically to cyclin D1 (AD5) that was derived from a human semi-synthetic scFv phage library was expressed in the soluble form in Escherichia coli (E. coli) HB2151 cells. To characterize AD5, soluble AD5 was purified successfully through ammonium sulfate precipitation and affinity chromatography from the culture supernatant of AD5/HB2151. ELISA assay revealed that purified soluble AD5 could specifically bind to human recombinant cyclin D1 with approximately (1.19±0.056) x 107 M-1 affinity constant and showed approximately 52% competitive inhibition with the anti-cyclin D1 polyclonal antibody for binding to cyclin D1 in vitro. These results suggest that the scFv antibody against cyclin D1 may be a novel potential tool for targeting cyclin D1 in cancer therapy and diagnosis. PMID:24127128

  20. Co-expression of Dsb proteins enables soluble expression of a single-chain variable fragment (scFv) against human type 1 insulin-like growth factor receptor (IGF-1R) in E. coli.

    PubMed

    Sun, Xue-Wen; Wang, Xiao-Hua; Yao, Yan-Bing

    2014-12-01

    Type 1 insulin-like growth factor receptor (IGF-1R) is a promising therapeutic target for cancer treatment. A single-chain variable fragment (scFv) against human IGF-1R forms inclusion body when expressed in periplasmic space of E. coli routinely. Here, we described that co-expression of appropriate disulfide bonds (Dsb) proteins known to catalyze the formation and isomerization of Dsb can markedly recover the soluble expression of target scFv in E. coli. A 50 % recovery in solubility of the scFv was observed upon co-expression of DsbC alone, and a maximum solubility (80 %) was obtained when DsbA and DsbC were co-expressed in combination. Furthermore, the soluble scFv present full antigen-binding activity with IGF-1R, suggesting its correct folding. This study also suggested that the selection of Dsb proteins should be tested case-by-case if the approach of co-expression of Dsb system is adopted to address the problem of insoluble expression of proteins carrying Dsb. PMID:25256416

  1. Production of recombinant single chain antibodies (scFv) in vegetatively reproductive Kalanchoe pinnata by in planta transformation.

    PubMed

    Jung, Yuchul; Rhee, Yong; Auh, Chung-Kyoon; Shim, Hyekyung; Choi, Jung-Jin; Kwon, Suk-Tae; Yang, Joo-Sung; Kim, Donggiun; Kwon, Myung-Hee; Kim, Yong-Sung; Lee, Sukchan

    2009-10-01

    We developed an asexual reproductive plant, Kalanchoe pinnata, as a new bioreactor for plant-based molecular farming using a newly developed transformation method. Leaf crenate margins were pin-pricked to infect the plant with the Agrobacterium strain LBA4404 and vacuum infiltration was also applied to introduce the target gene into the plants. Subsequently, the young mother leaf produced new clones at the leaf crenate margins without the need for time- and labor-consuming tissue culture procedures. The average transformation rates were approximately 77 and 84% for pin-prickling and vacuum-infiltration methods, respectively. To functionally characterize an introduced target protein, a nucleic acid hydrolyzing recombinant 3D8 scFv was selected and the plant based 3D8 scFv proteins were purified and analyzed. Based on abzyme analysis, the purified protein expressed with this system had catalytic activity and exhibited all of properties of the protein produced in an E. coli system. This result suggested that vegetatively reproductive K. pinnata can be a novel and potent bioreactor for bio-pharmaceutical proteins. PMID:19688214

  2. ImmunoPET imaging of B-cell lymphoma using 124I-anti-CD20 scFv dimers (diabodies).

    PubMed

    Olafsen, Tove; Sirk, Shannon J; Betting, David J; Kenanova, Vania E; Bauer, Karl B; Ladno, Waldemar; Raubitschek, Andrew A; Timmerman, John M; Wu, Anna M

    2010-04-01

    Rapid clearing engineered antibody fragments for immunoPET promise high sensitivity at early time points. Here, tumor targeting of anti-CD20 diabodies (scFv dimers) for detection of low-grade B-cell lymphomas were evaluated. In addition, the effect of linker length on oligomerization of the diabody was investigated. Four rituximab scFv variants in the V(L)-V(H) orientation with different linker lengths between the V domains (scFv-1, scFv-3, scFv-5, scFv-8), plus the scFv-5 with a C-terminal cysteine (Cys-Db) for site-specific modification were generated. The scFv-8 and Cys-Db were radioiodinated with (124)I for PET imaging, and biodistribution of (131)I-Cys-Db was carried out at 2, 4 10 and 20 h. The five anti-CD20 scFv variants were expressed as fully functional dimers. Shortening the linker to three or one residue did not produce higher order of multimers. Both (124)I-labeled scFv-8 and Cys-Db exhibited similar tumor targeting at 8 h post injection, with significantly higher uptakes than in control tumors (P < 0.05). At 20 h, less than 1% ID/g of (131)I-labeled Cys-Db was present in tumors and tissues. Specific tumor targeting and high contrast images were achieved with the anti-CD20 diabodies. These agents extend the repertoire of reagents that can potentially be used to improve detection of low-grade lymphomas. PMID:20053640

  3. ImmunoPET imaging of B-cell lymphoma using 124I-anti-CD20 scFv dimers (diabodies)

    PubMed Central

    Olafsen, Tove; Sirk, Shannon J.; Betting, David J.; Kenanova, Vania E.; Bauer, Karl B.; Ladno, Waldemar; Raubitschek, Andrew A.; Timmerman, John M.; Wu, Anna M.

    2010-01-01

    Rapid clearing engineered antibody fragments for immunoPET promise high sensitivity at early time points. Here, tumor targeting of anti-CD20 diabodies (scFv dimers) for detection of low-grade B-cell lymphomas were evaluated. In addition, the effect of linker length on oligomerization of the diabody was investigated. Four rituximab scFv variants in the VL–VH orientation with different linker lengths between the V domains (scFv-1, scFv-3, scFv-5, scFv-8), plus the scFv-5 with a C-terminal cysteine (Cys-Db) for site-specific modification were generated. The scFv-8 and Cys-Db were radioiodinated with 124I for PET imaging, and biodistribution of 131I-Cys-Db was carried out at 2, 4 10 and 20 h. The five anti-CD20 scFv variants were expressed as fully functional dimers. Shortening the linker to three or one residue did not produce higher order of multimers. Both 124I-labeled scFv-8 and Cys-Db exhibited similar tumor targeting at 8 h post injection, with significantly higher uptakes than in control tumors (P < 0.05). At 20 h, less than 1% ID/g of 131I-labeled Cys-Db was present in tumors and tissues. Specific tumor targeting and high contrast images were achieved with the anti-CD20 diabodies. These agents extend the repertoire of reagents that can potentially be used to improve detection of low-grade lymphomas. PMID:20053640

  4. Crystal structure of a 3B3 variant - A broadly neutralizing HIV-1 scFv antibody

    SciTech Connect

    Clark, K. Reed; Walsh, Scott T.R.

    2009-12-10

    We present the crystal structure determination of an anti-HIV-1 gp120 single-chain variable fragment antibody variant, 3B3, at 2.5 {angstrom} resolution. This 3B3 variant was derived from the b12 antibody, using phage display and site-directed mutagenesis of the variable heavy chain (V{sub H}) complementary-determining regions (CDRs). 3B3 exhibits enhanced binding affinity and neutralization activity against several cross-clade primary isolates of HIV-1 by interaction with the recessed CD4-binding site on the gp120 envelope protein. Comparison with the structures of the unbound and bound forms of b12, the 3B3 structure closely resembles these structures with minimal differences with two notable exceptions. First, there is a reorientation of the CDR-H3 of the V{sub H} domain where the primary sequences evolved from b12 to 3B3. The structural changes in CDR-H3 of 3B3, in light of the b12-gp120 complex structure, allow for positioning an additional Trp side chain in the binding interface with gp120. Finally, the second region of structural change involves two peptide bond flips in CDR-L3 of the variable light (VL) domain triggered by a point mutation in CDR-H3 of Q100eY resulting in changes in the intramolecular hydrogen bonding patterning between the VL and VH domains. Thus, the enhanced binding affinities and neutralization capabilities of 3B3 relative to b12 probably result from higher hydrophobic driving potential by burying more aromatic residues at the 3B3-gp120 interface and by indirect stabilization of intramolecular contacts of the core framework residues between the VL and VH domains possibly through more favorable entropic effect through the expulsion of water.

  5. Mapping the epitopes of a neutralizing antibody fragment directed against the lethal factor of Bacillus anthracis and cross-reacting with the homologous edema factor.

    PubMed

    Thullier, Philippe; Avril, Arnaud; Mathieu, Jacques; Behrens, Christian K; Pellequer, Jean-Luc; Pelat, Thibaut

    2013-01-01

    The lethal toxin (LT) of Bacillus anthracis, composed of the protective antigen (PA) and the lethal factor (LF), plays an essential role in anthrax pathogenesis. PA also interacts with the edema factor (EF, 20% identity with LF) to form the edema toxin (ET), which has a lesser role in anthrax pathogenesis. The first recombinant antibody fragment directed against LF was scFv 2LF; it neutralizes LT by blocking the interaction between PA and LF. Here, we report that scFv 2LF cross-reacts with EF and cross-neutralizes ET, and we present an in silico method taking advantage of this cross-reactivity to map the epitope of scFv 2LF on both LF and EF. This method identified five epitope candidates on LF, constituted of a total of 32 residues, which were tested experimentally by mutating the residues to alanine. This combined approach precisely identified the epitope of scFv 2LF on LF as five residues (H229, R230, Q234, L235 and Y236), of which three were missed by the consensus epitope candidate identified by pre-existing in silico methods. The homolog of this epitope on EF (H253, R254, E258, L259 and Y260) was experimentally confirmed to constitute the epitope of scFv 2LF on EF. Other inhibitors, including synthetic molecules, could be used to target these epitopes for therapeutic purposes. The in silico method presented here may be of more general interest. PMID:23741517

  6. In situ magnetic separation of antibody fragments from Escherichia coli in complex media

    PubMed Central

    2013-01-01

    Background In situ magnetic separation (ISMS) has emerged as a powerful tool to overcome process constraints such as product degradation or inhibition of target production. In the present work, an integrated ISMS process was established for the production of his-tagged single chain fragment variable (scFv) D1.3 antibodies (“D1.3”) produced by E. coli in complex media. This study investigates the impact of ISMS on the overall product yield as well as its biocompatibility with the bioprocess when metal-chelate and triazine-functionalized magnetic beads were used. Results Both particle systems are well suited for separation of D1.3 during cultivation. While the triazine beads did not negatively impact the bioprocess, the application of metal-chelate particles caused leakage of divalent copper ions in the medium. After the ISMS step, elevated copper concentrations above 120 mg/L in the medium negatively influenced D1.3 production. Due to the stable nature of the model protein scFv D1.3 in the biosuspension, the application of ISMS could not increase the overall D1.3 yield as was shown by simulation and experiments. Conclusions We could demonstrate that triazine-functionalized beads are a suitable low-cost alternative to selectively adsorb D1.3 fragments, and measured maximum loads of 0.08 g D1.3 per g of beads. Although copper-loaded metal-chelate beads did adsorb his-tagged D1.3 well during cultivation, this particle system must be optimized by minimizing metal leakage from the beads in order to avoid negative inhibitory effects on growth of the microorganisms and target production. Hereby, other types of metal chelate complexes should be tested to demonstrate biocompatibility. Such optimized particle systems can be regarded as ISMS platform technology, especially for the production of antibodies and their fragments with low stability in the medium. The proposed model can be applied to design future ISMS experiments in order to maximize the overall product yield

  7. Expression of V(H)-linker-V(L) orientation-dependent single-chain Fv antibody fragment derived from hybridoma 2E6 against aflatoxin B1 in Escherichia coli.

    PubMed

    Liu, Aiping; Ye, Yang; Chen, Weifeng; Wang, Xiaohong; Chen, Fusheng

    2015-02-01

    Aflatoxin B1 (AFB1) is a toxic secondary metabolic product, which threatens human and animal health. Antibody is a key factor for immunoassay against toxic stuff like AFB1, and single-chain Fv antibody fragment (scFv) has become a popular format of genetically engineered antibody. In this study, four hybridoma cell lines against AFB1 were obtained, and then scFvs 2E6 derived from hybridoma cell line 2E6 were constructed in different V(H)/V(L) orientations. Subsequently, scFvs 2E6 were expressed in E. coli BL21(DE3) mainly in the form of inclusion body. SDS-PAGE, Western blot and ELISA were employed to characterize scFvs 2E6. The results revealed that the yield of inclusion body of scFvs 2E6 in either V(H)/V(L) orientation was similar; however, only the scFv in V(H)-linker-V(L) orientation showed anti-AFB1 bioactivity after refolding. The present study underscores the importance of choosing optimal V(H)/V(L) orientation for scFv construction, and scFv may be favorable for immunoassays in food industry. PMID:25540048

  8. A synthetic antibody fragment targeting nicastrin affects assembly and trafficking of γ-secretase.

    PubMed

    Zhang, Xulun; Hoey, Robert; Koide, Akiko; Dolios, Georgia; Paduch, Marcin; Nguyen, Phuong; Wu, Xianzhong; Li, Yueming; Wagner, Steven L; Wang, Rong; Koide, Shohei; Sisodia, Sangram S

    2014-12-12

    The γ-secretase complex, composed of presenilin, nicastrin (NCT), anterior pharynx-defective 1 (APH-1), and presenilin enhancer 2 (PEN-2), is assembled in a highly regulated manner and catalyzes the intramembranous proteolysis of many type I membrane proteins, including Notch and amyloid precursor protein. The Notch family of receptors plays important roles in cell fate specification during development and in adult tissues, and aberrant hyperactive Notch signaling causes some forms of cancer. γ-Secretase-mediated processing of Notch at the cell surface results in the generation of the Notch intracellular domain, which associates with several transcriptional coactivators involved in nuclear signaling events. On the other hand, γ-secretase-mediated processing of amyloid precursor protein leads to the production of amyloid β (Aβ) peptides that play an important role in the pathogenesis of Alzheimer disease. We used a phage display approach to identify synthetic antibodies that specifically target NCT and expressed them in the single-chain variable fragment (scFv) format in mammalian cells. We show that expression of a NCT-specific scFv clone, G9, in HEK293 cells decreased the production of the Notch intracellular domain but not the production of amyloid β peptides that occurs in endosomal and recycling compartments. Biochemical studies revealed that scFvG9 impairs the maturation of NCT by associating with immature forms of NCT and, consequently, prevents its association with the other components of the γ-secretase complex, leading to degradation of these molecules. The reduced cell surface levels of mature γ-secretase complexes, in turn, compromise the intramembranous processing of Notch. PMID:25352592

  9. Structural Basis of Neutralization of the Major Toxic Component from the Scorpion Centruroides noxius Hoffmann by a Human-derived Single-chain Antibody Fragment

    SciTech Connect

    Canul-Tec, Juan Carlos; Riaño-Umbarila, Lidia; Rudiño-Piñera, Enrique; Becerril, Baltazar; Possani, Lourival D.; Torres-Larios, Alfredo

    2011-08-09

    It has previously been reported that several single-chain antibody fragments of human origin (scFv) neutralize the effects of two different scorpion venoms through interactions with the primary toxins of Centruroides noxius Hoffmann (Cn2) and Centruroides suffusus suffusus (Css2). Here we present the crystal structure of the complex formed between one scFv (9004G) and the Cn2 toxin, determined in two crystal forms at 2.5 and 1.9 {angstrom} resolution. A 15-residue span of the toxin is recognized by the antibody through a cleft formed by residues from five of the complementarity-determining regions of the scFv. Analysis of the interface of the complex reveals three features. First, the epitope of toxin Cn2 overlaps with essential residues for the binding of {beta}-toxins to its Na+ channel receptor site. Second, the putative recognition of Css2 involves mainly residues that are present in both Cn2 and Css2 toxins. Finally, the effect on the increase of affinity of previously reported key residues during the maturation process of different scFvs can be inferred from the structure. Taken together, these results provide the structural basis that explain the mechanism of the 9004G neutralizing activity and give insight into the process of directed evolution that gave rise to this family of neutralizing scFvs.

  10. Selection of single chain variable fragments (scFv) against Xylella fastidiosa subsp. pauca by phage display

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Xylella fastidiosa is a gram-negative member of the gamma proteobacteria. Xylella fastidiosa subsp pauca causes citrus variegated chlorosis in Brazil and enjoys ‘select agent’ status in the United States. Antibody based detection assays are commercially available for Xylella fastidiosa, and are ef...

  11. HER2-specific immunoligands engaging NKp30 or NKp80 trigger NK-cell-mediated lysis of tumor cells and enhance antibody-dependent cell-mediated cytotoxicity.

    PubMed

    Peipp, Matthias; Derer, Stefanie; Lohse, Stefan; Staudinger, Matthias; Klausz, Katja; Valerius, Thomas; Gramatzki, Martin; Kellner, Christian

    2015-10-13

    NK cells detect tumors through activating surface receptors, which bind self-antigens that are frequently expressed upon malignant transformation. To increase the recognition of tumor cells, the extracellular domains of ligands of the activating NK cell receptors NKp30, NKp80 and DNAM-1 (i.e. B7-H6, AICL and PVR, respectively) were fused to a single-chain fragment variable (scFv) targeting the human epidermal growth factor receptor 2 (HER2), which is displayed by various solid tumors. The resulting immunoligands, designated B7-H6:HER2-scFv, AICL:HER2-scFv, and PVR:HER2-scFv, respectively, bound HER2 and the addressed NK cell receptor. However, whereas B7-H6:HER2-scFv and AICL:HER2-scFv triggered NK cells to kill HER2-positive breast cancer cells at nanomolar concentrations, PVR:HER2-scFv was not efficacious. Moreover, NK cell cytotoxicity was enhanced synergistically when B7-H6:HER2-scFv or AICL:HER2-scFv were applied in combination with another HER2-specific immunoligand engaging the stimulatory receptor NKG2D. In contrast, no improvements were achieved by combining B7-H6:HER2-scFv with AICL:HER2-scFv. Additionally, B7-H6:HER2-scFv and AICL:HER2-scFv enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) by the therapeutic antibodies trastuzumab and cetuximab synergistically, with B7-H6:HER2-scFv exhibiting a higher efficacy. In summary, antibody-derived proteins engaging NKp30 or NKp80 may represent attractive biologics to further enhance anti-tumor NK cell responses and may provide an innovative approach to sensitize tumor cells for antibody-based immunotherapy. PMID:26392331

  12. HER2-specific immunoligands engaging NKp30 or NKp80 trigger NK-cell-mediated lysis of tumor cells and enhance antibody-dependent cell-mediated cytotoxicity

    PubMed Central

    Peipp, Matthias; Derer, Stefanie; Lohse, Stefan; Staudinger, Matthias; Klausz, Katja; Valerius, Thomas; Gramatzki, Martin; Kellner, Christian

    2015-01-01

    NK cells detect tumors through activating surface receptors, which bind self-antigens that are frequently expressed upon malignant transformation. To increase the recognition of tumor cells, the extracellular domains of ligands of the activating NK cell receptors NKp30, NKp80 and DNAM-1 (i.e. B7-H6, AICL and PVR, respectively) were fused to a single-chain fragment variable (scFv) targeting the human epidermal growth factor receptor 2 (HER2), which is displayed by various solid tumors. The resulting immunoligands, designated B7-H6:HER2-scFv, AICL:HER2-scFv, and PVR:HER2-scFv, respectively, bound HER2 and the addressed NK cell receptor. However, whereas B7-H6:HER2-scFv and AICL:HER2-scFv triggered NK cells to kill HER2-positive breast cancer cells at nanomolar concentrations, PVR:HER2-scFv was not efficacious. Moreover, NK cell cytotoxicity was enhanced synergistically when B7-H6:HER2-scFv or AICL:HER2-scFv were applied in combination with another HER2-specific immunoligand engaging the stimulatory receptor NKG2D. In contrast, no improvements were achieved by combining B7-H6:HER2-scFv with AICL:HER2-scFv. Additionally, B7-H6:HER2-scFv and AICL:HER2-scFv enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) by the therapeutic antibodies trastuzumab and cetuximab synergistically, with B7-H6:HER2-scFv exhibiting a higher efficacy. In summary, antibody-derived proteins engaging NKp30 or NKp80 may represent attractive biologics to further enhance anti-tumor NK cell responses and may provide an innovative approach to sensitize tumor cells for antibody-based immunotherapy. PMID:26392331

  13. Enhancement of antibody fragment secretion into the Escherichia coli periplasm by co-expression with the peptidyl prolyl isomerase, FkpA, in the cytoplasm.

    PubMed

    Levy, Raphael; Ahluwalia, Kiran; Bohmann, David J; Giang, Hoa M; Schwimmer, Lauren J; Issafras, Hassan; Reddy, Nithin B; Chan, Chung; Horwitz, Arnold H; Takeuchi, Toshihiko

    2013-08-30

    Improper protein folding or aggregation can frequently be responsible for low expression and poor functional activity of antibody fragments secreted into the Escherichia coli periplasm. Expression issues also can affect selection of antibody candidates from phage libraries, since antibody fragments displayed on phage also are secreted into the E. coli periplasm. To improve secretion of properly folded antibody fragments into the periplasm, we have developed a novel approach that involves co-expressing the antibody fragments with the peptidyl prolyl cis-trans isomerase, FkpA, lacking its signal sequence (cytFkpA) which consequently is expressed in the E. coli cytosol. Cytoplasmic expression of cytFkpA improved secretion of functional Fab fragments into the periplasm, exceeding even the benefits from co-expressing Fab fragments with native, FkpA localized in the periplasm. In addition, panning and subsequent screening of large Fab and scFv naïve phage libraries in the presence of cytFkpA significantly increased the number of unique clones selected, as well as their functional expression levels and diversity. PMID:23624043

  14. Gaussia Luciferase as a Genetic Fusion Partner with Antibody Fragments for Sensitive Immunoassay Monitoring of Clinical Biomarkers.

    PubMed

    Oyama, Hiroyuki; Morita, Izumi; Kiguchi, Yuki; Miyake, Sayaka; Moriuchi, Ayaka; Akisada, Tatsuki; Niwa, Toshifumi; Kobayashi, Norihiro

    2015-12-15

    In this study, we show the utility of Gaussia luciferase (GLuc), which is much smaller than previously found luciferases, as the fusion partner with artificial antibody species for developing sensitive immunoassay systems. As an example, we constructed a bioluminescent enzyme-linked immunosorbent assay (BL-ELISA) system determining the major glucocorticoid cortisol. A monoclonal antibody was newly elicited against a cortisol-albumin conjugate, and the genes encoding its variable domains (VH and VL) were cloned and combined to encode a single-chain Fv fragment (scFv). scFv was then linked to the wild-type GLuc gene or that encoding GLuc mutants reported to show improved emission kinetics and expressed in the periplasmic space of several Escherichia coli strains. Notably, the wild-type GLuc fusion protein (scFv-wtGLuc) showed the most suitable luminescent properties for BL-ELISAs. In our system, scFv-wtGLuc was reacted competitively with the analyte and immobilized cortisol moieties, and the bound GLuc activity was monitored with coelenterazine as the substrate. Successful batch-type luminescence detection was achieved using a plate reader without built-in injectors. The midpoint and limit of detection in a typical dose-response curve were 4.1 and 0.26 pg/assay, respectively, thus exhibiting much more sensitivity than conventional cortisol immunoassays. Serum cortisol levels (as the sum with cortisone) for healthy subjects, determined without any pretreatment, were compatible with reported reference ranges. The scFv-wtGLuc probe was stable over a year under storage as periplasmic extracts at -30 °C or with repeated freeze-thawing. These results suggest that GLuc fusions with antibody fragments might serve as useful and highly sensitive immunoassay probes in various clinical settings. PMID:26625180

  15. Production and characterization of a single-chain variable fragment linked alkaline phosphatase fusion protein for detection of O,O-diethyl organophosphorus pesticides in a one-step enzyme-linked immunosorbent assay

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A single-chain variable fragment (scFv) and alkaline phosphatase (AP) fusion protein for detection of O, O-diethyl organophosphorus pesticides (OPs) was produced and characterized. The scFv gene was prepared by cloning VL and VH genes from a hybridoma cell secreting monoclonal antibody with broad-s...

  16. Soluble Expression and Characterization of a New scFv Directed to Human CD123.

    PubMed

    Moradi-Kalbolandi, Shima; Davani, Dariush; Golkar, Majid; Habibi-Anbouhi, Mahdi; Abolhassani, Mohsen; Shokrgozar, Mohammad Ali

    2016-04-01

    Leukemic cancer stem cells (LSCs), as a unique cell population in acute myeloid leukemia (AML) marked by CD123 overexpression, are thought to play a key role in relapsed AML after chemotherapy. Thus, CD123 is considered as a particularly important target candidate for antibody-derived diagnosis and therapy. In the present work, we constructed an immunized murine antibody phage display library and isolated the functional anti-CD123 Single-chain fragment variable (scFv) clones. We also introduced fusing variable light (VL) and heavy (VH) chains with a new 18-amino acid residue linker as an alternative to conventional linkers. CD123-specific phage clones were progressively enriched through 4 rounds of biopanning, validated by phage ELISA, and anti-CD123 scFv clones with highest affinity were produced in Escherichia coli. The expression and purification of soluble scFv were verified by Western blot, and the results were indicative of the functionality of our proposed linker. The purified scFv specifically recognized CD123 by ELISA and flow cytometry, without any cross-reactivity with other related cell markers. Affinity of anti-CD123 scFv was measured to be 6.9 × 10(-7) M, using the competitive ELISA. Our work, therefore, provides a framework for future studies involving biological functions and applications of our anti-CD123 scFv. It also reveals the feasibility of high throughput methods to isolate biomarker-specific scFvs. PMID:26749295

  17. The antitumor efficacy of a novel adenovirus-mediated anti-p21Ras single chain fragment variable antibody on human cancers in vitro and in vivo.

    PubMed

    Yang, Ju-Lun; Pan, Xin-Yan; Zhao, Wen-Xing; Hu, Qi-Chan; Ding, Feng; Feng, Qiang; Li, Gui-Yun; Luo, Ying

    2016-03-01

    Activated ras genes are found in a large number of human tumors, and therefore are one of important targets for cancer therapy. This study investigated the antitumor effects of a novel single chain fragment variable antibody (scFv) against ras protein, p21Ras. The anti-p21Ras scFv gene was constructed by phage display library from hybridoma KGHR1, and then subcloned into replication-defective adenovirus vector to obtain recombinant adenovirus KGHV100. Human tumor cell lines with high expression of p21Ras SW480, MDA-MB‑231, OVCAR-3, BEL-7402, as well as tumor cell line with low expression of p21Ras, SKOV3, were employed to investigate antitumor effects in vitro and in vivo. Fluorescence microscopy demonstrated that KGHV100 was able to express intracellularly anti-p21Ras scFv antibody in cultured tumor cells and in transplantation tumor cells. MTT, Transwell, colony formation, and flow cytometry analysis showed that KGHV100 led to significant growth arrest in tumor cells with high p21Ras expression, and induced G0/G1 cell cycle arrest in the studied tumor cell lines. In vivo, KGHV100 significantly inhibited tumor growth following intratumoral injection, and the survival rates of the mice were higher than the control group. These results indicate that the adenovirus-mediated intracellular expression of the novel anti-p21Ras scFv exerted strong antitumoral effects, and may be a potential method for therapy of cancers with p21Ras overexpression. PMID:26780944

  18. Secretory signal peptide modification for optimized antibody-fragment expression-secretion in Leishmania tarentolae

    PubMed Central

    2012-01-01

    Background Secretory signal peptides (SPs) are well-known sequence motifs targeting proteins for translocation across the endoplasmic reticulum membrane. After passing through the secretory pathway, most proteins are secreted to the environment. Here, we describe the modification of an expression vector containing the SP from secreted acid phosphatase 1 (SAP1) of Leishmania mexicana for optimized protein expression-secretion in the eukaryotic parasite Leishmania tarentolae with regard to recombinant antibody fragments. For experimental design the online tool SignalP was used, which predicts the presence and location of SPs and their cleavage sites in polypeptides. To evaluate the signal peptide cleavage site as well as changes of expression, SPs were N-terminally linked to single-chain Fragment variables (scFv’s). The ability of L. tarentolae to express complex eukaryotic proteins with highly diverse post-translational modifications and its easy bacteria-like handling, makes the parasite a promising expression system for secretory proteins. Results We generated four vectors with different SP-sequence modifications based on in-silico analyses with SignalP in respect to cleavage probability and location, named pLTEX-2 to pLTEX-5. To evaluate their functionality, we cloned four individual scFv-fragments into the vectors and transfected all 16 constructs into L. tarentolae. Independently from the expressed scFv, pLTEX-5 derived constructs showed the highest expression rate, followed by pLTEX-4 and pLTEX-2, whereas only low amounts of protein could be obtained from pLTEX-3 clones, indicating dysfunction of the SP. Next, we analysed the SP cleavage sites by Edman degradation. For pLTEX-2, -4, and -5 derived scFv’s, the results corresponded to in-silico predictions, whereas pLTEX-3 derived scFv’s contained one additional amino-acid (AA). Conclusions The obtained results demonstrate the importance of SP-sequence optimization for efficient expression-secretion of sc

  19. Structural and functional characterization of a novel scFv anti-HSP60 of Strongyloides sp.

    PubMed Central

    Levenhagen, Marcelo Arantes; de Almeida Araújo Santos, Fabiana; Fujimura, Patrícia Tiemi; Caneiro, Ana Paula; Costa-Cruz, Julia Maria; Goulart, Luiz Ricardo

    2015-01-01

    Phage display is a powerful technology that selects specific proteins or peptides to a target. We have used Phage Display to select scFv (single-chain variable fragment) clones from a combinatorial library against total proteins of Strongyloides venezuelensis. After scFv characterization, further analysis demonstrated that this recombinant fragment of antibody was able to bind to an S. venezuelensis antigenic fraction of ~65 kDa, present in the body periphery and digestive system of infective larvae (L3), as demonstrated by immunofluorescence. Mass spectrometry results followed by bioinformatics analysis showed that this antigenic fraction was a heat shock protein 60 (HSP60) of Strongyloides sp. The selected scFv was applied in serodiagnosis by immune complexes detection in serum samples from individuals with strongyloidiasis using a sandwich enzyme-linked immunosorbent assay (ELISA), showing sensitivity of 97.5% (86.84–99.94), specificity of 98.81 (93.54–99.97), positive likelihood ratio of 81.60 and an area under the curve of 0.9993 (0.9973–1.000). Our study provided a novel monoclonal scFv antibody fragment which specifically bound to HSP60 of Strongyloides sp. and was applied in the development of an innovative serodiagnosis method for the human strongyloidiasis. PMID:25994608

  20. Isolation of Balamuthia mandrillaris-specific antibody fragments from a bacteriophage antibody display library.

    PubMed

    Siddiqui, Ruqaiyyah; Kulsoom, Huma; Lalani, Salima; Khan, Naveed Ahmed

    2016-07-01

    Balamuthia mandrillaris is a protist pathogen that can cause encephalitis with a mortality rate of more than 95%. Early diagnosis followed by aggressive treatment is a pre-requisite for successful prognosis. Current methods for identifying this organism rely on culture and microscopy, antibody-based methods using animals, or involve the use of molecular tools that are expensive. Here, we describe the isolation of antibody fragments that can be used for the unequivocal identification of B. mandrillaris. B. mandrillaris-specific antibody fragments were isolated from a bacteriophage antibody display library. Individual clones were studied by enzyme-linked immunosorbent assay, and immunofluorescence. Four antibody clones showed specific binding to B. mandrillaris. The usefulness of phage antibody display technology as a diagnostic tool for isolating antibody fragments against B. mandrillaris antigens and studying their biological role(s) is discussed further. PMID:27055361

  1. Overproduction of anti-Tn antibody MLS128 single-chain Fv fragment in Escherichia coli cytoplasm using a novel pCold-PDI vector.

    PubMed

    Subedi, Ganesh P; Satoh, Tadashi; Hanashima, Shinya; Ikeda, Akemi; Nakada, Hiroshi; Sato, Reiko; Mizuno, Mamoru; Yuasa, Noriyuki; Fujita-Yamaguchi, Yoko; Yamaguchi, Yoshiki

    2012-03-01

    Overproduction of recombinant proteins in Escherichia coli is often hampered by their failure to fold correctly, leading to their accumulation within inclusion bodies. To overcome the problem, a variety of techniques aimed at soluble expression have been developed including low temperature expression and/or fusion of soluble tags and chaperones. However, a general protocol for bacterial expression of disulfide bond-containing proteins has hitherto not been established. Single chain Fv fragments (scFvs) are disulfide bond-containing proteins often difficult to express in soluble forms in E. coli. We here examine in detail the E. coli expression of a scFv originating from an anti-carbohydrate MLS128 antibody as a model system. We combine three techniques: (1) tagging scFv with thioredoxin, DsbC and protein disulfide isomerase (PDI), (2) expressing the proteins at low temperature using the pCold vector system, and (3) using Origami E. coli strains with mutations in the thioredoxin reductase and glutathione reductase genes. We observed a high expression level of soluble MLS128-scFv in the Origami strain only when PDI is used as a tag. The recombinant protein retains full binding activity towards synthetic carbohydrate antigens. The developed "pCold-PDI" vector has potential for overproduction of other scFvs and disulfide-containing proteins in the Origami strains. PMID:22245752

  2. The novel anti-CD19 chimeric antigen receptors with humanized scFv (single-chain variable fragment) trigger leukemia cell killing.

    PubMed

    Qian, Liren; Li, Dan; Ma, Lie; He, Ting; Qi, Feifei; Shen, Jianliang; Lu, Xin-An

    2016-01-01

    The molecular design of CARs (Chimeric Antigen Receptors), especially the scFv, has been a major part to use of CAR-T cells for targeted adoptive immunotherapy. To address this issue, we chose a vector backbone encoding a second-generation CAR based on efficacy of a murine scFv-based CAR. Next, we generated a panel of humanized scFvs and tested in vitro for their ability to direct CAR-T cells to specifically lyse, proliferate, and secrete cytokines in response to antigen-bearing targets. Furthermore, in a xenograft model of lymphoma, human T cells expressing humanized scFvs exhibited the same anti-tumor efficacy as those expressing murine scFv and prolonged survival compared with cells expressing control CAR. Therefore, we uncovered CARs expressing humanized scFv domain that contribute the similar enhanced antileukemic efficacy and survival in tumor bearing mice. These results provide the basis for the future clinical studies of CAR-T cells transduced with humanized scFv directed to CD19. PMID:26996927

  3. DNA immunization combined with scFv phage display identifies antagonistic GCGR specific antibodies and reveals new epitopes on the small extracellular loops.

    PubMed

    van der Woning, Bas; De Boeck, Gitte; Blanchetot, Christophe; Bobkov, Vladimir; Klarenbeek, Alex; Saunders, Michael; Waelbroeck, Magali; Laeremans, Toon; Steyaert, Jan; Hultberg, Anna; De Haard, Hans

    2016-01-01

    The identification of functional monoclonal antibodies directed against G-protein coupled receptors (GPCRs) is challenging because of the membrane-embedded topology of these molecules. Here, we report the successful combination of llama DNA immunization with scFv-phage display and selections using virus-like particles (VLP) and the recombinant extracellular domain of the GPCR glucagon receptor (GCGR), resulting in glucagon receptor-specific antagonistic antibodies. By immunizing outbred llamas with plasmid DNA containing the human GCGR gene, we sought to provoke their immune system, which generated a high IgG1 response. Phage selections on VLPs allowed the identification of mAbs against the extracellular loop regions (ECL) of GCGR, in addition to multiple VH families interacting with the extracellular domain (ECD) of GCGR. Identifying mAbs binding to the ECL regions of GCGR is challenging because the large ECD covers the small ECLs in the energetically most favorable 'closed conformation' of GCGR. Comparison of Fab with scFv-phage display demonstrated that the multivalent nature of scFv display is essential for the identification of GCGR specific clones by selections on VLPs because of avid interaction. Ten different VH families that bound 5 different epitopes on the ECD of GCGR were derived from only 2 DNA-immunized llamas. Seven VH families demonstrated interference with glucagon-mediated cAMP increase. This combination of technologies proved applicable in identifying multiple functional binders in the class B GPCR context, suggesting it is a robust approach for tackling difficult membrane proteins. PMID:27211075

  4. Conversion of scFv peptide-binding specificity for crystal chaperone development

    SciTech Connect

    Pai, Jennifer C.; Culver, Jeffrey A.; Drury, Jason E.; Motani, Rakesh S.; Lieberman, Raquel L.; Maynard, Jennifer A.

    2012-02-07

    In spite of advances in protein expression and purification over the last decade, many proteins remain recalcitrant to structure determination by X-ray crystallography. One emerging tactic to obtain high-quality protein crystals for structure determination, particularly in the case of membrane proteins, involves co-crystallization with a protein-specific antibody fragment. Here, we report the development of new recombinant single-chain antibody fragments (scFv) capable of binding a specific epitope that can be introduced into internal loops of client proteins. The previously crystallized hexa-histidine-specific 3D5 scFv antibody was modified in the complementary determining region and by random mutagenesis, in conjunction with phage display, to yield scFvs with new biochemical characteristics and binding specificity. Selected variants include those specific for the hexa-histidine peptide with increased expression, solubility (up to 16.6 mg/ml) and sub-micromolar affinity, and those with new specificity for the EE hexa-peptide (EYMPME) and nanomolar affinity. Complexes of one such chaperone with model proteins harboring either an internal or a terminal EE tag were isolated by gel filtration. The 3.1 {angstrom} resolution structure of this chaperone reveals a binding surface complementary to the EE peptide and a {approx}52 {angstrom} channel in the crystal lattice. Notably, in spite of 85% sequence identity, and nearly identical crystallization conditions, the engineered scFv crystallizes in a different space group than the parent 3D5 scFv, and utilizes two new crystal contacts. These engineered scFvs represent a new class of chaperones that may eliminate the need for de novo identification of candidate chaperones from large antibody libraries.

  5. Repeated dosing of digoxin-fragmented antibody in preterm eclampsia.

    PubMed

    Adair, C D; Buckalew, V M; Kipikasa, J; Torres, C; Stallings, S P; Briery, C M

    2009-02-01

    Early onset eclampsia has significant morbidity and mortality for both the mother and fetus. No effective treatment exists at present except delivery and seizure prophylaxis with magnesium sulfate. We report the novel use of a fragmented ovine antibody against digoxin for the treatment of eclampsia. A 16-year-old primagravida at 29 weeks 5/7 days gestation presented with clinical diagnosis of eclampsia and was treated with compassionate off-label use of digoxin-fragmented ovine antibody (Digibind Glaxo Smith Kline, Research Triangle Park, NC, USA). Improvement of her underlying disorder during a 48 h treatment window was noted without adverse maternal or neonatal outcome. We suggest digoxin-fragmented ovine antibody as a possible intervention in preterm pregnancies complicated by pre-eclampsia or eclampsia. PMID:19177044

  6. Biotechnological applications of recombinant single-domain antibody fragments

    PubMed Central

    2011-01-01

    Background Single-domain antibody fragments possess structural features, such as a small dimension, an elevated stability, and the singularity of recognizing epitopes non-accessible for conventional antibodies that make them interesting for several research and biotechnological applications. Results The discovery of the single-domain antibody's potentials has stimulated their use in an increasing variety of fields. The rapid accumulation of articles describing new applications and further developments of established approaches has made it, therefore, necessary to update the previous reviews with a new and more complete summary of the topic. Conclusions Beside the necessary task of updating, this work analyses in detail some applicative aspects of the single-domain antibodies that have been overseen in the past, such as their efficacy in affinity chromatography, as co-crystallization chaperones, protein aggregation controllers, enzyme activity tuners, and the specificities of the unconventional single-domain fragments. PMID:21658216

  7. The human antibody fragment DIATHIS1 specific for CEACAM1 enhances natural killer cell cytotoxicity against melanoma cell lines in vitro.

    PubMed

    Dupuis, Maria L; Fiori, Valentina; Soriani, Alessandra; Ricci, Biancamaria; Dominici, Sabrina; Moricoli, Diego; Ascione, Alessandro; Santoni, Angela; Magnani, Mauro; Cianfriglia, Maurizio

    2015-01-01

    Several lines of evidence show that de novo expression of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is strongly associated with reduced disease-free survival of patients affected by metastatic melanoma. Previously published investigations report that homophilic interactions between CEACAM1 expressed on natural killer (NK) cells and tumors inhibit the NK cell-mediated killing independently of major histocompatibility complex class I recognition. This biological property can be physiologically relevant in metastatic melanoma because of the increased CEACAM1 expression observed on NK cells from some patients. Moreover, this inhibitory mechanism in many cases might hinder the efficacy of immunotherapeutic treatments of CEACAM1 malignancies because of tumor evasion by activated effector cells. In the present study, we designed an in vitro experimental model showing that the human single-chain variable fragment (scFv) DIATHIS1 specific for CEACAM1 is able to enhance the lytic machinery of NK cells against CEACAM1 melanoma cells. The coincubation of the scFv DIATHIS1 with CEACAM1 melanoma cells and NK-92 cell line significantly increases the cell-mediated cytotoxicity. Moreover, pretreatment of melanoma cells with scFv DIATHIS1 promotes the activation and the degranulation capacity of in vitro-expanded NK cells from healthy donors. It is interesting to note that the melanoma cell line MelC and the primary melanoma cells STA that respond better to DIATHIS1 treatment, express higher relative levels of CEACAM1-3L and CEACAM1-3S splice variants isoforms compared with Mel501 cells that are less responsive to DIATHIS1-induced NK cell-mediated cytotoxicity. Taken together, our results suggest that the fully human antibody fragment DIATHIS1 originated by biopanning approach from a phage antibody library may represent a relevant biotechnological platform to design and develop completely human antimelanoma therapeutics of biological origin. PMID:26448580

  8. The Human Antibody Fragment DIATHIS1 Specific for CEACAM1 Enhances Natural Killer Cell Cytotoxicity Against Melanoma Cell Lines In Vitro

    PubMed Central

    Dupuis, Maria L.; Soriani, Alessandra; Ricci, Biancamaria; Dominici, Sabrina; Moricoli, Diego; Ascione, Alessandro; Santoni, Angela; Magnani, Mauro; Cianfriglia, Maurizio

    2015-01-01

    Several lines of evidence show that de novo expression of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is strongly associated with reduced disease-free survival of patients affected by metastatic melanoma. Previously published investigations report that homophilic interactions between CEACAM1 expressed on natural killer (NK) cells and tumors inhibit the NK cell-mediated killing independently of major histocompatibility complex class I recognition. This biological property can be physiologically relevant in metastatic melanoma because of the increased CEACAM1 expression observed on NK cells from some patients. Moreover, this inhibitory mechanism in many cases might hinder the efficacy of immunotherapeutic treatments of CEACAM1+ malignancies because of tumor evasion by activated effector cells. In the present study, we designed an in vitro experimental model showing that the human single-chain variable fragment (scFv) DIATHIS1 specific for CEACAM1 is able to enhance the lytic machinery of NK cells against CEACAM1+ melanoma cells. The coincubation of the scFv DIATHIS1 with CEACAM1+ melanoma cells and NK-92 cell line significantly increases the cell-mediated cytotoxicity. Moreover, pretreatment of melanoma cells with scFv DIATHIS1 promotes the activation and the degranulation capacity of in vitro–expanded NK cells from healthy donors. It is interesting to note that the melanoma cell line MelC and the primary melanoma cells STA that respond better to DIATHIS1 treatment, express higher relative levels of CEACAM1-3L and CEACAM1-3S splice variants isoforms compared with Mel501 cells that are less responsive to DIATHIS1-induced NK cell–mediated cytotoxicity. Taken together, our results suggest that the fully human antibody fragment DIATHIS1 originated by biopanning approach from a phage antibody library may represent a relevant biotechnological platform to design and develop completely human antimelanoma therapeutics of biological origin. PMID

  9. Molecular engineering of high affinity single-chain antibody fragment for endothelial targeting of proteins and nanocarriers in rodents and humans.

    PubMed

    Greineder, Colin F; Hood, Elizabeth D; Yao, Anning; Khoshnejad, Makan; Brenner, Jake S; Johnston, Ian H; Poncz, Mortimer; Gottstein, Claudia; Muzykantov, Vladimir R

    2016-03-28

    Endothelial cells (EC) represent an important target for pharmacologic intervention, given their central role in a wide variety of human pathophysiologic processes. Studies in lab animal species have established that conjugation of drugs and carriers with antibodies directed to surface targets like the Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1, a highly expressed endothelial transmembrane protein) help to achieve specific therapeutic interventions in ECs. To translate such "vascular immunotargeting" to clinical practice, it is necessary to replace antibodies by advanced ligands that are more amenable to use in humans. We report the molecular design of a single chain variable antibody fragment (scFv) that binds with high affinity to human PECAM-1 and cross-reacts with its counterpart in rats and other animal species, allowing parallel testing in vivo and in human endothelial cells in microfluidic model. Site-specific modification of the scFv allows conjugation of protein cargo and liposomes, enabling their endothelial targeting in these models. This study provides a template for molecular engineering of ligands, enabling studies of drug targeting in animal species and subsequent use in humans. PMID:26855052

  10. Chaperone-Assisted Soluble Expression of a Humanized Anti-EGFR ScFv Antibody in E. Coli

    PubMed Central

    Veisi, Kamal; Farajnia, Safar; Zarghami, Nosratollah; Khoram Khorshid, Hamid Reza; Samadi, Nasser; Ahdi Khosroshahi, Shiva; Zarei Jaliani, Hossein

    2015-01-01

    Purpose: Formation of inclusion bodies is a considerable obstacle threatening the advantages of E. coli expression system to serve as the most common and easiest system in recombinant protein production. To solve this problem, several strategies have been proposed among which application of molecular chaperones is of remarkable consideration. The aim of this study was to evaluate the effects of molecular chaperones on soluble expression of aggregation-prone humanized single chain antibody. Methods: To increase the solubility of a humanized single chain antibody (hscFv), different chaperone plasmids including PG-tf2 (GroES- GroEL- tig), ptf16 (tig) and pGro7 (GroES- GroEL) were co-expressed in BL21 cells containing pET-22b- hscFv construct. The solubility of recombinant hscFv was analyzed by SDS-PAGE. After purification of soluble hscFv by Ni-NTA column, the biological activity and cytotoxicity of the recombinant protein were tested by ELISA and MTT assay, respectively. Results: SDS-PAGE analysis of the hscFv revealed that chaperone utility remarkably increased (up to 50%) the solubility of the protein. ELISA test and MTT assay analyses also confirmed the biological activity of the gained hscFv in reaction with A431 cells (OD value: 2.6) and inhibition of their proliferation, respectively. Conclusion: The results of this study revealed that co-expression of chaperones with hscFv leads to remarkable increase in the solubility of the recombinant hscFv, which could be of great consideration for large scale production of recombinant single chain antibodies. PMID:26793607

  11. Therapeutic Monoclonal Antibodies and Fragments: Bevacizumab.

    PubMed

    Klein, Ainat; Loewenstein, Anat

    2016-01-01

    Bevacizumab (Avastin) is a recombinant humanized monoclonal immunoglobulin antibody that has two antigen-binding domains and blocks all active forms of vascular endothelial growth factor-A. It was originally designed and is still in use as antitumor agent (for colorectal and non-small cell lung cancers). Besides inhibiting vessel growth and neovascularization, the drug promotes the regression of existing microvessels and induces 'normalization' of surviving mature vasculature, stabilizes vessels and prevents leakage. Its molecular weight is 149 kDa and its estimated terminal half-life is approximately 20 days for both men and women. The effectiveness and safety of bevacizumab was proven in retrospective and prospective controlled clinical trials for the treatment of neovascular age-related macular degeneration, neovascularization in proliferative diabetic retinopathy, diabetic macular edema, retinal vein occlusion and retinopathy of prematurity, especially for zone I. Uncontrolled trials have shown its effectiveness in various other conditions as myopic and uveitic choroidal neovascularization and neovascular glaucoma. There are no absolute contraindications to intravitreal injection though it is recommended to withhold treatment in patients who have recently suffered from a cardiovascular or cerebrovascular event and during pregnancy. Ocular complications from intravitreal use are usually mild and transient (corneal abrasion, chemosis, subconjunctival hemorrhage and vitreous hemorrhage). Bacterial endophthalmitis is rare (about 0.1%). New or progressive subretinal hemorrhages, tears of the retinal pigment epithelium and an increased incidence of geographic atrophy have also been reported. PMID:26502311

  12. Progress in programming antibody fragments to crystallize.

    PubMed

    Edmundson, A B; Borrebaeck, C A

    1998-01-01

    Completion of the X-ray analysis of the human B7-15A2 Fab opened a new vista (Immunotechnology 3, no. 4). In the crystal lattice, both the lambda-type light chain (CL domain) and gamma 1-type heavy chain (CH1 domain) participated in formation of antiparallel beta-pleated sheets with neighboring molecules related to the reference Fab by 2-fold axes. This observation evoked memories of the first description of this type of packing for human Bence-Jones (lambda chain) dimers 20 years ago (Ely K.R. et al. Biochemistry 1978;17:158-167). Reexamination of packing interactions in selected crystal systems revealed that the C domains of lambda and gamma 1 chains were structurally amenable to the formation of such cross-molecule beta-structures, but kappa chain CL domains were not. In the latter, a single proline residue disrupted the order of beta-strand 3-3 in the middle of the surface used in lambda and gamma 1 chains for intermolecular interactions with symmetry-related molecules. For the packing of Fv molecules, the VL domains are structurally well suited for analogous packing interactions through antiparallel 4-1 beta-strands in adjacent molecules. Such interactions have been shown to provide the driving force in the crystal packing of a human (Pot) Fv from an IgM-kappa cryoglobulin. Together, these observations suggest several avenues through which propensity to crystallize can be programmed into the designs of synthetic human Fabs, Fvs and single-chain antibodies. PMID:9530564

  13. Comparison of three microbial hosts for the expression of an active catalytic scFv.

    PubMed

    Robin, Sylvain; Petrov, Kliment; Dintinger, Thierry; Kujumdzieva, Anna; Tellier, Charles; Dion, Michel

    2003-01-01

    Antibodies represent an interesting protein framework on which catalytic functions can be grafted. In previous studies, we have reported the characterization of the catalytic antibody 4B2 obtained on the basis of the "bait and switch" strategy which catalyzes two different chemical reactions: the allylic isomerization of beta,gamma-unsaturated ketones and the Kemp elimination. We have cloned the antibody 4B2 and expressed it as a single-chain Fv (scFv) fragment in different expression systems, Escherichia coli and two yeasts species, in order to elicit the most suitable system to study its catalytic activity. The scFv4B2 was secreted as an active form in the culture medium of Pichia pastoris and Kluyveromyces lactis, which led respectively to 4 and 1.3mg/l after purification. In E. coli, different strategies were investigated to increase the cytoplasmic soluble fraction, which resulted, in all cases, in the expression of a low amount of functional antibodies. By contrast, substantial amount of scFv4B2 could be purified when it was expressed as inclusion bodies (12mg/l) and submitted to an in vitro refolding process. Its catalytic activity was measured and proved to be comparable to that of the whole IgG. However, the instability of the scFv4B2 in solution prevented from an exhaustive characterization of its activity and stabilization of this protein appears to be essential before designing strategies to improve its catalytic activity. PMID:12531284

  14. Directed evolution of an anti-prion protein scFv fragment to an affinity of 1 pM and its structural interpretation.

    PubMed

    Luginbühl, Béatrice; Kanyo, Zoltan; Jones, R Mark; Fletterick, Robert J; Prusiner, Stanley B; Cohen, Fred E; Williamson, R Anthony; Burton, Dennis R; Plückthun, Andreas

    2006-10-13

    Bovine spongiform encephalopathy (BSE) is a fatal neurodegenerative prion disease affecting cattle that is transmissible to humans, manifesting as a variant of Creutzfeldt-Jakob disease (vCJD) likely following the consumption of meat contaminated with BSE prions. High-affinity antibodies are a prerequisite for the development of simple, highly sensitive and non-invasive diagnostic tests that are able to detect even small amounts of the disease-associated PrP conformer (PrP(Sc)). We describe here the affinity maturation of a single-chain Fv antibody fragment with a binding affinity of 1 pM to a peptide derived from the unstructured region of bovine PrP (BoPrP (90-105)). This is the tightest peptide-binding antibody reported to date and may find useful application in diagnostics, especially when PrP(Sc) is pretreated by denaturation and/or proteolysis for peptide-like presentation. Several rounds of directed evolution and off-rate selection with ribosome display were performed using an antibody library generated from a single PrP binder with error-prone PCR and DNA-shuffling. As the correct determinations of affinities in this range are not straightforward, competition biosensor techniques and KinExA methods were both applied and compared. Structural interpretation of the affinity improvement was performed based on the crystal structure of the original prion binder in complex with the BoPrP (95-104) peptide by modeling the corresponding mutations. PMID:16962610

  15. Fast antibody fragment motion: flexible linkers act as entropic spring

    DOE PAGESBeta

    Stingaciu, Laura R.; Ivanova, Oxana; Ohl, Michael; Biehl, Ralf; Richter, Dieter

    2016-03-29

    A flexible linker region between three fragments allows antibodies to adjust their binding sites to an antigen or receptor. Using Neutron Spin Echo Spectroscopy we observed fragment motion on a timescale of 7 ns with motional amplitudes of about 1 nm relative to each other. The mechanistic complexity of the linker region can be described by a spring model with Brownian motion of the fragments in a harmonic potential. Displacements, timescale, friction and force constant of the underlying dynamics are accessed. The force constant exhibits a similar strength to an entropic spring, with friction of the fragment matching the unboundmore » state. The observed fast motions are fluctuations in pre-existing equilibrium configurations. In conclusion, the Brownian motion of domains in a harmonic potential is the appropriate model to examine functional hinge motions dependent on the structural topology and highlights the role of internal forces and friction to function.« less

  16. Fast antibody fragment motion: flexible linkers act as entropic spring

    PubMed Central

    Stingaciu, Laura R.; Ivanova, Oxana; Ohl, Michael; Biehl, Ralf; Richter, Dieter

    2016-01-01

    A flexible linker region between three fragments allows antibodies to adjust their binding sites to an antigen or receptor. Using Neutron Spin Echo Spectroscopy we observed fragment motion on a timescale of 7 ns with motional amplitudes of about 1 nm relative to each other. The mechanistic complexity of the linker region can be described by a spring model with Brownian motion of the fragments in a harmonic potential. Displacements, timescale, friction and force constant of the underlying dynamics are accessed. The force constant exhibits a similar strength to an entropic spring, with friction of the fragment matching the unbound state. The observed fast motions are fluctuations in pre-existing equilibrium configurations. The Brownian motion of domains in a harmonic potential is the appropriate model to examine functional hinge motions dependent on the structural topology and highlights the role of internal forces and friction to function. PMID:27020739

  17. Fast antibody fragment motion: flexible linkers act as entropic spring.

    PubMed

    Stingaciu, Laura R; Ivanova, Oxana; Ohl, Michael; Biehl, Ralf; Richter, Dieter

    2016-01-01

    A flexible linker region between three fragments allows antibodies to adjust their binding sites to an antigen or receptor. Using Neutron Spin Echo Spectroscopy we observed fragment motion on a timescale of 7 ns with motional amplitudes of about 1 nm relative to each other. The mechanistic complexity of the linker region can be described by a spring model with Brownian motion of the fragments in a harmonic potential. Displacements, timescale, friction and force constant of the underlying dynamics are accessed. The force constant exhibits a similar strength to an entropic spring, with friction of the fragment matching the unbound state. The observed fast motions are fluctuations in pre-existing equilibrium configurations. The Brownian motion of domains in a harmonic potential is the appropriate model to examine functional hinge motions dependent on the structural topology and highlights the role of internal forces and friction to function. PMID:27020739

  18. Development of a biotinylated broad-specificity single-chain variable fragment antibody and a sensitive immunoassay for detection of organophosphorus pesticides.

    PubMed

    Zhao, Fengchun; Tian, Yuan; Wang, Huimin; Liu, Jiye; Han, Xiao; Yang, Zhengyou

    2016-09-01

    Organophosphorus pesticides (OPs) are the most widely used pesticides in agriculture, and OP residues have been broadly reported in food and environmental samples. The aim of this study is to develop a recombinant antibody-based broad-specificity immunoassay for OPs. A phage display library was prepared from a mouse pre-immunized with a generic immunogen of OPs, and a single-chain variable fragment (scFv) antibody was selected. The selected scFv antibody was fused with biotin acceptor domain (BAD) and overexpressed as an inclusion body in Escherichia coli BL21 (DE3). Then, the protein was refolded by stepwise urea gradient dialysis and biotinylated in vitro by E. coli biotin ligase (BirA). Subsequently, the scFv-BAD protein was purified from the biotinylated system with high yield (66.7 mg L(-1)) and confirmed by SDS-PAGE and Western blot. Based on the biotinylated scFv-BAD, a sensitive and broad-specificity competitive indirect enzyme-linked immunosorbent assay (ciELISA) for detection of OPs was developed. The cross-reactivity (CR) studies demonstrated that the ciELISA described here exhibited the broadest detection spectrum for OPs up to now, and 30 OPs could be determined with 50 % inhibition value (IC50) values ranging from 19.4 to 515.2 ng mL(-1). Moreover, the developed ciELISA was used for the recovery study of the spiked samples and showed satisfactory recoveries. Graphical Abstract Schematic diagram of the development of biotinylated broad-specificity single-chain variable fragment antibody-based immunoassay for organophosphorus pesticides. PMID:27411546

  19. Negative effects of a disulfide bond mismatch in anti-rabies G protein single-chain antibody variable fragment FV57.

    PubMed

    Duan, Ye; Gu, Tiejun; Zhang, Xizhen; Jiang, Chunlai; Yuan, Ruosen; Li, Zhuang; Wang, Dandan; Chen, Xiaoxu; Wu, Chunlai; Chen, Yan; Wu, Yongge; Kong, Wei

    2014-06-01

    Rabies virus (RV) causes a fatal infectious disease requiring efficient post-exposure prophylaxis (PEP), which includes a rabies vaccine and rabies immunoglobulin (RIG). The single-chain antibody variable fragment (scFv), a small engineered antibody fragment derived from an antibody variable heavy chain and light chain, has the potential to replace the current application of RIG. In previous studies, we constructed and evaluated an anti-rabies virus G protein scFv (FV57) based on the monoclonal antibody CR57. Of the five cysteines in FV57, four are linked in intra-chain disulfide bonds (Cys-VH28/Cys-VH98 and Cys-VL16/Cys-VL84), and one is free (Cys-VL85). However, the thiol in Cys-VL85 neighboring Cys-VL84 in the CDR3 of the light chain is likely to mismatch with the thiol in Cys-VL16 during the renaturing process. In order to study effects of the mismatched disulfide bond, Cys-VL85 and Cys-VL84 of FV57 were mutated to serine to construct mutants FV57(VL85S) and FV57(VL84S). Furthermore, the disulfide bonds in the light chain of FV57, FV57(VL85S) and FV57(VL84S) were deleted by mutating Cys-VL16 to serine. All mutants were prepared and evaluated along with the original FV57. The results indicated that the mismatched disulfide bond of FV57 linking the light chain FR1 and CDR3 would confer deleterious negative effects on its activity against RV, likely due to spatial hindrance in the light chain CDR3. Moreover, avoidance of the disulfide bond mismatch provided an additional 30% protective efficacy against RV infection in the mouse RV challenge model. Thus, modifications of FV57 to eliminate the disulfide bond mismatch may provide a candidate therapeutic agent for effective PEP against rabies. PMID:24598312

  20. Monoclonal antibodies and Fc fragments for treating solid tumors.

    PubMed

    Eisenbeis, Andrea M; Grau, Stefan J

    2012-01-01

    Advances in biotechnology, better understanding of pathophysiological processes, as well as the identification of an increasing number of molecular markers have facilitated the use of monoclonal antibodies and Fc fragments in various fields in medicine. In this context, a rapidly growing number of these substances have also emerged in the field of oncology. This review will summarize the currently approved monoclonal antibodies used for the treatment of solid tumors with a focus on their clinical application, biological background, and currently ongoing trials. PMID:22291463

  1. Monoclonal antibodies and Fc fragments for treating solid tumors

    PubMed Central

    Eisenbeis, Andrea M; Grau, Stefan J

    2012-01-01

    Advances in biotechnology, better understanding of pathophysiological processes, as well as the identification of an increasing number of molecular markers have facilitated the use of monoclonal antibodies and Fc fragments in various fields in medicine. In this context, a rapidly growing number of these substances have also emerged in the field of oncology. This review will summarize the currently approved monoclonal antibodies used for the treatment of solid tumors with a focus on their clinical application, biological background, and currently ongoing trials. PMID:22291463

  2. Efficient silkworm expression of single-chain variable fragment antibody against ginsenoside Re using Bombyx mori nucleopolyhedrovirus bacmid DNA system and its application in enzyme-linked immunosorbent assay for quality control of total ginsenosides.

    PubMed

    Sakamoto, Seiichi; Pongkitwitoon, Benyakan; Nakamura, Seiko; Maenaka, Katsumi; Tanaka, Hiroyuki; Morimoto, Satoshi

    2010-09-01

    A single-chain variable fragment (scFv) antibody against ginsenoside Re (G-Re) have been successfully expressed in the silkworm larvae using Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system. The baculovirus donor vector for expression of scFv against G-Re (GRe-scFv) was constructed to contain honeybee melittin signal sequence to accelerate secretion of the recombinant GRe-scFv into the haemolymph of silkworm larvae. Functional recombinant GRe-scFv was purified by cation exchange chromatography followed by immobilized metal ion affinity chromatography. The yield of purified GRe-scFv was 6.5 mg per 13 silkworm larvae, which is equivalent to 650 mg/l of the haemolymph, exhibiting extremely higher yield than that expressed in Escherichia coli (1.7 mg/l of culture medium). It was revealed from characterization that GRe-scFv retained similar characteristic of the parental monoclonal antibody (MAb) against G-Re (MAb-4G10), making it possible to develop indirect competitive enzyme-linked immunosorbent assay (icELISA) for quality control of total ginsenosides in various ginsengs. The detectable range for calibration of G-Re by developed icELISA shows 0.05-10 microg/ml. These results clearly suggested that the silkworm expression system is quite useful for the expression of functional scFv that frequently required time- and cost-consuming re-folding when it expressed in E. coli. PMID:20592135

  3. An assay for the detection of grapevine leafroll-associated virus 3 using a single-chain fragment variable antibody.

    PubMed

    Cogotzi, Laura; Giampetruzzi, Annalisa; Nölke, Greta; Orecchia, Martin; Elicio, Vito; Castellano, Maria Antonietta; Martelli, Giovanni P; Fischer, Rainer; Schillberg, Stefan; Saldarelli, Pasquale

    2009-01-01

    Grapevine leafroll-associated virus 3 (GLRaV-3) is a major pathogen of grapevine. A previously described single-chain fragment variable (scFv) antibody (scFvLR3), directed against the coat protein (CP) of GLRaV-3, was expressed in Escherichia coli and used to develop a diagnostic ELISA kit. The antibody was fused to the light chain constant domain of human immunoglobulin to create the bivalent reagent C(L)-LR3, which was purified from the periplasmic fraction, giving a yield of ~5 mg/l. The sensitivity of the reagent against recombinant GLRaV-3 CP was 0.1 ng. The sensitivity, specificity and durability of the reagent was similar to a commercial kit. The C(L)-LR3 showed a weak cross-reaction in immune electron microscopy assays to GLRaV-1 and -7, but not with the phylogenetically more distant GLRaV-2. A fully recombinant kit was developed with the inclusion of a recombinant GLRaV-3 CP expressed in bacteria, thus avoiding problems associated with virus propagation and purification. This system represents a rapid, simple, sensitive and standardized diagnostic protocol for GLRaV-3 detection. PMID:19082687

  4. In Vivo Biodistribution, PET Imaging, and Tumor Accumulation of 86Y- and 111In-Antimindin/RG-1, Engineered Antibody Fragments in LNCaP Tumor–Bearing Nude Mice

    PubMed Central

    Schneider, Douglas W.; Heitner, Tara; Alicke, Bruno; Light, David R.; McLean, Kirk; Satozawa, Noboru; Parry, Gordon; Yoo, Jeongsoo; Lewis, Jason S.; Parry, Renate

    2016-01-01

    To optimize in vivo tissue uptake kinetics and clearance of engineered monoclonal antibody (mAb) fragments for radiotherapeutic and radiodiagnostic applications, we compared the biodistribution and tumor localization of four 111In- and 86Y-labeled antibody formats, derived from a single antimindin/RG-1 mAb, in a prostate tumor model. The IgG, diabody, single-chain variable domain (scFv), and novel miniantibody formats, composed of the human IgE-CH4 and a modified IgG1 hinge linked to scFv domains, were compared. Methods Antibodies were first derivatized with the bifunctional chelator CHX-A″-diethylenetriamine pentaacetic acid and then bound to the radiometal to create radiolabeled immunoconjugates. Human LNCaP xenografts were grown in nude mice, and 111In- or 86Y-labeled antibodies were administered intravenously. Tissues were harvested at different times, and the level of antibody deposition was determined by measuring radioactivity. Whole-body small-animal PET of mice receiving 86Y-labeled antibodies was performed at 6 time points and colocalized with simultaneous micro-CT imaging. Results The biodistributions of 111In and 86Y antibodies were quite similar. The blood, tumor, kidney, and liver tissues contained varying levels of radioactivity. The antibody accumulation in the tumor correlated with molecular size. The IgG steadily increased with time to 24.1 percentage injected dose per gram (%ID/g) at 48 h. The miniantibody accumulated at a similar rate to reach a lower level (14.2 %ID/g) at 48 h but with a higher tumor-to-blood ratio than the IgG. Tumor accumulation of the diabody peaked at 3 h, reaching a much lower level (3.7 %ID/g). A combination of rapid clearance and lower relative affinity of the scFv precluded deposition in the tumor. Small-animal PET results correlated well with the biodistribution results, with similar tumor localization patterns. Conclusion The larger antibody formats (IgG and miniantibody) gave higher tumor uptake levels than did the

  5. A polar ring endows improved specificity to an antibody fragment.

    PubMed

    Schaefer, Zachary P; Bailey, Lucas J; Kossiakoff, Anthony A

    2016-07-01

    Engineering monovalent Fab fragments into bivalent formats like IgGs or F(ab')2 can lead to aggregation presumably because of nonspecific off-target interactions that induce aggregation. In an effort to further understand the molecular determinants of nonspecific interactions for engineered antibodies and natively folded proteins in general, we focused on a synthetic Fab with low nanomolar affinity to histone chaperone Anti-silencing factor 1 (Asf1) that demonstrates off-target binding through low solubility (∼5 mg/mL) in the multivalent F(ab') 2 state. Here, we generated phage display-based shotgun scanning libraries to introduce aspartate as a negative design element into the antibody paratope. The antibody-combining site was amenable to aspartate substitution at numerous positions within the antigen binding loops and one variant, Tyr(L93) Asp/His(L94) Asp/Thr(H100b) Asp, possessed high solubility (>100 mg/ml). Furthermore, the mutations decreased nonspecific interactions measured by column interaction chromatography and ELISA in the multivalent antibody format while maintaining high affinity to the antigen. Structural determination of the antibody-antigen complex revealed that the aspartate-permissive residues formed a polar ring around the structural and functional paratope, recapitulating the canonical feature of naturally occurring protein-protein interactions. This observation may inform future strategies for the design and engineering of molecular recognition. PMID:27334407

  6. Construction of scFv that bind both fibronectin-binding protein A and clumping factor A of Stapylococcus aureus.

    PubMed

    Wang, Man; Zhang, Yan; Li, Benqiang; Zhu, Jianguo

    2015-06-01

    Bovine mastitis (BM) causes significant losses to the dairy industry. Vaccines against the causative agent of BM, Staphylococcus aureus, do not confer adequate protection. Because passive immunization with antibodies permits disease prevention, we constructed a recombinant single-chain antibody (scFv) against fibronectin-binding protein A (FnBPA) and clumping factor A (ClfA), two important virulence factors in S. aureus infection. The DNA coding sequences of the variable heavy (VH) and variable light (VL) domains of antibodies produced in the peripheral blood lymphocytes of cows with S. aureus-induced mastitis were obtained using reverse transcription and polymerase chain reaction, and the VH and VL cDNAs were assembled in-tandem using a DNA sequence encoding a (Gly4Ser)3 peptide linker. The scFv cDNAs were cloned into the pOPE101 plasmid for the expression of soluble scFv protein in Escherichia coli. The binding of the scFvs to both FnBPA and ClfA was confirmed using an indirect ELISA and Western blotting. The DNA sequences of the framework regions of the VH and VL domains were highly conserved, and the complementarity-determining regions displayed significant diversity, especially in CDR3 of the VH domain. These novel bovine antibody fragments may be useful as a therapeutic candidate for the prevention and treatment of S. aureus-induced bovine mastitis. PMID:25910693

  7. Large-Scale Purification of r28M: A Bispecific scFv Antibody Targeting Human Melanoma Produced in Transgenic Cattle

    PubMed Central

    Spiesberger, Katrin; Paulfranz, Florian; Egger, Anton; Reiser, Judith; Vogl, Claus; Rudolf-Scholik, Judith; Mayrhofer, Corina; Grosse-Hovest, Ludger; Brem, Gottfried

    2015-01-01

    Background 30 years ago, the potential of bispecific antibodies to engage cytotoxic T cells for the lysis of cancer cells was discovered. Today a variety of bispecific antibodies against diverse cell surface structures have been developed, the majority of them produced in mammalian cell culture systems. Beside the r28M, described here, no such bispecific antibody is known to be expressed by transgenic livestock, although various biologicals for medical needs are already harvested—mostly from the milk—of these transgenics. In this study we investigated the large-scale purification and biological activity of the bispecific antibody r28M, expressed in the blood of transgenic cattle. This tandem single-chain variable fragment antibody is designed to target human CD28 and the melanoma/glioblastoma-associated cell surface chondroitin sulfate proteoglycan 4 (CSPG4). Results With the described optimized purification protocol an average yield of 30 mg enriched r28M fraction out of 2 liters bovine plasma could be obtained. Separation of this enriched fraction by size exclusion chromatography into monomers, dimers and aggregates and further testing regarding the biological activity revealed the monomer fraction as being the most appropriate one to continue working with. The detailed characterization of the antibody’s activity confirmed its high specificity to induce the killing of CSPG4 positive cells. In addition, first insights into tumor cell death pathways mediated by r28M-activated peripheral blood mononuclear cells were gained. In consideration of possible applications in vivo we also tested the effect of the addition of different excipients to r28M. Conclusion Summing up, we managed to purify monomeric r28M from bovine plasma in a large-scale preparation and could prove that its biological activity is unaffected and still highly specific and thus, might be applicable for the treatment of melanoma. PMID:26469402

  8. Potent and broad neutralizing activity of a single chain antibody fragment against cell-free and cell-associated HIV-1

    PubMed Central

    Borges, Andrew Rosa; Ptak, Roger G; Wang, Yanping; Dimitrov, Antony S; Alam, S. Munir; Wieczorek, Lindsay; Bouma, Peter; Fouts, Timothy; Jiang, Shibo; Polonis, Victoria R; Haynes, Barton F; Quinnan, Gerald V; Montefiori, David C; Dimitrov, Dimiter S

    2010-01-01

    Several human monoclonal antibodies (hmAbs) exhibit relatively potent and broad neutralizing activity against HIV-1, but there has not been much success in using them as potential therapeutics. We have previously hypothesized and demonstrated that small engineered antibodies can target highly conserved epitopes that are not accessible by full-size antibodies. However, their potency has not been comparatively evaluated with known HIV-1-neutralizing hmAbs against large panels of primary isolates. We report here the inhibitory activity of an engineered single chain antibody fragment (scFv), m9, against several panels of primary HIV-1 isolates from group M (clades A–G) using cell-free and cell-associated virus in cell line-based assays. M9 was much more potent than scFv 17b, and more potent than or comparable to the best-characterized broadly neutralizing hmAbs IgG1 b12, 2G12, 2F5 and 4e10. It also inhibited cell-to-cell transmission of HIV-1 with higher potency than enfuvirtide (t-20, Fuzeon). M9 competed with a sulfated CCR5 N-terminal peptide for binding to gp120-CD4 complex, suggesting an overlapping epitope with the coreceptor binding site. M9 did not react with phosphatidylserine (pS) and cardiolipin (CL), nor did it react with a panel of autoantigens in an antinuclear autoantibody (ANA) assay. We further found that escape mutants resistant to m9 did not emerge in an immune selection assay. these results suggest that m9 is a novel anti-HIV-1 candidate with potential therapeutic or prophylactic properties, and its epitope is a new target for drug or vaccine development. PMID:20305395

  9. Exceptionally long CDR3H of bovine scFv antigenized with BoHV-1 B-epitope generates specific immune response against the targeted epitope.

    PubMed

    Pasman, Yfke; Soliman, Caroline; Ramsland, Paul A; Kaushik, Azad K

    2016-09-01

    We discovered that some bovine antibodies are amongst the largest known to exist due to the presence of an exceptionally long CDR3H (≥49 amino acids) with multiple cysteines that provide a unique knob and stalk structure to the antigen binding site. The large CDR3H size, unlike mouse and human, provides a suitable platform for antigenization with large configurational B-epitopes. Here we report the identification of a B-epitope on the gC envelope protein of bovine herpes virus type-1 (BoHV-1) recognized by a bovine IgG1 antibody. The identified 156 amino acid long gC fragment (gC156) was expressed as a recombinant protein. Subsequently, a functional scFv fragment with a 61 amino-acid long CDR3H (scFv1H12) was expressed such that gC156 was grafted into the CDR3H, replacing the "knob" region (gC156scFv1H12 or Ag-scFv). Importantly, the Ag-scFv could be recognized by a neutralizing antibody fragment (scFv3-18L), which suggests that the engraftment of gC156 into the CDR3H of 1H12 maintained the native conformation of the BoHV-1 B-epitope. A 3D model of gC156 was generated using fold-recognition approaches and this was grafted onto the CDR3H stalk of the 1H12 Fab crystal structure to predict the 3D structure of the Ag-scFv. The grafted antigen in Ag-scFv is predicted to have a compact conformation with the ability to protrude into the solvent. Upon immunization of bovine calves, the antigenized scFv (gC156scFv1H12) induced a higher antibody response as compared to free recombinant gC156. These observations suggest that antigenization of bovine scFv with an exceptionally long CDR3H provides a novel approach to developing the next generation of vaccines against infectious agents that require induction of protective humoral immunity. PMID:27497190

  10. Generation of scFv specific to human VEGFR-3 from the neutralizing mAb BDD073.

    PubMed

    Chen, Hao; Wang, Chaoqun; Gao, Yuan; Gao, Jianen; Zhou, Xiaping; Cai, Zhiming; Sun, Qihong

    2015-01-01

    In our previous study, we have produced a neutralizing mAb of vascular endothelial growth factor receptor 3 (VEGFR-3), specifically BDD073, which could inhibit angiogenesis in the CAM model. However, the clinical application of BDD073 is restricted due to its mouse origin, which might cause human anti-mouse antibody reactions. Herein, we generated functional recombinant single-chain variable fragments (scFv) from mAb BDD073 producing mouse hybridoma cells. The scFv gene containing variable regions of heavy and light chains of BDD073 was cloned into an expression vector with trx tag and expressed in Escherichia coli BL21 (DE3). The recombinant scFv was purified and refolded with Ni-NTA agarose metal affinity column. The bacterially expressed scFv showed moderate potency and specificity to the human VEGFR-3. It may serve as a potential candidate of anti-VEGFR3 treatment for biotechnological and therapeutic applications. PMID:25428897

  11. Development of single chain variable fragment (scFv) antibodies against Xylella fastidiosa subsp. pauca by phage display

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Xylella fastidiosa is a member of the gamma proteobacteria. It is fastidious, insect-vectored and xylem-limited and causes a variety of diseases, some severe, on a wide range of economically important perennial crops, including grape and citrus. Xylella fastidiosa subsp pauca causes citrus variegat...

  12. Generation of a Single Chain Antibody Variable Fragment (scFv) to Sense Selectively RhoB Activation

    PubMed Central

    Chinestra, Patrick; Olichon, Aurélien; Medale-Giamarchi, Claire; Lajoie-Mazenc, Isabelle; Gence, Rémi; Inard, Cyril; Ligat, Laetitia; Faye, Jean-Charles; Favre, Gilles

    2014-01-01

    Determining the cellular level of activated form of RhoGTPases is of key importance to understand their regulatory functions in cell physiopathology. We previously reported scFvC1, that selectively bind to the GTP-bound form of RhoA, RhoB and RhoC. In this present study we generate, by molecular evolution, a new phage library to isolate scFvs displaying high affinity and selectivity to RhoA and RhoB. Using phage display affinity maturation against the GTP-locked mutant RhoAL63, we isolated scFvs against RhoA active conformation that display Kd values at the nanomolar range, which corresponded to an increase of affinity of three orders of magnitude compared to scFvC1. Although a majority of these evolved scFvs remained selective towards the active conformation of RhoA, RhoB and RhoC, we identified some scFvs that bind to RhoA and RhoC but not to RhoB activated form. Alternatively, we performed a substractive panning towards RhoB, and isolated the scFvE3 exhibiting a 10 times higher affinity for RhoB than RhoA activated forms. We showed the peculiar ability of scFvE3 to detect RhoB but not RhoA GTP-bound form in cell extracts overexpressing Guanine nucleotide Exchange Factor XPLN as well as in EGF stimulated HeLa cells. Our results demonstrated the ability of scFvs to distinguish RhoB from RhoA GTP-bound form and provide new selective tools to analyze the cell biology of RhoB GTPase regulation. PMID:25365345

  13. A Strategy for Generating a Broad-Spectrum Monoclonal Antibody and Soluble Single-Chain Variable Fragments against Plant Potyviruses.

    PubMed

    Liu, Han-Lin; Lin, Wei-Fang; Hu, Wen-Chi; Lee, Yung-An; Chang, Ya-Chun

    2015-10-01

    Potyviruses are major pathogens that often cause mixed infection in calla lilies. To reduce the time and cost of virus indexing, a detection method for the simultaneous targeting of multiple potyviruses was developed by generating a broad-spectrum monoclonal antibody (MAb) for detecting the greatest possible number of potyviruses. The conserved 121-amino-acid core regions of the capsid proteins of Dasheen mosaic potyvirus (DsMV), Konjak mosaic potyvirus (KoMV), and Zantedeschia mild mosaic potyvirus (ZaMMV) were sequentially concatenated and expressed as a recombinant protein for immunization. After hybridoma cell fusion and selection, one stable cell line that secreted a group-specific antibody, named C4 MAb, was selected. In the reaction spectrum test, the C4 MAb detected at least 14 potyviruses by indirect enzyme-linked immunosorbent assay (I-ELISA) and Western blot analysis. Furthermore, the variable regions of the heavy (VH) and light (VL) chains of the C4 MAb were separately cloned and constructed as single-chain variable fragments (scFvs) for expression in Escherichia coli. Moreover, the pectate lyase E (PelE) signal peptide of Erwinia chrysanthemi S3-1 was added to promote the secretion of C4 scFvs into the medium. According to Western blot analysis and I-ELISA, the soluble C4 scFv (VL-VH) fragment showed a binding specificity similar to that of the C4 MAb. Our results demonstrate that a recombinant protein derived from fusion of the conserved regions of viral proteins has the potential to produce a broad-spectrum MAb against a large group of viruses and that the PelE signal peptide can improve the secretion of scFvs in E. coli. PMID:26209665

  14. A Strategy for Generating a Broad-Spectrum Monoclonal Antibody and Soluble Single-Chain Variable Fragments against Plant Potyviruses

    PubMed Central

    Liu, Han-Lin; Lin, Wei-Fang; Hu, Wen-Chi; Lee, Yung-An

    2015-01-01

    Potyviruses are major pathogens that often cause mixed infection in calla lilies. To reduce the time and cost of virus indexing, a detection method for the simultaneous targeting of multiple potyviruses was developed by generating a broad-spectrum monoclonal antibody (MAb) for detecting the greatest possible number of potyviruses. The conserved 121-amino-acid core regions of the capsid proteins of Dasheen mosaic potyvirus (DsMV), Konjak mosaic potyvirus (KoMV), and Zantedeschia mild mosaic potyvirus (ZaMMV) were sequentially concatenated and expressed as a recombinant protein for immunization. After hybridoma cell fusion and selection, one stable cell line that secreted a group-specific antibody, named C4 MAb, was selected. In the reaction spectrum test, the C4 MAb detected at least 14 potyviruses by indirect enzyme-linked immunosorbent assay (I-ELISA) and Western blot analysis. Furthermore, the variable regions of the heavy (VH) and light (VL) chains of the C4 MAb were separately cloned and constructed as single-chain variable fragments (scFvs) for expression in Escherichia coli. Moreover, the pectate lyase E (PelE) signal peptide of Erwinia chrysanthemi S3-1 was added to promote the secretion of C4 scFvs into the medium. According to Western blot analysis and I-ELISA, the soluble C4 scFv (VL-VH) fragment showed a binding specificity similar to that of the C4 MAb. Our results demonstrate that a recombinant protein derived from fusion of the conserved regions of viral proteins has the potential to produce a broad-spectrum MAb against a large group of viruses and that the PelE signal peptide can improve the secretion of scFvs in E. coli. PMID:26209665

  15. Synthesis and pre-clinical evaluation of an 18F-labeled single-chain antibody fragment for PET imaging of epithelial ovarian cancer

    PubMed Central

    Sharma, Sai Kiran; Wuest, Melinda; Way, Jenilee D; Bouvet, Vincent R; Wang, Monica; Wuest, Frank R

    2016-01-01

    Anti-CA125 antibodies have been used in immunoassays to quantify levels of shed antigen in the serum of patients who are under surveillance for epithelial ovarian cancer (EOC). However, there is currently no molecular imaging probe in the clinic for the assessment of CA125 expression in vivo. The present study describes the development of an 18F-labeled single-chain variable fragment (scFv) for PET imaging of CA125 in preclinical EOC models. Anti-CA125 scFv was derived from MAb-B43.13 by recombinant expression of the fragment in E.coli. Fragment scFv-B43.13 was purified via immobilized metal affinity chromatography and characterized for antigen binding via immuno-staining and flow cytometry. Prosthetic group N-succinimidyl 4-[18F]fluorobenzoate ([18F]SFB) was used for radiolabeling of scFv-B43.13. Preclinical ovarian cancer models were developed based on ovarian cancer cell lines OVCAR3 (CA125-positive) and SKOV3 (CA125-negative) in NIH-III mice. The radiopharmacological profile of 18F-labeled scFv-B43.13 ([18F]FBz-scFv-B43.13) was studied with PET. [18F]FBz-scFv-B43.13 was prepared in radiochemical yields of 3.7 ± 1.8% (n = 5) at an effective specific activity of 3.88 ± 0.76 GBq/µmol (n = 5). The radiotracer demonstrated selective uptake in CA125-positive OVCAR3 cells and virtually no uptake in CA125-negative SKOV3 cells. Standardized uptake values (SUV) of radioactivity uptake in OVCAR3 tumors was 0.5 (n = 3) and 0.3 (n = 2) in SKOV3 tumors after 60 min post injection (p.i.). PMID:27508105

  16. Synthesis and pre-clinical evaluation of an (18)F-labeled single-chain antibody fragment for PET imaging of epithelial ovarian cancer.

    PubMed

    Sharma, Sai Kiran; Wuest, Melinda; Way, Jenilee D; Bouvet, Vincent R; Wang, Monica; Wuest, Frank R

    2016-01-01

    Anti-CA125 antibodies have been used in immunoassays to quantify levels of shed antigen in the serum of patients who are under surveillance for epithelial ovarian cancer (EOC). However, there is currently no molecular imaging probe in the clinic for the assessment of CA125 expression in vivo. The present study describes the development of an (18)F-labeled single-chain variable fragment (scFv) for PET imaging of CA125 in preclinical EOC models. Anti-CA125 scFv was derived from MAb-B43.13 by recombinant expression of the fragment in E.coli. Fragment scFv-B43.13 was purified via immobilized metal affinity chromatography and characterized for antigen binding via immuno-staining and flow cytometry. Prosthetic group N-succinimidyl 4-[(18)F]fluorobenzoate ([(18)F]SFB) was used for radiolabeling of scFv-B43.13. Preclinical ovarian cancer models were developed based on ovarian cancer cell lines OVCAR3 (CA125-positive) and SKOV3 (CA125-negative) in NIH-III mice. The radiopharmacological profile of (18)F-labeled scFv-B43.13 ([(18)F]FBz-scFv-B43.13) was studied with PET. [(18)F]FBz-scFv-B43.13 was prepared in radiochemical yields of 3.7 ± 1.8% (n = 5) at an effective specific activity of 3.88 ± 0.76 GBq/µmol (n = 5). The radiotracer demonstrated selective uptake in CA125-positive OVCAR3 cells and virtually no uptake in CA125-negative SKOV3 cells. Standardized uptake values (SUV) of radioactivity uptake in OVCAR3 tumors was 0.5 (n = 3) and 0.3 (n = 2) in SKOV3 tumors after 60 min post injection (p.i.). PMID:27508105

  17. Single cell analysis applied to antibody fragment production with Bacillus megaterium: development of advanced physiology and bioprocess state estimation tools

    PubMed Central

    2011-01-01

    Background Single cell analysis for bioprocess monitoring is an important tool to gain deeper insights into particular cell behavior and population dynamics of production processes and can be very useful for discrimination of the real bottleneck between product biosynthesis and secretion, respectively. Results Here different dyes for viability estimation considering membrane potential (DiOC2(3), DiBAC4(3), DiOC6(3)) and cell integrity (DiBAC4(3)/PI, Syto9/PI) were successfully evaluated for Bacillus megaterium cell characterization. It was possible to establish an appropriate assay to measure the production intensities of single cells revealing certain product secretion dynamics. Methods were tested regarding their sensitivity by evaluating fluorescence surface density and fluorescent specific concentration in relation to the electronic cell volume. The assays established were applied at different stages of a bioprocess where the antibody fragment D1.3 scFv production and secretion by B. megaterium was studied. Conclusions It was possible to distinguish between live, metabolic active, depolarized, dormant, and dead cells and to discriminate between high and low productive cells. The methods were shown to be suitable tools for process monitoring at single cell level allowing a better process understanding, increasing robustness and forming a firm basis for physiology-based analysis and optimization with the general application for bioprocess development. PMID:21496219

  18. A Nanotechnology-Based Platform for Extending the Pharmacokinetic and Binding Properties of Anti-methamphetamine Antibody Fragments

    PubMed Central

    Nanaware-Kharade, Nisha; Thakkar, Shraddha; Gonzalez III, Guillermo A.; Peterson, Eric C.

    2015-01-01

    To address the need for effective medications to aid in the treatment of methamphetamine (METH) abuse, we used a nanotechnology approach to customize the in vivo behavior of an anti-METH single chain antibody (scFv7F9Cys). Anti-METH scFv7F9Cys was conjugated to dendrimer nanoparticles via a polyethylene glycol (PEG) linker to generate high-order conjugates termed dendribodies. We found that the high affinity (KD = 6.2 nM) and specificity for METH was unchanged after nanoparticle conjugation. The dendribodies were administered in an i.v. bolus to male Sprague Dawley rats after starting a s.c. infusion of METH. The PCKN values for clearance and volume of distribution of scFv7F9Cys after conjugation to dendrimers decreased 45 and 1.6-fold respectively, and the terminal elimination half-life increased 20-fold. Organ distribution of scFv7F9Cys and dendribody in blood and urine agreed well with the PCKN data. Renal clearance appeared to be the major route of elimination for both experimental medications. We have thus successfully developed a novel multivalent METH-binding nanomedicine by conjugating multiple anti-METH scFvs to dendrimer nanoparticles, extending the scFv half-life from 1.3 (±0.3) to 26 (±2.6) hr. These data suggest that the dendribody design could be a feasible platform for generating multivalent antibodies with customizable PCKN profiles. PMID:26159352

  19. A Nanotechnology-Based Platform for Extending the Pharmacokinetic and Binding Properties of Anti-methamphetamine Antibody Fragments.

    PubMed

    Nanaware-Kharade, Nisha; Thakkar, Shraddha; Gonzalez, Guillermo A; Peterson, Eric C

    2015-01-01

    To address the need for effective medications to aid in the treatment of methamphetamine (METH) abuse, we used a nanotechnology approach to customize the in vivo behavior of an anti-METH single chain antibody (scFv7F9Cys). Anti-METH scFv7F9Cys was conjugated to dendrimer nanoparticles via a polyethylene glycol (PEG) linker to generate high-order conjugates termed dendribodies. We found that the high affinity (KD = 6.2 nM) and specificity for METH was unchanged after nanoparticle conjugation. The dendribodies were administered in an i.v. bolus to male Sprague Dawley rats after starting a s.c. infusion of METH. The PCKN values for clearance and volume of distribution of scFv7F9Cys after conjugation to dendrimers decreased 45 and 1.6-fold respectively, and the terminal elimination half-life increased 20-fold. Organ distribution of scFv7F9Cys and dendribody in blood and urine agreed well with the PCKN data. Renal clearance appeared to be the major route of elimination for both experimental medications. We have thus successfully developed a novel multivalent METH-binding nanomedicine by conjugating multiple anti-METH scFvs to dendrimer nanoparticles, extending the scFv half-life from 1.3 (± 0.3) to 26 (± 2.6) hr. These data suggest that the dendribody design could be a feasible platform for generating multivalent antibodies with customizable PCKN profiles. PMID:26159352

  20. Anti-CD20 single chain variable antibody fragment-apolipoprotein A-I chimera containing nanodisks promote targeted bioactive agent delivery to CD20-positive lymphomas.

    PubMed

    Crosby, Natasha M; Ghosh, Mistuni; Su, Betty; Beckstead, Jennifer A; Kamei, Ayako; Simonsen, Jens B; Luo, Bing; Gordon, Leo I; Forte, Trudy M; Ryan, Robert O

    2015-08-01

    A fusion protein comprising an α-CD20 single chain variable fragment (scFv) antibody, a spacer peptide, and human apolipoprotein (apo) A-I was constructed and expressed in Escherichia coli. The lipid interaction properties intrinsic to apoA-I as well as the antigen recognition properties of the scFv were retained by the chimera. scFv•apoA-I was formulated into nanoscale reconstituted high-density lipoprotein particles (termed nanodisks; ND) and incubated with cultured cells. α-CD20 scFv•apoA-I ND bound to CD20-positive non-Hodgkins lymphoma (NHL) cells (Ramos and Granta) but not to CD20-negative T lymphocytes (i.e., Jurkat). Binding to NHL cells was partially inhibited by pre-incubation with rituximab, a monoclonal antibody directed against CD20. Confocal fluorescence microscopy analysis of Granta cells following incubation with α-CD20 scFv•apoA-I ND formulated with the intrinsically fluorescent hydrophobic polyphenol, curcumin, revealed α-CD20 scFv•apoA-I localizes to the cell surface, while curcumin off-loads and gains entry to the cell. Compared to control incubations, viability of cultured NHL cells was decreased upon incubation with α-CD20 scFv•apoA-I ND harboring curcumin. Thus, formulation of curcumin ND with α-CD20 scFv•apoA-I as the scaffold component confers cell targeting and enhanced bioactive agent delivery, providing a strategy to minimize toxicity associated with chemotherapeutic agents. PMID:25994015

  1. Single-chain Fv fragment antibodies selected from an intrabody library as effective mono- or bivalent reagents for in vitro protein detection.

    PubMed

    Desplancq, Dominique; Rinaldi, Anne-Sophie; Stoessel, Audrey; Sibler, Annie-Paule; Busso, Didier; Oulad-Abdelghani, Mustapha; Van Regenmortel, Marc H; Weiss, Etienne

    2011-06-30

    In spite of their many potential applications, recombinant antibody molecules selected by phage display are rarely available commercially, one reason being the absence of robust bacterial expression systems that yield sufficient quantities of reagents for routine applications. We previously described the construction and validation of an intrabody library that allows the selection of single-chain Fv (scFv) fragments solubly expressed in the cytoplasm. Here, we show that it is possible to obtain monomeric scFvs binding specifically to human papillomavirus type 16 E6 and cellular gankyrin oncoproteins in quantities higher than 0.5 g/L of shake-flask culture in E. coli cytoplasm after auto-induction. In addition, stable bivalent scFvs of increased avidity were produced by tagging the scFvs with the dimeric glutathione-S-transferase enzyme (GST). These minibody-like molecules were further engineered by fusion with green fluorescent protein (GFPuv), leading to high yield of functional bivalent fluorescent antibody fragments. Our results demonstrate that scFvs selected from an intrabody library can be engineered into cost-effective bivalent reagents suitable for many biomedical and industrial applications. PMID:21501618

  2. Characterization of Tumor-Avid Antibody Fragments Genetically Engineered for Mono-Specific Radionuclide Chelation

    SciTech Connect

    Quinn, T.P.

    2003-12-31

    The successful clinical application of targeted-radiopharmaceuticals depends on the development of molecules that optimize tumor specific radionuclide deposition and minimize non-specific organ irradiation. To this end, this proposal outlines a research effort to identify and evaluate novel antibodies and antibody fragments that bind breast tumors. The tumor-avid antibodies will be investigated for as imaging and therapeutic agents and to gain a better understanding of the pharmacokinetics and metabolism of radiolabeled tumor-avid antibody fragments through the use of site-specifically labeled molecules. Antibodies or antibody fragments, that bind breast carcinoma carbohydrate antigens, will be obtained from hybridoma or bacteriophage library screening. More specifically, antibody fragments that bind the carcinoma-associated Thomsen-Friedenreich (T) antigen will be radiolabeled with {sup 99m}Tc and {sup 188}Re at a natural amino acid chelation site and will be investigated in vivo for their abilities to target human breast tumors. In addition, site-specific radiolabeled antibody fragments will be biosynthesized using misacylated suppressor tRNAs. Homogeneously radiolabeled populations of antibody fragments will be used to investigate the effects of radionuclide location and chelation chemistries on their biodistribution and metabolism. It is hypothesized that site-specifically radiolabeled antibody fragments will possess enhanced tumor imaging and therapeutic properties due to optimal label location and conjugation chemistries. New insights into the factors that govern antibody metabolism in vivo are also expected from this work. Results from these studies should enhance our ability to design and synthesize radiolabeled antibody fragments that have improved pharmacokinetic properties. The studies in this proposal involve basic research into the development of antibody-based radiopharmaceuticals, with the ultimate goal of application in humans. This type of basic

  3. Production in yeast of pseudotype virus-like particles harboring functionally active antibody fragments neutralizing the cytolytic activity of vaginolysin

    PubMed Central

    2011-01-01

    Background Recombinant antibodies can be produced in different formats and different expression systems. Single chain variable fragments (scFvs) represent an attractive alternative to full-length antibodies and they can be easily produced in bacteria or yeast. However, the scFvs exhibit monovalent antigen-binding properties and short serum half-lives. The stability and avidity of the scFvs can be improved by their multimerization or fusion with IgG Fc domain. The aim of the current study was to investigate the possibilities to produce in yeast high-affinity scFv-Fc proteins neutralizing the cytolytic activity of vaginolysin (VLY), the main virulence factor of Gardnerella vaginalis. Results The scFv protein derived from hybridoma cell line producing high-affinity neutralizing antibodies against VLY was fused with human IgG1 Fc domain. Four different variants of anti-VLY scFv-Fc fusion proteins were constructed and produced in yeast Saccharomyces cerevisiae. The non-tagged scFv-Fc and hexahistidine-tagged scFv-Fc proteins were found predominantly as insoluble aggregates and therefore were not suitable for further purification and activity testing. The addition of yeast α-factor signal sequence did not support secretion of anti-VLY scFv-Fc but increased the amount of its intracellular soluble form. However, the purified protein showed a weak VLY-neutralizing capability. In contrast, the fusion of anti-VLY scFv-Fc molecules with hamster polyomavirus-derived VP2 protein and its co-expression with VP1 protein resulted in an effective production of pseudotype virus-like particles (VLPs) that exhibited strong VLY-binding activity. Recombinant scFv-Fc molecules displayed on the surface of VLPs neutralized VLY-mediated lysis of human erythrocytes and HeLa cells with high potency comparable to that of full-length antibody. Conclusions Recombinant scFv-Fc proteins were expressed in yeast with low efficiency. New approach to display the scFv-Fc molecules on the surface of

  4. Cloning and expression of an anti-LDL(-) single-chain variable fragment, and its inhibitory effect on experimental atherosclerosis

    PubMed Central

    Kazuma, Soraya M; Cavalcante, Marcela F; Telles, Andréia ER; Maranhão, Andrea Queiroz; Abdalla, Dulcineia SP

    2013-01-01

    The in vivo modified forms of low-density lipoprotein (LDL) are important for the formation of foam cells and as mediators of the immuno-inflammatory process involved in the progression of atherosclerosis. Electronegative LDL, LDL(-), is a LDL subfraction with pro-inflammatory properties that is present in human blood. To investigate possible atheroprotective effects, an anti-LDL(-) single-chain variable fragment (scFv) was expressed in the methylotrophic yeast Pichia pastoris and its activity was evaluated in vitro against macrophages and in experimental atherosclerosis in Ldlr-/-mice. The recombinant 2C7 scFv was produced in a yield of 9.5 mg of protein/L. The specificity and affinity of purified 2C7 scFv against LDL(-) was confirmed by ELISA. To assess the activity of 2C7 scFv on foam cell formation, RAW 264.7 macrophages were exposed to LDL(-) in the presence or absence of 2C7 scFv. The 2C7 scFv inhibited the uptake of LDL(-) by macrophages in a dose-dependent manner, and internalization of LDL(-) by these cells was found to be mediated by the CD36 and CD14 receptor. In addition, compared with untreated cells, lipid accumulation in macrophages was decreased, and the expression of Cd36, Tlr-4 and Cox-2 was downregulated in macrophages treated with 2C7 scFv. Importantly, compared with untreated mice, the treatment of Ldlr-/- mice with 2C7 scFv decreased the atherosclerotic lesion area at the aortic sinus. In conclusion, our data show that 2C7 scFv inhibits foam cell formation and atherosclerotic plaque development by modulating the expression of genes relevant to atherogenesis. These results encourage further use of this antibody fragment in the development of new therapeutic strategies that neutralize the pro-atherogenic effects of LDL(-). PMID:23924793

  5. Gigantoxin-4-4D5 scFv is a novel recombinant immunotoxin with specific toxicity against HER2/neu-positive ovarian carcinoma cells.

    PubMed

    Lv, Xinxin; Zhang, Jian; Xu, Rui; Dong, Yuguo; Sun, Aiyou; Shen, Yaling; Wei, Dongzhi

    2016-07-01

    Immunotoxins are a new class of antibody-targeted therapy in clinical development. Traditional immunotoxins that are constructed from the toxins of plants or bacteria need to be internalized to the cytoplasm and thus have limited antitumor efficacy. In the present study, we combined a recently reported sea anemone cytolysin Gigantoxin-4 with an anti-HER2/neu single-chain variable fragment 4D5 scFv to construct a novel immunotoxin. We fused a SUMO tag to the N-terminus of Gigantoxin-4-4D5 scFv and it was successfully expressed in Escherichia coli strain BL21 (DE3) in a soluble form. After purification, the purity of Gigantoxin-4-4D5 scFv reached 96 % and the yield was 14.3 mg/L. Our results demonstrated that Gigantoxin-4-4D5 scFv exerted a highly cytotoxic effect on the HER2/neu-positive ovarian carcinoma SK-OV-3 cell line. And the hemolytic activity was weaker, making it safe for normal cells. The results of immunofluorescence analysis showed that this novel immunotoxin could specifically bind to SK-OV-3 cells with no recognition of human embryonic kidney 293 cells. Scanning electron microscope observations and extracellular lactate dehydrogenase activity indicated that it could induce necrosis in SK-OV-3 cells by disrupting the cell membrane. Moreover, it could also mediate apoptosis of SK-OV-3 cells. PMID:27063011

  6. Novel Antibody Vectors for Imaging

    PubMed Central

    Olafsen, Tove; Wu, Anna M.

    2010-01-01

    Non-invasive molecular imaging approaches include nuclear, optical, MRI, CT, ultrasound and photoacoustic imaging, which require accumulation of a signal delivered by a probe at the target site. Monoclonal antibodies (mAbs) are high affinity molecules that can be used for specific, high signal delivery to cell surface molecules. However, their long circulation time in blood makes them unsuitable as imaging probes. Efforts to improve antibodies pharmacokinetics without compromising affinity and specificity have been made through protein engineering. Antibody variants that differ in antigen binding sites and size have been generated and evaluated as imaging probes to target tissues of interest. Fast clearing fragments such as single-chain Fv (scFv; 25 kDa) with one antigen binding site (monovalent) demonstrated low accumulation in tumors due the low exposure time to the target. Using scFv as building block to produce larger, bivalent fragments such as scFv dimers (diabodies, 50 kDa) and scFv-fusion proteins (80 kDa minibodies and 105 kDa scFv-Fc) resulted in higher tumor accumulation due to their longer residence time in blood. Imaging studies with these fragments following radiolabeling have demonstrated excellent, high contrast images in gamma cameras and PET scanners. Several studies have also investigated antibody fragments conjugated to fluorescence (near infrared dyes), bioluminescence (luciferases) and quantum dots for optical imaging and iron oxides nanoparticles for MRI. However, these studies indicate that there are several factors that influence successful targeting and imaging. These include stability of the antibody fragment, the labeling chemistry (direct or indirect), whether critical residues are modified, the number of antigen expressed on the cell, and whether the target has a rapid recycling rate or internalizes upon binding. The preclinical data presented are compelling and it is evident that antibody-based molecular imaging tracers will play an

  7. High-level production in Pichia pastoris of an anti-p185HER-2 single-chain antibody fragment using an alternative secretion expression vector.

    PubMed

    Gurkan, Cemal; Symeonides, Stefan N; Ellar, David J

    2004-02-01

    The methylotrophic yeast Pichia pastoris has become a highly popular expression host for the recombinant production of a wide variety of proteins. Initial success with this system was greatly facilitated by the development of versatile expression vectors that were almost exclusively based on the strong, tightly regulated promoter of the P. pastoris major alcohol oxidase gene ( AOX1 ). For example, pIB4 is an Escherichia coli - P. pastoris shuttle vector that also uses the AOX1 promoter to allow intracellular expression of endogenous and foreign genes in the latter organism. Since the eukaryotic advantages of P. pastoris would be best harnessed through the secretory targeting of the recombinant proteins, we modified the pIB4 vector by adding the Saccharomyces cerevisiae alpha-factor secretion signal immediately upstream of its multiple cloning site. Here we describe the construction of this modified vector, pIB4alpha, and its successful use for the high-level expression and secretion of a functional single-chain antibody fragment (scFv), C6.5, which targets p185(HER-2), a cell-surface glycoprotein overexpressed in about 30% of human breast and ovarian cancers. The PCR strategy used for the subcloning of the C6.5 construct into pIB4alpha also introduced a short DNA sequence coding for a C-terminal hexahistidine tag, which allowed subsequent purification of the secreted scFv, by immobilized-metal-affinity chromatography, to a yield of 70 mg x l(-1) of shake-flask culture. In conclusion, our results suggest that the secretion expression vector pIB4alpha not only complements the original pIB4 vector for intracellular expression in P. pastoris, but might also constitute an attractive alternative to the commercially available secretion expression vectors. PMID:12962542

  8. Targeting immune effector molecules to human tumor cells through genetic delivery of 5T4-specific scFv fusion proteins.

    PubMed

    Myers, Kevin A; Ryan, Matthew G; Stern, Peter L; Shaw, David M; Embleton, M Jim; Kingsman, Susan M; Carroll, Miles W

    2002-11-01

    Although several clinical trials have shown beneficial effects by targeting tumor-associated antigens (TAAs) with monoclonal antibodies, a number of issues, including poor penetration of the tumor mass and human antimouse antibody responses, remain. The use of recombinant single-chain Fv (scFv) fragments has the potential to address these and other issues while allowing the addition of different effector functions. To develop therapeutic strategies that recruit both humoral and cellular arms of the immune response, we have constructed chimeric proteins linking either the human IgG1 Fc domain or the extracellular domain of murine B7.1 to a scFv specific for the oncofetal glycoprotein, 5T4. This TAA is expressed by a wide variety of carcinomas and is associated with metastasis and poorer clinical outcome. We have engineered retroviral constructs that produce fusion proteins able to interact simultaneously with both 5T4-positive cells and with the receptor/ligands of the immune effector moieties. Genetic delivery through a murine leukemia virus vector to 5T4-positive tumor cells results in the secreted scFv fusion protein binding to the cell surface. Furthermore, the scFv-HIgG1 fusion protein is able to direct lysis of 5T4-expressing human tumor cell lines through antibody-dependent cell cytotoxicity, indicating its potential as a gene therapy for human cancers. PMID:12386827

  9. pMINERVA: A donor-acceptor system for the in vivo recombineering of scFv into IgG molecules.

    PubMed

    Batonick, M; Kiss, M M; Fuller, E P; Magadan, C M; Holland, E G; Zhao, Q; Wang, D; Kay, B K; Weiner, M P

    2016-04-01

    Phage display is the most widely used method for selecting binding molecules from recombinant antibody libraries. However, validation of the phage antibodies often requires early production of the cognate full-length immunoglobulin G (IgG). The conversion of phage library outputs to a full immunoglobulin via standard subcloning is time-consuming and limits the number of clones that can be evaluated. We have developed a novel system to convert scFvs from a phage display vector directly into IgGs without any in vitro subcloning steps. This new vector system, named pMINERVA, makes clever use of site-specific bacteriophage integrases that are expressed in Escherichia coli and intron splicing that occurs within mammalian cells. Using this system, a phage display vector contains both bacterial and mammalian regulatory regions that support antibody expression in E. coli and mammalian cells. A single-chain variable fragment (scFv) antibody is expressed on the surface of bacteriophage M13 as a genetic fusion to the gpIII coat protein. The scFv is converted to an IgG that can be expressed in mammalian cells by transducing a second E. coli strain. In that strain, the phiC31 recombinase fuses the heavy chain constant domain from an acceptor plasmid to the heavy chain variable domain and introduces controlling elements upstream of the light chain variable domain. Splicing in mammalian cells removes a synthetic intron containing the M13 gpIII gene to produce the fusion of the light chain variable domain to the constant domain. We show that phage displaying a scFv and recombinant IgGs generated using this system are expressed at wild-type levels and retain normal function. Use of the pMINERVA completely eliminates the labor-intensive subcloning and DNA sequence confirmation steps currently needed to convert a scFv into a functional IgG Ab. PMID:26851519

  10. Photoluminescence detection of 2,4,6-trinitrotoluene (TNT) binding on diatom frustule biosilica functionalized with an anti-TNT monoclonal antibody fragment.

    PubMed

    Zhen, Le; Ford, Nicole; Gale, Debra K; Roesijadi, Guritno; Rorrer, Gregory L

    2016-05-15

    A selective and label-free biosensor for detection of the explosive compound 2,4,6-trinitrotoluene (TNT) in aqueous solution was developed based on the principle of photoluminescence quenching of upon immunocomplex formation with antibody-functionalized diatom frustule biosilica. The diatom frustule is an intricately nanostructured, highly porous biogenic silica material derived from the shells of microscopic algae called diatoms. This material emits strong visible blue photoluminescence (PL) upon UV excitation. PL-active frustule biosilica was isolated from cultured cells of the marine diatom Pinnularia sp. and functionalized with a single chain variable fragment (scFv) derived from an anti-TNT monoclonal antibody. When TNT was bound to the anti-TNT scFv-functionalized diatom frustule biosilica, the PL emission from the biosilica was partially quenched due to the electrophilic nature of the nitro (-NO2) groups on the TNT molecule. The dose-response curve for immunocomplex formation of TNT on the scFv-functionalized diatom frustule biosilica had a half-saturation binding constant of 6.4 ± 2.4·10(-8)M and statistically-significant measured detection limit of 3.5·10(-8)M. The binding and detection were selective for TNT and TNB (trinitrobenzene) but not RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) or 2,6-DNT (2,6-dinitrotoluene). PMID:26774089

  11. Selection of a human butyrylcholinesterase-like antibody single-chain variable fragment resistant to AChE inhibitors from a phage library expressed in E. coli.

    PubMed

    Podestà, Adriano; Rossi, Serena; Massarelli, Ilaria; Carpi, Sara; Adinolfi, Barbara; Fogli, Stefano; Bianucci, Anna Maria; Nieri, Paola

    2014-01-01

    Organophosphates are potent poisoning agents that cause severe cholinergic toxicity. Current treatment has been reported to be unsatisfactory and novel antidotes are needed. In this study, we used a single-chain variable fragment (scFv) library to select a recombinant antibody fragment (WZ1-14.2.1) with butyrylcholinesterase-like catalytic activity by using an innovative method integrating genetic selection and the bait-and-switch strategy. Ellman assay demonstrated that WZ1-14.2.1 has Michaelis-Menten kinetics in the hydrolysis of all the three substrates used, acetylthiocholine, propionylthiocholine and butyrylthiocholine. Notably, the catalytic activity was resistant to the following acetylcholinesterase inhibitors: neostigmine, iso-OMPA, chlorpyrifos oxon, dichlorvos, and paraoxon ethyl. Otherwise, the enzymatic activity of WZ1-14.2.1 was inhibited by the selective butyrylcholinesterase inhibitor, ethopropazine, and by the Ser-blocking agent phenylmethanesuphonyl fluoride. A hypothetical 3D structure of the WZ1-14.2.1 catalytic site, compatible with functional results, is proposed on the basis of a molecular modeling analysis. PMID:24675419

  12. Selection of a human butyrylcholinesterase-like antibody single-chain variable fragment resistant to AChE inhibitors from a phage library expressed in E. coli

    PubMed Central

    Podestà, Adriano; Rossi, Serena; Massarelli, Ilaria; Carpi, Sara; Adinolfi, Barbara; Fogli, Stefano; Bianucci, Anna Maria; Nieri, Paola

    2014-01-01

    Organophosphates are potent poisoning agents that cause severe cholinergic toxicity. Current treatment has been reported to be unsatisfactory and novel antidotes are needed. In this study, we used a single-chain variable fragment (scFv) library to select a recombinant antibody fragment (WZ1–14.2.1) with butyrylcholinesterase-like catalytic activity by using an innovative method integrating genetic selection and the bait-and-switch strategy. Ellman assay demonstrated that WZ1–14.2.1 has Michaelis-Menten kinetics in the hydrolysis of all the three substrates used, acetylthiocholine, propionylthiocholine and butyrylthiocholine. Notably, the catalytic activity was resistant to the following acetylcholinesterase inhibitors: neostigmine, iso-OMPA, chlorpyrifos oxon, dichlorvos, and paraoxon ethyl. Otherwise, the enzymatic activity of WZ1–14.2.1 was inhibited by the selective butyrylcholinesterase inhibitor, ethopropazine, and by the Ser-blocking agent phenylmethanesuphonyl fluoride. A hypothetical 3D structure of the WZ1–14.2.1 catalytic site, compatible with functional results, is proposed on the basis of a molecular modeling analysis. PMID:24675419

  13. Effect of radiochemical modification on biodistribution of scFvD2B antibody fragment recognising prostate specific membrane antigen.

    PubMed

    Frigerio, Barbara; Benigni, Fabio; Luison, Elena; Seregni, Ettore; Pascali, Claudio; Fracasso, Giulio; Morlino, Sara; Valdagni, Riccardo; Mezzanzanica, Delia; Canevari, Silvana; Figini, Mariangela

    2015-11-01

    Antibody-based reagents represent a promising strategy as clinical diagnostic tools. Prostate cancer (PCa) is the second-leading cause of death in males in the Western population. There is a presently unmet need for accurate diagnostic tool to localize and define the extent of both primary PCa and occult recurrent disease. One of the most suitable targets for PCa is the prostate-specific membrane antigen (PSMA) recognised by the monoclonal antibody D2B that we re-shaped into the single chain Fv (scFv format). Aim of this study was to evaluate in preclinical in vivo models the target specificity of scFvD2B after labelling with different radionuclides. (111)In radiolabelling was performed via the chelator Bz-NOTA, and (131)I radioiodination was performed using iodogen. The potential for molecular imaging and the biological behaviour of the radiolabelled scFvD2B were evaluated in mice bearing two subcutaneous PCa isogenic cell lines that differed only in PSMA expression. Biodistribution studies were performed at 3, 9, 15 and 24h after injection to determine the optimal imaging time point. A significant kidney accumulation, as percentage of injected dose of tissue (%ID/g), was observed for (111)In-scFvD2B at 3h after injection (45%ID/g) and it was maintained up to 24h (26%ID/g). By contrast, kidney accumulation of (131)I-scFvD2B was only marginally (0.3%ID/g at 24h). At the optimal time point defined between 15h and 24h, regardless of the radionuclide used, the scFvD2B was able to localize significantly better in the PSMA expressing tumours compared to the negative control; with (131)I-scFvD2B yielding a significantly better target/background ratio compared to (111)In-scFvD2B. These data suggest that, besides antigen specificity, chemical modification may affect antibody fragment biodistribution. PMID:26404855

  14. Expression, purification and characterization of a human single-chain Fv antibody fragment fused with the Fc of an IgG1 targeting a rabies antigen in Pichia pastoris.

    PubMed

    Wang, Ding-ding; Su, Man-man; Sun, Yan; Huang, Shu-lin; Wang, Ju; Yan, Wei-qun

    2012-11-01

    Because the demand for rabies post exposure prophylaxis (PEP) treatment has increased exponentially in recent years, the limited supply of human and equine rabies immunoglobulin (HRIG and ERIG) has failed to provide an adequate amount of the required passive immune component in PEP in countries where canine rabies is endemic. The replacement of HRIG and ERIG with a potentially cheaper and efficacious alternative biological for the treatment of rabies in humans, therefore, remains a high priority. In this study, we set out to assess a human single-chain Fv antibody fragment fused with the Fc of an IgG1 targeting a rabies antigen to develop a product that can be used as a component of the PEP cocktail. We cloned the ScFv fragment from a human ScFv library that was established previously and inserted this fragment into the expression vector pPICZαC/Fc. An active recombinant ScFv-Fc fusion protein was successfully expressed in Pichia pastoris. The production of ScFv-Fc was optimized and scaled up in an 80L fermentor with yields exceeding 60mg/L. The ScFv-Fc protein was purified to more than 95% purity using a two-step scheme: ammonium sulfate fractionation and Protein A Sepharose CL-4B. The ScFv-Fc fusion protein neutralized rabies virus in a standard in vivo neutralization assay in which the virus was incubated with the ScFv-Fc molecules before intracranial inoculation in mice. Our results suggest that functional antibodies can be produced in P. pastoris and that ScFv-Fc fusion proteins have the potential to serve as therapeutic candidates. PMID:22982755

  15. Single-chain antibody-fragment M6P-1 possesses a mannose 6-phosphate monosaccharide-specific binding pocket that distinguishes N-glycan phosphorylation in a branch-specific manner†.

    PubMed

    Blackler, Ryan J; Evans, Dylan W; Smith, David F; Cummings, Richard D; Brooks, Cory L; Braulke, Thomas; Liu, Xinyu; Evans, Stephen V; Müller-Loennies, Sven

    2016-02-01

    The acquisition of mannose 6-phosphate (Man6P) on N-linked glycans of lysosomal enzymes is a structural requirement for their transport from the Golgi apparatus to lysosomes mediated by the mannose 6-phosphate receptors, 300 kDa cation-independent mannose 6-phosphate receptor (MPR300) and 46 kDa cation-dependent mannose 6-phosphate receptor (MPR46). Here we report that the single-chain variable domain (scFv) M6P-1 is a unique antibody fragment with specificity for Man6P monosaccharide that, through an array-screening approach against a number of phosphorylated N-glycans, is shown to bind mono- and diphosphorylated Man6 and Man7 glycans that contain terminal αMan6P(1 → 2)αMan(1 → 3)αMan. In contrast to MPR300, scFv M6P-1 does not bind phosphodiesters, monophosphorylated Man8 or mono- or diphosphorylated Man9 structures. Single crystal X-ray diffraction analysis to 2.7 Å resolution of Fv M6P-1 in complex with Man6P reveals that specificity and affinity is achieved via multiple hydrogen bonds to the mannose ring and two salt bridges to the phosphate moiety. In common with both MPRs, loss of binding was observed for scFv M6P-1 at pH values below the second pKa of Man6P (pKa = 6.1). The structures of Fv M6P-1 and the MPRs suggest that the change of the ionization state of Man6P is the main driving force for the loss of binding at acidic lysosomal pH (e.g. lysosome pH ∼ 4.6), which provides justification for the evolution of a lysosomal enzyme transport pathway based on Man6P recognition. PMID:26503547

  16. Engineering of a recombinant trivalent single-chain variable fragment antibody directed against rabies virus glycoprotein G with improved neutralizing potency.

    PubMed

    Turki, Imène; Hammami, Akil; Kharmachi, Habib; Mousli, Mohamed

    2014-02-01

    Human and equine rabies immunoglobulins are currently available for passive immunization against rabies. However, these are hampered by the limited supply and some drawbacks. Advances in antibody engineering have led to overcome issues of clinical applications and to improve the protective efficacy. In the present study, we report the generation of a trivalent single-chain Fv (scFv50AD1-Fd), that recognizes the rabies virus glycoprotein, genetically fused to the trimerization domain of the bacteriophage T4 fibritin, termed 'foldon' (Fd). scFv50AD1-Fd was expressed as soluble recombinant protein in bacterial periplasmic space and purified through affinity chromatography. The molecular integrity and stability were analyzed by polyacrylamide gradient-gel electrophoresis, size-exclusion chromatography and incubation in human sera. The antigen-binding properties of the trimeric scFv were analyzed by direct and competitive-ELISA. Its apparent affinity constant was estimated at 1.4 ± 0.25 × 10(9)M(-1) and was 75-fold higher than its monovalent scFv (1.9 ± 0.68 × 10(7)M(-1)). The scFv50AD1-Fd neutralized rabies virus in a standard in vitro and in vivo neutralization assay. We showed a high neutralization activity up to 75-fold compared with monovalent format and the WHO standard serum. The gain in avidity resulting from multivalency along with an improved biological activity makes the trivalent scFv50AD1-Fd construct an important reagent for rabies protection. The antibody engineering approach presented here may serve as a strategy for designing a new generation of anti-rabies for passive immunotherapy. PMID:24091293

  17. Antibody-Mediated Pathogen Resistance in Plants.

    PubMed

    Peschen, Dieter; Schillberg, Stefan; Fischer, Rainer

    2016-01-01

    The methods described in this chapter were developed in order to produce transgenic plants expressing pathogen-specific single-chain variable fragment (scFv) antibodies fused to antifungal peptides (AFPs), conferring resistance against fungal pathogens. We describe the selection from a phage display library of avian scFv antibodies that recognize cell surface proteins on fungi from the genus Fusarium, and the construction of scFv-AFP fusion protein constructs followed by their transient expression in tobacco (Nicotiana spp.) plants and stable expression in Arabidopsis thaliana plants. Using these techniques, the antibody fusion with the most promising in vitro activity can be used to generate transgenic plants that are resistant to pathogens such as Fusarium oxysporum f. sp. matthiolae. PMID:26614296

  18. Two ScFv antibody libraries derived from identical VL-VH framework with different binding site designs display distinct binding profiles.

    PubMed

    Huovinen, Tuomas; Syrjänpää, Markku; Sanmark, Hanna; Brockmann, Eeva-Christine; Azhayev, Alex; Wang, Qi; Vehniäinen, Markus; Lamminmäki, Urpo

    2013-10-01

    In directed evolution experiments, a single randomization scheme of an antibody gene does not provide optimal diversity for recognition of all sizes of antigens. In this study, we have expanded the recognition potential of our universal library, termed ScFvP, with a second distinct diversification scheme. In the second library, termed ScFvM, diversity was designed closer to the center of the antigen binding site in the same antibody framework as earlier. Also, the CDR-H3 loop structures were redesigned to be shorter, 5-12 aa and mostly without the canonical salt bridge between Arg106H and Asp116H to increase the flexibility of the loop and to allow more space in the center of the paratope for binding smaller targets. Antibodies were selected from the two libraries against various antigens separately and as a mixture. The origin and characteristics of the retrieved antibodies indicate that complementary diversity results in complementary functionality widening the spectrum of targets amenable for selection. PMID:23966567

  19. Nanocell targeting using engineered bispecific antibodies

    PubMed Central

    Taylor, Karin; Howard, Christopher B; Jones, Martina L; Sedliarou, Ilya; MacDiarmid, Jennifer; Brahmbhatt, Himanshu; Munro, Trent P; Mahler, Stephen M

    2015-01-01

    There are many design formats for bispecific antibodies (BsAbs), and the best design choice is highly dependent on the final application. Our aim was to engineer BsAbs to target a novel nanocell (EnGeneIC Delivery Vehicle or EDVTMnanocell) to the epidermal growth factor receptor (EGFR). EDVTMnanocells are coated with lipopolysaccharide (LPS), and BsAb designs incorporated single chain Fv (scFv) fragments derived from an anti-LPS antibody (1H10) and an anti-EGFR antibody, ABX-EGF. We engineered various BsAb formats with monovalent or bivalent binding arms and linked scFv fragments via either glycine-serine (G4S) or Fc-linkers. Binding analyses utilizing ELISA, surface plasmon resonance, bio-layer interferometry, flow cytometry and fluorescence microscopy showed that binding to LPS and to either soluble recombinant EGFR or MDA-MB-468 cells expressing EGFR, was conserved for all construct designs. However, the Fc-linked BsAbs led to nanocell clumping upon binding to EDVTMnanocells. Clumping was eliminated when additional disulfide bonds were incorporated into the scFv components of the BsAbs, but this resulted in lower BsAb expression. The G4S-linked tandem scFv BsAb format was the optimal design with respect to EDV binding and expression yield. Doxorubicin-loaded EDVTMnanocells actively targeted with tandem scFv BsAb in vivo to MDA-MB-468-derived tumors in mouse xenograft models enhanced tumor regression by 40% compared to passively targeted EDVTMnanocells. BsAbs therefore provide a functional means to deliver EDVTMnanocells to target cells. PMID:25523746

  20. Nanocell targeting using engineered bispecific antibodies.

    PubMed

    Taylor, Karin; Howard, Christopher B; Jones, Martina L; Sedliarou, Ilya; MacDiarmid, Jennifer; Brahmbhatt, Himanshu; Munro, Trent P; Mahler, Stephen M

    2015-01-01

    There are many design formats for bispecific antibodies (BsAbs), and the best design choice is highly dependent on the final application. Our aim was to engineer BsAbs to target a novel nanocell (EnGeneIC Delivery Vehicle or EDV(TM)nanocell) to the epidermal growth factor receptor (EGFR). EDV(TM)nanocells are coated with lipopolysaccharide (LPS), and BsAb designs incorporated single chain Fv (scFv) fragments derived from an anti-LPS antibody (1H10) and an anti-EGFR antibody, ABX-EGF. We engineered various BsAb formats with monovalent or bivalent binding arms and linked scFv fragments via either glycine-serine (G4S) or Fc-linkers. Binding analyses utilizing ELISA, surface plasmon resonance, bio-layer interferometry, flow cytometry and fluorescence microscopy showed that binding to LPS and to either soluble recombinant EGFR or MDA-MB-468 cells expressing EGFR, was conserved for all construct designs. However, the Fc-linked BsAbs led to nanocell clumping upon binding to EDV(TM)nanocells. Clumping was eliminated when additional disulfide bonds were incorporated into the scFv components of the BsAbs, but this resulted in lower BsAb expression. The G4S-linked tandem scFv BsAb format was the optimal design with respect to EDV binding and expression yield. Doxorubicin-loaded EDV(TM)nanocells actively targeted with tandem scFv BsAb in vivo to MDA-MB-468-derived tumors in mouse xenograft models enhanced tumor regression by 40% compared to passively targeted EDV(TM)nanocells. BsAbs therefore provide a functional means to deliver EDV(TM)nanocells to target cells. PMID:25523746

  1. Antibody-based fluorescent and fluorescent ratiometric indicators for detection of phosphotyrosine.

    PubMed

    Huynh Nhat, Kim Phuong; Watanabe, Takayoshi; Yoshikoshi, Kensuke; Hohsaka, Takahiro

    2016-08-01

    Fluorescent indicators for protein phosphorylation are very important in not only fundamental biology but also biomedical applications. In this study, we developed novel fluorescent and fluorescent ratiometric indicators for detection of phosphotyrosine (pTyr) derivatives. A single-chain antibody variable fragment (scFv) against phosphotyrosine was fluorescent-labeled by incorporation of tetramethylrhodamine (TAMRA)-linked nonnatural amino acid at the N- or C-terminus. The TAMRA-labeled scFv showed fluorescence enhancement upon addition of pTyr-containing peptides based on antigen-dependent fluorescence quenching effect on TAMRA. The TAMRA-labeled scFv was further fused with enhanced green fluorescent protein (EGFP) to generate a double-labeled scFv for pTyr. In the absence of antigen, fluorescence resonance energy transfer (FRET) occurred from EGFP to TAMRA but TAMRA was quenched. The antigen-binding removed the quenching of TAMRA while FRET occurred without altering its efficiency. As a result of the FRET and antigen-dependent fluorescence quenching effect, the double-labeled scFv exhibited fluorescence ratio enhancement upon the antigen-binding. The fluorescent and fluorescent ratiometric indicators obtained in this study will become a novel tool for analysis of protein phosphorylation. Moreover, this strategy utilizes antibody derivatives, and therefore, can be easily applied to other antigen-antibody pairs to generate fluorescent ratiometric indicators for various target molecules. PMID:26896314

  2. High-level expression, purification, and characterization of bifunctional ScFv-9R fusion protein.

    PubMed

    Zhang, Xiguang; Xie, Jiasen; Sun, Yan; Xu, Huijing; Du, Tonghua; Liu, Zixuan; Chen, Jinhui; Zheng, Zhong; Liu, Keqiang; Zhang, Jizhou; Kan, Mujie; Li, Xiaokun; Xiao, Yechen

    2014-06-01

    Fibroblast growth factor receptor 3 (FGFR3) is a noted proto-oncogene involved in the pathogenesis of many tumors, so more and more studies focus on the potential use of receptor kinase inhibitor and therapeutic antibodies against FGFR3. In this study, we designed a novel fusion protein containing the single-chain Fv (ScFv) against FGFR3 and 9-arginine, denoted as ScFv-9R. To achieve the high-level production and soluble expression, ScFv and ScFv-9R were fused with small ubiquitin-related modifier (Sumo) by polymerase chain reaction and expressed in Escherichia coli BL21 (DE3). The recombinant bacteria was induced by 0.5 mM isopropyl-β-D-thiogalactopyranoside for 20 h at 20 °C; supernatants of Sumo-ScFv was harvested and purified by DEAE Sepharose FF and Ni-NTA orderly, and supernatants of Sumo-ScFv-9R was harvested and purified by Ni-NTA. After cleaved by the Sumo protease, the recombinant ScFv or ScFv-9R was released from the fusion protein, respectively. The purity of ScFv or ScFV-9R was shown to be higher than 90 %, and their yield reached 3-5 mg per liter of bacterial culture. In vitro data showed that ScFV-9R can attenuate the phosphorylation of FGFR3 and ERK in the absence or presence of FGF9. Gel retardation assay showed that 1 μg of ScFv-9R could efficiently bind to about 4 pmol siRNA. Fluorescent microscope analysis showed that ScFv-9R can efficiently bind and deliver siRNA into RT112 cells. In conclusion, we use Sumo fusion system to acquire high-level production, soluble expression, and bifunctional activity of ScFv-9R in E. coli. Our results also revealed that ScFv-9R, as a novel carrier, may have potential applications in antitumor studies and pharmaceutical development. PMID:24519456

  3. Anti-sulfotyrosine antibodies

    DOEpatents

    Bertozzi, Carolyn R.; Kehoe, John; Bradbury, Andrew M.

    2009-09-15

    The invention provides anti-sulfotyrosine specific antibodies capable of detecting and isolating polypeptides that are tyrosine-sulfated. The sulfotyrosine antibodies and antibody fragments of the invention may be used to discriminate between the non-sulfated and sulfated forms of such proteins, using any number of immunological assays, such ELISAs, immunoblots, Western Blots, immunoprecipitations, and the like. Using a phage-display system, single chain antibodies (scFvs) were generated and screened against tyrosine-sulfated synthetic peptide antigens, resulting in the isolation of scFvs that specifically recognize sulfotyrosine-containing peptides and/or demonstrate sulfotyrosine-specific binding in tyrosine sulfated proteins. The VH and VL genes from one such sulfotyrosine-specific scFv were employed to generate a full length, sulfotyrosine-specific immunoglobulin.

  4. Preparation of Recombinant Human Monoclonal Antibody Fab Fragments Specific for Entamoeba histolytica

    PubMed Central

    Tachibana, Hiroshi; Cheng, Xun-Jia; Watanabe, Katsuomi; Takekoshi, Masataka; Maeda, Fumiko; Aotsuka, Satoshi; Kaneda, Yoshimasa; Takeuchi, Tsutomu; Ihara, Seiji

    1999-01-01

    Genes coding for human antibody Fab fragments specific for Entamoeba histolytica were cloned and expressed in Escherichia coli. Lymphocytes were separated from the peripheral blood of a patient with an amebic liver abscess. Poly(A)+ RNA was isolated from the lymphocytes, and then genes coding for the light chain and Fd region of the heavy chain were amplified by a reverse transcriptase PCR. The amplified DNA fragments were ligated with a plasmid vector and were introduced into Escherichia coli. Three thousand colonies were screened for the production of antibodies to E. histolytica HM-1:IMSS by an indirect fluorescence-antibody (IFA) test. Lysates from five Escherichia coli clones were positive. Analysis of the DNA sequences of the five clones showed that three of the five heavy-chain sequences and four of the five light-chain sequences differed from each other. When the reactivities of the Escherichia coli lysates to nine reference strains of E. histolytica were examined by the IFA test, three Fab fragments with different DNA sequences were found to react with all nine strains and another Fab fragment was found to react with seven strains. None of the four human monoclonal antibody Fab fragments reacted with Entamoeba dispar reference strains or with other enteric protozoan parasites. These results indicate that the bacterial expression system reported here is effective for the production of human monoclonal antibodies specific for E. histolytica. The recombinant human monoclonal antibody Fab fragments may be applicable for distinguishing E. histolytica from E. dispar and for use in the serodiagnosis of amebiasis. PMID:10225840

  5. High affinity anti-Internalin B VHH antibody fragments isolated from naturally and artificially immunized repertoires.

    PubMed

    Gene, Robert W; Kumaran, Jyothi; Aroche, Cristina; van Faassen, Henk; Hall, J Christopher; MacKenzie, C Roger; Arbabi-Ghahroudi, Mehdi

    2015-01-01

    The need for rapid and easy technologies for the detection of food-borne and environmental pathogens is essential for safeguarding the health of populations. Furthermore, distribution of tainted food and water can have consequences which can affect whole economies. Antibodies and antibody fragments have been historically used in detection platforms due to their antigen specificity and robust physicochemical properties. In this study, we report the isolation and characterization of antibody fragments from the heavy chain antibody repertoire (VHH) of Camelidae which bind with specificity and high affinity to the Listeria monocytogenes invasin, Internalin B (InlB). To the best of our knowledge, this is the first report of anti-InlB VHHs from camelids. These anti-InlB VHHs were not cross-reactive to the structurally related Listeria invasin Internalin A (InlA) and are potential reagents to be used in the development of detection and medical technologies. PMID:25450000

  6. Fragmentation, labeling and biodistribution studies of KS1/4, a monoclonal antibody

    SciTech Connect

    Mohd, S.B.

    1987-01-01

    In this study, an IgG2a (KS1/4), a monoclonal antibody (MoAb) specific against a human lung adenocarcinoma (UCLA P-3) was successfully fragmented enzymatically to yield F(ab')/sub 2/ and Fab by using pepsin and papain, respectively. The kinetic of fragmentation of the MoAb was compared to that of human immunoglobulin G (IgG). A similar pattern of fragmentation was observed with both antibodies with a higher percentage yield of the F(ab')/sub 2/ and Fab obtained upon the fragmentation of the IgG by the enzymes. The KS1/4 and the two fragments were labeled with three different radionuclides, namely iodine-131, indium-111 and selenium-75. The radioiodination of the MoAb and the fragments was carried out by using a modified chloramine-T method. Radiometal labeling of the MoAb and the fragments with indium-111 was performed by using DTPA as a bifunctional chelating agent, while intrinsic labeling of the MoAb was done by culturing the hybridoma in the presence of /sup 75/Se-methionine. The biodistribution of the radiolabeled MoAb, F(ab')/sub 2/ and Fab fragments were performed by injecting the preparations intravenously into nude mice bearing human lung adenocarcinoma.

  7. Altered specificity of single-chain antibody fragments bound to pandemic H1N1-2009 influenza virus after conversion of the phage-bound to the soluble form

    PubMed Central

    2012-01-01

    Background In 2009, a novel influenza A/H1N1 virus (H1N1pdm) quickly spread worldwide and co-circulated with then-existing seasonal H1N1 virus (sH1N1). Distinguishing between these 2 viruses was necessary to better characterize the epidemiological properties of the emergent virus, including transmission patterns, pathogenesis, and anti-influenza drug resistance. This situation prompted us to develop a point-of-care virus differentiation system before entering the 2009–2010 influenza season. Aiming to establish H1N1pdm-specific detection tools rapidly, we employed phage display libraries to select H1N1pdm-specific single-chain variable fragments (scFvs). Findings Human single-fold scFv libraries (Tomlinson I + J) underwent selection for the ability to bind H1N1pdm virus particles. Three rounds of panning brought 1152 phage-bound scFvs, of which 58 clones reacted with H1N1pdm specifically or preferentially over sH1N1 in an enzyme-linked immunosorbent assay (ELISA). After conversion of the scFvs to soluble form, 7 clones demonstrating high/stable expression were finally obtained. However, all the soluble scFvs except No. 29 were found to have lost their specificity/preference for H1N1pdm in ELISA. The specificity/preference of No. 29 was also confirmed by immunofluorescence assay and immunoprecipitation, and the viral nucleoprotein was identified by ELISA as its target protein. The change in specificity associated with scFv conversion from phage-bound to soluble form could be due to loss of phage scaffold pIII protein, which likely provides structural support for the scFv antigen-binding site. It is also possible that the similar antigenic properties of H1N1pdm and sH1N1 led to the observed alterations in scFv specificity. Discussion Using a phage display library, we obtained 7 soluble scFv clones reactive against H1N1pdm; however, only 1 showed specificity/preference toward H1N1pdm. Our results confirmed that using phage display libraries was highly

  8. Composition and method for detecting cancer with technetium labeled antibody fragments

    SciTech Connect

    Burchiel, S. W.; Crockford, D. R.; Rhodes, B. A.

    1984-10-23

    F(ab')/sub 2/ or Fab fragments of antibodies to: (a) human chorionic gonadotropin (hCG), hCG alpha subunit, hCG beta subunit, or an hCG-like material; or (b) other tumor specific or tumor associated molecules, to include carcinoembryonic antigen (CEA), alpha fetoprotein (AFP), human melanoma associated antigens, human sarcoma associated antigens or other antigens, are radiolabeled with technetium-99m (Tc-99m). When the F(ab')/sub 2/ or Fab fragments of antibody to such tumor associated antigens are injected intravenously into a patient, the radiolabeled composition accumulates at tumor sites. The accumulation of the cancer seeking radiopharmaceutical at tumor sites permits detection by external gamma scintigraphy. Thus, the composition is useful in the monitoring, localization and detection of cancer in the body. In an alternative composition, a double antibody approach to tumor localization using radiolabeled F(ab')/sub 2/ or Fab fragments is utilized. In this approach, a tumor specific antibody in the form of IgG, F(ab')/sub 2/ or Fab is first administered to a patient intravenously. Following a sufficient period of time, a second antibody in the form of F(ab')/sub 2/ or Fab is administered. The second antibody is radiolabeled with Tc-99m and has the property that it is reactive with the first antibody. This double antibody method has the advantage over a single antibody approach in that smaller tumors can be localized and detected and that the total amount of radioactive trace localized at the cancer site is increased.

  9. Antibody Derived Peptides for Detection of Ebola Virus Glycoprotein

    PubMed Central

    López-Pacheco, Felipe; Pérez-Chavarría, Roberto; González-Vázquez, Juan Carlos; González-González, Everardo; Trujillo-de Santiago, Grissel; Ponce-Ponce de León, César Alejandro; Zhang, Yu Shrike; Dokmeci, Mehmet Remzi; Khademhosseini, Ali; Alvarez, Mario Moisés

    2015-01-01

    Background Current Ebola virus (EBOV) detection methods are costly and impractical for epidemic scenarios. Different immune-based assays have been reported for the detection and quantification of Ebola virus (EBOV) proteins. In particular, several monoclonal antibodies (mAbs) have been described that bind the capsid glycoprotein (GP) of EBOV GP. However, the currently available platforms for the design and production of full-length mAbs are cumbersome and costly. The use of antibody fragments, rather than full-length antibodies, might represent a cost-effective alternative for the development of diagnostic and possibly even therapeutic alternatives for EBOV. Methods/Principal Findings We report the design and expression of three recombinant anti-GP mAb fragments in Escherichia coli cultures. These fragments contained the heavy and light variable portions of the three well-studied anti-GP full-length mAbs 13C6, 13F6, and KZ52, and are consequently named scFv-13C6, scFv-13F6, and Fab-KZ52, respectively. All three fragments exhibited specific anti-GP binding activity in ELISA experiments comparable to that of full-length anti-GP antibodies (i.e., the same order of magnitude) and they are easily and economically produced in bacterial cultures. Conclusion/Significance Antibody fragments might represent a useful, effective, and low cost alternative to full-length antibodies in Ebola related capture and diagnostics applications. PMID:26489048

  10. Engineering Venom’s Toxin-Neutralizing Antibody Fragments and Its Therapeutic Potential

    PubMed Central

    Alvarenga, Larissa M.; Zahid, Muhammad; di Tommaso, Anne; Juste, Matthieu O.; Aubrey, Nicolas; Billiald, Philippe; Muzard, Julien

    2014-01-01

    Serum therapy remains the only specific treatment against envenoming, but anti-venoms are still prepared by fragmentation of polyclonal antibodies isolated from hyper-immunized horse serum. Most of these anti-venoms are considered to be efficient, but their production is tedious, and their use may be associated with adverse effects. Recombinant antibodies and smaller functional units are now emerging as credible alternatives and constitute a source of still unexploited biomolecules capable of neutralizing venoms. This review will be a walk through the technologies that have recently been applied leading to novel antibody formats with better properties in terms of homogeneity, specific activity and possible safety. PMID:25153256

  11. Association of selenocysteine transfer RNA fragments with serum antibody response to Mycoplasma spp. in beef cattle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective was to identify transfer RNA fragments (tRFs) associated with a serum antibody response to Mycoplasma spp. in beef cattle. Serum from sixteen beef calves was collected at three points: in summer after calves were born, in fall at weaning, and in the following spring. All sera collected...

  12. Toward in vivo imaging of heart disease using a radiolabeled single-chain Fv fragment targeting tenascin-C.

    PubMed

    Kobayashi, Norihiro; Odaka, Kenichi; Uehara, Tomoya; Imanaka-Yoshida, Kyoko; Kato, Yoshinori; Oyama, Hiroyuki; Tadokoro, Hiroyuki; Akizawa, Hiromichi; Tanada, Shuji; Hiroe, Michiaki; Fukumura, Toshimitsu; Komuro, Issei; Arano, Yasushi; Yoshida, Toshimichi; Irie, Toshiaki

    2011-12-01

    Antibodies specific to a particular target molecule can be used as analytical reagents, not only for in vitro immunoassays but also for noninvasive in vivo imaging, e.g., immunoscintigraphies. In the latter case, it is important to reduce the size of antibody molecules in order to achieve suitable in vivo "diagnostic kinetics" and generate higher-resolution images. For these purposes, single-chain Fv fragments (scFvs; M(r) < 30 kDa) have greater potential than intact immunoglobulins (~150 kDa) or Fab (or Fab') fragments (~50 kDa). Our recent observation of enhanced tenascin-C (Tnc) expression at sites of cardiac repair after myocardial infarction prompted us to develop a radiolabeled scFv against Tnc for in vivo imaging of heart disease. We cloned the genes encoding the heavy and light chain variable domains of the mouse anti-Tnc monoclonal antibody 4F10, and combined them to create a single gene. The resulting scFv-4F10 gene was expressed in E. coli cells to produce soluble scFv proteins. scFv-4F10 has an affinity for Tnc (K(a) = 3.5 × 10(7) M(-1)), similar to the Fab fragment of antibody 4F10 (K(a) = 1.3 × 10(7) M(-1)) and high enough to be of practical use. A cysteine residue was then added to the C-terminus to achieve site-specific (111)In labeling via a chelating group. The resulting (111)In-labeled scFv was administered to a rat model of acute myocardial infarction. Biodistribution and quantitative autoradiographic studies indicated higher uptake of the radioactivity at the infarcted myocardium than the noninfarcted one. Single photon emission computed tomography (SPECT) provided in vivo cardiac images that coincided with the ex vivo observations. Our results will promote advances in diagnostic strategies for heart disease. PMID:22074352

  13. Detection of experimental myocarditis by monoclonal antimyosin antibody, Fab fragment

    SciTech Connect

    Rezkalla, S.; Kloner, R.A.; Khaw, B.A.; Haber, E.; Fallon, J.T.; Smith, F.E.; Khatib, R.

    1989-02-01

    The purpose of this study was to determine whether monoclonal antimyosin Fab (antigen binding fragment) was capable of labeling hearts with experimental coxsackievirus myocarditis, and to determine whether Fab could be used for detecting myocardial damage in either early or chronic phases of the disease. Sixty-five, 3-week-old cesarean-derived 1 (CD 1) mice were divided into two groups: group I (noninfected animals) and group II (infected with coxsackievirus B3). Mice from each group were killed on days 7, 17, 30, or 90 of infection. Forty-eight hours before killing, mice were injected with monoclonal I-125 antimyosin, Fab (25 microCi/injection) and radioactivity was counted in the heart. Selected heart sections were also examined by autoradiography. Heart radioactivity, count/m/mg (m +/- SEM) on days 7, 17, 30, and 90 of infection was 10.8 +/- 1.7, 21.3 +/- 1.1, 11.2 +/- 3.4, and 12.4 +/- 1.5 for group I, versus 36.7 +/- 8.0 (p less than 0.01), 50.0 +/- 4.5 (p less than 0.001), 33.4 +/- 16.1 (p = NS), and 40.6 +/- 8.5 (p less than 0.01) for group II, respectively. Autoradiography revealed focal uptake within areas of necrotic myocardium. We conclude that I125 Fab may be useful in detecting myocardial damage in the experimental model of murine myocarditis up to day 90 of infection.

  14. Fusogenics: a recombinant immunotoxin-based screening platform to select internalizing tumor-specific antibody fragments.

    PubMed

    Cizeau, Jeannick; Torres, Marianne G P; Cowling, Sharla G; Stibbard, Stacy; Premsukh, Arjune; Entwistle, Joycelyn; MacDonald, Glen C

    2011-01-01

    Antibody-based therapeutics play a vital role in the treatment of certain cancers; however, despite commercial success, various strategies are being pursued to increase their potency and hence improve patient outcomes. The use of antibodies to deliver a cytotoxic payload offers a promising alternative for more efficacious therapies. Immunotoxins are composed of an internalizing antibody fragment linked to a bacterial or plant toxin. Once internalized, the payload, such as Pseudomonas exotoxin A (PE), blocks protein synthesis and induces apoptosis. Typically, immunotoxins are developed by first isolating a tumor-specific antibody, which is then either chemically linked to a toxin or reengineered as a fusion protein. Here, the authors describe the development of Fusogenics, an immunotoxin-based screening method that selects internalizing tumor-specific antibodies using a functional assay. Selected immune library clones were characterized and shown to be selective against normal tissues and specific to tumor tissues. In summary, the Fusogenics immunotoxin platform represents a unique, single-step selection approach combining specificity and functionality to isolate novel internalizing tumor-specific antibody fragments with potential for direct clinical application in the treatment of cancer. PMID:21131595

  15. Development and Preclinical Testing of a High Affinity Single Chain Antibody against (+)-Methamphetamine

    PubMed Central

    Peterson, Eric C.; Laurenzana, Elizabeth M.; Atchley, William T.; Hendrickson, Howard; Owens, S. Michael

    2009-01-01

    Chronic or excessive (+)-methamphetamine (METH) use often leads to addiction and toxicity to critical organs like the brain. With medical treatment as a goal, a novel single chain variable fragment (scFv) against METH was engineered from anti-METH monoclonal antibody mAb6H4 (IgG, κ light chain, KD = 11 nM) and found to have similar ligand affinity (KD = 10 nM) and specificity as mAb6H4. The anti-METH scFv (scFv6H4) was cloned, expressed in yeast, purified and formulated as a naturally occurring mixture of monomer (~75%) and dimer (~25%). To test the in vivo efficacy of the scFv6H4, male Sprague Dawley rats (n=5) were implanted with 3-day sc osmotic pumps delivering 3.2 mg/kg/day METH. After reaching steady-state METH concentrations, an i.v. dose of scFv6H4 (36.5 mg/kg, equimolar to the METH body burden) was administered along with a [3H]-scFv6H4 tracer. Serum pharmacokinetic (PCKN) analysis of METH and [3H]-scFv6H4 showed that the scFv6H4 caused an immediate 65-fold increase in the METH concentrations and a 12-fold increase in the serum METH area under the concentration-time curve from 0–480 min after scFv6H4 administration. The scFv6H4 monomer was quickly cleared or converted to multivalent forms with an apparent t1/2λz of 5.8 min. In contrast, the larger scFv6H4 multivalent forms (dimers, trimers, etc.) showed a much longer t1/2λz (228 min), and the significantly increased METH serum molar concentrations correlated directly with scFv6H4 serum molar concentrations. Considered together these data suggested that the scFv6H4 multimers (and not the monomer) were responsible for the prolonged redistribution of METH into the serum. PMID:18192498

  16. Efficient expression of single chain variable fragment antibody against paclitaxel using the Bombyx mori nucleopolyhedrovirus bacmid DNA system and its characterizations.

    PubMed

    Yusakul, Gorawit; Sakamoto, Seiichi; Tanaka, Hiroyuki; Morimoto, Satoshi

    2016-07-01

    A single chain variable fragment (scFv), the smallest unit of functional recombinant antibody, is an attractive format of recombinant antibodies for various applications due to its small fragment and possibility of genetic engineering. Hybridoma clone 3A3 secreting anti-paclitaxel monoclonal antibody was used to construct genes encoding its variable domains of heavy (VH) and light (VL) chains. The VH and VL domains were linked to be the PT-scFv3A3 using flexible peptide linker in a format of VH-(GGGGS)5-VL. The PT-scFv3A3 was primarily expressed using the pET28a(+) vector in the Escherichia coli system, and was then further expressed by using the Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system. Interestingly, the reactivity of PT-scFv3A3 expressed in the hemolymph of B. mori using the BmNPV bacmid DNA system was much higher than that expressed in the E. coli system. Using indirect competitive enzyme-linked immunosorbent assay (icELISA), the PT-scFv3A3 (B. mori) reacted not only with immobilized paclitaxel, but also with free paclitaxel in a concentration-dependent manner, with the linear range of free paclitaxel between 0.156 and 5.00 µg/ml. The PT-scFv3A3 (B. mori) exhibited less cross-reactivity (%) than its parental MAb clone 3A3 against paclitaxel-related compounds, including docetaxel (31.1 %), 7-xylosyltaxol (22.1 %), baccatin III (<0.68 %), 10-deacetylbaccatin III (<0.68 %), 1-hydroxybaccatin I (<0.68 %), and 1-acetoxy-5-deacetylbaccatin I (<0.68 %). With the exception of cephalomannine, the cross-reactivity was slightly increased to 8.50 %. The BmNPV bacmid DNA system was a highly efficient expression system of active PT-scFv3A3, which is applicable for PT-scFv3A3-based immunoassay of paclitaxel. In addition, the PT-scFv3A3 can be applied to evaluate its neutralizing property of paclitaxel or docetaxel toxicity. PMID:26940321

  17. Neutralizing human anti crotoxin scFv isolated from a nonimmunized phage library.

    PubMed

    Cardoso, D F; Nato, F; England, P; Ferreira, M L; Vaughan, T J; Mota, I; Mazie, J C; Choumet, V; Lafaye, P

    2000-04-01

    Combinatorial phage display technology offers a new possibility for making human antibodies which could be used in immune therapy. We explored the use of this technology to make human scFvs specific for crotoxin, the main toxic component of the venom of the South-American rattlesnake Crotalus durissus terrificus. Crotoxin, a phospholipase A2 neurotoxin constituted by the association of two subunits, exerts its lethal action by blocking neuromuscular transmission. This is the first report of human anticrotoxin scFvs (scFv 1, scFv 6 and scFv 8) isolated from a naive library of more than 1010 scFv clones with in vivo neutralizing activity. Nevertheless, differences are observed at the level of biological and immunological effects. Only scFv 8 is able to reduce the myotoxicity induced by crotoxin and scFv 1 is capable of altering the in vitro enzymatic activity of this toxin. All three scFvs recognize a region of one subunit located at the junction with the other one. Moreover these scFvs share strong amino acid homologies at the level of either the heavy or the light chain. Taken together, our results suggest that the use of human anticrotoxin scFvs may lead to a new and less aggressive passive immune therapy against poisoning by the venom of Crotalus durissus terrificus. PMID:10736105

  18. PET imaging of osteosarcoma in dogs using a fluorine-18-labeled monoclonal antibody fab fragment

    SciTech Connect

    Page, R.L.; Garg, P.K.; Gard, S. ||

    1994-09-01

    Four dogs with histologically confirmed osteogenic sarcoma were studied with PET following intravenous injection of the {sup 18}F-labeled Fab fragment of TP-3, a monoclonal antibody specific for human and canine osteosarcomas. The antibody fragment was labeled using the N-succinimidyl (8-(4{prime}-({sup 18}F)fluorobenzyl)amino)suberate acylation agent. Blood clearance of activity was biphasic in all dogs but half-times were variable (T{sub 1/2{beta}} = 2-13 hr). Catabolism of labeled Fab was reflected by the decrease in protein-associated activity in serum from more than 90% at 1 min to 60%-80% at 4 hr. PET images demonstrated increased accumulation of {sup 18}F at the primary tumor site relative to normal contralateral bone in one dog as early as 15 min after injection. Biopsies obtained after euthanasia indicated higher uptake at the edges of the tumor as observed on the PET scans. Tumor uptake was 1-3 x 10{sup -3}% injected dose/g, a level similar to that reported for other Fab fragments in human tumors. In the three dogs with metastatic disease, early PET images reflected activity in the blood pool but later uptake was observed in suspected metastatic sites. These results, although preliminary, suggest that PET imaging of {sup 18}F-labeled antibody fragments is feasible and that dogs with spontaneous tumors could be a valuable model for preclinical research with radioimmunoconjugates. 34 refs., 6 figs., 2 tabs.

  19. Interference of HCV replication by cell penetrable human monoclonal scFv specific to NS5B polymerase

    PubMed Central

    Thueng-in, Kanyarat; Thanongsaksrikul, Jeeraphong; Jittavisutthikul, Surasak; Seesuay, Watee; Chulanetra, Monrat; Sakolvaree, Yuwaporn; Srimanote, Potjanee; Chaicumpa, Wanpen

    2014-01-01

    A new class of hepatitis C virus (HCV)-targeted therapeutics that is safe, broadly effective and can cope with virus mutations is needed. The HCV's NS5B is highly conserved and different from human protein, and thus it is an attractive target for anti-HCV therapeutics development. In this study, NS5B bound-phage clones selected from a human single chain variable antibody fragment (scFv) phage display library were used to transform appropriate E. coli bacteria. Two scFv inhibiting HCV polymerase activity were selected. The scFvs were linked to a cell penetrating peptide to make cell penetrable scFvs. The transbodies reduced the HCV RNA and infectious virus particles released into the culture medium and inside hepatic cells transfected with a heterologous HCV replicon. They also rescued the innate immune response of the transfected cells. Phage mimotope search and homology modeling/molecular docking revealed the NS5B subdomains and residues bound by the scFvs. The scFv mimotopes matched residues of the NS5B, which are important for nucleolin binding during HCV replication, as well as residues that interconnect the fingers and thumb domains for forming a polymerase active groove. Both scFvs docked on several residues at the thumb armadillo-like fold that could be the polymerase interactive sites of other viral/host proteins for the formation of the replication complex and replication initiation. In conclusion, human transbodies that inhibited HCV RdRp activity and HCV replication and restored the host innate immune response were produced. They are potentially future interferon-free anti-HCV candidates, particularly in combination with other cognates that are specific to NS5B epitopes and other HCV enzymes. PMID:25517317

  20. Characterization of a recombinant humanized anti-cocaine monoclonal antibody and its Fab fragment.

    PubMed

    Kirley, Terence L; Norman, Andrew B

    2015-01-01

    Variations of post-translational modifications are important for stability and in vivo behavior of therapeutic antibodies. A recombinant humanized anti-cocaine monoclonal antibody (h2E2) was characterized for heterogeneity of N-linked glycosylation and disulfide bonds. In addition, charge heterogeneity, which is partially due to the presence or absence of C-terminal lysine on the heavy chains, was examined. For cocaine overdose therapy, Fab fragments may be therapeutic, and thus, a simplified method of generation, purification, and characterization of the Fab fragment generated by Endoproteinase Lys-C digestion was devised. Both the intact h2E2 antibody and purified Fab fragments were analyzed for their affinities for cocaine and 2 of its metabolites, benzoylecgonine and cocaethylene, by fluorescence quenching of intrinsic antibody tyrosine and tryptophan fluorescence resulting from binding of these drugs. Binding constants obtained from fluorescence quenching measurements are in agreement with recently published radioligand and ELISA binding assays. The dissociation constants determined for the h2E2 monoclonal and its Fab fragment are approximately 1, 5, and 20 nM for cocaethylene, cocaine, and benzoylecgonine, respectively. Tryptophan fluorescence quenching (emission at 330 nm) was measured after either excitation of tyrosine and tryptophan (280 nm) or selective excitation of tryptophan alone (295 nm). More accurate binding constants are obtained using tryptophan selective excitation at 295 nm, likely due to interfering absorption of cocaine and metabolites at 280 nm. These quenching results are consistent with multiple tryptophan and tyrosine residues in or near the predicted binding location of cocaine in a previously published 3-D model of this antibody's variable region. PMID:25692880

  1. Characterization of a recombinant humanized anti-cocaine monoclonal antibody and its Fab fragment

    PubMed Central

    Kirley, Terence L; Norman, Andrew B

    2015-01-01

    Variations of post-translational modifications are important for stability and in vivo behavior of therapeutic antibodies. A recombinant humanized anti-cocaine monoclonal antibody (h2E2) was characterized for heterogeneity of N-linked glycosylation and disulfide bonds. In addition, charge heterogeneity, which is partially due to the presence or absence of C-terminal lysine on the heavy chains, was examined. For cocaine overdose therapy, Fab fragments may be therapeutic, and thus, a simplified method of generation, purification, and characterization of the Fab fragment generated by Endoproteinase Lys-C digestion was devised. Both the intact h2E2 antibody and purified Fab fragments were analyzed for their affinities for cocaine and 2 of its metabolites, benzoylecgonine and cocaethylene, by fluorescence quenching of intrinsic antibody tyrosine and tryptophan fluorescence resulting from binding of these drugs. Binding constants obtained from fluorescence quenching measurements are in agreement with recently published radioligand and ELISA binding assays. The dissociation constants determined for the h2E2 monoclonal and its Fab fragment are approximately 1, 5, and 20 nM for cocaethylene, cocaine, and benzoylecgonine, respectively. Tryptophan fluorescence quenching (emission at 330 nm) was measured after either excitation of tyrosine and tryptophan (280 nm) or selective excitation of tryptophan alone (295 nm). More accurate binding constants are obtained using tryptophan selective excitation at 295 nm, likely due to interfering absorption of cocaine and metabolites at 280 nm. These quenching results are consistent with multiple tryptophan and tyrosine residues in or near the predicted binding location of cocaine in a previously published 3-D model of this antibody's variable region. PMID:25692880

  2. Improved fluoroquinolone detection in ELISA through engineering of a broad-specific single-chain variable fragment binding simultaneously to 20 fluoroquinolones.

    PubMed

    Wen, Kai; Nölke, Greta; Schillberg, Stefan; Wang, Zhanhui; Zhang, Suxia; Wu, Congming; Jiang, Haiyang; Meng, Hui; Shen, Jianzhong

    2012-07-01

    Fluoroquinolones (FQs) are a group of synthetic, broad-spectrum antibacterial agents. Due to its extensive use in animal industry and aquaculture, residues of these antibiotics and the emergence of bacteria resistant to FQs have become a major public health issue. To prepare a generic antibody capable of recognizing nearly all FQs, a single-chain variable fragment (scFv) was generated from the murine hybridoma cells C49H1 producing a FQ-specific monoclonal antibody. This scFv was characterized by indirect competitive enzyme-linked immunosorbent assay (ciELISA), and it showed identical binding properties to parental monoclonal antibody: it was capable of recognizing 17 of 20 targeted FQs below maximum residue limits, except for sarafloxacin (SAR), difloxacin (DIF), and trovafloxacin (TRO) which are highly concerned members in the FQs family. In order to broaden the specificity of this scFv to SAR and its analogues (DIF and TRO), protein homology modeling and antibody-ligands docking analysis were employed to identify the potential key amino acid residues involved in hapten antibody. A mutagenesis phage display library was generated by site directed mutagenesis randomizing five aminoacid residues in the third heavy-chain complementarity determining region. After one round of panning against biotinylated norfloxacin (NOR) and four rounds of panning against biotinylated SAR, scFv variants we screened showed up to 10-fold improved IC(50) against SAR, DIF, and TRO in ciELISA while the specificity against other FQs was fully retained. PMID:22549819

  3. Diagnosis of and therapy for solid tumors with radiolabeled antibodies and immune fragments

    SciTech Connect

    Carrasquillo, J.A.; Krohn, K.A.; Beaumier, P.; McGuffin, R.W.; Brown, J.P.; Hellstroem, K.E.; Hellstroem, I.; Larson, S.M.

    1984-01-01

    Antibodies which are directed against human tumor-associated antigens can potentially be used as carriers of radioactivity for in vivo diagnosis (radioimmunodetection) or treatment (radioimmunotherapy) of solid tumors, including colon, hepatoma, cholangiocarcinoma, and melanoma. Murine monoclonal antibodies (MOAB), produced by the hybridoma technique of Kohler and Milstein, are replacing conventional heterosera as sources of antibodies, because MOAB can be produced in large quantities as reproducible reagents with homogeneous binding properties. We have studied human melanoma using MOAB IgG and Fab fragments that recognize the human melanoma-associated antigens p97 and ''high-molecular-weight antigen''. Both antigens are found in the membrane of melanomas at much larger concentrations than in normal adult tissues. We have performed radioimmunodetection studies with whole immunoglobulin and have detected 88% of lesions greater than 1.5 cm. We have used Fab fragments for radioimmunotherapy and have found that large doses of radiolabeled antibodies (up to 342 mCi) can be repetitively given to patients without excessive end-organ toxicity. Two of three patients treated with high-dose radiolabeled antimelanoma Fab showed an effect from the treatment. Although both technical and biologic problems remain, the use of radiolabeled antibodies that are directed against tumor-associated antigens holds future promise as a new therapeutic approach to solid tumors that are resistant to conventional therapy.

  4. Antibody response by cultured spleen fragments from carrier-primed mice to hapten-protein conjugates.

    PubMed

    Hurme, M; Nakamura, I; Kaartinen, M; Mäkelä, O

    1975-01-01

    Hapten-protein conjugates stimulated very poor anti-hapten responses in mouse spleen fragment cultures from unimmunized mice, whereas hapten coupled to type III pneumococcal polysaccharide or polylysine induced good responses. When the donors of the fragments were primed with the carrier protein, hapten-protein conjugates induced a strong anti-hapten response. Both the true primary and the carrier-primed response in vitro consisted mainly of IgA antibodies of 9-13S. In carrier-primed responses also IgM was produced at the beginning and IgG at the end of those responses. PMID:1080285

  5. Nanoparticles Modified With Tumor-targeting scFv Deliver siRNA and miRNA for Cancer Therapy

    PubMed Central

    Chen, Yunching; Zhu, Xiaodong; Zhang, Xiaoju; Liu, Bin; Huang, Leaf

    2010-01-01

    Targeted delivery of RNA-based therapeutics for cancer therapy remains a challenge. We have developed a LPH (liposome-polycation-hyaluronic acid) nanoparticle formulation modified with tumor-targeting single-chain antibody fragment (scFv) for systemic delivery of small interfering RNA (siRNA) and microRNA (miRNA) into experimental lung metastasis of murine B16F10 melanoma. The siRNAs delivered by the scFv targeted nanoparticles efficiently downregulated the target genes (c-Myc/MDM2/VEGF) in the lung metastasis. Two daily intravenous injections of the combined siRNAs in the GC4-targeted nanoparticles significantly reduced the tumor load in the lung. miRNA-34a (miR-34a) induced apoptosis, inhibited survivin expression, and downregulated MAPK pathway in B16F10 cells. miR-34a delivered by the GC4-targeted nanoparticles significantly downregulated the survivin expression in the metastatic tumor and reduced tumor load in the lung. When miR-34a and siRNAs were co-formulated in GC4-targeted nanoparticles, an enhanced anticancer effect was observed. PMID:20606648

  6. The effect of internalizing human single chain antibody fragment on liposome targeting to epithelioid and sarcomatoid mesothelioma

    PubMed Central

    Iyer, Arun K.; Su, Yang; Feng, Jinjin; Lan, Xiaoli; Zhu, Xiaodong; Liu, Yue; Gao, Dongwei; Seo, Youngho; VanBrocklin, Henry F.; Broaddus, V. Courtney; Liu, Bin; He, Jiang

    2011-01-01

    Immunoliposomes (ILs) anchored with internalizing human antibodies capable of targeting all subtypes of mesothelioma can be useful for targeted imaging and therapy of this malignant disease. The objectives of this study were to evaluate both the in vitro and in vivo tumor targeted internalization of novel internalizing human single chain antibody (scFv) anchored ILs on both epithelioid (M28) and sarcomatoid (VAMT-1) subtypes of human mesothelioma. ILs were prepared by post-insertion of mesothelioma-targeting human scFv (M1) onto preformed liposomes and radiolabeled with 111In (111In-IL-M1), along with control non-targeted liposomes (111In-CL). Incubation of 111In-IL-M1 with M28, VAMT-1, and a control non-tumorigenic cell-line (BPH-1) at 37°C for 24 h revealed efficient binding and rapid internalization of ILs into both subtypes of tumor cells but not into the BPH-1 cells; internalization accounted for approximately 81-94% of total cell accumulation in mesothelioma cells compared to 37-55% in control cells. In tumor bearing mice intravenous (i.v.) injection of 111In-IL-M1 led to remarkable tumor accumulation: 4 % and 4.7% injected dose per gram (% ID/g) for M28 and VAMT-1 tumors, respectively, 48 h after injection. Furthermore, tumor uptake of 111In-IL-M1 in live xenograft animal models was verified by single photon emission computed tomography (SPECT/CT). In contrast, i.v. injection of 111In-CL in tumor-bearing mice revealed very low uptake in both subtypes of mesothelioma, 48 h after injection. In conclusion, M1 scFv-anchored ILs showed selective tumor targeting and rapid internalization into both epithelioid and sarcomatoid subtypes of human mesothelioma, demonstrating its potential as a promising vector for enhanced tumor drug targeting. PMID:21255833

  7. Binding of a monoclonal antibody and its Fab fragment to supported phospholipid monolayers measured by total internal reflection fluorescence microscopy.

    PubMed Central

    Pisarchick, M L; Thompson, N L

    1990-01-01

    The association of an anti-dinitrophenyl monoclonal antibody and its Fab fragment with supported phospholipid monolayers composed of a mixture of dipalmitoylphosphatidylcholine and dinitrophenyl-conjugated dipalmitoylphosphatidylethanolamine has been characterized with total internal reflection fluorescence microscopy. The surface densities of bound antibodies were measured as a function of the antibody and Fab solution concentrations, and as a function of the solution concentration of dinitrophenylglycine. The apparent association constant of Fab fragments with surface-associated haptens was approximately 10-fold lower than the association constant for haptens in solution, and the apparent surface association constant for intact antibodies was only approximately 10-fold higher than the constant for Fab fragments. Data analysis with simple theoretical models indicated that, at most antibody surface densities, 50-90% of membrane-associated intact antibodies were attached to the surface by two antigen binding sites. PMID:2291943

  8. Isolation of Llama Antibody Fragments for Prevention of Dandruff by Phage Display in Shampoo

    PubMed Central

    Dolk, Edward; van der Vaart, Marcel; Lutje Hulsik, David; Vriend, Gert; de Haard, Hans; Spinelli, Silvia; Cambillau, Christian; Frenken, Leon; Verrips, Theo

    2005-01-01

    As part of research exploring the feasibility of using antibody fragments to inhibit the growth of organisms implicated in dandruff, we isolated antibody fragments that bind to a cell surface protein of Malassezia furfur in the presence of shampoo. We found that phage display of llama single-domain antibody fragments (VHHs) can be extended to very harsh conditions, such as the presence of shampoo containing nonionic and anionic surfactants. We selected several VHHs that bind to the cell wall protein Malf1 of M. furfur, a fungus implicated in causing dandruff. In addition to high stability in the presence of shampoo, these VHHs are also stable under other denaturing conditions, such as high urea concentrations. Many of the stable VHHs were found to contain arginine at position 44. Replacement of the native amino acid at position 44 with arginine in the most stable VHH that lacked this arginine resulted in a dramatic further increase in the stability. The combination of the unique properties of VHHs together with applied phage display and protein engineering is a powerful method for obtaining highly stable VHHs that can be used in a wide range of applications. PMID:15640220

  9. Affinity maturation of single-chain variable fragment specific for aflatoxin B(1) using yeast surface display.

    PubMed

    Min, Won-Ki; Kim, Sung-Gun; Seo, Jin-Ho

    2015-12-01

    As aflatoxin B1 is one of the most toxic mycotoxins, it is important to detect and to quantify aflatoxin B1 accurately by immunological methods. To enhance aflatoxin B1-binding affinity of the single-chain variable fragment, yeast surface display technique combined with fluorescence-activated cell sorting was applied. A randomly mutated scFv library was subjected to 4 rounds of fluorescence-activated cell sorting, resulting in isolation of 5 scFv variants showing an affinity improvement compared to the parental wild type scFv. The best scFv with a 9-fold improvement in affinity for aflatoxin B1 exhibited similar specificity to the monoclonal antibody. Most of the mutations in scFv-M37 were located outside of the canonical antigen-contact loops, suggesting that its affinity improvement might be driven by an allosteric effect inducing scFv-M37 to form a more favorable binding pocket for aflatoxin B1 than the wild type scFv. PMID:26041237

  10. Characterization of neutralizing monoclonal antibodies directed against tetanus toxin fragment C.

    PubMed

    Yousefi, Mehdi; Tahmasebi, Fathollah; Younesi, Vahid; Razavi, Alireza; Khoshnoodi, Jalal; Bayat, Ali Ahmad; Abbasi, Ebrahim; Rabbani, Hodjatallah; Jeddi-Tehrani, Mahmood; Shokri, Fazel

    2014-01-01

    Clostridium tetani causes a life-threatening infectious disease by production of tetanus neurotoxin (TeNT), a 150 kDa molecule composed of light (LC) and heavy chain (HC) polypeptides. The TeNT HC contains an N-terminal domain critical for LC translocation and a C-terminal toxin receptor-binding domain known as fragment C. Despite extensive investigations on epitope specificity of anti-TeNT antibodies, the immunodominant neutralizing epitopes of the toxin are poorly defined. This study describes the generation and characterization of four monoclonal antibodies (MAb) specific for TeNT. The characteristics of each MAb were explored in terms of isotype, specificity, affinity, and immuno-globulin heavy chain variable region (IGHV) gene usage using ELISA, Western blotting, and sequencing techniques. The toxin neutralizing activity of the MAbs was also investigated using the in vitro GT1b neutralizing assay. The data demonstrated that all MAbs bind to tetanus toxin and toxoid. Sub-fragments binding analysis showed that two MAbs react with fragment C, one with both fragment C and LC, and one with LC. Only the two fragment C-specific MAbs were able to neutralize the toxin. Sequencing of the expressed VH and VL genes revealed rearrangements of various VH and VL gene segments in all hybridoma clones. Clonality of the hybridomas was also confirmed by a competition assay that showed recognition of distinct epitopes by these MAbs. The results suggest the importance of TeNT fragment C in terms of immunogenicity and toxin neutralization activity. PMID:23369087

  11. A Tat-grafted anti-nucleic acid antibody acquires nuclear-localization property and a preference for TAR RNA

    SciTech Connect

    Jeong, Jong-Geun; Kim, Dong-Sik; Kim, Yong-Sung; Kwon, Myung-Hee

    2011-03-18

    Highlights: {yields} We generate '{sub H3}Tat-3D8' by grafting Tat{sub 48-60} peptide to VH CDR of 3D8 scFv antibody. {yields} {sub H3}Tat-3D8 antibody retains nucleic acid binding and hydrolyzing activities. {yields} {sub H3}Tat-3D8 acquires a preference for TAR RNA structure. {yields} Properties of Tat{sub 48-60} is transferred to an antibody via Tat-grafting into a CDR. -- Abstract: The 3D8 single chain variable fragment (3D8 scFv) is an anti-nucleic acid antibody that can hydrolyze nucleic acids and enter the cytosol of cells without reaching the nucleus. The Tat peptide, derived from the basic region of the HIV-1 Tat protein, translocates to cell nuclei and has TAR RNA binding activity. In this study, we generated a Tat-grafted antibody ({sub H3}Tat-3D8) by replacing complementarity-determining region 3 (CDR3) within the VH domain of the 3D8 scFv with a Tat{sub 48-60} peptide (GRKKRRQRRRPPQ). {sub H3}Tat-3D8 retained the DNA-binding and DNA-hydrolyzing activity of the scFv, and translocated to the nuclei of HeLa cells and preferentially recognized TAR RNA. Thus, the properties associated with the Tat peptide were transferred to the antibody via Tat-grafting without loss of the intrinsic DNA-binding and hydrolyzing activities of the 3D8 scFv antibody.

  12. Cholestatic Liver Disease after Rituximab and Adalimumab and the Possible Role of Cross-Reacting Antibodies to Fab 2 Fragments

    PubMed Central

    Koetter, Ina; Schwab, Matthias; Fritz, Peter; Kimmel, Martin; Alscher, M. Dominik; Braun, Niko

    2013-01-01

    Background Millions of patients are treated with therapeutic monoclonal antibodies (Tmabs) for miscellaneous diseases. We investigated sera from six patients who received immune globulin, from one patient with refractory anti-neutrophil-cytoplasmic antibody (ANCA)-associated granulomatosis with polyangiitis (GPA) who developed two episodes of acute cholestatic liver disease, one after treatment with rituximab and a second after adalimumab and a healthy control group. Methods Three sera from the patient and six sera from patients who received immune globulin were analyzed for antibodies to rituximab and adalimumab by ELISA. Additionally, sera from the patients and from nine healthy blood donors were coated with the Fab fragment of an unrelated humanized monoclonal antibody, with human Fc proteins as well as a mouse IgG globulin. Results Viral serology for hepatitis A, B, C and autoantibodies specific for autoimmune liver disorders were negative. In all three sera from the patient antibodies to rituximab could be detected, but also antibodies to adalimumab were present even at time points when the patient had not yet received adalimumab, indicating cross reactivity between both substances. Testing against an unrelated human Fab fragment revealed positive results, indicating that the patient had antibodies against human Fab fragments in general. The Fc proteins were negative, and patients’ sera did also not react with mouse IgG globulins. Remarkably, 2 out of 5 patients which were treated with immune globulin had antibodies against human Fab fragments in general whereas in none of the samples from healthy controls antibodies to Fab fragment could be detected. Conclusion This is the first study demonstrating cholestatic liver disease induced by two different Tmabs. Cross - reacting antibodies to Fab2 fragments in general are probably involved. Further studies must show if these Fab2 antibodies in general are related with drug-induced side effects and accelerated drug

  13. Kinetic analysis of a monoclonal therapeutic antibody and its single-chain homolog by surface plasmon resonance.

    PubMed

    Patel, Rekha; Andrien, Bruce A

    2010-01-01

    Monoclonal antibodies (mAbs) and antibody fragments have become an emerging class of therapeutics since 1986. Their versatility enables them to be engineered for optimal efficiency and decreased immunogenicity, and the path to market has been set by recent regulatory approvals. One of the initial criteria for success of any protein or antibody therapeutic is to understand its binding characteristics to the target antigen. Surface plasmon resonance (SPR) has been widely used and is an important tool for ligand-antigen binding characterization. In this work, the binding kinetics of a recombinant mAb and its single-chain antibody homolog, single-chain variable fragment (scFv), was analyzed by SPR. These two proteins target the same antigen. The binding kinetics of the mAb (bivalent antibody) and scFv (monovalent scFv) for this antigen was analyzed along with an assessment of the thermodynamics of the binding interactions. Alternative binding configurations were investigated to evaluate potential experimental bias because theoretically experimental binding configuration should have no impact on binding kinetics. Self-association binding kinetics in the proteins' respective formulation solutions and antigen epitope mapping were also evaluated. Functional characterization of monoclonal and single-chain antibodies has become just as important as structural characterization in the biotechnology field. PMID:19720041

  14. PEGylation of antibody fragments greatly increases their local residence time following delivery to the respiratory tract.

    PubMed

    Koussoroplis, Salome Juliette; Paulissen, Geneviève; Tyteca, Donatienne; Goldansaz, Hadi; Todoroff, Julie; Barilly, Céline; Uyttenhove, Catherine; Van Snick, Jacques; Cataldo, Didier; Vanbever, Rita

    2014-08-10

    Inhalation aerosols offer a targeted therapy for respiratory diseases. However, the therapeutic efficacy of inhaled biopharmaceuticals is limited by the rapid clearance of macromolecules in the lungs. The aim of this research was to study the effects of the PEGylation of antibody fragments on their local residence time after administration to the respiratory tract. We demonstrate that the conjugation of a two-armed 40-kDa polyethylene glycol (PEG) chain to anti-interleukin-17A (IL-17A) F(ab')2 and anti-IL-13 Fab' greatly prolonged the presence of these fragments within the lungs of mice. The content of PEGylated antibody fragments within the lungs plateaued up to 4h post-delivery, whereas the clearance of unconjugated proteins started immediately after administration. Forty-eight hours post-delivery, F(ab')2 and Fab' contents in the lungs had decreased to 10 and 14% of the dose initially deposited, respectively. However, this value was 40% for both PEG40-F(ab')2 and PEG40-Fab'. The prolonged pulmonary residency of the anti-IL-17A PEG40-F(ab')2 translated into an improved efficacy in reducing lung inflammation in a murine model of house dust mite-induced lung inflammation. We demonstrate that PEGylated proteins were principally retained within the lung lumen rather than the nasal cavities or lung parenchyma. In addition, we report that PEG increased pulmonary retention of antibody fragments through mucoadhesion and escape from alveolar macrophages rather than increased hydrodynamic size or improved enzymatic stability. The PEGylation of proteins might find broad application in the local delivery of therapeutic proteins to diseased airways. PMID:24845126

  15. Antibody fragment-conjugated polymeric micelles incorporating platinum drugs for targeted therapy of pancreatic cancer.

    PubMed

    Ahn, Jooyeon; Miura, Yutaka; Yamada, Naoki; Chida, Tsukasa; Liu, Xueying; Kim, Ahram; Sato, Ryuta; Tsumura, Ryo; Koga, Yoshikatsu; Yasunaga, Masahiro; Nishiyama, Nobuhiro; Matsumura, Yasuhiro; Cabral, Horacio; Kataoka, Kazunori

    2015-01-01

    Antibody-mediated therapies including antibody-drug conjugates (ADCs) have shown much potential in cancer treatment by tumor-targeted delivery of cytotoxic drugs. However, there is a limitation of payloads that can be delivered by ADCs. Integration of antibodies to drug-loaded nanocarriers broadens the applicability of antibodies to a wide range of therapeutics. Herein, we developed antibody fragment-installed polymeric micelles via maleimide-thiol conjugation for selectively delivering platinum drugs to pancreatic tumors. By tailoring the surface density of maleimide on the micelles, one tissue factor (TF)-targeting Fab' was conjugated to each carrier. Fab'-installed platinum-loaded micelles exhibited more than 15-fold increased cellular binding within 1 h and rapid cellular internalization compared to non-targeted micelles, leading to superior in vitro cytotoxicity. In vivo, Fab'-installed micelles significantly suppressed the growth of pancreatic tumor xenografts for more than 40 days, outperforming non-targeted micelles and free drugs. These results indicate the potential of Fab'-installed polymeric micelles for efficient drug delivery to solid tumors. PMID:25477168

  16. Functional expression of a single-chain antibody to ErbB-2 in plants and cell-free systems

    PubMed Central

    Galeffi, Patrizia; Lombardi, Alessio; Pietraforte, Immacolata; Novelli, Flavia; Di Donato, Monica; Sperandei, Maria; Tornambé, Andrea; Fraioli, Rocco; Martayan, Aline; Natali, Pier Giorgio; Benevolo, Maria; Mottolese, Marcella; Ylera, Francisco; Cantale, Cristina; Giacomini, Patrizio

    2006-01-01

    Background Aberrant signaling by ErbB-2 (HER 2, Neu), a member of the human Epidermal Growth Factor (EGF) receptor family, is associated with an aggressive clinical behaviour of carcinomas, particularly breast tumors. Antibodies targeting the ErbB-2 pathway are a preferred therapeutic option for patients with advanced breast cancer, but a worldwide deficit in the manufacturing capacities of mammalian cell bioreactors is foreseen. Methods Herein, we describe a multi-platform approach for the production of recombinant Single chain Fragments of antibody variable regions (ScFvs) to ErbB-2 that involves their functional expression in (a) bacteria, (b) transient as well as stable transgenic tobacco plants, and (c) a newly developed cell-free transcription-translation system. Results An ScFv (ScFv800E6) was selected by cloning immunoglobulin sequences from murine hybridomas, and was expressed and fully functional in all the expression platforms, thereby representing the first ScFv to ErbB-2 produced in hosts other than bacteria and yeast. ScFv800E6 was optimized with respect to redox synthesis conditions. Different tags were introduced flanking the ScFv800E6 backbone, with and without spacer arms, including a novel Strep II tag that outperforms conventional streptavidin-based detection systems. ScFv800E6 was resistant to standard chemical radiolabeling procedures (i.e. Chloramine T), displayed a binding ability extremely similar to that of the parental monovalent Fab' fragment, as well as a flow cytometry performance and an equilibrium binding affinity (Ka approximately 2 × 108 M-1) only slightly lower than those of the parental bivalent antibody, suggesting that its binding site is conserved as compared to that of the parental antibody molecule. ScFv800E6 was found to be compatible with routine reagents for immunohistochemical staining. Conclusion ScFv800E6 is a useful reagent for in vitro biochemical and immunodiagnostic applications in oncology, and a candidate for

  17. Production, purification and biological characterization of mono-PEGylated anti-IL-17A antibody fragments.

    PubMed

    Koussoroplis, Salome-Juliette; Heywood, Sam; Uyttenhove, Catherine; Barilly, Céline; Van Snick, Jacques; Vanbever, Rita

    2013-09-15

    The aim of this study was to maximize the yield of the production of mono-PEGylated anti-interleukin-17A (anti-IL-17A) antibody fragments using large (≥ 20 kDa) polyethylene glycol (PEG) chains. Particular attention was paid to selectively yield mono-PEGylated species to maintain the maximum possible functionality and to simplify the purification. Neutralization of IL-17A by antibody constructs might find application for the treatment of bronchial hyperreactivity. Amino-directed and sulfhydryl-directed PEGylation of the native antibody fragments were compared. The former was selected as it produced the most interesting construct in terms of yield and preservation of biological activity. In particular, the F(ab')2-PEG conjugate with one 40 kDa branched PEG prepared in this study was produced at a 42% yield. The conjugate presented only a slight decrease in its binding activity and in its in vitro inhibitory potency offering interesting perspectives for in vivo studies. PMID:23850622

  18. Isolation and characterization of antibody fragments selective for toxic oligomeric tau.

    PubMed

    Tian, Huilai; Davidowitz, Eliot; Lopez, Patricia; He, Ping; Schulz, Philip; Moe, James; Sierks, Michael R

    2015-03-01

    Oligomeric tau species are important in the onset and progression of Alzheimer's disease (AD), as they are neurotoxic and can propagate tau-tangle pathology. Therefore, reagents that selectively recognize different key morphologies of tau are needed to help define the role of tau in AD and related diseases. We utilized a biopanning protocol that combines the binding diversity of phage-displayed antibody libraries with the powerful imaging capability of atomic force microscopy to isolate single-chain antibody fragments (scFvs) that selectively bind toxic oligomeric tau. We isolated 3 different antibody fragments that bind oligomeric but not monomeric or fibrillar tau. The scFvs differentiate brain tissue homogenates of both 3×TG and tau-AD mice from wild-type mice, detecting oligomeric tau at much earlier ages than when neurofibrillary tangles are typically detected. The scFvs also distinguish human postmortem AD brain tissue from cognitively normal postmortem human brain tissue, demonstrating the potential of this approach for developing biomarkers for early detection and progression of AD. PMID:25616912

  19. Proteomic differences in recombinant CHO cells producing two similar antibody fragments.

    PubMed

    Sommeregger, Wolfgang; Mayrhofer, Patrick; Steinfellner, Willibald; Reinhart, David; Henry, Michael; Clynes, Martin; Meleady, Paula; Kunert, Renate

    2016-09-01

    Chinese hamster ovary (CHO) cells are the most commonly used mammalian hosts for the production of biopharmaceuticals. To overcome unfavorable features of CHO cells, a lot of effort is put into cell engineering to improve phenotype. "Omics" studies investigating elevated growth rate and specific productivities as well as extracellular stimulus have already revealed many interesting engineering targets. However, it remains largely unknown how physicochemical properties of the recombinant product itself influence the host cell. In this study, we used quantitative label-free LC-MS proteomic analyses to investigate product-specific proteome differences in CHO cells producing two similar antibody fragments. We established recombinant CHO cells producing the two antibodies, 3D6 and 2F5, both as single-chain Fv-Fc homodimeric antibody fragments (scFv-Fc). We applied three different vector strategies for transgene delivery (i.e., plasmid, bacterial artificial chromosome, recombinase-mediated cassette exchange), selected two best performing clones from transgene variants and transgene delivery methods and investigated three consecutively passaged cell samples by label-free proteomic analysis. LC-MS-MS profiles were compared in several sample combinations to gain insights into different aspects of proteomic changes caused by overexpression of two different heterologous proteins. This study suggests that not only the levels of specific product secretion but the product itself has a large impact on the proteome of the cell. Biotechnol. Bioeng. 2016;113: 1902-1912. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. PMID:26913574

  20. Isolation and characterization of antibody fragments selective for toxic oligomeric tau

    PubMed Central

    Tian, Huilai; Davidowitz, Eliot; Lopez, Patricia; He, Ping; Schulz, Philip; Moe, James; Sierks, Michael R.

    2014-01-01

    Oligomeric tau species are important in the onset and progression of Alzheimer’s Disease (AD) as they are neurotoxic and can propagate tau tangle pathology. Therefore reagents that selectively recognize different key morphologies of tau are needed to help define the role of tau in AD and related diseases. We utilized a biopanning protocol that combines the binding diversity of phage-displayed antibody libraries with the powerful imaging capability of atomic force microscopy (AFM) to isolate single chain antibody fragments (scFvs) that selectively bind toxic oligomeric tau. We isolated three different antibody fragments that bind oligomeric but not monomeric or fibrillar tau. The scFvs differentiate brain tissue homogenates of both 3×TG and tau-AD mice from wild type mice, detecting oligomeric tau at much earlier ages than when neurofibrillary tangles are typically detected. The scFvs also distinguish human post-mortem AD brain tissue from cognitively normal post-mortem human brain tissue demonstrating the potential of this approach for developing biomarkers for early detection and progression of AD. PMID:25616912

  1. Mutations in Antibody Fragments Modulate Allosteric Response Via Hydrogen-Bond Network Fluctuations.

    PubMed

    Srivastava, Amit; Tracka, Malgorzata B; Uddin, Shahid; Casas-Finet, Jose; Livesay, Dennis R; Jacobs, Donald J

    2016-05-10

    A mechanical perturbation method that locally restricts conformational entropy along the protein backbone is used to identify putative allosteric sites in a series of antibody fragments. The method is based on a distance constraint model that integrates mechanical and thermodynamic viewpoints of protein structure wherein mechanical clamps that mimic substrate or cosolute binding are introduced. Across a set of six single chain-Fv fragments of the anti-lymphotoxin-β receptor antibody, statistically significant responses are obtained by averaging over 10 representative structures sampled from a molecular dynamics simulation. As expected, the introduced clamps locally rigidify the protein, but long-ranged increases in both rigidity and flexibility are also frequently observed. Expanding our analysis to every molecular dynamics frame demonstrates that the allosteric responses are modulated by fluctuations within the hydrogen-bond network where the native ensemble is comprised of conformations that both are, and are not, affected by the perturbation in question. Population shifts induced by the mutations alter the allosteric response by adjusting which hydrogen-bond networks are the most probable. These effects are compared using response maps that track changes across each single chain-Fv fragment, thus providing valuable insight into how sensitive allosteric mechanisms are to mutations. PMID:27166802

  2. Single Chain Variable Fragment Against Aβ Expressed in Baculovirus Inhibits Abeta Fibril Elongation and Promotes its Disaggregation

    PubMed Central

    Fang, Fang; Song, Lin-Lin; Jiao, Yue-Ying; Wang, He; Peng, Xiang-Lei; Zheng, Yan-Peng; Wang, Jun; He, Jin-Sheng; Hung, Tao

    2015-01-01

    Alzheimer’s disease (AD) is the most common form of age-related dementia, and the most urgent problem is that it is currently incurable. Amyloid-β (Aβ) peptide is believed to play a major role in the pathogenesis of AD. We previously reported that an Aβ N-terminal amino acid targeting monoclonal antibody (MAb), A8, inhibits Aβ fibril formation and has potential as an immunotherapy for AD based on a mouse model. To further study the underlying mechanisms, we tested our hypothesis that the single chain fragment variable (scFv) without the Fc fragment is capable of regulating either Aβ aggregation or disaggregation in vitro. Here, a model of cell-free Aβ “on-pathway” aggregation was established and identified using PCR, Western blot, ELISA, transmission electron microscopy (TEM) and thioflavin T (ThT) binding analyses. His-tagged A8 scFvs was cloned and solubly expressed in baculovirus. Our data demonstrated that the Ni-NTA agarose affinity-purified A8 scFv inhibited the forward reaction of “on-pathway” aggregation and Aβ fibril maturation. The effect of A8 scFv on Aβ fibrillogenesis was markedly more significant when administered at the start of the Aβ folding reaction. Furthermore, the results also showed that pre-formed Aβ fibrils could be disaggregated via incubation with purified A8 scFv, which suggested that A8 scFv is involved in the reverse reaction of Aβ aggregation. Therefore, A8 scFv was capable of both inhibiting fibrillogenesis and disaggregating matured fibrils. Our present study provides valuable insight into the regulators of ultrastructural dynamics of cell-free “on-pathway” Aβ aggregation and will assist in the development of therapeutic strategies for AD. PMID:25919299

  3. Recombinant human antibodies: linkage of an Fab fragment from a combinatorial library to an Fc fragment for expression in mammalian cell culture.

    PubMed

    Bender, E; Woof, J M; Atkin, J D; Barker, M D; Bebbington, C R; Burton, D R

    1993-04-01

    The combinatorial phage library approach to immunoglobulin repertoire cloning recently made it possible to isolate gene fragments encoding human immunoglobulin G1 Fabs binding with high affinity to specific antigens. Here we describe the construction of genes encoding whole human anti-tetanus toxoid antibodies based on one of these gene fragments and the efficient expression of these constructs by co-transfection of separate heavy and light chain vectors into a Chinese hamster ovary cell line constitutively expressing a viral transactivator protein. This system will be generally useful for the rapid analysis of recombinant antibodies derived from combinatorial libraries. PMID:8518367

  4. Conjugation of R-Phycoerythrin to a Polyclonal Antibody and F (ab')2 Fragment of a Polyclonal Antibody by Two Different Methods.

    PubMed

    Mahmoudian, Jafar; Jeddi-Tehrani, Mahmood; Rabbani, Hodjattallah; Mahmoudi, Ahmad Reza; Akhondi, Mohammad Mehdi; Zarnani, Amir Hassan; Goli, Leila Balaei; Babaei, Mahdokht; Ghods, Roya

    2010-04-01

    R-Phycoerythrin (R-PE), a fluorescent protein from phycobiliprotein family, is isolated from red algae. Conjugation of antibodies to R-PE facilitates multiple fluorescent staining methods. In the present study polyclonal antibodies and polyclonal F(ab')2 fragment antibodies were conjugated to R-PE by two different methods. The efficiency of the methods was evaluated using Immunocytochemistry (ICC) and Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE). In the first conjugation method, PE was attached to SMCC linker followed by conjugation of antibody to PE-SMCC. In the second method, SH groups were added onto R-PE molecule, while the antibody was attached to SPDP linker. Then, the antibody-SPDP molecule was conjugated to R-PE. Our results showed that the two conjugation methods did not have any abrogative effects on the antibody binding activity. PMID:23407609

  5. Anti Transglutaminase Antibodies Cause Ataxia in Mice

    PubMed Central

    Boscolo, Sabrina; Lorenzon, Andrea; Sblattero, Daniele; Florian, Fiorella; Stebel, Marco; Marzari, Roberto; Not, Tarcisio; Aeschlimann, Daniel; Ventura, Alessandro; Hadjivassiliou, Marios; Tongiorgi, Enrico

    2010-01-01

    Background Celiac disease (CD) is an autoimmune gastrointestinal disorder characterized by the presence of anti-transglutaminase 2 (TG2) and anti-gliadin antibodies. Amongst the neurological dysfunctions associated with CD, ataxia represents the most common one. Methods We analyzed by immunohistochemistry, the anti-neural reactivity of the serum from 20 CD patients. To determine the role of anti-TG2 antibodies in ataxia, two anti-TG2 single chain variable fragments (scFv), isolated from a phage-display IgA antibody library, were characterized by immunohistochemistry and ELISA, and injected in mice to study their effects on motor coordination. We found that 75% of the CD patient population without evidence of neurological involvement, has circulating anti-neural IgA and/or IgG antibodies. Two anti-TG2 scFvs, cloned from one CD patient, stained blood vessels but only one reacted with neurons. This anti-TG2 antibody showed cross reactivity with the transglutaminase isozymes TG3 and TG6. Intraventricular injection of the anti-TG2 or the anti-TG2/3/6 cross-reactive scFv provoked transient, equally intensive ataxia in mice. Conclusion The serum from CD patients contains anti-TG2, TG3 and TG6 antibodies that may potentially cause ataxia. PMID:20300628

  6. High efficiency recovery and epitope specific sorting of an scFv yeast display library

    SciTech Connect

    Siegel, Robert W.; Coleman, James R.; Miller, Keith D.; Feldhaus, Michael

    2004-03-01

    In order to more productively utilize the rich source of antigen specific reagents present in the previously described non-immune scFv yeast display library (Feldhaus et al., 2003) one must be able to efficiently isolate and characterize clones within the library. To this end, we have developed and validated a magnetic bead sorting technique utilizing the Miltenyi MacsTm system to recover greater than 90% of the antigen specific clones present in the library. In combination with flow cytometry, we rapidly reduced diversity and enriched for antigen specific clones in three rounds of selection. Furthermore, we demonstrate the use of pre-existing monoclonal antibodies (mAbs) for antigen labeling and subsequent flow cytometric sorting and characterization of epitope specific scFv. Combining these two improvements in library screening allowed isolation and characterization of 3 epitope specific scFv (including a previously uncharacterized epitope) to a 6 kd protein, epidermal growth factor EGF.

  7. Use of solid-phase double-antibody radioimmunoassay to identify species from small skeletal fragments.

    PubMed

    Ubelaker, Douglas H; Lowenstein, Jerold M; Hood, Darden G

    2004-09-01

    Protein radioimmunoassay (pRIA) offers the potential to identify species in small skeletal fragments submitted as forensic evidence. The technique consists of protein extraction followed by a solid-phase double-antibody radioimmunoassay using controls of antisera (raised in rabbits) and radioactive (iodine-125) antibody of rabbit gamma globulin (produced in donkeys). Species determination results from evaluation of radioactivity uptake. To demonstrate the potential of this technique, six known bone samples (three human and three nonhuman, including one from a deer [Odocoileus virginianus]) were submitted for blind analysis. pRIA correctly distinguished the human from the nonhuman samples. Using 200 mg or less of each sample, species of the deer specimen was identified correctly, given the choices of cow, deer, dog, goat, and pig. PMID:15461091

  8. Optimizing Radiolabeled Engineered Anti-p185HER2 Antibody Fragments for In vivo Imaging

    PubMed Central

    Olafsen, Tove; Kenanova, Vania E.; Sundaresan, Gobalakrishnan; Anderson, Anne-Line; Crow, Desiree; Yazaki, Paul J.; Li, Lin; Press, Michael F.; Gambhir, Sanjiv S.; Williams, Lawrence E.; Wong, Jeffrey Y.C.; Raubitschek, Andrew A.; Shively, John E.; Wu, Anna M.

    2014-01-01

    We have recently described the in vivo properties of an iodinated anti-p185HER2 engineered antibody fragment [minibody (scFv-CH3)2; 80 kDa], made from the internalizing 10H8 monoclonal antibody. Although the 10H8 minibody showed excellent binding to the target in vitro, only modest tumor uptake [5.6 ± 1.7% injected dose per gram (ID/g) of tissue] was achieved in nude mice bearing MCF7/HER2 breast cancer tumors. Here, in an attempt to improve targeting, the 10H8 minibody was conjugated to 1,4,7,10-tetraazacyclododecane-N, N′, N″, N‴-tetraacetic acid (DOTA), radio metal labeled, and evaluated in vivo. The tumor uptake of 111In-DOTA 10H8 minibody was 5.7 ± 0.1% ID/g, similar to the radioiodinated 10H8 minibody. However, in addition to the expected liver clearance, the kidneys had unexpectedly high activity (34.0 ± 4.0% ID/g). A minibody derived from a second anti-p185HER2 antibody (trastuzumab; hu4D5v8) was also made. Tumor uptakes, evaluated by quantitative microPET using 64Cu-DOTA hu4D5v8 minibody, were 4.2 ± 0.5% ID/g. Furthermore, in non-tumor-bearing mice, 111In-DOTA hu4D5v8 minibody exhibited similar elevated uptake in the kidneys (28.4 ± 6.5% ID/g). Immunohistochemical staining of kidneys from non-tumor-bearing mice showed strong specific staining of the proximal tubules, and Western blot analysis of kidney lysate confirmed the presence of cross-reactive antigen. To further improve tumor uptake and normal tissue distribution, a larger hu4D5v8 fragment [(scFv-CH2-CH3)2; 105 kDa] was made, engineered to exhibit rapid clearance kinetics. This fragment, when evaluated by microPET, exhibited improved tumor targeting (12.2 ± 2.4% ID/g) and reduced kidney uptake (13.1 ± 1.5% ID/g). Thus, by manipulating the size and format of anti-p185 antibody fragments, the kidney activity was reduced and high or low expression of p185HER2 in xenografts could be distinguished by microPET imaging. PMID:15994969

  9. Engineered antibody fragments for immuno-PET imaging of endogenous CD8+ T cells in vivo

    PubMed Central

    Tavaré, Richard; McCracken, Melissa N.; Zettlitz, Kirstin A.; Knowles, Scott M.; Salazar, Felix B.; Olafsen, Tove; Witte, Owen N.; Wu, Anna M.

    2014-01-01

    The noninvasive detection and quantification of CD8+ T cells in vivo are important for both the detection and staging of CD8+ lymphomas and for the monitoring of successful cancer immunotherapies, such as adoptive cell transfer and antibody-based immunotherapeutics. Here, antibody fragments are constructed to target murine CD8 to obtain rapid, high-contrast immuno-positron emission tomography (immuno-PET) images for the detection of CD8 expression in vivo. The variable regions of two anti-murine CD8-depleting antibodies (clones 2.43 and YTS169.4.2.1) were sequenced and reformatted into minibody (Mb) fragments (scFv-CH3). After production and purification, the Mbs retained their antigen specificity and bound primary CD8+ T cells from the thymus, spleen, lymph nodes, and peripheral blood. Importantly, engineering of the parental antibodies into Mbs abolished the ability to deplete CD8+ T cells in vivo. The Mbs were subsequently conjugated to S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid for 64Cu radiolabeling. The radiotracers were injected i.v. into antigen-positive, antigen-negative, immunodeficient, antigen-blocked, and antigen-depleted mice to evaluate specificity of uptake in lymphoid tissues by immuno-PET imaging and ex vivo biodistribution. Both 64Cu-radiolabeled Mbs produced high-contrast immuno-PET images 4 h postinjection and showed specific uptake in the spleen and lymph nodes of antigen-positive mice. PMID:24390540

  10. Structural modifications of monoclonal antibodies following direct versus indirect labelling with 99Tcm: does fragmentation really occur?

    PubMed

    Behr, T; Becker, W; Hannappel, E; Wolf, F

    1994-11-01

    In this study, the influence of direct and indirect 99Tcm-labelling on the molecular structural integrity of monoclonal antibodies and other immunoglobulin preparations was investigated. Molecular composition of antibody preparations [two IgG monoclonal antibodies, one F(ab')2 fragment (all directly labelled), one indirectly labelled polyclonal human immunoglobulin preparation] and of serum samples after antibody injection were studied using polyacrylamide gel electrophoresis (PAGE; non-reducing and reducing conditions) and gel filtration chromatography. With PAGE, depending on the conditions used, a variety of lower molecular weight products could be detected. When analysing the same antibody preparations by gel filtration chromatography, all complete antibody preparations appeared as homogenous proteins of IgG molecular weight (150 kD). In F(ab')2 fragments, some further fragmentation to Fab' was noticed. Neither in vitro nor in vivo (serum) evidence of smaller fragments could be detected by gel filtration, despite their presence in PAGE. We therefore conclude that through the reductive step of direct 99Tcm-labelling, interchain disulphide linkages are broken but the polypeptide chains of complete IgG remain associated by non-covalent linkages, whereas (F(ab')2 is fragmented further to form essentially Fab'. The protein-denaturating conditions of PAGE (even if performed non-reducingly) seem to produce artifacts, not representing the real in vivo condition. PAGE results should therefore be interpreted only with great care. PMID:7870392

  11. A collagen-binding EGFR antibody fragment targeting tumors with a collagen-rich extracellular matrix

    PubMed Central

    Liang, Hui; Li, Xiaoran; Wang, Bin; Chen, Bing; Zhao, Yannan; Sun, Jie; Zhuang, Yan; Shi, Jiajia; Shen, He; Zhang, Zhijun; Dai, Jianwu

    2016-01-01

    Many tumors over-express collagen, which constitutes the physical scaffold of tumor microenvironment. Collagen has been considered to be a target for cancer therapy. The collagen-binding domain (CBD) is a short peptide, which could bind to collagen and achieve the sustained release of CBD-fused proteins in collagen scaffold. Here, a collagen-binding EGFR antibody fragment was designed and expressed for targeting the collagen-rich extracellular matrix in tumors. The antibody fragment (Fab) of cetuximab was fused with CBD (CBD-Fab) and expressed in Pichia pastoris. CBD-Fab maintained antigen binding and anti-tumor activity of cetuximab and obtained a collagen-binding ability in vitro. The results also showed CBD-Fab was mainly enriched in tumors and had longer retention time in tumors in A431 s.c. xenografts. Furthermore, CBD-Fab showed a similar therapeutic efficacy as cetuximab in A431 xenografts. Although CBD-Fab hasn’t showed better therapeutic effects than cetuximab, its smaller molecular and special target may be applicable as antibody–drug conjugates (ADC) or immunotoxins. PMID:26883295

  12. Single-Chain Antibody Library

    DOE Data Explorer

    Baird, Cheryl

    Researchers at Pacific Northwest National Laboratory (PNNL) have constructed a nonimmune library consisting of 109 human antibody scFv fragments, which have been cloned and expressed on the surface of yeast. Nanomolar-affinity scFvs are routinely obtained by magnetic bead screening and flow cytometric sorting. The yeast library can be amplified 1010 fold without measurable loss of clonal diversity. This allows for indefinite expansion of the library. All scFv clones can be assessed directly on the yeast cell surface by immunofluorescent labeling and flow cytometry, obviating separate subcloning, expression, and purification steps. The ability to use multiplex library screening demonstrates the utility of this approach for high-throughput antibody isolation for proteomic applications. The yeast library may be used for research projects or teaching performed for U.S. Government purposes only. If you would like to request an aliquot of the single-chain antibody library for your research, please print and fill out the Materials Transfer Agreement (MTA) [PDF, 20K]. The website provides the contact information for mailing the MTA. [copied from http://www.sysbio.org/dataresources/singlechain.stm

  13. Evaluation of selectivity in homologous multimodal chromatographic systems using in silico designed antibody fragment libraries.

    PubMed

    Karkov, Hanne Sophie; Woo, James; Krogh, Berit Olsen; Ahmadian, Haleh; Cramer, Steven M

    2015-12-24

    This study describes the in silico design, surface property analyses, production and chromatographic evaluations of a diverse set of antibody Fab fragment variants. Based on previous findings, we hypothesized that the complementarity-determining regions (CDRs) constitute important binding sites for multimodal chromatographic ligands. Given that antibodies are highly diversified molecules and in particular the CDRs, we set out to examine the generality of this result. For this purpose, four different Fab fragments with different CDRs and/or framework regions of the variable domains were identified and related variants were designed in silico. The four Fab variant libraries were subsequently generated by site-directed mutagenesis and produced by recombinant expression and affinity purification to enable examination of their chromatographic retention behavior. The effects of geometric re-arrangement of the functional moieties on the multimodal resin ligands were also investigated with respect to Fab variant retention profiles by comparing two commercially available multimodal cation-exchange ligands, Capto MMC and Nuvia cPrime, and two novel multimodal ligand prototypes. Interestingly, the chromatographic data demonstrated distinct selectivity trends between the four Fab variant libraries. For three of the Fab libraries, the CDR regions appeared as major binding sites for all multimodal ligands. In contrast, the fourth Fab library displayed a distinctly different chromatographic behavior, where Nuvia cPrime and related multimodal ligand prototypes provided markedly improved selectivity over Capto MMC. Clearly, the results illustrate that the discriminating power of multimodal ligands differs between different Fab fragments. The results are promising indications that multimodal chromatography using the appropriate multimodal ligands can be employed in downstream bioprocessing for challenging selective separation of product related variants. PMID:26654254

  14. Polynucleotides encoding anti-sulfotyrosine antibodies

    SciTech Connect

    Bertozzi, Carolyn R.; Kehoe, John; Bradbury, Andrew M.

    2011-01-11

    The invention provides anti-sulfotyrosine specific antibodies capable of detecting and isolating polypeptides that are tyrosine-sulfated. The sulfotyrosine antibodies and antibody fragments of the invention may be used to discriminate between the non-sulfated and sulfated forms of such proteins, using any number of immunological assays, such ELISAs, immunoblots, Western Blots, immunoprecipitations, and the like. Using a phage-display system, single chain antibodies (scFvs) were generated and screened against tyrosine-sulfated synthetic peptide antigens, resulting in the isolation of scFvs that specifically recognize sulfotyrosine-containing peptides and/or demonstrate sulfotyrosine-specific binding in tyrosine sulfated proteins. The VH and VL genes from one such sulfotyrosine-specific scFv were employed to generate a full length, sulfotyrosine-specific immunoglobulin.

  15. [Molecular dynamics of immune complex of photoadduct-containing DNA with Fab-Anti-DNA antibody fragment].

    PubMed

    Akberova, N I; Zhmurov, A A; Nevzorova, T A; Litvinov, R I

    2016-01-01

    Antibodies to DNA play an important role in the pathogenesis of autoimmune diseases. The elucidation of structural mechanisms of both the antigen recognition and the interaction of anti-DNA antibodies with DNA will help to understand the role of DNA-containing immune complexes in various pathologies and can provide a basis for new treatment modalities. Moreover, the DNA-antibody complex is an analog of specific intracellular DNA-protein interactions. In this work, we used in silico molecular dynamic simulations of bimolecular complexes of the dsDNA segment containing the Fab fragment of an anti-DNA antibody to obtain the detailed thermodynamic and structural characteristics of dynamic intermolecular interactions. Using computationally modified crystal structure of the Fab-DNA complex (PDB ID: 3VW3), we studied the equilibrium molecular dynamics of the 64M-5 antibody Fab fragment associated with the dsDNA fragment containing the thymine dimer, the product of DNA photodamage. Amino acid residues that constitute paratopes and the complementary nucleotide epitopes for the Fab-DNA construct were identified. Stacking and electrostatic interactions were found to play the main role in mediating the most specific antibody-dsDNA contacts, while hydrogen bonds were less significant. These findings may shed light on the formation and properties of pathogenic anti-DNA antibodies in autoimmune diseases, such as systemic lupus erythematosus associated with skin photosensitivity and DNA photodamage. PMID:27414790

  16. Human antibody response to fragments A and B of diphtheria toxin and a synthetic peptide of amino acid residues 141-157 of fragment A.

    PubMed Central

    Perera, V. Y.; Corbel, M. J.

    1990-01-01

    Examination of a selection of serum samples from adults from two regions of England showed that 50% of men in the 16-24 years and over 55 years age groups had high titres of antibody to diphtheria toxin (DT). In contrast, only 11% of women aged 16 to over 55 years had high titres of antibody to DT. All human antisera with high anti-DT titres reacted with a synthetic peptide (SP) corresponding to the amino acids 141-157 of DT fragment A, with sera from men aged 35 to over 55 years showing the highest titres. High antibody titres to fragment A paralleled those to SP in both sexes. Titres of antibody to DT fragment B were highest in individuals with high titres to DT. In sera from both sexes immunoglobulin G1 was the predominant subclass reactive with all three antigens. However, both IgG1 and IgG4 and to a lesser extent IgG2 and IgG3 were present in immunoglobulin concentrates. Images Fig. 1 Fig. 2 PMID:2249709

  17. Screening, expression, and characterization of an anti-human oxidized low-density lipoprotein single-chain variable fragment.

    PubMed

    Kumano-Kuramochi, Miyuki; Fujimura, Takashi; Komba, Shiro; Maeda-Yamamoto, Mari; Machida, Sachiko

    2016-09-01

    Increased levels of oxidized low-density lipoprotein (OxLDL) in the blood circulation are correlated with atherosclerosis. Monoclonal antibody-based detection systems have been reported for OxLDL. We identified novel single-chain variable fragments (scFvs) having affinity for human OxLDL and related ligands. We constructed an scFv library from nonimmunized human spleen mRNA. Two types (γ+κ and μ+λ) of scFv phage libraries were enriched by biopanning, and five scFv clones with affinity for OxLDL were identified. The γκ5 scFv, which showed the highest affinity for OxLDL, was cloned into pET-22b(+) and expressed in Escherichia coli BL21(DE3). γκ5, expressed as an inclusion body in BL21(DE3), was refolded and purified. The specificity and sensitivity of γκ5 were analyzed using enzyme-linked immunosorbent assays (ELISAs). The γκ5 scFv showed affinity for OxLDL and acetylated LDL. The sensitivity of γκ5 to low concentrations (1-2 μg/mL) of OxLDL was higher than that to AcLDL and LDL. Finally, we developed a sandwich ELISA using γκ5 and CTLD14 (a lectin-like OxLDL receptor-1 ligand recognition region), which allowed specific detection of OxLDL at a level below 0.1 μg/mL. Our results indicated that the γκ5 scFv was a promising molecule for the detection of modified LDL at very low concentrations. PMID:27038672

  18. Generating Recombinant Antibodies against Putative Biomarkers of Retinal Injury

    PubMed Central

    Kierny, Michael R.; Cunningham, Thomas D.; Bouhenni, Rachida A.; Edward, Deepak P.; Kay, Brian K.

    2015-01-01

    Candidate biomarkers, indicative of disease or injury, are beginning to overwhelm the process of validation through immunological means. Recombinant antibodies developed through phage-display offer an alternative means of generating monoclonal antibodies faster than traditional immunization of animals. Peptide segments of putative biomarkers of laser induced injury in the rabbit, discovered through mass spectrometry, were used as targets for a selection against a library of phage-displayed human single-chain variable fragment (scFv) antibodies. Highly specific antibodies were isolated to four of these unique peptide sequences. One antibody against the retinal protein, Guanine Nucleotide-Binding Protein Beta 5 (GBB5), had a dissociation constant ~300 nM and recognized the full-length endogenous protein in retinal homogenates of three different animal species by western blot. Alanine scanning of the peptide target identified three charged and one hydrophobic amino acid as the critical binding residues for two different scFvs. To enhance the utility of the reagent, one scFv was dimerized through a Fragment-crystallizable hinge region (i.e., Fc) and expressed in HEK-293 cells. This dimeric reagent yielded a 25-fold lower detection limit in western blots. PMID:25902199

  19. Flow-Cytometric Isolation of Human Antibodies from a Nonimmune Saccharomyces cerevisiae Surface Display Library

    SciTech Connect

    Feldhaus, Michael ); Siegel, Robert W. ); Opresko, Lee ); Coleman, James R. ); Feldhaus, Jane M. ); Yeung, Yik A.; Cochran, Jennifer R.; Heinzelman, Peter; Colby, David; Swers, Jeffrey; Graff, Christilyn; Wiley, H Steven ); Wittrup, K D.

    2003-02-28

    A nonimmune library of 109 human antibody scFv fragments has been cloned and expressed on the surface of yeast, and nanomolar-affinity scFvs routinely obtained by magnetic bead screening and flow cytometric sorting. The yeast library can be amplified 1010-fold without measurable loss of clonal diversity, enabling effectively indefinite expansion of the library. The expression, stability, and antigen binding properties of more than 50 isolated scFv clones were assessed directly on the yeast cell surface by immunofluorescent labeling and flow cytometry, obviating separate subcloning, expression, and purification steps and thereby expediting the isolation of novel affinity reagents. The ability to use multiplex library screening demonstrates the utility of this approach for high throughput antibody isolation for proteomics applications.

  20. Preparation and diagnostic use of a novel recombinant single-chain antibody against rabies virus glycoprotein.

    PubMed

    Yuan, Ruosen; Chen, Xiaoxu; Chen, Yan; Gu, Tiejun; Xi, Hualong; Duan, Ye; Sun, Bo; Yu, Xianghui; Jiang, Chunlai; Liu, Xintao; Wu, Chunlai; Kong, Wei; Wu, Yongge

    2014-02-01

    Rabies virus (RABV) causes a fatal infectious disease, but effective protection may be achieved with the use of rabies immunoglobulin and a rabies vaccine. Virus-neutralizing antibodies (VNA), which play an important role in the prevention of rabies, are commonly evaluated by the RABV neutralizing test. For determining serum VNA levels or virus titers during the RABV vaccine manufacturing process, reliability of the assay method is highly important and mainly dependent on the diagnostic antibody. Most diagnostic antibodies are monoclonal antibodies (mAbs) made from hybridoma cell lines and are costly and time consuming to prepare. Thus, production of a cost-effective mAb for determining rabies VNA levels or RABV titers is needed. In this report, we describe the prokaryotic production of a RABV-specific single-chain variable fragment (scFv) protein with a His-tag (scFv98H) from a previously constructed plasmid in a bioreactor, including the purification and refolding process as well as the functional testing of the protein. The antigen-specific binding characteristics, affinity, and relative affinity of the purified protein were tested. The scFv98H antibody was compared with a commercial RABV nucleoprotein mAb for assaying the VNA level of anti-rabies serum samples from different sources or testing the growth kinetics of RABV strains for vaccine manufactured in China. The results indicated that scFv98H may be used as a novel diagnostic tool to assay VNA levels or virus titers and may be used as an alternative for the diagnostic antibody presently employed for these purposes. PMID:24241896

  1. Noninvasive brain cancer imaging with a bispecific antibody fragment, generated via click chemistry.

    PubMed

    Luo, Haiming; Hernandez, Reinier; Hong, Hao; Graves, Stephen A; Yang, Yunan; England, Christopher G; Theuer, Charles P; Nickles, Robert J; Cai, Weibo

    2015-10-13

    Early diagnosis remains a task of upmost importance for reducing cancer morbidity and mortality. Successful development of highly specific companion diagnostics targeting aberrant molecular pathways of cancer is needed for sensitive detection, accurate diagnosis, and opportune therapeutic intervention. Herein, we generated a bispecific immunoconjugate [denoted as Bs-F(ab)2] by linking two antibody Fab fragments, an anti-epidermal growth factor receptor (EGFR) Fab and an anti-CD105 Fab, via bioorthogonal "click" ligation of trans-cyclooctene and tetrazine. PET imaging of mice bearing U87MG (EGFR/CD105(+/+)) tumors with (64)Cu-labeled Bs-F(ab)2 revealed a significantly enhanced tumor uptake [42.9 ± 9.5 percentage injected dose per gram (%ID/g); n = 4] and tumor-to-background ratio (tumor/muscle ratio of 120.2 ± 44.4 at 36 h postinjection; n = 4) compared with each monospecific Fab tracer. Thus, we demonstrated that dual targeting of EGFR and CD105 provides a synergistic improvement on both affinity and specificity of (64)Cu-NOTA-Bs-F(ab)2. (64)Cu-NOTA-Bs-F(ab)2 was able to visualize small U87MG tumor nodules (<5 mm in diameter), owing to high tumor uptake (31.4 ± 10.8%ID/g at 36 h postinjection) and a tumor/muscle ratio of 76.4 ± 52.3, which provided excellent sensitivity for early detection. Finally, we successfully confirmed the feasibility of a ZW800-1-labeled Bs-F(ab)2 for near-infrared fluorescence imaging and image-guided surgical resection of U87MG tumors. More importantly, our rationale can be used in the construction of other disease-targeting bispecific antibody fragments for early detection and diagnosis of small malignant lesions. PMID:26417085

  2. Noninvasive brain cancer imaging with a bispecific antibody fragment, generated via click chemistry

    PubMed Central

    Luo, Haiming; Hernandez, Reinier; Hong, Hao; Graves, Stephen A.; Yang, Yunan; England, Christopher G.; Theuer, Charles P.; Nickles, Robert J.; Cai, Weibo

    2015-01-01

    Early diagnosis remains a task of upmost importance for reducing cancer morbidity and mortality. Successful development of highly specific companion diagnostics targeting aberrant molecular pathways of cancer is needed for sensitive detection, accurate diagnosis, and opportune therapeutic intervention. Herein, we generated a bispecific immunoconjugate [denoted as Bs-F(ab)2] by linking two antibody Fab fragments, an anti-epidermal growth factor receptor (EGFR) Fab and an anti-CD105 Fab, via bioorthogonal “click” ligation of trans-cyclooctene and tetrazine. PET imaging of mice bearing U87MG (EGFR/CD105+/+) tumors with 64Cu-labeled Bs-F(ab)2 revealed a significantly enhanced tumor uptake [42.9 ± 9.5 percentage injected dose per gram (%ID/g); n = 4] and tumor-to-background ratio (tumor/muscle ratio of 120.2 ± 44.4 at 36 h postinjection; n = 4) compared with each monospecific Fab tracer. Thus, we demonstrated that dual targeting of EGFR and CD105 provides a synergistic improvement on both affinity and specificity of 64Cu-NOTA-Bs-F(ab)2. 64Cu-NOTA-Bs-F(ab)2 was able to visualize small U87MG tumor nodules (<5 mm in diameter), owing to high tumor uptake (31.4 ± 10.8%ID/g at 36 h postinjection) and a tumor/muscle ratio of 76.4 ± 52.3, which provided excellent sensitivity for early detection. Finally, we successfully confirmed the feasibility of a ZW800-1–labeled Bs-F(ab)2 for near-infrared fluorescence imaging and image-guided surgical resection of U87MG tumors. More importantly, our rationale can be used in the construction of other disease-targeting bispecific antibody fragments for early detection and diagnosis of small malignant lesions. PMID:26417085

  3. Design and construction of a new human naïve single-chain fragment variable antibody library, IORISS1.

    PubMed

    Pasello, Michela; Zamboni, Silvia; Mallano, Alessandra; Flego, Michela; Picci, Piero; Cianfriglia, Maurizio; Scotlandi, Katia

    2016-04-20

    Human monoclonal antibodies are a powerful tool with increasingly successful exploitations and the single chain fragment variable format can be considered the building block for the implementation of more complex and effective antibody-based constructs. Phage display is one of the best and most efficient methods to isolate human antibodies selected from an efficient and variable phage display library. We report a method for the construction of a human naïve single-chain variable fragment library, termed IORISS1. Many different sets of oligonucleotide primers as well as optimized electroporation and ligation reactions were used to generate this library of 1.2×10(9) individual clones. The key difference is the diversity of variable gene templates, which was derived from only 15 non-immunized human donors. The method described here, was used to make a new human naïve single-chain fragment variable phage display library that represents a valuable source of diverse antibodies that can be used as research reagents or as a starting point for the development of therapeutics. Using biopanning, we determined the ability of IORISS1 to yield antibodies. The results we obtained suggest that, by using an optimized protocol, an efficient phage antibody library can be generated. PMID:26945728

  4. Multiparameter optimization method and enhanced production of secreted recombinant single-chain variable fragment against the HIV-1 P17 protein from Escherichia coli by fed-batch fermentation.

    PubMed

    Paopang, Porntip; Kasinrerk, Watchara; Tayapiwatana, Chatchai; Seesuriyachan, Phisit; Butr-Indr, Bordin

    2016-04-01

    The single-chain fragment variable (scFv) was used to produce a completely functional antigen-binding fragment in bacterial systems. The advancements in antibody engineering have simplified the method of producing Fv fragments and made it more efficient and generally relevant. In a previous study, the scFv anti HIV-1 P17 protein was produced by a batch production system, optimized by the sequential simplex optimization method. This study continued that work in order to enhance secreted scFv production by fed-batch cultivation, which supported high volumetric productivity and provided a large amount of scFvs for diagnostic and therapeutic research. The developments in cell culture media and process parameter settings were required to realize the maximum production of cells. This study investigated the combined optimization methods, Plackett-Burman design (PBD) and sequential simplex optimization, with the aim of optimize feed medium. Fed-batch cultivation with an optimal feeding rate was determined. The result demonstrated that a 20-mL/hr feeding rate of the optimized medium can increase cell growth, total protein production, and scFv anti-p17 activity by 4.43, 1.48, and 6.5 times more than batch cultivation, respectively. The combined optimization method demonstrated novel power tools for the optimization strategy of multiparameter experiments. PMID:25831436

  5. Antibody fragment recognition layers for surface plasmon resonance biosensing: a parametric study

    NASA Astrophysics Data System (ADS)

    Magalhães, André; Bordeira, Sandro; Almeida, Ana Cristina; Fontes, Vanessa; Costa, Maria João L.; Fonseca, Luís P.; da Fonseca, João Garcia

    2009-02-01

    A comparative study is reported regarding the use of two different surface plasmon resonance (SPR) biosensors, a homemade SPR grating biosensor and a reference prism coupled biosensor, to perform quantification of C-reactive protein (CRP) in human blood serum. Surface functionalization was conducted using anti-CRP fragments immobilized directly on gold. Adsorption time optimization for the antibody fragments monolayer, non-specific binding (NSB) resistance evaluation and CRP detection were conducted, with better results achieved by the grating biosensor on all topics, namely less functionalization time, higher resistance to NSB and wider CRP dynamic concentration range. A study regarding comparison between continuous flow and surface coating immobilization is also reported in this work. We have shown that surface coating immobilization achieves similar NSB resistance and CRP detection results, allowing a 75% assay cost reduction by lower solution volume requirement. Results suggest that the coating immobilization technique is the best suited to be used in further studies in order to obtain a viable immunosensor for CRP and other biomarkers detection in complex biological fluids.

  6. Structural design of disialoganglioside GD2 and CD3-bispecific antibodies to redirect T cells for tumor therapy

    PubMed Central

    Cheng, Ming; Ahmed, Mahiuddin; Xu, Hong; Cheung, Nai-Kong V.

    2015-01-01

    Antibody based immunotherapy has proven efficacy for patients with high risk neuroblastoma. However, despite being the most efficient tumoricidal effectors, T cells are underutilized because they lack Fc receptors. Using a monovalent single chain fragment (ScFv) platform, we engineered tandem scFv bispecific antibodies (BsAbs) that specifically target disialoganglioside (GD2) on tumor cells and CD3 on T cells. Structural variants of BsAbs were constructed and ranked based on binding to GD2, and on competency in inducing T cell mediated tumor cytotoxicity. In vitro thermal stability and binding measurements were used to characterize each of the constructs, and in silico molecular modeling was used to show how the orientation of the variable region heavy (VH) and light (VL) chains of the anti-GD2 ScFv could alter the conformations of key residues responsible for high affinity binding. We showed that the VH-VL orientation, the (GGGGS)3 linker, disulfide bond stabilization of scFv, when combined with an affinity matured mutation provided the most efficient BsAb to direct T cells to lyse GD2 positive tumor cells. In vivo, the optimized BsAb could efficiently inhibit melanoma and neuroblastoma xenograft growth. These findings provide preclinical validation of a structure-based method to assist in designing BsAb for T-cell mediated therapy. PMID:24895182

  7. Nebulized anti-IL-13 monoclonal antibody Fab' fragment reduces allergen-induced asthma.

    PubMed

    Hacha, Jonathan; Tomlinson, Kate; Maertens, Ludovic; Paulissen, Geneviève; Rocks, Natacha; Foidart, Jean-Michel; Noel, Agnès; Palframan, Roger; Gueders, Maud; Cataldo, Didier D

    2012-11-01

    IL-13 is a prototypic T helper type 2 cytokine and a central mediator of the complex cascade of events leading to asthmatic phenotype. Indeed, IL-13 plays key roles in IgE synthesis, bronchial hyperresponsiveness, mucus hypersecretion, subepithelial fibrosis, and eosinophil infiltration. We assessed the potential efficacy of inhaled anti-IL-13 monoclonal antibody Fab' fragment on allergen-induced airway inflammation, hyperresponsiveness, and remodeling in an experimental model of allergic asthma. Anti-IL-13 Fab' was administered to mice as a liquid aerosol generated by inExpose inhalation system in a tower allowing a nose-only exposure. BALB/c mice were treated by PBS, anti-IL-13 Fab', or A33 Fab' fragment and subjected to ovalbumin exposure for 1 and 5 weeks (short-term and long-term protocols). Our data demonstrate a significant antiasthma effect after nebulization of anti-IL-13 Fab' in a model of asthma driven by allergen exposure as compared with saline and nonimmune Fab fragments. In short- and long-term protocols, administration of the anti-IL-13 Fab' by inhalation significantly decreased bronchial responsiveness to methacholine, bronchoalveolar lavage fluid eosinophilia, inflammatory cell infiltration in lung tissue, and many features of airway remodeling. Levels of proinflammatory mediators and matrix metalloprotease were significantly lower in lung parenchyma of mice treated with anti-IL-13 Fab'. These data demonstrate that an inhaled anti-IL-13 Fab' significantly reduces airway inflammation, hyperresponsiveness, and remodeling. Specific neutralization of IL-13 in the lungs using an inhaled anti-IL-13 Fab' could represent a novel and effective therapy for the treatment of asthma. PMID:22904197

  8. Serotype- and serogroup-specific detection of African horsesickness virus using phage displayed chicken scFvs for indirect double antibody sandwich ELISAs.

    PubMed

    van Wyngaardt, Wouter; Mashau, Cordelia; Wright, Isabel; Fehrsen, Jeanni

    2013-01-01

    There is an ongoing need for standardized, easily renewable immunoreagents for detecting African horsesickness virus (AHSV). Two phage displayed single-chain variable fragment (scFv) antibodies, selected from a semi-synthetic chicken antibody library, were used to develop double antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISAs) to detect AHSV. In the DAS-ELISAs, the scFv previously selected with directly immobilized AHSV-3 functioned as a serotype-specific reagent that recognized only AHSV-3. In contrast, the one selected with AHSV-8 captured by IgG against AHSV-3 recognized all nine AHSV serotypes but not the Bryanston strain of equine encephalosis virus. Serving as evidence for its serogroup-specificity. These two scFvs can help to rapidly confirm the presence of AHSV while additional serotype-specific scFvs may simplify AHSV serotyping. PMID:23388433

  9. Construction of a rationally designed antibody platform for sequencing-assisted selection.

    PubMed

    Larman, H Benjamin; Xu, George Jing; Pavlova, Natalya N; Elledge, Stephen J

    2012-11-01

    Antibody discovery platforms have become an important source of both therapeutic biomolecules and research reagents. Massively parallel DNA sequencing can be used to assist antibody selection by comprehensively monitoring libraries during selection, thus greatly expanding the power of these systems. We have therefore constructed a rationally designed, fully defined single-chain variable fragment (scFv) library and analysis platform optimized for analysis with short-read deep sequencing. Sequence-defined oligonucleotide libraries encoding three complementarity-determining regions (L3 from the light chain, H2 and H3 from the heavy chain) were synthesized on a programmable microarray and combinatorially cloned into a single scFv framework for molecular display. Our unique complementarity-determining region sequence design optimizes for protein binding by utilizing a hidden Markov model that was trained on all antibody-antigen cocrystal structures in the Protein Data Bank. The resultant ~10(12)-member library was produced in ribosome-display format, and comprehensively analyzed over four rounds of antigen selections by multiplex paired-end Illumina sequencing. The hidden Markov model scFv library generated multiple binders against an emerging cancer antigen and is the basis for a next-generation antibody production platform. PMID:23064642

  10. Quantitation of imaging with I-131-F(ab')/sub 2/ fragments of monoclonal antibody in patients

    SciTech Connect

    Moldofsky, P.J.; Hammond, N.D.; Mulhern, C.B. Jr.

    1984-01-01

    Iodine-131 labeled F(ab')/sub 2/ fragments of monoclonal antibody (IgG/sub 2a/ immunoglobulin with specificity for a cell surface antigen of colon carcinoma) have been used for quantitative imaging of tumor in 27 patients. Activity of I-131 F(ab')/sub 2/ fragments localized in tumor and in liver was quantitated using a modification of the method of Thomas SR, employing computer-acquired conjugate views (i.e. 180 opposed) to eliminate need for tumor or organ depth and tissue attenuation. The method was validated with an abdominal imaging phantom showing accuracy of +/- 10%. Quantitation indicates that activity reaches a peak in tumor at 48-72 hours and the ratio of activity in hepatic metastases to activity in liver peaks at approximately 72 hours. Mean activity in tumor was less than 0.01% of the administered dose per gram of tumor at any imaging time from 24 to 168 hours, while mean activity in surrounding liver was less than .002% of administered dose per gram of liver at any imaging time. Liver activity decreased monotonically with time, showing no peak activity. This non-invasive method of quantitating the distribution of F(ab')/sub 2/ fragments of monoclonal antibody in patients has proven accurate by comparison with phantom simulation. This type of quantitation is necessary for evaluating optimal imaging time, comparing relative utility of various antibodies and has use for therapeutic applications of monoclonal antibody fragments.

  11. Phase analysis in single-chain variable fragment production by recombinant Pichia pastoris based on proteomics combined with multivariate statistics.

    PubMed

    Fujiki, Yuya; Kumada, Yoichi; Kishimoto, Michimasa

    2015-08-01

    The proteomics technique, which consists of two-dimensional gel electrophoresis (2-DE), peptide mass fingerprinting (PMF), gel image analysis, and multivariate statistics, was applied to the phase analysis of a fed-batch culture for the production of a single-chain variable fragment (scFv) of an anti-C-reactive protein (CRP) antibody by Pichia pastoris. The time courses of the fed-batch culture were separated into three distinct phases: the growth phase of the batch process, the growth phase of the fed-batch process, and the production phase of the fed-batch process. Multivariate statistical analysis using 2-DE gel image analysis data clearly showed the change in the culture phase and provided information concerning the protein expression, which suggested a metabolic change related to cell growth and production during the fed-batch culture. Furthermore, specific proteins, such as alcohol oxidase, which is strongly related to scFv expression, and proteinase A, which could biodegrade scFv in the latter phases of production, were identified via the PMF method. The proteomics technique provided valuable information about the effect of the methanol concentration on scFv production. PMID:25636980

  12. Method for preparation of single chain antibodies

    SciTech Connect

    Cheung, Nai-Kong V.; Guo, Hong-fen

    2012-04-03

    This invention provides a method for identifying cells expressing a target single chain antibody (scFv) directed against a target antigen from a collection of cells that includes cells that do not express the target scFv, comprising the step of combining the collection of cells with an anti-idiotype directed to an antibody specific for the target antigen and detecting interaction, if any, of the anti-idiotype with the cells, wherein the occurrence of an interaction identifies the cell as one which expresses the target scFv. This invention also provides a method for making a single chain antibody (scFv) directed against an antigen, wherein the selection of clones is made based upon interaction of those clones with an appropriate anti-idiotype, and heretofore inaccessible scFv so made. This invention provides the above methods or any combination thereof. Finally, this invention provides various uses of these methods.

  13. Proteins of the cystic fibrosis respiratory tract. Fragmented immunoglobulin G opsonic antibody causing defective opsonophagocytosis.

    PubMed Central

    Fick, R B; Naegel, G P; Squier, S U; Wood, R E; Gee, J B; Reynolds, H Y

    1984-01-01

    In the disease cystic fibrosis (CF), pulmonary infection with Pseudomonas aeruginosa is a common clinical complication that determines most morbidity and almost all excess mortality. We postulated that in this disease a defect in Pseudomonas-reactive IgG antibodies may contribute to chronic Pseudomonas infections. Bronchoalveolar lavages were performed upon 13 patients with CF, 7 patients with chronic bronchitis characterized by recurrent Pseudomonas infections, and 4 normal volunteers. The levels of various proteins important to host defenses and proteases were determined; enzyme inhibition studies were performed. CF respiratory immunoglobulin levels were significantly elevated when compared with both normals and patients with chronic bronchitis (P less than 0.05). Albumin and transferrin levels were decreased in the CF lung fluids. CF elastolytic activity was strikingly elevated (means = 6.02 micrograms/mg total protein) and the inhibitory profile suggested such activity resembled a serine-proteinase. Alpha-1-antitrypsin antigenic levels were not altered in CF respiratory fluids. There was a tendency for the lavage IgG to fall as elastase levels rose (r = -0.29). IgG opsonins for two Pseudomonas immunotypes were isolated with affinity chromatography for functional and immunochemical studies. Bacterial phagocytic rates in the presence of these Pseudomonas-reactive IgG opsonins derived from CF lavage fluid were depressed (0.3% uptake/unit time) when compared with similarly titered positive controls (uptake = 1.3%/unit time, P less than 0.001). Additionally, normal pulmonary macrophage intracellular killing of Pseudomonas was severely altered in the presence of opsonins derived from CF respiratory fluids. At some time points, less than 30% of the bacteria were killed. CF IgG opsonins contain a cleavage fragment (100,000 D, 5S sedimentation coefficient) with antigenic determinants similar to the Fab portion of IgG. The presence of such a fragment was inversely

  14. A humanized anti-M2 scFv shows protective in vitro activity against influenza

    SciTech Connect

    Bradbury, Andrew M; Velappan, Nileena; Schmidt, Jurgen G

    2008-01-01

    M2 is one of the most conserved influenza proteins, and has been widely prospected as a potential universal vaccine target, with protection predominantly mediated by antibodies. In this paper we describe the creation of a humanized single chain Fv from 14C2, a potent monoclonal antibody against M2. We show that the humanized scFv demonstrates similar activity to the parental mAb: it is able to recognize M2 in its native context on cell surfaces and is able to show protective in vitro activity against influenza, and so represents a potential lead antibody candidate for universal prophylactic or therapeutic intervention in influenza.

  15. Use of 18F-2-Fluorodeoxyglucose to Label Antibody Fragments for Immuno-Positron Emission Tomography of Pancreatic Cancer

    PubMed Central

    2015-01-01

    We generated 18F-labeled antibody fragments for positron emission tomography (PET) imaging using a sortase-mediated reaction to install a trans-cyclooctene-functionalized short peptide onto proteins of interest, followed by reaction with a tetrazine-labeled-18F-2-deoxyfluoroglucose (FDG). The method is rapid, robust, and site-specific (radiochemical yields > 25%, not decay corrected). The availability of 18F-2-deoxyfluoroglucose avoids the need for more complicated chemistries used to generate carbon–fluorine bonds. We demonstrate the utility of the method by detecting heterotopic pancreatic tumors in mice by PET, using anti-Class II MHC single domain antibodies. We correlate macroscopic PET images with microscopic two-photon visualization of the tumor. Our approach provides easy access to 18F-labeled antibodies and their fragments at a level of molecular specificity that complements conventional 18F-FDG imaging. PMID:26955657

  16. Purification of antibody and antibody-fragment from E. coli homogenate using 6,9-diamino-2-ethoxyacridine lactate as precipitation agent.

    PubMed

    Persson, Josefine; Lester, Philip

    2004-08-01

    To obtain a more efficient purification process for antibody fragments from an Escherichia coli homogenate, the precipitant, Ethodin (6,9-diamino-2-ethoxyacridine lactate) was introduced to the homogenate. By adding the precipitant a drastic reduction of host cell protein was obtained. The majority of the proteins were recovered in a precipitate with the cell debris, while the antibody or antibody-fragment was recovered in the clarified supernatant. In addition, DNA was also efficiently precipitated when using Ethodin as a precipitation agent. The improved purity of the clarified extract obtained by using the precipitant allows for the use of smaller chromatography columns and may reduce the number of chromatographic steps required in the recovery process. The effect of Ethodin concentration, pH, temperature, and conductivity were investigated. The investigation was performed on two different antibody-fragments, e.g., F(ab')(2) molecules and a full-length antibody produced in E. coli. The two F(ab')(2) proteins were F(ab')(2)A and F(ab')(2)B, which have a similar molecular mass (100 kDa) but different isoelectric points (pIs), i.e., 8.9 and 7.5, respectively. The full-length antibody, Ab (the full IgG form of F(ab')(2)B) has a pI of 7.8 and molecular mass of 150 kDa. The investigation showed that the highest purification factors were obtained at neutral pH, low conductivity, and Ethodin concentrations of 0.6%. PMID:15281116

  17. Construction and Screening of Antigen Targeted Immune Yeast Surface Display Antibody Libraries

    SciTech Connect

    Miller, Keith D.; Pefaur, Noah B.; Baird, Cheryl L.

    2008-07-01

    These protocols describe a yeast surface display-based process for the rapid selection of antibodies from immunized mice, eliminating the need for creating and screening hybridoma fusions. A yeast surface display library of single-chain antibody fragments (scFvs) is created from antigen-binding B cells from the splenocytes of immunized mice. The antigen targeted library is then screened for antigen specific scFv by magnetic activated cell sorting (MACS) and fluorescence activated cell sorting (FACS). Library construction and screening can be accomplished in as little as 2 weeks resulting in a panel of scFvs specific for the target antigen.

  18. Construction and Screening of Antigen Targeted Immune Yeast Surface Display Antibody Libraries

    SciTech Connect

    Miller, Keith D.; Pefaur, Noah B.; Baird, Cheryl L.

    2009-08-02

    These protocols describe a yeast surface display-based process for the rapid selection of antibodies from immunized mice, eliminating the need for creating and screening hybridoma fusions. A yeast surface display library of single-chain antibody fragments (scFvs) is created from antigen-binding B cells from the splenocytes of immunized mice. The antigen targeted library is then screened for antigen specific scFv by magneticactivated cell sorting (MACS) and fluorescence-activated cell sorting (FACS). Library construction and screening can be accomplished in as little as 2 weeks, resulting in a panel of scFvs specific for the target antigen.

  19. Characterization of Poliovirus Neutralization Escape Mutants of Single-Domain Antibody Fragments (VHHs)

    PubMed Central

    Schotte, Lise; Thys, Bert; Strauss, Mike; Filman, David J.; Rombaut, Bart

    2015-01-01

    To complete the eradication of poliovirus and to protect unvaccinated people subsequently, the development of one or more antiviral drugs will be necessary. A set of five single-domain antibody fragments (variable parts of the heavy chain of a heavy-chain antibody [VHHs]) with an in vitro neutralizing activity against poliovirus type 1 was developed previously (B. Thys, L. Schotte, S. Muyldermans, U. Wernery, G. Hassanzadeh-Ghassabeh, and B. Rombaut, Antiviral Res 87:257–264, 2010, http://dx.doi.org/10.1016/j.antiviral.2010.05.012), and their mechanisms of action have been studied (L. Schotte, M. Strauss, B. Thys, H. Halewyck, D. J. Filman, M. Bostina, J. M. Hogle, and B. Rombaut, J Virol 88:4403–4413, 2014, http://dx.doi.org/10.1128/JVI.03402-13). In this study, neutralization escape mutants were selected for each VHH. Sequencing of the P1 region of the genome showed that amino acid substitutions are found in the four viral proteins of the capsid and that they are located both in proximity to the binding sites of the VHHs and in regions further away from the canyon and hidden beneath the surface. Characterization of the mutants demonstrated that they have single-cycle replication kinetics that are similar to those of their parental strain and that they are all drug (VHH) independent. Their resistant phenotypes are stable, as they do not regain full susceptibility to the VHH after passage over HeLa cells in the absence of VHH. They are all at least as stable as the parental strain against heat inactivation at 44°C, and three of them are even significantly (P < 0.05) more resistant to heat inactivation. The resistant variants all still can be neutralized by at least two other VHHs and retain full susceptibility to pirodavir and 35-1F4. PMID:26014941

  20. Intein-mediated one-step purification of Escherichia coli secreted human antibody fragments.

    SciTech Connect

    Wu, Wan-Yi; Miller, Keith D.; Coolbaugh, Michael; Wood, David W.

    2011-02-25

    In this work, we apply self-cleaving affinity tag technology to several target proteins secreted into the Escherichia coli periplasm, including two with disulfide bonds. The target proteins were genetically fused to a self-cleaving chitin-binding domain intein tag for purification via a chitin agarose affinity resin. By attaching the intein-tagged fusion genes to the PelB secretion leader sequence, the tagged target proteins were secreted to the periplasmic space and could be recovered in active form by simple osmotic shock. After chitin-affinity purification, the target proteins were released from the chitin-binding domain tag via intein self-cleaving. This was induced by a small change in pH from 8.5 to 6.5 at room temperature, allowing direct elution of the cleaved target protein from the chitin affinity resin. The target proteins include the E. coli maltose-binding protein and b-lactamase enzyme, as well as two human antibody fragments that contain disulfide bonds. In all cases, the target proteins were purified with good activity and yield, without the need for refolding. Overall, this work demonstrates the compatibility of the DI-CM intein with the PelB secretion system in E. coli, greatly expanding its potential to more complex proteins.

  1. Selection and characterization of a human neutralizing antibody to human fibroblast growth factor-2

    SciTech Connect

    Tao, Jun; Xiang, Jun-Jian; Li, Dan; Deng, Ning; Wang, Hong; Gong, Yi-Ping

    2010-04-09

    Compelling evidences suggest that fibroblast growth factor-2 (FGF-2) plays important roles in tumor growth, angiogenesis and metastasis. Molecules blocking the FGF-2 signaling have been proposed as anticancer agents. Through screening of a human scFv phage display library, we have isolated several human single-chain Fv fragments (scFvs) that bind to human FGF-2. After expression and purification in bacteria, one scFv, named 1A2, binds to FGF-2 with a high affinity and specificity, and completes with FGF-2 binding to its receptor. This 1A2 scFv was then cloned into the pIgG1 vector and expressed in 293T cells. The purified hIgG1-1A2 antibody showed a high binding affinity of 8 x 10{sup -9} M to rhFGF-2. In a set of vitro assays, it inhibited various biological activities of FGF-2 such as the proliferation, migration and tube formation of human umbilical vein endothelial cells. More importantly, hIgG1-1A2 antibody also efficiently blocked the growth while inducing apoptosis of glioma cells. For the first time, we generated a human anti-FGF-2 antibody with proven in vitro anti-tumor activity. It may therefore present a new therapeutic candidate for the treatment of cancers that are dependent on FGF-2 signaling for growth and survival.

  2. Phage display-based on-slide selection of tumor-specific antibodies on formalin-fixed paraffin-embedded human tissue biopsies.

    PubMed

    Ten Haaf, Andre; Pscherer, Sibylle; Fries, Katharina; Barth, Stefan; Gattenlöhner, Stefan; Tur, Mehmet Kemal

    2015-08-01

    Phage display is an effective method for the generation of target-specific human antibodies. Standard phage display panning use purified proteins, antigen-transfected cells or tumor cell lines as target structure to generate specific antibodies. However, recombinant proteins can be difficult to express and purify in their native conformation and suitable cell lines are not always available. Additionally the antigen expression profile may change during cultivation and thus differ from the malignant cells in patient. Here we describe a method for the selection of specific antibodies from phage display libraries by panning against formalin-fixed paraffin-embedded (FFPE) tissue biopsies immobilized on glass slides, using small cell lung cancer (SCLC) as a case study. The human Tomlinson single-chain variable fragment (scFv) phage libraries I and J were panned against SCLC FFPE tissue slides for positive selection and healthy lung tissue for subtraction. The specificity of the selected scFv antibodies was confirmed in vitro by ELISA on immobilized SCLC cell membranes, by flow cytometry using the SCLC cell lines NCI-H69, NCI-H82 and DMS 273, and ex vivo against tissue microarrays containing 35 different SCLC samples and 20 types of normal organs. We monitored the internalization of three selected scFv antibodies and fused them with Pseudomonas exotoxin A (ETA') to produce immunotoxins whose cytotoxicity was confirmed by cell viability and apoptosis assays on different SCLC cell lines, achieving IC50 values of up to 23nM. The selection of SCLC-specific scFv antibodies by panning against FFPE tissue slides circumvents the challenges of using purified antigens or cell lines for antibody selection. PMID:26045318

  3. A new type of pseudothrombocytopenia: EDTA-mediated agglutination of platelets bearing Fab fragments of a chimaeric antibody.

    PubMed

    Christopoulos, C G; Machin, S J

    1994-07-01

    In vitro agglutination of platelets leading to low automated platelet counts was observed in EDTA-anticoagulated blood from human volunteers receiving infusions of Fab fragments of a chimaeric monoclonal antibody to platelet glycoprotein IIb-IIIa. This pseudothrombocytopenia depended on the presence of chimaeric Fab on the platelet surface and was not seen when sodium citrate was used as anticoagulent. Preliminary evidence suggests that this phenomenon might be mediated by immunoglobulin G reactive with the human component of the chimaeric Fab. It is important to exclude pseudothrombocytopenia when low automated platelet counts are reported in association with the administration of chimaeric anti-platelet antibodies. PMID:7993813

  4. Maturation Pathways of Cross-Reactive HIV-1 Neutralizing Antibodies

    PubMed Central

    Xiao, Xiaodong; Chen, Weizao; Feng, Yang; Dimitrov, Dimiter S.

    2009-01-01

    Several human monoclonal antibodies (hmAbs) and antibody fragments, including the best characterized in terms of structure-function b12 and Fab X5, exhibit relatively potent and broad HIV-1 neutralizing activity. However, the elicitation of b12 or b12-like antibodies in vivo by vaccine immunogens based on the HIV-1 envelope glycoprotein (Env) has not been successful. B12 is highly divergent from the closest corresponding germline antibody while X5 is less divergent. We have hypothesized that the relatively high degree of specific somatic hypermutations may preclude binding of the HIV-1 envelope glycoprotein (Env) to closest germline antibodies, and that identifying antibodies that are intermediates in the pathways to maturation could help design novel vaccine immunogens to guide the immune system for their enhanced elicitation. In support of this hypothesis we have previously found that a germline-like b12 (monovalent and bivalent scFv as an Fc fusion protein or IgG) lacks measurable binding to an Env as measured by ELISA with a sensitivity in the μM range [1]; here we present evidence confirming and expanding these findings for a panel of Envs. In contrast, a germline-like scFv X5 bound Env with high (nM) affinity. To begin to explore the maturation pathways of these antibodies we identified several possible b12 intermediate antibodies and tested their neutralizing activity. These intermediate antibodies neutralized only some HIV-1 isolates and with relatively weak potency. In contrast, germline-like scFv X5 neutralized a subset of the tested HIV-1 isolates with comparable efficiencies to that of the mature X5. These results could help explain the relatively high immunogenicity of the coreceptor binding site on gp120 and the abundance of CD4-induced (CD4i) antibodies in HIV-1-infected patients (X5 is a CD4i antibody) as well as the maturation pathway of X5. They also can help identify antigens that can bind specifically to b12 germline and intermediate antibodies

  5. Affinity maturation of T-cell receptor-like antibodies for Wilms tumor 1 peptide greatly enhances therapeutic potential

    PubMed Central

    Zhao, Qi; Ahmed, Mahiuddin; Tassev, Dimiter V.; Hasan, Aisha; Kuo, Tzu-Yun; Guo, Hong-fen; O’Reilly, Richard J.; Cheung, Nai-Kong V.

    2016-01-01

    WT1126 (RMFPNAPYL) is a human leukocyte antigen-A2 (HLA-A2) restricted peptide derived from Wilms tumor protein (WT1), which is widely expressed in a broad spectrum of leukemias, lymphomas and solid tumors. A novel T-cell-receptor (TCR)-like single chain variable fragment (scFv) antibody specific for the T cell epitope consisting of the WT1/HLA-A2 complex was isolated from a human scFv phage library. This scFv was affinity-matured by mutagenesis combined with yeast display, and structurally analyzed using a homology model. This monovalent scFv showed a 100-fold affinity improvement (dissociation constant [KD]= 3nM) and exquisite specificity towards its targeted epitope or HLA-A2+/WT1+ tumor cells. Bivalent scFv-huIgG1-Fc fusion protein demonstrated an even higher avidity (KD = 2pM) binding to the T cell epitope and to tumor targets, and was capable of mediating antibody-dependent cell-mediated cytotoxicity or tumor lysis by chimeric antigen receptor (CAR)-expressing human T or NK-92-MI transfected cells. This antibody demonstrated specific and potent cytotoxicity in vivo towards WT1-positive leukemia xenograft that was HLA-A2 restricted. In summary, T cell epitopes can provide novel targets for antibody-based therapeutics. By combining phage and yeast displays and scFv-Fc fusion platforms, a strategy for developing high affinity TCR-like antibodies could be rapidly explored for potential clinical development. PMID:25987253

  6. Injectable formulations for an intravitreal sustained-release application of a novel single-chain VEGF antibody fragment.

    PubMed

    Asmus, Lutz R; Grimshaw, John P A; Richle, Philipp; Eicher, Barbara; Urech, David M; Gurny, Robert; Möller, Michael

    2015-09-01

    Sustained-release formulations of a single-chain anti-VEGF-A antibody fragment were investigated in vitro toward their potential use for intravitreal applications. The hydrophobic polyester hexylsubstituted poly(lactic acid) (hexPLA) was selected as the sustained-release excipient for its biodegradability and semi-solid aggregate state, allowing an easy and mild formulation procedure. The lyophilized antibody fragment ESBA903 was micronized and incorporated into the liquid polymer matrix by cryo-milling, forming homogeneous and injectable suspensions. The protein showed excellent compatibility with the hexPLA polymer and storage stability at 4°C for 10 weeks. Additionally, hexPLA shielded the incorporated active substance from the surrounding medium, resulting in a better stability of ESBA903 inside the polymer than after its release in the buffer solution. Formulations of ESBA903 with hexPLA having drug loadings between 1.25% and 5.0% and polymer molecular weights of 1500 g/mol, 2500 g/mol, 3500 g/mol and 5000 g/mol were investigated regarding their in vitro release. All formulations except with the highest molecular weight formed spherical depots in aqueous buffer solutions and released the antibody fragment for at least 6-14 weeks. The polymer viscosity derived from the molecular weight strongly influenced the release rate, while the drug loading had minor influence, allowing customization of the release profile and the daily drug release. Size exclusion chromatography and SDS-PAGE revealed that the antibody fragment structure was kept intact during incorporation and release from the liquid matrix. Furthermore, the released protein monomer maintained its high affinity to human VEGF-A, as measured by surface plasmon resonance analysis. Formulations of ESBA903 in hexPLA meet the basic needs to be used for intravitreal sustained-release applications in age-related macular degeneration treatment. PMID:25779352

  7. Preparation of a Chicken scFv to Analyze Gentamicin Residue in Animal Derived Food Products.

    PubMed

    Li, Cui; He, Jinxin; Ren, Hao; Zhang, Xiaoying; Du, Enqi; Li, Xinping

    2016-04-01

    Chicken is an ideal model for simplified recombinant antibody library generation. It has been rarely been reported to apply chicken single-chain variable fragments (scFvs) in immunoassays for the detection of antibiotic and chemical contaminants in animal food products. In this study, the scFvs (S-1 and S-5) were isolated from a phage display library derived from a hyperimmunized chicken. The checker board titration revealed that the optimum concentrations of S-1 and S-5 were 0.78 μg/mL and 0.44 μg/mL respectively, to obtain OD450 around 1.0 at 5 μg/mL of Gent-OVA coating concentration. Both S-1 and S-5 exhibited negligible cross reactivity with kanamycin and amikacin. The 50% inhibitory concentration (IC50) of S-1 and S-5 were 12.418 ng/mL and 14.674 ng/mL respectively. In the indirect competitive ELISA (ic-ELISA), the limits of detection for S-1 and S-5 were 0.147 ng/mL and 0.219 ng/mL respectively. The mean recovery for Gent ranged from 60.91% to 118.09% with no more than 10.35% relative standard deviation (RSD) between the intra-assay and the inter-assay. These results indicate the chicken scFv based ic-ELISA method is suitable for the detection of Gent residue in animal derived edible tissues and milk. PMID:26980703

  8. Production of a recombinant antibody specific for i blood group antigen, a mesenchymal stem cell marker.

    PubMed

    Hirvonen, Tia; Suila, Heli; Tiitinen, Sari; Natunen, Suvi; Laukkanen, Marja-Leena; Kotovuori, Annika; Reinman, Mirka; Satomaa, Tero; Alfthan, Kaija; Laitinen, Saara; Takkinen, Kristiina; Räbinä, Jarkko; Valmu, Leena

    2013-10-01

    Multipotent mesenchymal stem/stromal cells (MSCs) offer great promise for future regenerative and anti-inflammatory therapies. Panels of functional and phenotypical markers are currently used in characterization of different therapeutic stem cell populations from various sources. The i antigen (linear poly-N-acetyllactosamine) from the Ii blood group system has been suggested as a marker for MSCs derived from umbilical cord blood (UCB). However, there are currently no commercially available antibodies recognizing the i antigen. In the present study, we describe the use of antibody phage display technology to produce recombinant antibodies recognizing a structure from the surface of mesenchymal stem cells. We constructed IgM phage display libraries from the lymphocytes of a donor with an elevated serum anti-i titer. Antibody phage display technology is not dependent on immunization and thus allows the generation of antibodies against poorly immunogenic molecules, such as carbohydrates. Agglutination assays utilizing i antigen-positive red blood cells (RBCs) from UCB revealed six promising single-chain variable fragment (scFv) antibodies, three of which recognized epitopes from the surface of UCB-MSCs in flow cytometric assays. The amino acid sequence of the VH gene segment of B12.2 scFv was highly similar to the VH4.21 gene segment required to encode anti-i specificities. Further characterization of binding properties revealed that the binding of B12.2 hyperphage was inhibited by soluble linear lactosamine oligosaccharide. Based on these findings, we suggest that the B12.2 scFv we have generated is a prominent anti-i antibody that recognizes i antigen on the surface of both UCB-MSCs and RBCs. This binder can thus be utilized in UCB-MSC detection and isolation as well as in blood group serology. PMID:24083089

  9. Generation of recombinant monoclonal antibodies to study structure-function of envelope protein VP28 of white spot syndrome virus from shrimp

    SciTech Connect

    Wang Yuzhen; Zhang Xiaohua; Yuan Li; Xu Tao; Rao Yu; Li Jia; Dai Heping

    2008-08-08

    White spot syndrome virus (WSSV) is a major pathogen in shrimp aquaculture. VP28 is one of the most important envelope proteins of WSSV. In this study, a recombinant antibody library, as single-chain fragment variable (scFv) format, displayed on phage was constructed using mRNA from spleen cells of mice immunized with full-length VP28 expressed in Escherichia coli. After several rounds of panning, six scFv antibodies specifically binding to the epitopes in the N-terminal, middle, and C-terminal regions of VP28, respectively, were isolated from the library. Using these scFv antibodies as tools, the epitopes in VP28 were located on the envelope of the virion by immuno-electron microscopy. Neutralization assay with these antibodies in vitro suggested that these epitopes may not be the attachment site of WSSV to host cell receptor. This study provides a new way to investigate the structure and function of the envelope proteins of WSSV.

  10. Human monoclonal ScFv specific to NS1 protein inhibits replication of influenza viruses across types and subtypes.

    PubMed

    Yodsheewan, Rungrueang; Maneewatch, Santi; Srimanote, Potjanee; Thueng-In, Kanyarat; Songserm, Thaweesak; Dong-Din-On, Fonthip; Bangphoomi, Kunan; Sookrung, Nitat; Choowongkomon, Kiattawee; Chaicumpa, Wanpen

    2013-10-01

    Currently, there is a need of new anti-influenza agents that target influenza virus proteins other than ion channel M2 and neuraminidase. Non-structural protein-1 (NS1) is a highly conserved multifunctional protein which is indispensable for the virus replication cycle. In this study, fully human single chain antibody fragments (HuScFv) that bound specifically to recombinant and native NS1 were produced from three huscfv-phagemid transformed Escherichia coli clones (nos. 3, 10 and 11) selected from a human ScFv phage display library. Western blot analysis, mimotope searching/epitope identification, homology modeling/molecular docking and phage mimotope ELISA inhibition indicated that HuScFv of clone no. 3 reacted with NS1 R domain important for host innate immunity suppression; HuScFv of clone nos. 10 and 11 bound to E domain sites necessary for NS1 binding to the host eIF4GI and CPSF30, respectively. The HuScFv of all clones could enter the influenza virus infected cells and interfered with the NS1 activities leading to replication inhibition of viruses belonging to various heterologous A subtypes and type B by 2-64-fold as semi-quantified by hemagglutination assay. Influenza virus infected cells treated with representative HuScFv (clone 10) had up-expression of IRF3 and IFN-β genes by 14.75 and 4.95-fold, respectively, in comparison with the controls, indicating that the antibodies could restore the host innate immune response. The fully human single chain antibodies have high potential for developing further as a safe (adjunctive) therapeutic agent for mitigating, if not abrogating, severe symptoms of influenza. PMID:23928258

  11. Anti-MET immunoPET for non-small cell lung cancer using novel fully human antibody fragments

    PubMed Central

    Li, Keyu; Tavaré, Richard; Zettlitz, Kirstin A.; Mumenthaler, Shannon M.; Mallick, Parag; Zhou, Yu; Marks, James D.; Wu, Anna M.

    2014-01-01

    MET, the receptor of hepatocyte growth factor, plays important roles in tumorigenesis and drug resistance in numerous cancers including non-small cell lung cancer. As increasing numbers of MET inhibitors are being developed for clinical applications, antibody fragment based immuno-positron emission tomography (immunoPET) has the potential to rapidly quantify in vivo MET expression levels for drug response evaluation and patient stratification for these targeted therapies. Here, fully human single-chain variable fragments (scFvs) isolated from a phage display library were re-formatted into bivalent cys-diabodies (scFv-cys dimers) with affinities to MET ranging from 0.7 nM to 5.1 nM. The candidate with the highest affinity, H2, was radiolabeled with 89Zr for immunoPET studies targeting non-small cell lung cancer xenografts: low MET expressing Hcc827 and the gefitinib-resistant Hcc827-GR6 with 4-fold MET over-expression. ImmunoPET at as early as 4 hours post injection produced high contrast images, and ex vivo biodistribution analysis at 20 hours post injection showed about 2-fold difference in tracer uptake levels between the parental and resistant tumors (p < 0.01). Further immunoPET studies using a larger fragment, the H2 minibody (scFv-CH3 dimer) produced similar results at later time points. Two of the antibody clones (H2 and H5) showed in vitro growth inhibitory effects on MET-dependent gefitinib-resistant cell lines, while no effects were observed on resistant lines lacking MET activation. In conclusion, these fully human antibody fragments inhibit MET-dependent cancer cells and enable rapid immunoPET imaging to assess MET expression levels, showing potential for both therapeutic and diagnostic applications. PMID:25143449

  12. Neonatal Immunization with Respiratory Syncytial Virus Glycoprotein Fragment Induces Protective Immunity in the Presence of Maternal Antibodies in Mice

    PubMed Central

    Noh, Youran; Shim, Byoung-Shik; Cheon, In Su; Rho, Semi; Kim, Hee Joo; Choi, Youngjoo; Kang, Chang-Yuil; Chang, Jun

    2013-01-01

    Abstract Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infections in infants and the elderly worldwide. The significant morbidity and mortality associated with this infection underscores the urgent need for development of RSV vaccine. In this study, we first show that intranasal administration of RSV glycoprotein core fragment (Gcf) to neonatal mice can induce systemic humoral immune responses and protective immunity against RSV without causing lung eosinophilia, although antibody response was shifted to a Th2 response. Next, we examined whether the presence of maternal anti-RSV antibodies would affect the responsiveness and protection efficacy of Gcf in newborn mice, since infants can possess RSV-specific maternal antibodies due to frequent RSV re-infections to adults. Intranasal administration of Gcf induced antibody response and increased IFNγ secretion and protected mice against RSV challenge without severe lung eosinophilia, even in the presence of high levels of RSV-specific maternal antibodies. Thus, our findings suggest that Gcf may be an effective and safe RSV vaccine during the neonatal period. PMID:23869549

  13. Monoclonal Antibody Fragments for Targeting Therapeutics to Growth Plate Cartilage | NCI Technology Transfer Center | TTC

    Cancer.gov

    The NICHD seeks statements of capability or interest from parties interested in collaborative research to co-develop, evaluate, or commercialize treatment of skeletal disorders using targeting antibodies.

  14. Gluten-specific antibodies of celiac disease gut plasma cells recognize long proteolytic fragments that typically harbor T-cell epitopes.

    PubMed

    Dørum, Siri; Steinsbø, Øyvind; Bergseng, Elin; Arntzen, Magnus Ø; de Souza, Gustavo A; Sollid, Ludvig M

    2016-01-01

    This study aimed to identify proteolytic fragments of gluten proteins recognized by recombinant IgG1 monoclonal antibodies generated from single IgA plasma cells of celiac disease lesions. Peptides bound by monoclonal antibodies in complex gut-enzyme digests of gluten treated with the deamidating enzyme transglutaminase 2, were identified by mass spectrometry after antibody pull-down with protein G beads. The antibody bound peptides were long deamidated peptide fragments that contained the substrate recognition sequence of transglutaminase 2. Characteristically, the fragments contained epitopes with the sequence QPEQPFP and variants thereof in multiple copies, and they typically also harbored many different gluten T-cell epitopes. In the pull-down setting where antibodies were immobilized on a solid phase, peptide fragments with multivalent display of epitopes were targeted. This scenario resembles the situation of the B-cell receptor on the surface of B cells. Conceivably, B cells of celiac disease patients select gluten epitopes that are repeated multiple times in long peptide fragments generated by gut digestive enzymes. As the fragments also contain many different T-cell epitopes, this will lead to generation of strong antibody responses by effective presentation of several distinct T-cell epitopes and establishment of T-cell help to B cells. PMID:27146306

  15. Gluten-specific antibodies of celiac disease gut plasma cells recognize long proteolytic fragments that typically harbor T-cell epitopes

    PubMed Central

    Dørum, Siri; Steinsbø, Øyvind; Bergseng, Elin; Arntzen, Magnus Ø.; de Souza, Gustavo A.; Sollid, Ludvig M.

    2016-01-01

    This study aimed to identify proteolytic fragments of gluten proteins recognized by recombinant IgG1 monoclonal antibodies generated from single IgA plasma cells of celiac disease lesions. Peptides bound by monoclonal antibodies in complex gut-enzyme digests of gluten treated with the deamidating enzyme transglutaminase 2, were identified by mass spectrometry after antibody pull-down with protein G beads. The antibody bound peptides were long deamidated peptide fragments that contained the substrate recognition sequence of transglutaminase 2. Characteristically, the fragments contained epitopes with the sequence QPEQPFP and variants thereof in multiple copies, and they typically also harbored many different gluten T-cell epitopes. In the pull-down setting where antibodies were immobilized on a solid phase, peptide fragments with multivalent display of epitopes were targeted. This scenario resembles the situation of the B-cell receptor on the surface of B cells. Conceivably, B cells of celiac disease patients select gluten epitopes that are repeated multiple times in long peptide fragments generated by gut digestive enzymes. As the fragments also contain many different T-cell epitopes, this will lead to generation of strong antibody responses by effective presentation of several distinct T-cell epitopes and establishment of T-cell help to B cells. PMID:27146306

  16. SPR Biosensor for the Detection of L. monocytogenes using Phage Displayed Antibody

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Whole cells of Listeria monocytogenes were detected with a compact, surface plasmon resonance (SPR) sensor using a phage-displayed scFv antibody to the virulence factor ActA for biorecognition. Phage Lm P4:A8, expressing the scFv antibody fused to the pIII surface protein was immobilized to the se...

  17. Affinity improvement by fine tuning of single-chain variable fragment against aflatoxin B1.

    PubMed

    Min, Won-Ki; Na, Kang-In; Yoon, Jung-Hyun; Heo, Yoon-Jee; Lee, Daesang; Kim, Sung-Gun; Seo, Jin-Ho

    2016-10-15

    Aflatoxin B1 (AFB1) produced in Aspergillus flavus is a major hepatocarcinogen found in foods and feed. For effective immunological detection of AFB1 at low concentrations, the development of high affinity antibody for AFB1 is required. Previously, an affinity-maturated single-chain variable fragment containing 6 mutations (scFv-M37) was isolated from an artificial mutagenic library, which showed a 9-fold higher affinity than its wild type scFv. In this study, the effect of the 6 mutated residues on the affinity improvement was characterized using surface plasmon resonance analysis, which identified a deleterious mutation (VH-A110T) located on a framework region of the scFv-M37. The back mutation of VH-A110T resulted in a 3.2-fold affinity improvement, which was attributed to decrease of dissociation rate constant (kd) in interaction between AFB1 and the back mutant scFv. The biophysical analyses using circular dichroism and gel filtration revealed that the back mutation of VH-A110T caused a subtle conformational change of the scFv toward tighter binding to AFB1. PMID:27173568

  18. Intracellular interactome of secreted antibody Fab fragment in Pichia pastoris reveals its routes of secretion and degradation.

    PubMed

    Pfeffer, Martin; Maurer, Michael; Stadlmann, Johannes; Grass, Josephine; Delic, Marizela; Altmann, Friedrich; Mattanovich, Diethard

    2012-03-01

    Protein translation, translocation, folding, processing, and secretion in eukaryotic cells are complex and not always straightforward processes, e.g., different routes of secretion and degradation exist. Formation of malfolded proteins in the endoplasmic reticulum (ER) can be one of the major bottlenecks for recombinant protein production. In this regard, an in-depth analysis of the interactions of a secreted protein during its pathway through the cell may be beneficial, as realized in this study for the methylotrophic yeast Pichia pastoris. The antibody fragment Fab3H6 used here is the anti-idiotype to the HIV neutralizing antibody 2F5 and is known to be intracellularly degraded in significant amounts when expressed in P. pastoris. The interactome of Fab3H6 was analyzed by using a pull-down mass spectrometry approach, and 23 proteins were found to bind specifically to the antibody fragment. Those allowed concluding that Fab3H6 is post-translationally translocated into the ER and degraded via the proteasome as well as the vacuole. In line with this, the expression of Fab3H6 increased the proteasomal activities by over 20%. Partial inhibition of the proteasome resulted in a significant increase of extracellular Fab3H6. Thus, it seems that ER quality control overshoots its requirements for the recombinant protein expressed and that more than just terminally malfolded protein is degraded by ER-associated degradation. This work will further facilitate our understanding how recombinant proteins behave in the secretory pathway. PMID:22350260

  19. In Vitro Neutralisation of Rotavirus Infection by Two Broadly Specific Recombinant Monovalent Llama-Derived Antibody Fragments

    PubMed Central

    Aladin, Farah; Einerhand, Alexandra W. C.; Bouma, Janneke; Bezemer, Sandra; Hermans, Pim; Wolvers, Danielle; Bellamy, Kate; Frenken, Leon G. J.; Gray, Jim; Iturriza-Gómara, Miren

    2012-01-01

    Rotavirus is the main cause of viral gastroenteritis in young children. Therefore, the development of inexpensive antiviral products for the prevention and/or treatment of rotavirus disease remains a priority. Previously we have shown that a recombinant monovalent antibody fragment (referred to as Anti-Rotavirus Proteins or ARP1) derived from a heavy chain antibody of a llama immunised with rotavirus was able to neutralise rotavirus infection in a mouse model system. In the present work we investigated the specificity and neutralising activity of two llama antibody fragments, ARP1 and ARP3, against 13 cell culture adapted rotavirus strains of diverse genotypes. In addition, immunocapture electron microscopy (IEM) was performed to determine binding of ARP1 to clinical isolates and cell culture adapted strains. ARP1 and ARP3 were able to neutralise a broad variety of rotavirus serotypes/genotypes in vitro, and in addition, IEM showed specific binding to a variety of cell adapted strains as well as strains from clinical specimens. These results indicated that these molecules could potentially be used as immunoprophylactic and/or immunotherapeutic products for the prevention and/or treatment of infection of a broad range of clinically relevant rotavirus strains. PMID:22403728

  20. A phage-displayed chicken single-chain antibody fused to alkaline phosphatase detects Fusarium pathogens and their presence in cereal grains.

    PubMed

    Hu, Zu-Quan; Li, He-Ping; Zhang, Jing-Bo; Huang, Tao; Liu, Jin-Long; Xue, Sheng; Wu, Ai-Bo; Liao, Yu-Cai

    2013-02-18

    Fusarium and its poisonous mycotoxins are distributed worldwide and are of particular interest in agriculture and food safety. A simple analytical method to detect pathogens is essential for forecasting diseases and controlling mycotoxins. This article describes a proposed method for convenient and sensitive detection of Fusarium pathogens that uses the fusion of single-chain variable fragment (scFv) and alkaline phosphatase (AP). A highly reactive scFv antibody specific to soluble cell wall-bound proteins (SCWPs) of F. verticillioides was selected from an immunized chicken phagemid library by phage display. The antibody was verified to bind on the surface of ungerminated conidiospores and mycelia of F. verticillioides. The scFv-AP fusion was constructed, and soluble expression in bacteria was confirmed. Both the antibody properties and enzymatic activity were retained, and the antigen-binding capacity of the fusion was enhanced by the addition of a linker. Surface plasmon resonance measurements confirmed that the fusion displayed 4-fold higher affinity compared with the fusion's parental scFv antibody. Immunoblot analyses showed that the fusion had good binding capacity to the components from SCWPs of F. verticillioides, and enzyme-linked immunosorbent assays revealed that the detection limit of the fungus was below 10(-2) μg mL(-1), superior to the scFv antibody. The fusion protein was able to detect fungal concentrations as low as 10(-3) mg g(-1) of maize grains in both naturally and artificially contaminated samples. Thus, the fusion can be applied in rapid and simple diagnosis of Fusarium contamination in field and stored grain or in food. PMID:23374219

  1. Advancing the global proteome survey platform by using an oriented single chain antibody fragment immobilization approach.

    PubMed

    Säll, Anna; Persson, Helena; Ohlin, Mats; Borrebaeck, Carl A K; Wingren, Christer

    2016-09-25

    Increasing the understanding of a proteome and how its protein composition is affected by for example different diseases, such as cancer, has the potential to improve strategies for early diagnosis and therapeutics. The Global Proteome Survey or GPS is a method that combines mass spectrometry and affinity enrichment with the use of antibodies. The technology enables profiling of complex proteomes in a species independent manner. The sensitivity of GPS, and other methods relying on affinity enrichment, is largely affected by the activity of the exploited affinity reagent. We here present an improvement of the GPS platform by utilizing an antibody immobilization approach which ensures a controlled immobilization process of the antibody to the magnetic bead support. More specifically, we make use of an antibody format that enables site-directed biotinylation and use this in combination with streptavidin coated magnetic beads. The performance of the expanded GPS platform was evaluated by profiling yeast proteome samples. We demonstrate that the oriented antibody immobilization strategy increases the ability of the GPS platform and results in larger fraction of functional antibodies. Additionally, we show that this new antibody format enabled in-solution capture, i.e. immobilization of the antibodies after sample incubation. A workflow has been established that permit the use of an oriented immobilization strategy for the GPS platform. PMID:26703809

  2. ESCRT-mediated Uptake and Degradation of Brain-targeted α-synuclein Single Chain Antibody Attenuates Neuronal Degeneration In Vivo

    PubMed Central

    Spencer, Brian; Emadi, Sharareh; Desplats, Paula; Eleuteri, Simona; Michael, Sarah; Kosberg, Kori; Shen, Jay; Rockenstein, Edward; Patrick, Christina; Adame, Anthony; Gonzalez, Tania; Sierks, Michael; Masliah, Eliezer

    2014-01-01

    Parkinson's disease and dementia with Lewy bodies are neurodegenerative disorders characterized by accumulation of α-synuclein (α-syn). Recently, single-chain fragment variables (scFVs) have been developed against individual conformational species of α-syn. Unlike more traditional monoclonal antibodies, these scFVs will not activate or be endocytosed by Fc receptors. For this study, we investigated an scFV directed against oligomeric α-syn fused to the LDL receptor-binding domain from apolipoprotein B (apoB). The modified scFV showed enhanced brain penetration and was imported into neuronal cells through the endosomal sorting complex required for transport (ESCRT) pathway, leading to lysosomal degradation of α-syn aggregates. Further analysis showed that the scFV was effective at ameliorating neurodegenerative pathology and behavioral deficits observed in the mouse model of dementia with Lewy bodies/Parkinson's disease. Thus, the apoB modification had the effect of both increasing accumulation of the scFV in the brain and directing scFV/α-syn complexes for degradation through the ESCRT pathway, leading to improved therapeutic potential of immunotherapy. PMID:25008355

  3. A Novel Human scFv Library with Non-Combinatorial Synthetic CDR Diversity

    PubMed Central

    Kang, Seungmin; Kim, Wankyu; Shim, Hyunbo

    2015-01-01

    The present work describes the construction and validation of a human scFv library with a novel design approach to synthetic complementarity determining region (CDR) diversification. The advantage of synthetic antibody libraries includes the possibility of exerting fine control over factors like framework sequences, amino acid and codon usage, and CDR diversity. However, random combinatorial synthesis of oligonucleotides for CDR sequence diversity also produces many clones with unnatural sequences and/or undesirable modification motifs. To alleviate these issues, we designed and constructed a novel semi-synthetic human scFv library with non-combinatorial, pre-designed CDR diversity and a single native human framework each for heavy, kappa, and lambda chain variable domains. Next-generation sequencing analysis indicated that the library consists of antibody clones with highly nature-like CDR sequences and the occurrence of the post-translational modification motifs is minimized. Multiple unique clones with nanomolar affinity could be isolated from the library against a number of target antigens, validating the library design strategy. The results demonstrate that it is possible to construct a functional antibody library using low, non-combinatorial synthetic CDR diversity, and provides a new strategy for the design of antibody libraries suitable for demanding applications. PMID:26484868

  4. Galactosylated streptavidin for improved clearance of biotinylated intact and F(ab')2 fragments of an anti-tumour antibody.

    PubMed Central

    Marshall, D.; Pedley, R. B.; Melton, R. G.; Boden, J. A.; Boden, R.; Begent, R. H.

    1995-01-01

    Persistence of high levels of radiolabelled antibody in the circulation is a major limitation of radioimmunotherapy. Biotinylation of the radiolabelled anti-tumour antibody followed by administration of streptavidin is known to give much improved tumour to blood ratios as the radioantibody is complexed and subsequently cleared via the reticuloendothelial system, although prolonged splenic uptake is a problem. We have investigated the effect on the clearance pattern and tumour localisation of a 125I-labelled biotinylated anti-CEA antibody (A5B7) after administration of a galactosylated form of streptavidin (gal-streptavidin) in nude mice bearing a human colon carcinoma xenograft. Fifteen minutes to 1 h after gal-streptavidin administration the complexes were cleared via the liver alone (as opposed to liver and spleen after native streptavidin). Twenty-four hours after administration of gal-streptavidin, the tumour to blood ratio for biotinylated A5B7 IgG increased from 2.9 to 13.2 and for biotinylated F(ab')2 fragments an increase from 4.9 to 33.2 was achieved. The reduction in tumour accumulation of F(ab')2 24 h after injection of the clearing agent was less than that seen with intact antibody. Injection of asialofetuin inhibited clearance, confirming that removal of the gal-streptavidin-biotinylated antibody complexes from the blood was via the asialoglycoprotein receptor on liver hepatocytes. Therefore, galactosylation of the streptavidin clearing agent allows rapid removal of radiolabelled biotinylated antibodies via the liver asialoglycoprotein receptor, as opposed to the reticuloendothelial system. Images Figure 5 Figure 6 Figure 7 PMID:7529526

  5. Recombinant anti-CD20 antibody fragments for microPET imaging of B-cell lymphoma

    PubMed Central

    Olafsen, Tove; Betting, David; Kenanova, Vania E.; Salazar, Felix B.; Clarke, Pat; Said, Jonathan; Raubitschek, Andrew A.; Timmerman, John M.; Wu, Anna M.

    2010-01-01

    The CD20 cell surface antigen is expressed at high levels by over 90% of B cell non-Hodgkin lymphomas (NHL), and is the target of the anti-CD20 monoclonal antibody rituximab. To provide more sensitive, tumor-specific positron emission tomography (PET) imaging of NHL, we sought to develop PET imaging agents targeting CD20. Methods Two recombinant anti-CD20 rituximab fragments, a minibody (scFv-CH3 dimer, 80 kDa) and a modified scFv-Fc fragment (105 kDa), designed to clear rapidly, were generated. Both fragments were radiolabeled with 124I, and the minibody was additionally radiometal labeled with 64Cu following conjugation to 1,4,7,10-tetraazacyclododecane-N,N’,N’’,N’’’-tetraacetic acid (DOTA). The radioiodinated fragments and the radiometal labeled minibody were evaluated in mice as microPET imaging agents for in vivo imaging of human CD20-expressing lymphomas. Results Rapid and specific localization to CD20-positive tumors was observed with the radioiodinated fragments. However, their tumor uptakes and blood activities differed, resulting in different levels of contrast in the images. The best candidate was the minibody, with superior uptake (2-fold higher than the scFv-Fc) in CD20-positive tumor and low uptake in CD20-negative tumor. Positive tumor to negative tumor ratios were 7.0(±3.1) and 3.9(±0.7) for the minibody and scFv-Fc, respectively at 21 hours. About a 5-fold lower ratio was achieved with the 64Cu-DOTA-minibody at 19 hours due to higher residual background activity in CD20 negative tumor. Conclusion Radioiodinated minibody and scFv-Fc fragment produced excellent, high-contrast images in vivo. These new immunoPET agents may prove useful for the imaging CD20 positive lymphomas in preclinical models and in humans with NHL. PMID:19690034

  6. Effects of an amyloid-beta 1-42 oligomers antibody screened from a phage display library in APP/PS1 transgenic mice

    PubMed Central

    Wang, Jianping; Li, Nan; Ma, Jun; Gu, Zhiqiang; Yu, Lie; Fu, Xiaojie; Liu, Xi; Wang, Jian

    2016-01-01

    We screened anti-Aβ1-42 antibodies from a human Alzheimer’s disease (AD) specific single chain variable fragment (scFv) phage display library and assessed their effects in APP/PS1 transgenic mice. Reverse transcription-PCR was used to construct the scFv phage display library, and screening identified 11A5 as an anti-Aβ1-42 antibody. We mixed 11A5 and the monoclonal antibody 6E10 with Aβ1-42 and administered the mixture to Sprague-Dawley rats via intracerebroventricular injection. After 30 days, rats injected with the antibody/ Aβ1-42 mixture and those injected with Aβ1-42 alone were tested on the Morris water maze. We also injected 11A5 and 6E10 into APP/PS1 transgenic mice and assessed the concentrations of Aβ in brain and peripheral blood by ELISA at 1-month intervals for 3 months. Finally we evaluated behavior changes in the Morris water maze. Rats injected with Aβ1-42 and mixed antibodies showed better performance in the Morris water maze than did rats injected with Aβ1-42 alone. In APP/PS1 transgenic mice, Aβ concentration was lower in the brains of the antibody-treated group than in the control group, but higher in the peripheral blood. The antibody-treated mice also exhibited improved behavioral performance in the Morris water maze. In conclusion, anti-Aβ1-42 antibodies (11A5) screened from the human scFv antibody phage display library promoted the efflux or clearance of Aβ1-42 and effectively decreased the cerebral Aβ burden in an AD mouse model. PMID:26820640

  7. Antibodies against Venom of the Snake Deinagkistrodon acutus

    PubMed Central

    Lee, Yu-Ching; Liang, Meng-Huei; Leu, Sy-Jye; Lin, Liang-Tzung; Chiang, Jen-Ron

    2015-01-01

    Snake venom protein from Deinagkistrodon acutus (DA protein), one of the major venomous species in Taiwan, causes hemorrhagic symptoms that can lead to death. Although horse-derived antivenin is a major treatment, relatively strong and detrimental side effects are seen occasionally. In our study, yolk immunoglobulin (IgY) was purified from eggs, and DA protein was recognized using Western blotting and an enzyme-linked immunosorbent assay (ELISA), similar to therapeutic horse antivenin. The ELISA also indicated that specific IgY antibodies were elicited after the fifth booster, plateaued, and lasted for at least 3 months. To generate monoclonal single-chain variable fragment (scFv) antibodies, we used phage display technology to construct two libraries with short or long linkers, containing 6.24 × 108 and 5.28 × 108 transformants, respectively. After four rounds of biopanning, the eluted phage titer increased, and the phage-based ELISA indicated that the specific clones were enriched. Nucleotide sequences of 30 individual clones expressing scFv were analyzed and classified into four groups that all specifically recognized the DA venom protein. Furthermore, based on mass spectrometry, the scFv-bound protein was deduced to be snake venom metalloproteinase proteins. Most importantly, both IgY and mixed scFv inhibited the lethal effect in mice injected with the minimum lethal dosage of the DA protein. We suggest that together, these antibodies could be applied to the development of diagnostic agents or treatments for snakebite envenomation in the future. PMID:26475102

  8. Absence of SV40 antibodies or DNA fragments in prediagnostic mesothelioma serum samples.

    PubMed

    Kjaerheim, Kristina; Røe, Oluf Dimitri; Waterboer, Tim; Sehr, Peter; Rizk, Raeda; Dai, Hong Yan; Sandeck, Helmut; Larsson, Erik; Andersen, Aage; Boffetta, Paolo; Pawlita, Michael

    2007-06-01

    The rhesus monkey virus Simian Virus 40 (SV40) is a member of the polyomavirus family. It was introduced inadvertently to human populations through contaminated polio vaccine during the years 1956-1963, can induce experimental tumors in animals and transform human cells in culture. SV40 DNA has been identified in mesothelioma and other human tumors in some but not all studies. We tested prediagnostic sera from 49 mesothelioma cases and 147 matched controls for antibodies against the viral capsid protein VP1 and the large T antigen of SV40 and of the closely related human polyomaviruses BK and JC, and for SV40 DNA. Cases and controls were identified among donors to the Janus Serum Bank, which was linked to the Cancer Registry of Norway. Antibodies were analyzed by recently developed multiplex serology based on recombinantly expressed fusions of glutathione-S transferase with viral proteins as antigens combined with fluorescent bead technology. BKV and JCV specific antibodies cross- reactive with SV40 were preabsorbed with the respective VP1 proteins. Sera showing SV40 reactivity after preabsorption with BKV and JCV VP1 were further analyzed in SV40 neutralization assays. SV40 DNA was analyzed by SV40 specific polymerase chain reactions. The odds ratio for being a case when tested positive for SV40 VP1 in the antibody capture assay was 1.5 (95% CI 0.6-3.7) and 2.0 (95% CI 0.6-7.0) when only strongly reactive sera where counted as positive. Although some sera could neutralize SV40, preabsorption with BKV and JCV VP1 showed for all such sera that this neutralizing activity was due to cross-reacting antibodies and did not represent truly SV40-specific antibodies. No viral DNA was found in the sera. No significant association between SV40 antibody response in prediagnostic sera and risk of mesothelioma was seen. PMID:17315193

  9. A human and a mouse anti-idiotypic antibody specific for human T14(+) anti-DNA antibodies reconstructed by phage display.

    PubMed

    Leung, D T; Yam, N W; Chui, Y L; Wong, K C; Lim, P L

    2000-09-19

    Little is known about human anti-idiotypic antibodies. Phage display methodology was used to reconstruct these antibodies from lupus patients, which recognize a subset (T14(+)) of anti-DNA antibodies. Antigen-specific B cells were isolated from the blood using a peptide based on a complementarity determining region (V(H)CDR3) of the prototypic T14(+) antibody. cDNA fragments of the V(H) and V(L) genes prepared from the cells were expressed as phage displayed single chain Fv (scFv) fragments using the pCANTAB-5E phagemid vector. From a reactive clone obtained, the Ig genes used were identified to be V(H)3, D5-D3, J(H)4b, V(kappa)I and J(kappa)2. The heavy chain was highly mutated, especially in CDR3, which bears mutations mostly of the replacement type; this region is also unusual in being extremely long due to a D-D fusion. In contrast, a mouse hybridoma antibody, made to the same T14(+) peptide and transformed as a scFv fragment, uses a short V(H)CDR3 comprising five amino acids, three of which are tyrosines. Tyrosines may be important for antigen binding because two of these also exist in the human V(H)CDR3. The light chains of both antibodies may also contribute to the specificity of the protein, because their V(L) segments, including the CDRs, are highly homologous to each other. PMID:11024298

  10. Potent neutralization of VEGF biological activities with a fully human antibody Fab fragment directed against VEGF receptor 2

    SciTech Connect

    Miao, H.-Q. . E-mail: hua-quan.miao@imclone.com; Hu, Kun; Jimenez, Xenia; Navarro, Elizabeth; Zhang, Haifan; Lu Dan; Ludwig, Dale L.; Balderes, Paul; Zhu Zhenping . E-mail: zhenping.zhu@imclone.com

    2006-06-23

    Compelling evidence suggest that vascular endothelial growth factor (VEGF) and its receptors, especially receptor 2 (VEGFR2, or kinase insert domain-containing receptor, KDR), play a critical role in angiogenesis under both physiological and pathological conditions, including cancer and angiogenic retinopathies such as age-related macular degeneration (AMD). To this end, inhibition of angiogenesis with antagonists to either VEGF or KDR has yielded significant therapeutic efficacy both in preclinical studies in animal models and in clinical trials in patients with cancer and AMD. We previously reported the identification of a high affinity, fully human anti-KDR antibody fragment, 1121B Fab, through a highly stringent affinity maturation process with a Fab originally isolated from a naive human antibody phage display library. In this study, we demonstrate that 1121B Fab is able to strongly block KDR/VEGF interaction, resulting in potent inhibition of an array of biological activities of VEGF, including activation of the receptor and its signaling pathway, intracellular calcium mobilization, and migration and proliferation of endothelial cells. Taken together, our data lend strong support to the further development of 1121B Fab fragment as an anti-angiogenesis agent in both cancer and angiogenic retinopathies.

  11. Kinetic Characterisation of a Single Chain Antibody against the Hormone Abscisic Acid: Comparison with Its Parental Monoclonal

    PubMed Central

    Badescu, George O.; Marsh, Andrew; Smith, Timothy R.; Thompson, Andrew J.; Napier, Richard M.

    2016-01-01

    A single-chain Fv fragment antibody (scFv) specific for the plant hormone abscisic acid (ABA) has been expressed in the bacterium Escherichia coli as a fusion protein. The kinetics of ABA binding have been measured using surface plasmon resonance spectrometry (BIAcore 2000) using surface and solution assays. Care was taken to calculate the concentration of active protein in each sample using initial rate measurements under conditions of partial mass transport limitation. The fusion product, parental monoclonal antibody and the free scFv all have low nanomolar affinity constants, but there is a lower dissociation rate constant for the parental monoclonal resulting in a three-fold greater affinity. Analogue specificity was tested and structure-activity binding preferences measured. The biologically-active (+)-ABA enantiomer is recognised with an affinity three orders of magnitude higher than the inactive (-)-ABA. Metabolites of ABA including phaseic acid, dihydrophaseic acid and deoxy-ABA have affinities over 100-fold lower than that for (+)-ABA. These properties of the scFv make it suitable as a sensor domain in bioreporters specific for the naturally occurring form of ABA. PMID:27023768

  12. Engineering an Anti-Transferrin Receptor ScFv for pH-Sensitive Binding Leads to Increased Intracellular Accumulation

    PubMed Central

    Tillotson, Benjamin J.; Goulatis, Loukas I.; Parenti, Isabelle; Duxbury, Elizabeth; Shusta, Eric V.

    2015-01-01

    The equilibrium binding affinity of receptor-ligand or antibody-antigen pairs may be modulated by protonation of histidine side-chains, and such pH-dependent mechanisms play important roles in biological systems, affecting molecular uptake and trafficking. Here, we aimed to manipulate cellular transport of single-chain antibodies (scFvs) against the transferrin receptor (TfR) by engineering pH-dependent antigen binding. An anti-TfR scFv was subjected to histidine saturation mutagenesis of a single CDR. By employing yeast surface display with a pH-dependent screening pressure, scFvs having markedly increased dissociation from TfR at pH 5.5 were identified. The pH-sensitivity generally resulted from a central cluster of histidine residues in CDRH1. When soluble, pH-sensitive, scFv clone M16 was dosed onto live cells, the internalized fraction was 2.6-fold greater than scFvs that lacked pH-sensitive binding and the increase was dependent on endosomal acidification. Differences in the intracellular distribution of M16 were also observed consistent with an intracellular decoupling of the scFv M16-TfR complex. Engineered pH-sensitive TfR binding could prove important for increasing the effectiveness of TfR-targeted antibodies seeking to exploit endocytosis or transcytosis for drug delivery purposes. PMID:26713870

  13. Internalization and recycling of ALCAM/CD166 detected by a fully human single-chain recombinant antibody.

    PubMed

    Piazza, Tiziana; Cha, Emanuela; Bongarzone, Italia; Canevari, Silvana; Bolognesi, Andrea; Polito, Letizia; Bargellesi, Antonio; Sassi, Francesca; Ferrini, Silvano; Fabbi, Marina

    2005-04-01

    Activated leukocyte cell adhesion molecule (ALCAM/CD166), a member of the immunoglobulin superfamily with five extracellular immunoglobulin-like domains, promotes heterophilic (ALCAM-CD6) and homophilic (ALCAM-ALCAM) cell-cell interactions. Here we describe a fully human single-chain antibody fragment (scFv) directed to ALCAM/CD166. We selected the I/F8 scFv from a phage display library of human V-gene segments by cell panning and phage internalization into IGROV-I human ovary carcinoma cells. The I/F8 specificity was identified as ALCAM/CD166 by matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) peptide mass fingerprinting of the I/F8-immunoprecipitated protein. The I/F8 scFv reacts with the human, monkey and murine ALCAM/CD166 molecule, indicating that the recognized epitope is highly conserved. The I/F8 scFv completely abolished binding of both ALCAM/Fc and CD6/Fc soluble ligands, whereas it did not compete with the anti-ALCAM/CD166 murine monoclonal antibodies J4-81 and 3A6 and therefore recognizes a different epitope. Engagement through I/F8 scFv, 3A6 monoclonal antibody or CD6/Fc ligand induced ALCAM/CD166 internalization, with a kinetics slower than that of transferrin in the same cells. Newly internalized I/F8-ALCAM complexes colocalized with clathrin but not with caveolin and we demonstrated, using surface biotinylation and recycling assays, that endocytosed ALCAM/CD166 recycles back to the cell surface. Such an endocytic pathway allows the efficient delivery of an I/F8 scFv-saporin immunotoxin into tumor cells, as the conjugates are able to selectively kill cell lines expressing ALCAM/CD166. Altogether these data provide evidence of the suitability of the I/F8 scFv for further functional analysis of ALCAM/CD166 and intracellular delivery of effector moieties. PMID:15769845

  14. Serrumab: a human monoclonal antibody that counters the biochemical and immunological effects of Tityus serrulatus venom.

    PubMed

    Pucca, Manuela Berto; Zoccal, Karina Furlan; Roncolato, Eduardo Crosara; Bertolini, Thaís Barboza; Campos, Lucas Benício; Cologna, Camila Takeno; Faccioli, Lúcia Helena; Arantes, Eliane Candiani; Barbosa, José Elpidio

    2012-01-01

    In Brazil, the species Tityus serrulatus is responsible for the most severe cases of scorpion envenomation. There is currently a need for new scorpion anti-venoms that are more effective and less harmful. This study attempted to produce human monoclonal antibodies capable of inhibiting the activity of T. serrulatus venom (TsV), using the Griffin.1 library of human single-chain fragment-variable (scFv) phage antibodies. Four rounds of phage antibody selection were performed, and the round with the highest phage antibody titer was chosen for the production of monoclonal phage antibodies and for further analysis. The scFv 2A, designated serrumab, was selected for the production and purification of soluble antibody fragments. In a murine peritoneal macrophage cell line (J774.1), in vitro assays of the cytokines interleukin (IL)-6, tumor necrosis factor (TNF)-α, and IL-10 were performed. In male BALB/c mice, in vivo assays of plasma urea, creatinine, aspartate transaminase, and glucose were performed, as well as of neutrophil recruitment and leukocyte counts. It was found that serrumab inhibited the TsV-induced increases in the production of IL-6, TNFα, and IL-10 in J774.1 cells. The in vivo inhibition assay showed that serrumab also prevented TsV-induced increases in the plasma levels of urea, creatinine, aspartate transaminase, and glucose, as well as preventing the TsV-induced increase in neutrophil recruitment. The results indicate that the human monoclonal antibody serrumab is a candidate for inclusion in a mixture of specific antibodies to the various toxins present in TsV. Therefore, serrumab shows promise for use in the production of new anti-venom. PMID:22424317

  15. Crystal Structure of the Fab Fragment of an Anti-ofloxacin Antibody and Exploration of Its Specific Binding.

    PubMed

    He, Kuo; Du, Xinjun; Sheng, Wei; Zhou, Xiaonan; Wang, Junping; Wang, Shuo

    2016-03-30

    The limited knowledge on the mechanism of interactions between small contaminants and the corresponding antibodies greatly inhibits the development of enzyme-linked immunosorbent assay methods. In this study, the crystal structure of a Fab fragment specific for ofloxacin was obtained. On the basis of the crystal characteristics, the modeling of the interactions between ofloxacin and the Fab revealed that TYR31 and HIS99 of the heavy chain and MET20 and GLN79 of the light chain formed a hydrophobic region and that SER52 and ALA97 of the heavy chain and TYR35 of the light chain formed a salt bridge and two hydrogen bonds for specific binding. The key roles of SER52 and ALA97 were further confirmed by site-directed mutation. A specificity analysis using 14 ofloxacin analogues indicates that the length of the bond formed between the piperazine ring and the antibody plays key roles in specific recognition. This work helps to clarify the mechanisms through which antibodies recognize small molecules and improve immune detection methods. PMID:26963935

  16. Optimizing the expression of a monoclonal antibody fragment under the transcriptional control of the Escherichia coli lac promoter.

    PubMed

    Donovan, R S; Robinson, C W; Glick, B R

    2000-06-01

    The expression of a monoclonal antibody Fab fragment in Escherichia coli strain RB791/pComb3, induced with either lactose or isopropyl-beta-D-thiogalactoside (IPTG), was compared to determine if lactose might provide an inexpensive alternative to induction with IPTG. Induction of Fab expression imposed a metabolic load on the recombinant cells, resulting in lower final cell yields compared to the non-induced controls. An IPTG concentration of 0.05 mM was sufficient to achieve maximal expression of soluble Fab protein when inducing in the early-, mid-, or late-log phases of batch cultures grown using either glucose or glycerol as a carbon source. The largest overall yield of Fab fragments when using 0.05 mM IPTG was achieved by increasing the final yield of cells through glycerol feeding following induction in late-log phase. Lactose was as effective as IPTG for inducing Fab expression in E. coli RB791/pComb3. The greatest overall level of Fab expression was found when cells grown on glycerol were induced with 2 g/L lactose in late-log phase. Since the cost of 0.05 mM of IPTG is significantly greater than the cost of 2 g/L lactose, lactose provides an inexpensive alternative to IPTG for inducing the expression of Fab fragments, and possibly other recombinant proteins, from the E. coli lac promoter. PMID:10913975

  17. Cloning of scFv from hybridomas using a rational strategy: Application as a receptor to sensitive detection microcystin-LR in water.

    PubMed

    Zhang, Xiuyuan; He, Kuo; Zhao, Ruiping; Wang, Lixia; Jin, Yandan

    2016-10-01

    Single chain variable fragment (scFv), containing of heavy and light chains (VH and VL) joined by a short peptide linker, has been used widely for immunodetection. Nevertheless, cloning functional variable genes is still a bottle neck for the scFv generation technology. Here, a rational strategy for cloning and selecting variable region genes from an anti-microcystin-LR hybridoma was devised, then the functional VH and VL genes were recloned and assembled to scFv using splicing overlap extension PCR. The resulting scFv gene was recombinantly expressed as a soluble scFv-alkaline phosphatase fusion protein (scFv-AP) by vector PLIP6/GN. Then an indirect competitive chemiluminescent enzyme immunoassay (ic-CLEIA) for detection of microcystin-LR was developed. The half-maximum inhibition concentrations (IC50) and limits of detection (LODs, IC15) were 0.81 ± 0.04 μgL(-1) and 0.13 ± 0.03 μgL(-1), respectively. With the mean coefficient of variation lowing 8%, the mean recovery in intra-assay and inter-assay were 100.06% and 96.46%, The proposed strategy should be useful for generation scFv in a rapid and simple way. PMID:27380224

  18. [Construction of anti-B7-H4-scFv library and screening and identification of anti-B7-H4-scFv].

    PubMed

    Shao, Luanluan; Xu, Chaochao; Ji, Hongshuai; Mao, Weiping; Wang, Yingying; Liu, Xiaoqian; Zhu, Yanyan

    2016-09-01

    Objective To construct the ribosome display library of anti-B7-H4 extracellular domain, and select the antibody with high specificity. Methods The cDNA of B7-H4 extracellular domain was amplified from A549 cells by reverse transcription PCR (RT-PCR). To express ectodomains of B7-H4, the sequence of B7-H4 gene, which encodes the B7-H4 extracellular domains, was inserted into plasmid pET-28a(+). The purified recombinant protein of B7-H4 extracellular domain was used to immunize BALB/c mice. The total RNA was extracted from the spleen of BALB/c mice which had been immunized with B7-H4 recombinant protein. The genes of VH, Vκ and VH/Vκ were amplified separately by RT-PCR and splicing by overlap extension PCR (SOE-PCR). The gene of VH/ Vκ was ligated into pUM19-T vector and the ligated sample was transformed into competent E.coli DH5α. The resulting plasmid was isolated and then subjected to sequencing to verify the gene sequence. TNT(R)T7 Quick for PCR DNA kit was used to translate and screen the anti-B7-H4-scFv in vitro from the ribosome display library. Western blotting and an indirect ELISA were performed to detect the specificity of anti-B7-H4-scFv. Results The right sequences of VH, Vκ and VH/Vκ were acquired, which were 439, 680 and 1098 bp in length, respectively. The analysis of specificity demonstrated that the anti-B7-H4-scFv screened from the ribosome display library had a high specific combining ability with B7-H4. Conclusion The experiment has successively constructed the ribosome display library of anti-B7-H4 extracellular domain, and selected the anti-B7-H4-scFv which has a high specific binding ability with recombinant protein of B7-H4 extracelluar domain. PMID:27609584

  19. Generation of recombinant antibody fragments with toxin-neutralizing potential in loxoscelism.

    PubMed

    Karim-Silva, Sabrina; Moura, Juliana de; Noiray, Magali; Minozzo, Joao Carlos; Aubrey, Nicolas; Alvarenga, Larissa M; Billiald, Philippe

    2016-08-01

    Loxosceles spider bites often lead to serious envenomings and no definite therapy has yet been established. In such a context, it is of interest to consider an antibody-based targeted therapy. We have previously prepared a murine monoclonal IgG (LiMab7) that binds to 32-35kDa components of Loxosceles intermedia venom and neutralizes the dermonecrotic activity of the venom. Here, we re-engineered LiMab7 into a recombinant diabody. The protein was produced in bacteria and then it was functionally characterized. It proved to be efficient at neutralizing sphingomyelinase and hemolytic activities of the crude venom despite the slightly altered binding kinetic constants and the limited stability of the dimeric configuration. This is the first report of a specific recombinant antibody for a next-generation of Loxosceles antivenoms. PMID:27288291

  20. Structure of a human monoclonal antibody Fab fragment against gp41 of human immunodeficiency virus type

    NASA Technical Reports Server (NTRS)

    He, X. M.; Ruker, F.; Casale, E.; Carter, D. C.

    1992-01-01

    The three-dimensional structure of a human monoclonal antibody (Fab), which binds specifically to a major epitope of the transmembrane protein gp41 of the human immunodeficiency virus type 1, has been determined by crystallographic methods to a resolution of 2.7 A. It has been previously determined that this antibody recognizes the epitope SGKLICTTAVPWNAS, belongs to the subclass IgG1 (kappa), and exhibits antibody-dependent cellular cytotoxicity. The quaternary structure of the Fab is in an extended conformation with an elbow bend angle between the constant and variable domains of 175 degrees. Structurally, four of the hypervariable loops can be classified according to previously recognized canonical structures. The third hypervariable loops of the heavy (H3) and light chain (L3) are structurally distinct. Hypervariable loop H3, residues 102H-109H, is unusually extended from the surface. The complementarity-determining region forms a hydrophobic binding pocket that is created primarily from hypervariable loops L3, H3, and H2.

  1. Control of Established Colon Cancer Xenografts Using a Novel Humanized Single Chain Antibody-Streptococcal Superantigen Fusion Protein Targeting the 5T4 Oncofetal Antigen

    PubMed Central

    Patterson, Kelcey G.; Dixon Pittaro, Jennifer L.; Bastedo, Peter S.; Hess, David A.; Haeryfar, S. M. Mansour; McCormick, John K.

    2014-01-01

    Superantigens (SAgs) are microbial toxins that cross-link T cell receptors with major histocompatibility class II (MHC-II) molecules leading to the activation of large numbers of T cells. Herein, we describe the development and preclinical testing of a novel tumor-targeted SAg (TTS) therapeutic built using the streptococcal pyrogenic exotoxin C (SpeC) SAg and targeting cancer cells expressing the 5T4 tumor-associated antigen (TAA). To inhibit potentially harmful widespread immune cell activation, a SpeC mutation within the high-affinity MHC-II binding interface was generated (SpeCD203A) that demonstrated a pronounced reduction in mitogenic activity, yet this mutant could still induce immune cell-mediated cancer cell death in vitro. To target 5T4+ cancer cells, we engineered a humanized single chain variable fragment (scFv) antibody to recognize 5T4 (scFv5T4). Specific targeting of scFv5T4 was verified. SpeCD203A fused to scFv5T4 maintained the ability to activate and induce immune cell-mediated cytotoxicity of colorectal cancer cells. Using a xenograft model of established human colon cancer, we demonstrated that the SpeC-based TTS was able to control the growth and spread of large tumors in vivo. This required both TAA targeting by scFv5T4 and functional SAg activity. These studies lay the foundation for the development of streptococcal SAgs as ‘next-generation’ TTSs for cancer immunotherapy. PMID:24736661

  2. Control of established colon cancer xenografts using a novel humanized single chain antibody-streptococcal superantigen fusion protein targeting the 5T4 oncofetal antigen.

    PubMed

    Patterson, Kelcey G; Dixon Pittaro, Jennifer L; Bastedo, Peter S; Hess, David A; Haeryfar, S M Mansour; McCormick, John K

    2014-01-01

    Superantigens (SAgs) are microbial toxins that cross-link T cell receptors with major histocompatibility class II (MHC-II) molecules leading to the activation of large numbers of T cells. Herein, we describe the development and preclinical testing of a novel tumor-targeted SAg (TTS) therapeutic built using the streptococcal pyrogenic exotoxin C (SpeC) SAg and targeting cancer cells expressing the 5T4 tumor-associated antigen (TAA). To inhibit potentially harmful widespread immune cell activation, a SpeC mutation within the high-affinity MHC-II binding interface was generated (SpeCD203A) that demonstrated a pronounced reduction in mitogenic activity, yet this mutant could still induce immune cell-mediated cancer cell death in vitro. To target 5T4+ cancer cells, we engineered a humanized single chain variable fragment (scFv) antibody to recognize 5T4 (scFv5T4). Specific targeting of scFv5T4 was verified. SpeCD203A fused to scFv5T4 maintained the ability to activate and induce immune cell-mediated cytotoxicity of colorectal cancer cells. Using a xenograft model of established human colon cancer, we demonstrated that the SpeC-based TTS was able to control the growth and spread of large tumors in vivo. This required both TAA targeting by scFv5T4 and functional SAg activity. These studies lay the foundation for the development of streptococcal SAgs as 'next-generation' TTSs for cancer immunotherapy. PMID:24736661

  3. Utilisation of antibody microarrays for the selection of specific and informative antibodies from recombinant library binders of unknown quality.

    PubMed

    Kibat, Janek; Schirrmann, Thomas; Knape, Matthias J; Helmsing, Saskia; Meier, Doris; Hust, Michael; Schröder, Christoph; Bertinetti, Daniela; Winter, Gerhard; Pardes, Khalid; Funk, Mia; Vala, Andrea; Giese, Nathalia; Herberg, Friedrich W; Dübel, Stefan; Hoheisel, Jörg D

    2016-09-25

    Many diagnostic and therapeutic concepts require antibodies of high specificity. Recombinant binder libraries and related selection approaches allow the efficient isolation of antibodies against almost every target of interest. Nevertheless, it cannot be guaranteed that selected antibodies perform well and interact specifically enough with analytes unless an elaborate characterisation is performed. Here, we present an approach to shorten this process by combining the selection of suitable antibodies with the identification of informative target molecules by means of antibody microarrays, thereby reducing the effort of antibody characterisation by concentrating on relevant molecules. In a pilot scheme, a library of 456 single-chain variable fragment (scFv) binders to 134 antigens was used. They were arranged in a microarray format and incubated with the protein content of clinical tissue samples isolated from pancreatic ductal adenocarcinoma and healthy pancreas, as well as recurrent and non-recurrent bladder tumours. We observed significant variation in the expression of the E3 ubiquitin-protein ligase (CHFR) as well as the glutamate receptor interacting protein 2 (GRIP2), for example, always with more than one of the scFvs binding to these targets. Only the relevant antibodies were then characterised further on antigen microarrays and by surface plasmon resonance experiments so as to select the most specific and highest affinity antibodies. These binders were in turn used to confirm a microarray result by immunohistochemistry analysis. PMID:26709003

  4. Crystallization and preliminary X-ray structure determination of jack bean urease with a bound antibody fragment.

    PubMed

    Sheridan, Louisa; Wilmot, Carrie M; Cromie, Karen D; van der Logt, Paul; Phillips, Simon E V

    2002-02-01

    Urease allows organisms to use exogenous and internally generated urea as a nitrogen source, by catalyzing the hydrolysis of urea to form ammonia and carbon dioxide. Urease may also participate in the systemic nitrogen-transport pathways and possibly acts as a toxic defence protein. Jack bean urease (JBU) was the first nickel-metalloenzyme identified and was crystallized as early as 1926. Despite this, the structure has not yet been determined. An antibody fragment, Fv, that has a high affinity for JBU has been used to aid crystallization. The complex, which retains full enzyme activity, forms very small crystals that diffract weakly to 3.3 A. The crystals belong to the rhombohedral space group R32, with unit-cell parameters a = b = 228.6, c = 130.9 A. The structure of the urease molecule has been solved by molecular replacement using the structure of homogenous enzyme from Klebsiella aerogenes as a search model. PMID:11807281

  5. Coexpression of molecular chaperones to enhance functional expression of anti-BNP scFv in the cytoplasm of Escherichia coli for the detection of B-type natriuretic peptide.

    PubMed

    Maeng, Bo Hee; Nam, Dong Hyun; Kim, Yong Hwan

    2011-06-01

    Molecular chaperones are a ubiquitous family of cellular proteins that mediate the correct folding of other target polypeptides. In our previous study, the recombinant anti-BNP scFv, which has promising applications for diagnostic, prognostic, and therapeutic monitoring of heart failure, was expressed in the cytoplasm of Escherichia coli. However, when the anti-BNP scFv was expressed, 73.4% of expressed antibodies formed insoluble inclusion bodies. In this study, molecular chaperones were coexpressed with anti-BNP scFv with the goal of improving the production of functional anti-BNP in the cytoplasm of E. coli. Five sets of molecular chaperones were assessed for their effects on the production of active anti-BNP scFv. These sets included the following: trigger factor (TF); groES/groEL; groES/groEL/TF; dnaK/dnaJ/grpE; groES/groEL/dnaK/dnaJ/grpE. Of these chaperones, the coexpression of anti-BNP scFv with the groES/groEL chaperones encoded in plasmid pGro7 exhibited the most efficient functional expression of anti-BNP scFv as an active form. Coexpressed with the groES/groEL chaperones, 64.9% of the total anti-BNP scFv was produced in soluble form, which is 2.4 times higher scFv than that of anti-BNP scFv expressed without molecular chaperones, and the relative binding activity was 1.5-fold higher. The optimal concentration of L-arabinose required for induction of the groES/groEL chaperone set was determined to be 1.0 mM and relative binding activity was 3.5 times higher compared with that of no induction with L-arabinose. In addition, soluble anti-BNP scFv was increased from 11.5 to 31.4 μg/ml with optimized inducer concentration (1.0 mM L-arabinose) for the coexpression of the groES/groEL chaperones. These results demonstrate that the functional expression of anti-BNP scFv can be improved by coexpression of molecular chaperones, as molecular chaperones can identify and help to refold improperly folded anti-BNP scFv. PMID:25187138

  6. Expression of bioactive anti-CD20 antibody fragments and induction of ER stress response in Arabidopsis seeds.

    PubMed

    Wang, Dezhong; Ma, Jisheng; Sun, Difei; Li, Haiyan; Jiang, Chao; Li, Xiaokun

    2015-08-01

    Seed-based expression system is an attractive platform for the production of recombinant proteins in molecular farming. Despite the many advantages of molecular farming, little is known about the effect of the different subcellular accumulation of recombinant proteins on the endoplasmic reticulum (ER) quality control system in host plants. In this study, we analyzed the expression of anti-CD20 antibody fragments in seeds of Arabidopsis thaliana (ecotype Columbia) and corresponding glycosylation mutants, and evaluated the influence of three different signal sequences on the expression levels of scFv-Fc of C2B8. The highest protein accumulation level, with a maximum of 6.12 % total soluble proteins, was observed upon fusing proteins to the signal peptide of Arabidopsis seed storage albumin 2. The ER stress responses in developing seeds at 13 days post-anthesis were also compared across different transgenic lines under normal and heat shock conditions. Based on the gene expression profiles of ER stress transducers, our results suggest that accumulation of antibody fragments in the ER exerts more stress on ER homeostasis. In addition, quantitative PCR results also implicate enhanced activation of ER-associated degradation in transgenic lines. Last but not the least, we also demonstrate the anti-tumor potency of plant-derived proteins by showing the anti-tumor activity of purified scFv-Fc proteins against Daudi cells. Together, our data implies that better understanding of the interaction between exogenous protein production and the cellular quality control system of the host plant is necessary for the development of an optimal expression strategy that will be especially beneficial to commercial protein manufacturing. PMID:25957150

  7. Extreme scale-down of expanded bed adsorption: Purification of an antibody fragment directly from recombinant E. coli culture.

    PubMed

    Willoughby, Nik; Martin, Phillip; Titchener-Hooker, Nigel

    2004-09-01

    Scale-down is a methodology that combines the use of very small volumes of process fluid in dedicated devices to predict accurately the behaviour of process-scale biotechnological unit operations and for the production of comparable material for use in further devices which, taken together, facilitate the mimic of a complete full-scale process. This article provides the rationale behind the development of a small-scale mimic and demonstrates the use of a highly scaled-down expanded bed to predict hydrodynamic, kinetic, and adsorptive performance using less than 5-mL sample volumes. Data acquired on a specially developed 1.9 mm ID column was compared with that obtained in a standard 25 mm ID column. A homogenised E. coli system expressing an antibody fragment (F(ab)) adsorbed onto an rProtein A matrix was used to characterise the full adsorptive performance. Breakthrough curve studies using BSA in buffer were used to characterise binding kinetics. Performance at the two scales was comparable both in terms of expansion, axial dispersion, binding isotherms, and elution behaviour of the antibody fragment. The eluted F(ab) material was further purified by ion exchange chromatography to demonstrate the similarity between the profile of the product material obtained at both scales. The high level of scale-down (approximately 200-fold) provides for rapid process evaluation early in development, where material is at a premium and where a fast appreciation of the likely merits of one process strategy will lead to greater confidence in process selection and more robust flowsheets. PMID:15352062

  8. Datasets of a novel bivalent single chain antibody constructed by overlapping oligonucleotide annealing method targeting human CD123.

    PubMed

    Moradi-Kalbolandi, Shima; Habibi-Anbouhi, Mahdi; Golkar, Majid; Behdani, Mahdi; Rezaei, Gashin; Ghazizadeh, Leila; Abolhassani, Mohsen; Shokrgozar, Mohammad Ali

    2016-09-01

    Current therapies for acute myeloid leukemia (AML), are associated with high relapse rates. Hence, development of new therapeutic strategies is crucial to circumvent this problem. Bivalent antibody technology has been used to engineer novel antibody fragments with increased avidity, by assembling two scFv in a single molecule. Here, we present accompanying data from construction and characterization experiments of a biscFv antibody targeting CD123, the most important biomarker of leukemic cancer stem cells which play a key role in relapsed AML after chemotherapy. Data in this article are related to the research paper "Development of a novel engineered antibody targeting human CD123" Moradi-Kalbolandi S. et al. (2016) [1]. PMID:27536714

  9. Specific Conjugation of the Hinge Region for Homogeneous Preparation of Antibody Fragment-Drug Conjugate: A Case Study for Doxorubicin-PEG-anti-CD20 Fab' Synthesis.

    PubMed

    Zhou, Zhan; Zhang, Jing; Zhang, Yan; Ma, Guanghui; Su, Zhiguo

    2016-01-20

    Conventional preparation strategies for antibody-drug conjugates (ADCs) result in heterogeneous products with various molecular sizes and species. In this study, we developed a homogeneous preparation strategy by site-specific conjugation of the anticancer drug with an antibody fragment. The model drug doxorubicin (DOX) was coupled to the Fab' fragment of anti-CD20 IgG at its permissive sites through a heterotelechelic PEG linker, generating an antibody fragment-drug conjugate (AFDC). Anti-CD20 IgG was digested and reduced specifically with β-mercaptoethylamine to generate the Fab' fragment with two free mercapto groups in its hinge region. Meanwhile, DOX was conjugated with α-succinimidylsuccinate ω-maleimide polyethylene glycol (NHS-PEG-MAL) to form MAL-PEG-DOX, which was subsequently linked to the free mercapto containing Fab' fragment to form a Fab'-PEG-DOX conjugate. The dual site-specific bioconjugation was achieved through the combination of highly selective reduction of IgG and introduction of heterotelechelic PEG linker. The resulting AFDC provides an utterly homogeneous product, with a definite ratio of one fragment to two drugs. Laser confocal microscopy and cell ELISA revealed that the AFDC could accumulate in the antigen-positive Daudi tumor cell. In addition, the Fab'-PEG-DOX retained appreciable targeting ability and improved antitumor activity, demonstrating an excellent therapeutic effect on the lymphoma mice model for better cure rate and significantly reduced side effects. PMID:26700095

  10. Specific Targeting of a Naturally Presented Osteosarcoma Antigen, Papillomavirus Binding Factor Peptide, Using an Artificial Monoclonal Antibody*

    PubMed Central

    Tsukahara, Tomohide; Emori, Makoto; Murata, Kenji; Hirano, Takahisa; Muroi, Norihiro; Kyono, Masanori; Toji, Shingo; Watanabe, Kazue; Torigoe, Toshihiko; Kochin, Vitaly; Asanuma, Hiroko; Matsumiya, Hiroshi; Yamashita, Keiji; Himi, Tetsuo; Ichimiya, Shingo; Wada, Takuro; Yamashita, Toshihiko; Hasegawa, Tadashi; Sato, Noriyuki

    2014-01-01

    Osteosarcoma is a rare but highly malignant tumor occurring most frequently in adolescents. The prognosis of non-responders to chemotherapy is still poor, and new treatment modalities are needed. To develop peptide-based immunotherapy, we previously identified autologous cytotoxic T lymphocyte-defined osteosarcoma antigen papillomavirus binding factor (PBF) in the context of HLA-B55 and the cytotoxic T lymphocyte epitope (PBF A2.2) presented by HLA-A2. PBF and HLA class I are expressed in ∼90 and 70% of various sarcomas, respectively. However, the expression status of peptide PBF A2.2 presented by HLA-A2 on osteosarcoma cells has remained unknown because it is difficult to generate a specific probe that reacts with the HLA·peptide complex. For detection and qualification of the HLA-A*02:01·PBF A2.2 peptide complex on osteosarcoma cells, we tried to isolate a single chain variable fragment (scFv) antibody directed to the HLA-*A0201·PBF A2.2 complex using a naïve scFv phage display library. As a result, scFv clone D12 with high affinity (KD = 1.53 × 10−9 m) was isolated. D12 could react with PBF A2.2 peptide-pulsed T2 cells and HLA-A2+PBF+ osteosarcoma cell lines and simultaneously demonstrated that the HLA·peptide complex was expressed on osteosarcoma cells. In conclusion, scFv clone D12 might be useful to select candidate patients for PBF A2.2 peptide-based immunotherapy and develop antibody-based immunotherapy. PMID:24962571

  11. Intranuclear Delivery of a Novel Antibody-Derived Radiosensitizer Targeting the DNA-Dependent Protein Kinase Catalytic Subunit

    SciTech Connect

    Xiong Hairong; Lee, Robert J.; Haura, Eric B.; Edwards, John G.; Dynan, William S.; Li Shuyi

    2012-07-01

    Purpose: To inhibit DNA double-strand break repair in tumor cells by delivery of a single-chain antibody variable region fragment (ScFv 18-2) to the cell nucleus. ScFv 18-2 binds to a regulatory region of the DNA-dependent protein kinase (DNA-PK), an essential enzyme in the nonhomologous end-joining pathway, and inhibits DNA end-joining in a cell-free system and when microinjected into single cells. Development as a radiosensitizer has been limited by the lack of a method for intranuclear delivery to target cells. We investigated a delivery method based on folate receptor-mediated endocytosis. Methods and Materials: A recombinant ScFv 18-2 derivative was conjugated to folate via a scissile disulfide linker. Folate-ScFv 18-2 was characterized for its ability to be internalized by tumor cells and to influence the behavior of ionizing radiation-induced repair foci. Radiosensitization was measured in a clonogenic survival assay. Survival curves were fitted to a linear-quadratic model, and between-group differences were evaluated by an F test. Sensitization ratios were determined based on mean inhibitory dose. Results: Human KB and NCI-H292 lung cancer cells treated with folate-conjugated ScFv 18-2 showed significant radiosensitization (p < 0.001). Sensitization enhancement ratios were 1.92 {+-} 0.42 for KB cells and 1.63 {+-} 0.13 for NCI-H292 cells. Studies suggest that treatment inhibits repair of radiation-induced DSBs, as evidenced by the persistence of {gamma}-H2AX-stained foci and by inhibition of staining with anti-DNA-PKcs phosphoserine 2056. Conclusions: Folate-mediated endocytosis is an effective method for intranuclear delivery of an antibody-derived DNA repair inhibitor.

  12. A Highly Functional Synthetic Phage Display Library Containing over 40 Billion Human Antibody Clones

    PubMed Central

    Weber, Marcel; Bujak, Emil; Putelli, Alessia; Villa, Alessandra; Matasci, Mattia; Gualandi, Laura; Hemmerle, Teresa; Wulhfard, Sarah; Neri, Dario

    2014-01-01

    Several synthetic antibody phage display libraries have been created and used for the isolation of human monoclonal antibodies. The performance of antibody libraries, which is usually measured in terms of their ability to yield high-affinity binding specificities against target proteins of interest, depends both on technical aspects (such as library size and quality of cloning) and on design features (which influence the percentage of functional clones in the library and their ability to be used for practical applications). Here, we describe the design, construction and characterization of a combinatorial phage display library, comprising over 40 billion human antibody clones in single-chain fragment variable (scFv) format. The library was designed with the aim to obtain highly stable antibody clones, which can be affinity-purified on protein A supports, even when used in scFv format. The library was found to be highly functional, as >90% of randomly selected clones expressed the corresponding antibody. When selected against more than 15 antigens from various sources, the library always yielded specific and potent binders, at a higher frequency compared to previous antibody libraries. To demonstrate library performance in practical biomedical research projects, we isolated the human antibody G5, which reacts both against human and murine forms of the alternatively spliced BCD segment of tenascin-C, an extracellular matrix component frequently over-expressed in cancer and in chronic inflammation. The new library represents a useful source of binding specificities, both for academic research and for the development of antibody-based therapeutics. PMID:24950200

  13. Novel human 3-domain disulfide-stabilized antibody fragment against glycoprotein of rabies virus.

    PubMed

    Cai, Kun; Wang, Hui; Bao, Shizhong; Shi, Jing; Hou, Xiaojun; Gao, Xiang; Liu, Hao; Yin, Jun

    2008-04-01

    Mutated disulfide bond sites VH (Cys44) and VL (Cys100) were constructed in variable domains (Fvs) of the human anti-glycoprotein antigen of the rabies virus (anti-GPRV), and the light chain variable (VL) and heavy chain variable (VH) fragments were linked using the heavy chain constant region 1 (CH1) of the human immunoglobulin (Ig) to successfully construct a 3-domain disulfide-stabilized fragment of variables (3d-dsFv). 3d-dsFv was mainly expressed as an inclusion body. After refolding by the conventional dilution method, 3d-dsFv was purified using a nickel-nitrilotriacetic acid (Ni-NTA) column. Enyzme-linked immunosorbent assay (ELISA) was used to determine the binding activity of 3d-dsFv to GPRV. Flow cytometry studies and rapid fluorescent focus inhibition test were used to evaluate the function of 3d-dsFv. The results showed that the stability of 3d-dsFv was improved notably in some aspects such as thermal kinetics, ability to withstand urea denaturation, etc. 3d-dsFv could bind specially to infective cells and the GPRV. The titration of 3d-dsFv to RV-CVS is 83.3 IU/mg, and it can easily reach 2.5IU/mL, which is the value suggested by the WHO as effective for neutralization titration of the rabies virus. PMID:18424153

  14. Neutralization Analysis of a Chicken Single-Chain Variable Fragment Derived from an Immune Antibody Library Against Infectious Bronchitis Virus.

    PubMed

    Lin, Yuan; Li, Benqiang; Ye, Jiaxin; Wang, Man; Wang, Jianhua; Zhang, Ying; Zhu, Jianguo

    2015-09-01

    Avian infectious bronchitis virus (IBV), which is prevalent in many countries causing severe economic loss to the poultry industry, causes infectious bronchitis (IB) in birds. Recombinant single-chain variable fragments (scFvs) have been proven to effectively inhibit many viruses, both in vitro and in vivo, and they could be a potential diagnostic and therapeutic reagent to control IB. In this study, six anti-IBV chicken scFvs, ZL.10, ZL.64, ZL.78, ZL.80, ZL.138, and ZL.256, were obtained by screening random clones from an immune antibody library. An analysis of nucleotide sequences revealed that they represented distinctive genetic sequences and greatly varied in complementarity-determining region three of the heavy chain. Neutralization tests showed that ZL.10, which bound the S1 protein in western blots, inhibited the formation of syncytia in Vero cells 48 h post IBV infection and decreased the transcriptional level of nucleoprotein mRNA to 17.2%, while the other five scFvs, including ZL.78 and ZL.256, that bound the N protein did not. In conclusion, the results suggested that specific and neutralizing chicken scFvs against IBV, which can be safe and economical antibody reagents, can be produced in vitro through prokaryotic expression. PMID:26090700

  15. Methods of preparing and using single chain anti-tumor antibodies

    SciTech Connect

    Cheung, Nai-Kong; Guo, Hong-Fen

    2010-02-23

    This invention provides a method for identifying cells expressing a target single chain antibody (scFv) directed against a target antigen from a collection of cells that includes cells that do not express the target scFv, comprising the step of combining the collection of cells with an anti-idiotype directed to an antibody specific for the target antigen and detecting interaction, if any, of the anti-idiotype with the cells, wherein the occurrence of an interaction identifies the cell as one which expresses the target scFv. This invention also provides a method for making a single chain antibody (scFv) directed against an antigen, wherein the selection of clones is made based upon interaction of those clones with an appropriate anti-idiotype, and heretofore inaccessible scFv so made. This invention provides the above methods or any combination thereof. Finally, this invention provides various uses of these methods.

  16. Selective targeting of tumour neovasculature by a radiohalogenated human antibody fragment specific for the ED-B domain of fibronectin.

    PubMed

    Demartis, S; Tarli, L; Borsi, L; Zardi, L; Neri, D

    2001-04-01

    Angiogenesis is a characteristic feature of many aggressive tumours and other disorders. Antibodies capable of binding to new blood vessels, but not to mature vessels, could be used as selective targeting agents for immunoscintigraphic and radioimmunotherapeutic applications. Here we show that scFv(L19), a recombinant human antibody fragment with sub-nanomolar affinity for the ED-B domain of fibronectin, a marker of angiogenesis, can be stably labelled with iodine-125 and astatine-211 with full retention of immunoreactivity, using a trimethyl-stannyl benzoate bifunctional derivative. Biodistribution studies in mice bearing two different types of tumour grafted subcutaneously, followed by ex vivo micro-autoradiographic analysis, revealed that scFv(L19) rapidly localises around tumour blood vessels, but not around normal vessels. Four hours after intravenous injection of the stably radioiodinated scFv(L19), tumour to blood ratios were 6:1 in mice bearing the F9 murine teratocarcinoma and 9:1 in mice bearing an FE8 rat sarcoma. As expected, all other organs (including kidney) contained significantly less radioactivity than the tumour. Since the ED-B domain of fibronectin has an identical sequence in mouse and man, scFv(L19) is a pan-species antibody and the results presented here suggest clinical utility of radiolabelled scFv(L19) for the scintigraphic detection of angiogenesis in vivo. Furthermore, it should now be possible to investigate scFv(L19) for the selective delivery of 211At to the tumour neovasculature, causing the selective death of tumour endothelial cells and tumour collapse. PMID:11357506

  17. Crystal Structure of Snake Venom Acetylcholinesterase in Complex with Inhibitory Antibody Fragment Fab410 Bound at the Peripheral Site

    PubMed Central

    Bourne, Yves; Renault, Ludovic; Marchot, Pascale

    2015-01-01

    The acetylcholinesterase found in the venom of Bungarus fasciatus (BfAChE) is produced as a soluble, non-amphiphilic monomer with a canonical catalytic domain but a distinct C terminus compared with the other vertebrate enzymes. Moreover, the peripheral anionic site of BfAChE, a surface site located at the active site gorge entrance, bears two substitutions altering sensitivity to cationic inhibitors. Antibody Elec410, generated against Electrophorus electricus acetylcholinesterase (EeAChE), inhibits EeAChE and BfAChE by binding to their peripheral sites. However, both complexes retain significant residual catalytic activity, suggesting incomplete gorge occlusion by bound antibody and/or high frequency back door opening. To explore a novel acetylcholinesterase species, ascertain the molecular bases of inhibition by Elec410, and document the determinants and mechanisms for back door opening, we solved a 2.7-Å resolution crystal structure of natural BfAChE in complex with antibody fragment Fab410. Crystalline BfAChE forms the canonical dimer found in all acetylcholinesterase structures. Equally represented open and closed states of a back door channel, associated with alternate positions of a tyrosine phenol ring at the active site base, coexist in each subunit. At the BfAChE molecular surface, Fab410 is seated on the long Ω-loop between two N-glycan chains and partially occludes the gorge entrance, a position that fully reflects the available mutagenesis and biochemical data. Experimentally based flexible molecular docking supports a similar Fab410 binding mode onto the EeAChE antigen. These data document the molecular and dynamic peculiarities of BfAChE with high frequency back door opening, and the mode of action of Elec410 as one of the largest peptidic inhibitors targeting the acetylcholinesterase peripheral site. PMID:25411244

  18. Biokinetics of a radioiodinated antibreast carcinoma monoclonal antibody and fragment in humans

    SciTech Connect

    Zalutsky, M.R.; Noska, M.; Kaplan, W.D.; Hayes, D.; Colcher, D.; Schlom, J.; Kufe, D.

    1984-01-01

    Monoclonal antibody B6.2 is promising for the detection of breast cancers; it binds to >80% of human breast carcinoma (hbc) lines, its antigen is not seen in serum, and it localizes selectively in hbc xenografts in nude mice. The authors have studied the biokinetics of I-131 activity following injection of I-131-IgG and F(ab')/sub 2/ each in 4 patients (pts). The antibody was labeled using iodogen and tested for specific binding to hbc membrane extracts prior to injection. Pts received 0.6-1.1 mCi of I-131 and 50-100 ..mu..g of protein. Blood clearance of I-131 activity was biphasic with half times of 2 and 15.4 hrs for IgG; 1 and 30 hrs for F(ab')/sub 2/. Dehalogenation was noted: by 72 hrs post-injection, 22% (IgG) and 21% (F(ab')/sub 2/) of the injected dose of I-131 was found in the urine. In 2 pts receiving I-131 IgG, stomach uptake was 7-10% at 24 hrs. Protein associated activity in the blood was >90% for the first 8 hrs and gradually decreased to 79% (IgG) and 58% (F(ab')/sub 2/) at 48 hrs. High liver uptake, reported with other antibody systems, was not observed; <20% of the activity was seen in the liver at all time points for both proteins. In 1/4 pts receiving IgG and 4/4 receiving F(ab')/sub 2/, bone marrow uptake was clearly noted. In these pts, >20% of the activity present in blood was cell associated. This is not inconsistent with the observation that B6.2 binds to granulocytes in vitro. Increased binding to cells in the blood for F(ab')/sub 2/ probably accounts for the anomolously longer blood clearance half times observed for F(ab')/sub 2/ vs IgG and low liver accumulation most likely reflects the absence of hepatic or circulating B6.2 antigen.

  19. Antibody

    MedlinePlus

    An antibody is a protein produced by the body's immune system when it detects harmful substances, called antigens. Examples ... microorganisms (bacteria, fungi, parasites, and viruses) and chemicals. Antibodies may be produced when the immune system mistakenly ...

  20. Targeting melanoma with immunoliposomes coupled to anti-MAGE A1 TCR-like single-chain antibody

    PubMed Central

    Saeed, Mesha; van Brakel, Mandy; Zalba, Sara; Schooten, Erik; Rens, Joost AP; Koning, Gerben A; Debets, Reno; ten Hagen, Timo LM

    2016-01-01

    Therapy of melanoma using T-cells with genetically introduced T-cell receptors (TCRs) directed against a tumor-selective cancer testis antigen (CTA) NY-ESO1 demonstrated clear antitumor responses in patients without side effects. Here, we exploited the concept of TCR-mediated targeting through introduction of single-chain variable fragment (scFv) antibodies that mimic TCRs in binding major histocompatibility complex-restricted CTA. We produced scFv antibodies directed against Melanoma AntiGEn A1 (MAGE A1) presented by human leukocyte antigen A1 (HLA-A1), in short M1/A1, and coupled these TCR-like antibodies to liposomes to achieve specific melanoma targeting. Two anti-M1/A1 antibodies with different ligand-binding affinities were derived from a phage-display library and reformatted into scFvs with an added cysteine at their carboxyl termini. Protein production conditions, ie, bacterial strain, temperature, time, and compartments, were optimized, and following production, scFv proteins were purified by immobilized metal ion affinity chromatography. Batches of pure scFvs were validated for specific binding to M1/A1-positive B-cells by flow cytometry. Coupling of scFvs to liposomes was conducted by employing different conditions, and an optimized procedure was achieved. In vitro experiments with immunoliposomes demonstrated binding of M1/A1-positive B-cells as well as M1/A1-positive melanoma cells and internalization by these cells using flow cytometry and confocal microscopy. Notably, the scFv with nonenhanced affinity of M1/A1, but not the one with enhanced affinity, was exclusively bound to and internalized by melanoma tumor cells expressing M1/A1. Taken together, antigen-mediated targeting of tumor cells as well as promoting internalization of nanoparticles by these tumor cells is mediated by TCR-like scFv and can contribute to melanoma-specific targeting. PMID:27022262

  1. Purification, characterization, and biotinylation of single-chain antibodies.

    PubMed

    Kipriyanov, S M

    1998-01-01

    The variable region (Fv) portion of an antibody is comprised of the antibody V(H) and V(L) domains and is the smallest antibody fragment containing a complete antigen-binding site. To stabilize the association of the recombinant V(H) and V(L) domains, they have been linked in single-chain Fv constructs with a short peptide that bridges the approx 3.5 nm between the carboxy terminus of one domain and the ammo terminus of the other (1-3). An NMR comparison of the unlinked Fv fragment of the antibody McPC603 with the corresponding scFv containing a V(H)-(Gly(4)Ser)(3)-V(L) linker has shown no perturbation of the folding of the variable domains by the linker (4,5). In comparison to the much larger Fab', F(ab')(2), and IgG forms of monoclonal antibody (MAb) from which they are derived, scFvs have lower retention times in nontarget tissues, more rapid blood clearance, and better tumor penetration (6-8). ScFvs, therefore, represent potentially very useful molecules for the targeted delivery of drugs, toxins, or radionuclides to a tumor site. PMID:21390870

  2. STRUCTURE BASED DESIGN OF PROTEIN LIGANDS: A STUDY OF ANTIBODY-LIKE SCAFFOLDS TARGETED AGAINST THE ANTHRAX TOXIN

    SciTech Connect

    P. SHIFLETT; E. HONG-GELLER; ET AL

    2000-12-01

    We have adopted structure-based approaches to enhance the affinities of two single chain antibodies, scFv1 and scFv4, that bind to two different epitopes on the Protective Antigen (PA), a toxin from Bacillus anthracis. In one approach, we have modified scFv4 and re-engineered a novel antibody-like scaffold in which we have placed V{sub L} on the N terminus and V{sub H} on the C-terminus and joined them by a 10 amino-acid-long linker. This scaffold preserves the native V{sub L}-V{sub H} contact interface and the dispositions of the CDR loops. It binds to PA with 10 fold higher affinity than scFv4. In a second approach, we have created a bispecific ligand by covalently joining scFv1 and scFv4 by a flexible linker that supports simultaneous and synergistic binding of the two scFvs to PA. This bispecific scFv1-linker-scFv4 binds to PA with 10 fold higher affinity than the individual scFvs. The newly re-engineered antibody-like scaffold of scFv4 and scFv1-linker-scFv4 are expected to be potent inhibitors of PA binding to the host cells.

  3. Cytokine Activation by Antibody Fragments Targeted to Cytokine-Receptor Signaling Complexes.

    PubMed

    Kuruganti, Srilalitha; Miersch, Shane; Deshpande, Ashlesha; Speir, Jeffrey A; Harris, Bethany D; Schriewer, Jill M; Buller, R Mark L; Sidhu, Sachdev S; Walter, Mark R

    2016-01-01

    Exogenous cytokine therapy can induce systemic toxicity, which might be prevented by activating endogenously produced cytokines in local cell niches. Here we developed antibody-based activators of cytokine signaling (AcCS), which recognize cytokines only when they are bound to their cell surface receptors. AcCS were developed for type I interferons (IFNs), which induce cellular activities by binding to cell surface receptors IFNAR1 and IFNAR2. As a potential alternative to exogenous IFN therapy, AcCS were shown to potentiate the biological activities of natural IFNs by ∼100-fold. Biochemical and structural characterization demonstrates that the AcCS stabilize the IFN-IFNAR2 binary complex by recognizing an IFN-induced conformational change in IFNAR2. Using IFN mutants that disrupt IFNAR1 binding, AcCS were able to enhance IFN antiviral potency without activating antiproliferative responses. This suggests AcCS can be used to manipulate cytokine signaling for basic science and possibly for therapeutic applications. PMID:26546677

  4. Controlling Rotavirus-associated diarrhea: Could single-domain antibody fragments make the difference?

    PubMed

    Maffey, Lucia; Vega, Celina G; Parreño, Viviana; Garaicoechea, Lorena

    2015-01-01

    Group A Rotavirus (RVA) remains a leading cause of severe diarrhea and child mortality. The variable domain of camelid heavy chain antibodies (VHH) display potent antigen-binding capacity, have low production costs and are suitable for oral therapies. Two sets of anti-RVA VHHs have been developed: ARP1-ARP3; 2KD1-3B2. Here, we explore the potential of both sets as a prevention strategy complementary to vaccination and a treatment option against RVA-associated diarrhea in endangered populations. Both sets have been expressed in multiple production systems, showing extensive neutralizing capacity against strains of RVA in vitro. They were also tested in the neonatal mouse model with various degrees of success in preventing or treating RVA-induced diarrhea. Interestingly, mitigation of the symptoms was also achieved with freeze-dried ARP1, so that it could be applied in areas where cold chains are difficult to maintain. 3B2 was tested in a pre-clinical trial involving gnotobiotic piglets where it conferred complete protection against RVA-induced diarrhea. ARP1 was used in the first clinical trial for anti-RVA VHHs, successfully reducing stool output in infants with RVA diarrhea, with no detected side effects. PMID:26654700

  5. Genetically engineered multivalent single chain antibody constructs for cancer therapy

    SciTech Connect

    Surinder Batra, Ph D

    2006-02-27

    its tumor: normal tissue ratio for improved therapeutic index, we engineered a variety antibody constructs. These constructs were evaluated using novel approaches like special radionuclides, pretargeting and optimization. Due to the smaller size, the engineered antibody molecules should penetrate better throughout a tumor mass, with less dose heterogeneity, than is the case with intact IgG. Multivalent scFvs with an appropriate radionuclide, therefore, hold promising prospects for cancer therapy and clinical imaging in MAb-based radiopharmaceuticals. In addition, the human anti-mouse antibodies (HAMA) responses in patients against antibody-based therapy are usually directed against the immunoglobulin constant regions; however, anti-idiotypic responses can also be detected. The HAMA responses reduce the efficacy of treatment by removing the circulating antibody molecules, fragments, and possibly scFvs by altering the pharmacokinetic properties of the antibody. HAMA responses against divalent IgG, divalent Ig fragments, and possibly multimeric scFvs could cause immune complex formation with hypersensitivity or allergic reactions that could be harmful to patients. The use of small molecules, such as scFvs (monomeric as well as multimeric), with their shorter biological half-lives and the lack of the constant regions and humanized variable (binding regions) performed in our studies should reduce the development of HAMA. The generation of humanized and fully human scFvs should further reduce the development of HAMA. Specific accomplishments on the project are the production of large amounts of recombinant antibodies as they are required in large amounts for cancer diagnosis and therapy. A variety of single-chain Fv (scFv) constructs were engineered for the desired pharmacokinetic properties. Tetrameric and dimeric scFvs showed a two-fold advantage: (1) there was a considerable gain in avidity as compared to smaller fragments, and (2) the biological half-life was more

  6. Enhanced glutathione PEGylated liposomal brain delivery of an anti-amyloid single domain antibody fragment in a mouse model for Alzheimer's disease.

    PubMed

    Rotman, Maarten; Welling, Mick M; Bunschoten, Anton; de Backer, Maaike E; Rip, Jaap; Nabuurs, Rob J A; Gaillard, Pieter J; van Buchem, Mark A; van der Maarel, Silvère M; van der Weerd, Louise

    2015-04-10

    Treatment of neurodegenerative disorders such as Alzheimer's disease is hampered by the blood-brain barrier (BBB). This tight cerebral vascular endothelium regulates selective diffusion and active transport of endogenous molecules and xenobiotics into and out of the brain parenchyma. In this study, glutathione targeted PEGylated (GSH-PEG) liposomes were designed to deliver amyloid-targeting antibody fragments across the BBB into the brain. Two different formulations of GSH-PEG liposomes based on 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and egg-yolk phosphatidylcholine (EYPC) were produced. Both formulations encapsulate 15kDa amyloid beta binding llama single domain antibody fragments (VHH-pa2H). To follow the biodistribution of VHH-pa2H rather than the liposome, the antibody fragment was labeled with the radioisotope indium-111. To prolong the shelf life of the construct beyond the limit of radioactive decay, an active-loading method was developed to efficiently radiolabel the antibody fragments after encapsulation into the liposomes, with radiolabeling efficiencies of up to 68% after purification. The radiolabeled liposomes were administered via a single intravenous bolus injection to APPswe/PS1dE9 double transgenic mice, a mouse model of Alzheimer's disease, and their wildtype littermates. Both GSH-PEG DMPC and GSH-PEG EYPC liposomes significantly increased the standard uptake values (SUV) of VHH-pa2H in the blood of the animals compared to free VHH-pa2H. Encapsulation in GSH-PEG EYPC liposomes resulted in the highest increase in SUV in the brains of transgenic animals. Overall, these data provide evidence that GSH-PEG liposomes may be suitable for specific delivery of single domain antibody fragments over the BBB into the brain. PMID:25668771

  7. Immobilization strategies for single-chain antibody microarrays

    SciTech Connect

    Seurynck-Servoss, Shannon L.; Baird, Cheryl L.; Miller, Keith D.; Pefaur, Noah B.; Gonzalez, Rachel M.; Apiyo, David O.; Engelmann, Heather E.; Srinivastava, Sudhir; Kagan, Jacob; Rodland, Karin D.; Zangar, Richard C.

    2008-06-01

    Sandwich enzyme-linked immunosorbent assay (ELISA) microarrays have great potential for validating biomarkers of disease. ELISA relies on robust affinity reagents that retain activity when immobilized or when labeled for detection. Single-chain antibodies (scFv) are affinity reagents that have greater potential for high-throughput production than traditional immunoglobin G (IgG). Unfortunately, scFv are typically less stabile than IgG and not always suitable for use in sandwich ELISAs. We therefore investigated different immobilization strategies and scFv structural modifications to see if we could develop a more robust scFv reagent. Two promising strategies that emerged from these studies: 1) the precapture of epitope-tagged scFv using an anti-epitope antibody and 2) the direct printing of a thioredoxin/scFv fusion protein on glass slides. The use of either strategy improved the stability of immobilized scFv and increased the sensitivity of the scFv ELISA microarray assays, although the anti-epitope precapture method had a risk of reagent transfer. Using the direct printing method, we show that anti-PSA scFv are highly specific when tested against 21 different IgG-based assays. Finally, the scFv microarray PSA assay gave comparable results (R2 = 0.95) to a commercial 96-well ELISA when tested using serum samples. Overall, these results suggest that minor modifications of the scFv protein structure are sufficiently to produce reagents that are suitable for use in multiplex assay systems.

  8. A Fully Human Inhibitory Monoclonal Antibody to the Wnt Receptor RYK

    PubMed Central

    Parish, Clare L.; Takano, Elena A.; Fox, Stephen; Layton, Daniel; Nice, Edouard; Stacker, Steven A.

    2013-01-01

    RYK is an unusual member of the receptor tyrosine kinase (RTK) family that is classified as a putative pseudokinase. RYK regulates fundamental biological processes including cell differentiation, migration and target selection, axon outgrowth and pathfinding by transducing signals across the plasma membrane in response to the high affinity binding of Wnt family ligands to its extracellular Wnt inhibitory factor (WIF) domain. Here we report the generation and initial characterization of a fully human inhibitory monoclonal antibody to the human RYK WIF domain. From a naïve human single chain fragment variable (scFv) phage display library, we identified anti-RYK WIF domain–specific scFvs then screened for those that could compete with Wnt3a for binding. Production of a fully human IgG1κ from an inhibitory scFv yielded a monoclonal antibody that inhibits Wnt5a-responsive RYK function in a neurite outgrowth assay. This antibody will have immediate applications for modulating RYK function in a range of settings including development and adult homeostasis, with significant potential for therapeutic use in human pathologies. PMID:24058687

  9. A fully human inhibitory monoclonal antibody to the Wnt receptor RYK.

    PubMed

    Halford, Michael M; Macheda, Maria L; Parish, Clare L; Takano, Elena A; Fox, Stephen; Layton, Daniel; Nice, Edouard; Stacker, Steven A

    2013-01-01

    RYK is an unusual member of the receptor tyrosine kinase (RTK) family that is classified as a putative pseudokinase. RYK regulates fundamental biological processes including cell differentiation, migration and target selection, axon outgrowth and pathfinding by transducing signals across the plasma membrane in response to the high affinity binding of Wnt family ligands to its extracellular Wnt inhibitory factor (WIF) domain. Here we report the generation and initial characterization of a fully human inhibitory monoclonal antibody to the human RYK WIF domain. From a naïve human single chain fragment variable (scFv) phage display library, we identified anti-RYK WIF domain-specific scFvs then screened for those that could compete with Wnt3a for binding. Production of a fully human IgG1κ from an inhibitory scFv yielded a monoclonal antibody that inhibits Wnt5a-responsive RYK function in a neurite outgrowth assay. This antibody will have immediate applications for modulating RYK function in a range of settings including development and adult homeostasis, with significant potential for therapeutic use in human pathologies. PMID:24058687

  10. PET Imaging of CD105/Endoglin Expression with a 61/64Cu-Labeled Fab Antibody Fragment

    PubMed Central

    Zhang, Yin; Hong, Hao; Orbay, Hakan; Valdovinos, Hector F.; Nayak, Tapas R.; Theuer, Charles P.; Barnhart, Todd E.; Cai, Weibo

    2013-01-01

    Purpose The goal of this study was to generate and characterize the Fab fragment of TRC105, a monoclonal antibody that binds with high affinity to human and murine CD105 (i.e. endoglin), and investigate its potential for positron emission tomography (PET) imaging of tumor angiogenesis in a small animal model after 61/64Cu-labeling. Methods TRC105-Fab was generated by enzymatic papain digestion. The integrity and CD105 binding affinity of TRC105-Fab was evaluated before NOTA (i.e., 1,4,7-triazacyclononane-1,4,7-triacetic acid) conjugation and 61/64Cu-labeling. Serial PET imaging and biodistribution studies were carried out in the syngeneic 4T1 murine breast cancer model to quantify tumor targeting efficacy and normal organ distribution of 61/64Cu-NOTA-TRC105-Fab. Blocking studies with unlabeled TRC105 were performed to confirm CD105 specificity of the tracer in vivo. Immunofluorescence staining was also conducted to correlate tracer uptake in the tumor and normal tissues with CD105 expression. Results TRC105-Fab was produced with high purity through papain digestion of TRC105, as confirmed by SDS-PAGE, HPLC analysis, and mass spectrometry. 61/64Cu-labeling of NOTA-TRC105-Fab was achieved with ~50% yield (specific activity: ~44 GBq/µmol). PET imaging revealed rapid uptake of 64Cu-NOTA-TRC105-Fab in the 4T1 tumor (3.6 ± 0.4, 4.2 ± 0.5, 4.9 ± 0.3, 4.4 ± 0.7, and 4.6 ± 0.8 %ID/g at 0.5, 2, 5, 16, and 24 h post-injection respectively; n = 4). Since tumor uptake peaked soon after tracer injection, 61Cu-labeled TRC105-Fab was also able to provide tumor contrast at 3 and 8 h post-injection. CD105 specificity of the tracer was confirmed with blocking studies and histological examination. Conclusion Herein we report PET imaging of CD105 expression with 61/64Cu-NOTA-TRC105-Fab, which exhibited prominent and target specific uptake in the 4T1 tumor. The use of a Fab fragment led to much faster tumor uptake (which peaked at a few hours after tracer injection) compared to

  11. Production, purification, and characterization of scFv TNF ligand fusion proteins.

    PubMed

    Fick, Andrea; Wyzgol, Agnes; Wajant, Harald

    2012-01-01

    Single-chain variable fragments (scFvs) specific for tumor-associated cell surface antigens are the most broadly used reagents to direct therapeutic or diagnostic effector molecules, such as toxins, radioisotopes, and CD3-stimulating scFvs, to tumors. One novel class of effector molecules that can be targeted to tumors by scFvs are ligands of the tumor necrosis factor (TNF) family. Typically, these molecules have apoptosis inducing and/or immune stimulating properties and are therefore highly attractive for cancer treatment. N-terminal fusion of scFvs does not interfere with the receptor binding capabilities of TNF ligands and thus allows the straightforward generation of scFv TNF ligand fusion proteins. We report here a protocol for the purification of eukaryotically produced scFv TNF ligand fusion proteins based on affinity chromatography on anti-Flag agarose and further describe assays for the determination of the targeting index of this type of scFv-targeted proteins. PMID:22907375

  12. Interaction analysis of HIV-1 antibody 2G12 and Man9GlcNAc2 ligand: Theoretical calculations by fragment molecular orbital and MD methods

    NASA Astrophysics Data System (ADS)

    Koyama, Yuka; Ueno-Noto, Kaori; Takano, Keiko

    2013-07-01

    In HIV-1 infection, human antibody 2G12 is capable of recognizing the high-mannose glycans on the HIV-1 surface glycoprotein, gp120. To investigate the ligand binding mechanisms of antibody 2G12 with glycans aiming for the contribution to the medications, we carried out classical molecular dynamics (MD) simulations and ab initio fragment molecular orbital (FMO) calculations on the antibody 2G12 complex with its high-mannose ligand. We found that Mannose D1 of the ligand had the largest binding affinity with the antibody, which was well consistent with experimental reports. Furthermore, significant roles of Mannose 4 and 4‧ in the ligand binding were theoretically indicated.

  13. Labeling monoclonal antibodies and F(ab')2 fragments with the alpha-particle-emitting nuclide astatine-211: preservation of immunoreactivity and in vivo localizing capacity.

    PubMed Central

    Zalutsky, M R; Garg, P K; Friedman, H S; Bigner, D D

    1989-01-01

    alpha-Particles such as those emitted by 211At may be advantageous for radioimmunotherapy since they are radiation of high linear energy transfer, depositing high energy over a short distance. Here we describe a strategy for labeling monoclonal antibodies and F(ab')2 fragments with 211At by means of the bifunctional reagent N-succinimidyl 3-(trimethylstannyl)benzoate. An intact antibody, 81C6, and the F(ab')2 fragment of Me1-14 (both reactive with human gliomas) were labeled with 211At in high yield and with a specific activity of up to 4 mCi/mg in a time frame compatible with the 7.2-hr half-life of 211At. Quantitative in vivo binding assays demonstrated that radioastatination was accomplished with maintenance of high specific binding and affinity. Comparison of the biodistribution of 211At-labeled Me1-14 F(ab')2 to that of a nonspecific antibody fragment labeled with 211At and 131I in athymic mice bearing D-54 MG human glioma xenografts demonstrated selective and specific targeting of 211At-labeled antibody in this human tumor model. PMID:2476813

  14. Three-dimensional reconstruction of a co-complex of F-actin with antibody Fab fragments to actin's NH2 terminus.

    PubMed Central

    Orlova, A; Yu, X; Egelman, E H

    1994-01-01

    We have decorated F-actin with Fab fragments of antibodies to actin residues 1-7. These antibody fragments do not strongly affect the rigor binding of myosin S-1 to actin, but do affect the binding of S-1 to actin in the presence of nucleotide (DasGupta, G., and E. Reisler, 1989. J. Mol. Biol. 207:833-836; 1991. Biochemistry. 30:9961-9966; 1992. Biochemistry. 31:1836-1841). Although the binding constant is rather low, we estimate that we have achieved about 85% occupancy of the actin sites. Three-dimensional reconstructions from electron micrographs of both negatively stained and frozen-hydrated filaments show that the Fab fragment is bound at the location of the NH2 terminus in the model of Holmes et al. (Holmes, K.C., D. Popp, W. Gebhard, and W. Kabsch. 1990. Nature. 347:37-44) for F-actin, excluding very different orientations of the actin subunit in the filament. Most of the mass of the antibody is not visualized, which is due to the large mobility of the NH2 terminus in F-actin, differences in binding angle within the polyclonal antibody population, or a combination of both of these possibilities. Images FIGURE 1 FIGURE 5 FIGURE 7 FIGURE 10 PMID:8161679

  15. Module based antibody engineering: A novel synthetic REDantibody

    PubMed Central

    Markiv, Anatoliy; Anani, Bernard; Durvasula, Ravi V; Kang, Angray S

    2010-01-01

    We describe the facile generation of a stable recombinant antibody with intrinsic red fluorescent properties for qualitative and potentially quantitative immunofluorescence analysis. The REDantibody based on the X-ray crystallographic structures of the anti-sialyl-Tn antibody B72.3 and 3D model of the monomeric red fluorescent protein was designed to retain optimal spatial geometry between the C- and N- termini of the VH and VL chains respectively to mimic the domains interface pairing in antibody Fab fragments and to incorporate the red fluorescent protein as a bridging scaffold. The model was further validated by assembling a REDantibody based on CA19.9 the anti-sialylated Lewis (Le)a blood group antigen and 4D5-8 the anti-p185HER2 antibodies. The chimeric heavy and light chains containing red fluorescent protein as a bridge were correctly processed and secreted into E. coli periplasm for assembly and disulphide bond formation, further analysis revealed the molecules to be exclusively monomers. Purified anti-glycan proteins were used for an immunofluorescent analysis of Trypanosoma cruzi epimastigotes, and the anti-p185HER2 used to determine the binding properties. The REDantibody platform facilitates rapid generation of scFv chimeras that could be used for screening antibodies against cell surface markers. Furthermore, such modular assembly should permit the interchange of binding sites and of fluorophores to create robust panels of coloured antibodies. PMID:21055406

  16. Selection of Ceratitis capitata (Diptera: Tephritidae) Specific Recombinant Monoclonal Phage Display Antibodies for Prey Detection Analysis

    PubMed Central

    Monzó, César; Urbaneja, Alberto; Ximénez-Embún, Miguel; García-Fernández, Julia; García, José Luis; Castañera, Pedro

    2012-01-01

    Several recombinant antibodies against the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), one of the most important pests in agriculture worldwide, were selected for the first time from a commercial phage display library of human scFv antibodies. The specificity and sensitivity of the selected recombinant antibodies were compared with that of a rabbit polyclonal serum raised in parallel using a wide range of arthropod species as controls. The selected recombinant monoclonal antibodies had a similar or greater specificity when compared with classical monoclonal antibodies. The selected recombinant antibodies were successfully used to detect the target antigen in the gut of predators and the scFv antibodies were sequenced and compared. These results demonstrate the potential for recombinant scFv antibodies to be used as an alternative to the classical monoclonal antibodies or even molecular probes in the post-mortem analysis studies of generalist predators. PMID:23272105

  17. Co-Expression of Anti-Rotavirus Proteins (Llama VHH Antibody Fragments) in Lactobacillus: Development and Functionality of Vectors Containing Two Expression Cassettes in Tandem

    PubMed Central

    Günaydın, Gökçe; Álvarez, Beatriz; Lin, Yin; Hammarström, Lennart; Marcotte, Harold

    2014-01-01

    Rotavirus is an important pediatric pathogen, causing severe diarrhea and being associated with a high mortality rate causing approximately 500 000 deaths annually worldwide. Even though some vaccines are currently available, their efficacy is lower in the developing world, as compared to developed countries. Therefore, alternative or complementary treatment options are needed in the developing countries where the disease burden is the largest. The effect of Lactobacillus in promoting health and its use as a vehicle for delivery of protein and antibody fragments was previously shown. In this study, we have developed co-expression vectors enabling Lactobacillus paracasei BL23 to produce two VHH fragments against rotavirus (referred to as anti-rotavirus proteins 1 and 3, ARP1 and ARP3) as secreted and/or surface displayed products. ARP1 and ARP3 fragments were successfully co-expressed as shown by Western blot and flow cytometry. In addition, engineered Lactobacillus produced VHH antibody fragments were shown to bind to a broad range of rotavirus serotypes (including the human rotavirus strains 69M, Va70, F45, DS1, Wa and ST3 and simian rotavirus strains including RRV and SA11), by flow cytometry and ELISA. Hereby, we have demonstrated for the first time that when RRV was captured by one VHH displayed on the surface of co-expressor Lactobacillus, targeting other epitope was possible with another VHH secreted from the same bacterium. Therefore, Lactobacillus producing two VHH antibody fragments may potentially serve as treatment against rotavirus with a reduced risk of development of escape mutants. This co-expression and delivery platform can also be used for delivery of VHH fragments against a variety of mucosal pathogens or production of other therapeutic molecules. PMID:24781086

  18. A novel assay for monitoring internalization of nanocarrier coupled antibodies

    PubMed Central

    Nielsen, Ulrik B; Kirpotin, Dmitri B; Pickering, Edward M; Drummond, Daryl C; Marks, James D

    2006-01-01

    Background Discovery of tumor-selective antibodies or antibody fragments is a promising approach for delivering therapeutic agents to antigen over-expressing cancers. Therefore it is important to develop methods for the identification of target- and function specific antibodies for effective drug delivery. Here we describe a highly selective and sensitive method for characterizing the internalizing potential of multivalently displayed antibodies or ligands conjugated to liposomes into tumor cells. The assay requires minute amounts of histidine-tagged ligand and relies on the non-covalent coupling of these antibodies to fluorescent liposomes containing a metal ion-chelating lipid. Following incubation of cells with antibody-conjugated liposomes, surface bound liposomes are gently removed and the remaining internalized liposomes are quantitated based on fluorescence in a high throughput manner. We have termed this methodology "Chelated Ligand Internalization Assay", or CLIA. Results The specificity of the assay was demonstrated with different antibodies to the ErbB-2 and EGF receptors. Antibody-uptake correlated with receptor expression levels in tumor cell lines with a range of receptor expression. Furthermore, Ni-NTA liposomes containing doxorubicin were used to screen for the ability of antibodies to confer target-specific cytotoxicity. Using an anti-ErbB2 single chain Fv (scFv) (F5) antibody, cytotoxicity could be conferred to ErbB2-overexpressing cells; however, a poly(ethylene glycol)-linked lipid (DSPE-PEG-NTA-Ni) was necessary to allow for efficient loading of the drug and to reduce nonspecific drug leakage during the course of the assay. Conclusion The CLIA method we describe here represents a rapid, sensitive and robust assay for the identification and characterization of tumor-specific antibodies capable of high drug-delivery efficiency when conjugated to liposomal nanocarriers. PMID:17014727

  19. [One amino acid mutation in an anti-CD20 antibody fragment that affects the yield bacterial secretion and the affinity].

    PubMed

    Liu, Yin-Xing; Xiong, Dong-Sheng; Fan, Dong-Mei; Shao, Xiao-Feng; Xu, Yuan-Fu; Zhu, Zhen-Ping; Yang, Chun-Zheng

    2003-05-01

    Monoclonal antibodies (mAb) directed against CD20, either unmodified or in radiolabeled forms, have been successfully exploited in clinic as effective therapeutic agents in the management of non-Hodgkin's B-cell lymphoma. The antibody fragment is a potential agent in image and therapy of tumor. To further improve the soluble expression of anti-CD20 antibody Fab' fragment, PCR was used to mutate the anti-CD20 VL and VH genes and its biological activity was identified. The expression vector of chimeric antibody Fab' was constructed and expressed in E. coli. The data of mutant clone DNA sequence showed that the amino acid of light chain gene of the parent anti-CD20 antibody (H47) was successful mutated as Ser (GAG)-Asn (CAG). The soluble expression of mutated anti-CD20 Fab' (CD20-7) was 3.8 mg/g dry cell weight, while the parent (CD20-2) was 1.3 mg/g dry cell weight. The affinity constant Ka of CD20-7 was 2.2 x 10(9) L/mol. The primary results of competitive assays by FACS showed that CD20-7 could partially block the sites through which parent antibody (HI47) bind to Raji cells. There was difference in the Raji cells (CD20+)-binding activity between the mutant CD20-7 and parent CD20-2. The site mutation of anti-CD20 Fab' gene make it possible that the anti-CD20 antibody fragment was succeeded to obtain higher expression. In this thesis, we succeeded in completing mutation and expression of anti-CD20 Fab' genes, distinguishing its biological activity, and obtaining its highly expression. These period results will lay a foundation for development of other kind of anti-CD20 engineering antibody (for instance: Fab' Diabody and miniantibody), and make it possible for anti-CD20 antibody to be applied to tumor therapy in civil in the future. PMID:15969005

  20. Bone marrow dosimetry in rats using direct tissue counting after injection of radio-iodinated intact monoclonal antibodies or F(ab')2 fragments

    SciTech Connect

    Buchegger, F.; Chalandon, Y.; Pelegrin, A.; Hardman, N.; Mach, J.P. )

    1991-07-01

    Normal rats were injected intravenously with 131I- and 125I-labeled intact murine and chimeric mouse-human monoclonal antibodies directed against carcinoembryonic antigen or with the corresponding F(ab')2 fragments. At different times after injection, individual animals were killed and radioactivity of blood and major organs, including bones and bone marrow, was determined. Ratios comparing radioactivity concentration in different tissues with that of bone marrow were calculated and found to remain stable during several effective half-lives of the antibodies. Mean bone marrow radioactivity was 35% (range, 29%-40%) of that of blood and 126% (range, 108%-147%) of that of liver after injection of intact Mabs or F(ab')2 fragments. In nude rats bearing human colon carcinoma xenografts producing carcinoembryonic antigen, relative bone marrow radioactivity was slightly lower than that in normal rats.

  1. Advances in Immuno–Positron Emission Tomography: Antibodies for Molecular Imaging in Oncology

    PubMed Central

    Knowles, Scott M.; Wu, Anna M.

    2012-01-01

    Identification of cancer cell–surface biomarkers and advances in antibody engineering have led to a sharp increase in the development of therapeutic antibodies. These same advances have led to a new generation of radiolabeled antibodies and antibody fragments that can be used as cancer-specific imaging agents, allowing quantitative imaging of cell-surface protein expression in vivo. Immuno–positron emission tomography (immunoPET) imaging with intact antibodies has shown success clinically in diagnosing and staging cancer. Engineered antibody fragments, such as diabodies, minibodies, and single-chain Fv (scFv) –Fc, have been successfully employed for immunoPET imaging of cancer cell–surface biomarkers in preclinical models and are poised to bring same-day imaging into clinical development. ImmunoPET can potentially provide a noninvasive approach for obtaining target-specific information useful for titrating doses for radioimmunotherapy, for patient risk stratification and selection of targeted therapies, for evaluating response to therapy, and for predicting adverse effects, thus contributing to the ongoing development of personalized cancer treatment. PMID:22987087

  2. High contrast tumor imaging with radio-labeled antibody Fab fragments tailored for optimized pharmacokinetics via PASylation

    PubMed Central

    Mendler, Claudia T; Friedrich, Lars; Laitinen, Iina; Schlapschy, Martin; Schwaiger, Markus; Wester, Hans-Jürgen; Skerra, Arne

    2015-01-01

    Although antigen-binding fragments (Fabs) of antibodies constitute established tracers for in vivo radiodiagnostics, their functionality is hampered by a very short circulation half-life. PASylation, the genetic fusion with a long, conformationally disordered amino acid chain comprising Pro, Ala and Ser, provides a convenient way to expand protein size and, consequently, retard renal filtration. Humanized αHER2 and αCD20 Fabs were systematically fused with 100 to 600 PAS residues and produced in E. coli. Cytofluorimetric titration analysis on tumor cell lines confirmed that antigen-binding activities of the parental antibodies were retained. The radio-iodinated PASylated Fabs were studied by positron emission tomography (PET) imaging and biodistribution analysis in mouse tumor xenograft models. While the unmodified αHER2 and αCD20 Fabs showed weak tumor uptake (0.8% and 0.2% ID/g, respectively; 24 h p.i.) tumor-associated radioactivity was boosted with increasing PAS length (up to 9 and 26-fold, respectively), approaching an optimum for Fab-PAS400. Remarkably, 6- and 5-fold higher tumor-to-blood ratios compared with the unmodified Fabs were measured in the biodistribution analysis (48 h p.i.) for αHER2 Fab-PAS100 and Fab-PAS200, respectively. These findings were confirmed by PET studies, showing high imaging contrast in line with tumor-to-blood ratios of 12.2 and 5.7 (24 h p.i.) for αHER2 Fab-PAS100 and Fab-PAS200. Even stronger tumor signals were obtained with the corresponding αCD20 Fabs, both in PET imaging and biodistribution analysis, with an uptake of 2.8% ID/g for Fab-PAS100 vs. 0.24% ID/g for the unmodified Fab. Hence, by engineering Fabs via PASylation, plasma half-life can be tailored to significantly improve tracer uptake and tumor contrast, thus optimally matching reagent/target interactions. PMID:25484039

  3. PET Imaging of Abdominal Aortic Aneurysm with 64Cu-Labeled Anti-CD105 Antibody Fab Fragment

    PubMed Central

    Shi, Sixiang; Orbay, Hakan; Yang, Yunan; Graves, Stephen A.; Nayak, Tapas R.; Hong, Hao; Hernandez, Reinier; Luo, Haiming; Goel, Shreya; Theuer, Charles P.; Nickles, Robert J.; Cai, Weibo

    2015-01-01

    The critical challenge in abdominal aortic aneurysm (AAA) research is the accurate diagnosis and assessment of AAA progression. Angiogenesis is a pathological hallmark of AAA, and CD105 is highly expressed on newly formed vessels. Our goal was to use 64Cu-labeled anti-CD105 antibody Fab fragment for noninvasive assessment of angiogenesis in the aortic wall in a murine model of AAA. Methods Fab fragment of TRC105, a mAb that specifically binds to CD105, was generated by enzymatic papain digestion and conjugated to NOTA for 64Cu-labeling. Binding affinity/specificity of NOTA-TRC105-Fab was evaluated by flow cytometry and various ex vivo studies. BALB/c mice were anesthetized and treated with calcium phosphate to induce AAA, which underwent weekly PET scans using 64Cu-NOTA-TRC105-Fab. Biodistribution and autoradiography studies were also performed to confirm the accuracy of PET results. Results NOTA-TRC105-Fab exhibited high purity and specifically bound to CD105 in vitro. Uptake of 64Cu-NOTA-TRC105-Fab increased from a control level of 3.4 ± 0.1 to 9.5 ± 0.4 %ID/g at 6 h p.i. on Day 5, and decreased to 7.2 ± 1.4 %ID/g on Day 12 which correlated well with biodistribution and autoradiography studies (i.e. much higher tracer uptake in AAA than normal aorta). Of note, enhanced AAA contrast was achieved, due to the minimal background in the abdominal area of mice. Degradation of elastic fibers and highly expressed CD105 were observed in ex vivo studies. Conclusion 64Cu-NOTA-TRC105-Fab cleared rapidly through kidneys, which enabled noninvasive PET imaging of the aorta with enhanced contrast and showed increased angiogenesis (CD105 expression) during AAA. 64Cu-NOTA-TRC105-Fab PET may potentially be used for future diagnosis and prognosis of AAA. PMID:25883125

  4. Complete regression of a guinea pig hepatocarcinoma by immunotherapy with "tumor-immune" RNA or antibody to fibrin fragment E.

    PubMed

    Schlager, S I; Dray, S

    1976-01-01

    Two novel immunotherapeutic regimens were developed for a uniformly lethal, intradermally growing transplantable ascites variant (line 10) of a diethylnitrosamine-induced hepatoma in strain 2 guinea pigs. In an apparently tumor-specific immunotherapy model, 32 guinea pigs were cured by the injection into the tumor area, five or seven days after tumor challenge, of syngeneic or xenogeneic RNA extracts obtained from lymphoid tissues of line 10-immune strain 2 guinea pigs or rhesus monkeys, as part of a total regimen which included syngeneic nonsensitive peritoneal exudate cells injected prior to, and tumor-specific antigen injected after, the RNA. In another immunotherapy model, not tumor-specific, 18 strain 2 guinea pigs were cured by the injection into the tumor area, 6 and 16 days after tumor challenge, of antibody specific for fibrin fragment E (FFE), an essential component in the formation of a fibrin matrix considered to be important in tumor development. When therapy was delayed to 12 days in the RNA test system, or to 16 days in the anti-FFE test system, complete abrogation of the tumors did not occur. The long-term survival of the 50 successfully treated animals and their immunity to further tumor challenge indicated that both immunotherapeutic procedures had systemic effects. To test this further, line 10 cells were injected intradermally simultaneously at two sites and only one site was treated. When the one tumor location was treated with anti-FFE, complete regression of the treated tumor and a 30% retardation in the development of the untreated tumor were observed. When this tumor location was treated with the RNA regimen, complete regression of the tumors occurred at both the treated and the untreated sites. Optimal conditions for both immunotherapeutic models and their combination have yet to be establshed. Nonetheless, both immunotherapeutic regimens were more effective than any other immunotherapy thus far reported for this tumor, including the use

  5. Reducing heterophilic antibody interference in immunoassays using single chain antibodies

    SciTech Connect

    Baird, Cheryl L.; Tan, Ruimin; Fischer, Christopher J.; Victry, Kristin D.; Zangar, Richard C.; Rodland, Karin D.

    2011-12-15

    Sandwich ELISA microarrays have the potential to simultaneously quantify the levels of multiple diagnostic targets in a biological sample. However, as seen with traditional ELISA diagnostics, heterophilic antibodies (HA) in patient sera have the potential to cause interference in these assays. We demonstrate here that reducing the diagnostic capture antibody to its minimal functional unit, the variable heavy and light domains artificially connected with a short polypeptide linker (scFv), is an effective strategy for reducing the HA assay interference.

  6. Whole-body imaging of HER2/neu-overexpressing tumors using scFv-antibody conjugated quantum dots

    NASA Astrophysics Data System (ADS)

    Balalaeva, Irina V.; Zdobnova, Tatiana A.; Brilkina, Anna A.; Krutova, Irina M.; Stremovskiy, Oleg A.; Lebedenko, Elena N.; Vodeneev, Vladimir V.; Turchin, Ilya V.; Deyev, Sergey M.

    2010-02-01

    Semiconductor quantum dots (QDs) are widely used in different fields of bioscience and biotechnology due to their unique optical properties. QDs can be used as fluorescent markers for optical detection and monitoring of deeply located tumors in vivo after specific labeling achieved by conjugating of QDs with targeting molecules. In this work the possibilities of intravital tumor labeling with QDs and subsequent in vivo tumor imaging were estimated. The experiments were run on immunodeficient nu/nu mice bearing human breast carcinoma SKBR-3, overexpressing surface protein HER2/neu. We used quantum dots Qdot 705 ITK (Invitrogen, USA) linked to anti-HER2/neu 4D5 scFv antibody. Antibody scFv fragments as a targeting agent for directed delivery of fluorophores possess significant advantages over full-size antibodies due to their small size, lower cross-reactivity and immunogenicity. QDs were bound to 4D5 scFv by barnase-barstar system (bn-bst) analogous to the streptavidin-biotidin system. Whole-body images were obtained using diffuse fluorescence tomography (DFT) setup with low-frequency modulation and transilluminative configuration of scanning, created at the Institute of Applied Physics of RAS, Russia). DFT-results were confirmed ex vivo by confocal microscopy. We report the results of in vivo whole-body tumor imaging with QDs complexes as contrasting agents. Intravital images of QDs-labeled tumors were obtained using specific tumor cells targeting and fluorescence transilluminative imaging method, while "passive" QD-labeling failed to mark effectively the tumor.

  7. Using the local immune response from the natural buffalo host to generate an antibody fragment library that binds the early larval stages of Schistosoma japonicum.

    PubMed

    Hosking, Christopher G; Driguez, Patrick; McWilliam, Hamish E G; Ilag, Leodevico L; Gladman, Simon; Li, Yuesheng; Piedrafita, David; McManus, Donald P; Meeusen, Els N T; de Veer, Michael J

    2015-09-01

    Antibodies isolated from the local draining inguinal lymph node of field exposed-water buffaloes following challenge with Schistosoma japonicum cercariae showed high reactivity towards S. japonicum antigen preparations and bound specifically to formaldehyde-fixed S. japonicum schistosomules. Using this specific local immune response we produced a series of single-chain antibody Fv domain libraries from the same lymph nodes. Removal of phage that cross reacted with epitopes on adult parasites yielded a single-chain antibody Fv domain-phage library that specifically bound to whole formaldehyde-fixed and live S. japonicum schistosomules. DNA sequencing indicated clear enrichment of the single-chain antibody Fv domain library for buffalo B-cell complementarity determining regions post-selection for schistosomule binding. This study also revealed that long heavy chain complementarity determining regions appear to be an important factor when selecting for antibody binding fragments against schistosomule proteins. The selected single-chain antibody Fv domain-phage were used to probe a schistosome-specific protein microarray, which resulted in the recognition of many proteins expressed across all schistosome life-cycle stages. Following absorption to adult worms, the single-chain antibody Fv domain-phage library showed significantly reduced binding to most proteins, whilst two proteins (NCBI GenBank accession numbers AY915878 and AY815196) showed increased binding. We have thus developed a unique set of host derived single-chain antibody Fv domains comprising buffalo B-cell variable regions that specifically bind to early S. japonicum life-stages. PMID:26116907

  8. Effect of linker length between variable domains of single chain variable fragment antibody against daidzin on its reactivity.

    PubMed

    Yusakul, Gorawit; Sakamoto, Seiichi; Pongkitwitoon, Benyakan; Tanaka, Hiroyuki; Morimoto, Satoshi

    2016-07-01

    The peptide linker between variable domains of heavy (VH) and light (VL) chains is one of important factors that influence the characteristics of scFv, including binding activity and specificity against target antigen. The scFvs against daidzin (DZ-scFvs) with different linker lengths were constructed in the format of VH-(GGGGS)n-VL (n = 1, 3, 5, and 7). They were expressed in the hemolymph of silkworm larvae using the Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system, and their reactivity against daidzin and related compounds were evaluated using an indirect competitive enzyme-linked immunosorbent assay (icELISA), which is applicable for quantitative analysis of daidzin. The results showed that the reactivity of scFvs against daidzin was increased, whereas specificity slightly decreased when their peptide linker was lengthened. These results suggested that the linker length of DZ-scFvs contributes to its reactivity. In addition, the results emphasize that the linker length could control the reactivity of DZ-scFvs. PMID:27116996

  9. Discovery of Hapten-Specific scFv from a Phage Display Library and Applications for HER2-Positive Tumor Imaging

    PubMed Central

    2015-01-01

    In this study, an anti-hapten antibody (single chain Fv, scFv) against a hapten probe was developed as a unique reporter system for molecular imaging or therapy. The hapten peptide (histamine-succinyl-GSYK, Him) was synthesized for phage displayed scFv affinity selection and for conjugation with cypate (Cy-Him) for in vivo near-infrared (NIR) optical imaging. Hapten-specific scFvs were affinity selected from the human single fold phage display scFv libraries (Tomlinson I + J) with high specificity and affinity. Utilizing HER2 targeting as a model system, the highest affinity scFv (clone J42) was recombinantly fused to an anti-HER2 affibody (scFv-L-Aff) with no loss of affinity of either protein. The functionality of the hapten-scFv reporter system was tested in vitro with a HER2-positive human breast cancer cell line, SK-BR3, and in vivo with SK-BR3 xenografts. ScFv-L-Aff mediated the binding of the hapten to HER2 on SK-BR3 cells and from tissue from the SK-BR3 xenograft; however, scFv-L-Aff did not mediate uptake of the hapten in the SK-BR3 xenografted tumors, presumably due to rapid internalization of the HER2/scFv-L-Aff complex. Our results suggest that this hapten-peptide and anti-hapten scFv can be a universal reporter system in a wide range of imaging and therapeutic applications. PMID:24898150

  10. Conserved stem fragment from H3 influenza hemagglutinin elicits cross-clade neutralizing antibodies through stalk-targeted blocking of conformational change during membrane fusion.

    PubMed

    Gong, Xin; Yin, He; Shi, Yuhua; Guan, Shanshan; He, Xiaoqiu; Yang, Lan; Yu, Yongjiao; Kuai, Ziyu; Jiang, Chunlai; Kong, Wei; Wang, Song; Shan, Yaming

    2016-04-01

    Currently available influenza vaccines typically fail to elicit/boost broadly neutralizing antibodies due to the mutability of virus sequences and conformational changes during protective immunity, thereby limiting their efficacy. This problem needs to be addressed by further understanding the mechanisms of neutralization and finding the desired neutralizing site during membrane fusion. This study specifically focused on viruses of the H3N2 subtype, which have persisted as a principal source of influenza-related morbidity and mortality in humans since the 1968 influenza pandemic. Through sequence alignment and epitope prediction, a series of highly conserved stem fragments (spanning 47 years) were found and coupled to the Keyhole Limpet Hemocyanin (KLH) protein. By application of a combinatorial display library and crystal structure modeling, a stem fragment immunogen, located at the turning point of the HA neck undergoing conformational change during membrane fusion with both B- and T-cell epitopes, was identified. After synthesis of the optimal stem fragment using a multiple antigen peptide (MAP) system, strong humoral immune responses and cross-clade neutralizing activities against strains from the H3 subtype of group 2 influenza viruses after animal immunizations were observed. By detection of nuclear protein immunofluorescence with acid bypass treatment, antisera raised against MAP4 immunogens of the stem fragment showed the potential to inhibit the conformational change of HA in stem-targeted virus neutralization. The identification of this conserved stem fragment provides great potential for exploitation of this site of vulnerability in therapeutic and vaccine design. PMID:26875772

  11. A novel single-chain antibody redirects adenovirus to IL13Rα2-expressing brain tumors

    PubMed Central

    Kim, Julius W.; Young, Jacob S.; Solomaha, Elena; Kanojia, Deepak; Lesniak, Maciej S.; Balyasnikova, Irina V.

    2015-01-01

    The generation of a targeting agent that strictly binds to IL13Rα2 will significantly expand the therapeutic potential for the treatment of IL13Rα2-expressing cancers. In order to fulfill this goal, we generated a single-chain antibody (scFv47) from our parental IL13Rα2 monoclonal antibody and tested its binding properties. Furthermore, to demonstrate the potential therapeutic applicability of scFv47, we engineered an adenovirus by incorporating scFv47 as the targeting moiety in the viral fiber and characterized its properties in vitro and in vivo. The scFv47 binds to human recombinant IL13Rα2, but not to IL13Rα1 with a high affinity of 0.9 · 10−9 M, similar to that of the parental antibody. Moreover, the scFv47 successfully redirects adenovirus to IL13Rα2 expressing glioma cells both in vitro and in vivo. Our data validate scFv47 as a highly selective IL13Rα2 targeting agent and justify further development of scFv47-modified oncolytic adenovirus and other therapeutics for the treatment of IL13Rα2-expressing glioma and other malignancies. PMID:26656559

  12. A novel single-chain antibody redirects adenovirus to IL13Rα2-expressing brain tumors.

    PubMed

    Kim, Julius W; Young, Jacob S; Solomaha, Elena; Kanojia, Deepak; Lesniak, Maciej S; Balyasnikova, Irina V

    2015-01-01

    The generation of a targeting agent that strictly binds to IL13Rα2 will significantly expand the therapeutic potential for the treatment of IL13Rα2-expressing cancers. In order to fulfill this goal, we generated a single-chain antibody (scFv47) from our parental IL13Rα2 monoclonal antibody and tested its binding properties. Furthermore, to demonstrate the potential therapeutic applicability of scFv47, we engineered an adenovirus by incorporating scFv47 as the targeting moiety in the viral fiber and characterized its properties in vitro and in vivo. The scFv47 binds to human recombinant IL13Rα2, but not to IL13Rα1 with a high affinity of 0.9 · 10(-9) M, similar to that of the parental antibody. Moreover, the scFv47 successfully redirects adenovirus to IL13Rα2 expressing glioma cells both in vitro and in vivo. Our data validate scFv47 as a highly selective IL13Rα2 targeting agent and justify further development of scFv47-modified oncolytic adenovirus and other therapeutics for the treatment of IL13Rα2-expressing glioma and other malignancies. PMID:26656559

  13. Engineering, purification and applications of His-tagged recombinant antibody fragments with specificity for the major birch pollen allergen, bet v1.

    PubMed

    Flicker, S; Laffer, S; Steinberger, P; Alhani, B; Zhu, Y; Laukkanen, M L; Keinänen, K; Kraft, D; Valenta, R

    2000-01-01

    Type I allergy, an immunodisorder affecting almost 20% of the population worldwide, is based on the production of IgE antibodies against per se harmless allergens. We report the expression of hexahistidine-tagged antibody fragments (Fabs) with specificity for Bet v1, the major birch pollen allergen, in Escherichia coli. The cDNA coding for the heavy chain fragment of a mouse monoclonal anti-Bet v1 antibody, Bip 1, was engineered by PCR to contain a hexahistidine-encoding 3' end. The modified Bip1 heavy chain cDNA was co-expressed in E. coli XL-1 Blue with the Bip 1 light chain cDNA using the combinatorial plasmid pComb3H. His-tagged recombinant (r) Bip 1 Fabs were isolated by nickel affinity chromatography and rBip 1 Fabs without His-tag were purified via affinity to rBet v1. rBip 1 Fabs with and without His-tag bound specifically to rBet v1 and, like Bet v1 -specific human serum IgE and rabbit-anti rBet v1 antibodies, cross-reacted with Bet v1-related allergens in other plant-species (alder, oak, hazelnut). We demonstrate the usefulness of His-tagged rBip 1 Fabs (1) for the identification of pollen samples containing Bet v 1 by particle blotting, (2) forthe detection of Bet v1-specific IgE antibodies in human serum samples by sandwich ELISA and (3) for the quantification of Bet v1 in solution. Based on these examples we suggest to use rBip 1 Fabs for the detection of Bet v1 and Bet v1-related allergens in natural allergen sources for allergy prevention, as well as for the standardization of natural allergen extracts produced for diagnosis and immunotherapy of birch pollen allergy. PMID:10722049

  14. Construction and functional analysis of an anti-human cervical carcinoma/anti-human CD3 single-chain bispecific antibody.

    PubMed

    Wu, Hong; Yao, Li; Chou, Lin; Yang, Jin-Hua; Zhang, Yun-Xiu; Li, Xiao-Li; Shan, Bo-Er

    2016-07-01

    The aim of the present study was to construct a single-chain bispecific antibody (scBsAb) against cervical carcinoma and to investigate its biological activities. The scBsAb was constructed using a genetic cloning technique and antigen binding activities were detected by ELISA. The iodogen method was used to analyze the pharmacokinetics. The Rosette formation test was used to detect the binding ability between peripheral blood lymphocytes (PBLs) and Cs1213 cervical cancer cells. In addition, the MTT method was performed to detect the killing effect of PBLs. The molecular weight of the scBsAb was ~60 kDa. The antigen binding activities of scBsAbs were compared with the anti‑human cervical carcinoma antibody single‑chain Fv fragment (CSAs‑1 scFv) and anti‑cluster of differentiation (CD)3 scFv (P>0.05). In addition, a pharmacokinetics assay demonstrated that compared with the two corresponding scFvs, scBsAbs exhibited a significantly prolonged retention time in the body (P<0.01). In addition, the number of rosettes formed by PBLs and Cs1213 cells in the scBsAb group was markedly greater than that in the scFv groups or the RPMI‑1640 group (P<0.05 and P<0.01, respectively). The killing activity of PBLs against scBsAb‑mediated Cs1213 cells was significantly greater than that mediated by the other antibodies (P<0.05). When the concentration of scBsAb was 40 µg/ml, the killing rate was 64.5%. Thus, anti‑human cervical carcinoma/anti‑CD3 scBsAbs may possess two types of antigen binding activity, prolong the duration in vivo and improve the killing activity of PBLs against cancer cells. PMID:27220396

  15. Single Chain Variable Fragments Produced in Escherichia coli against Heat-Labile and Heat-Stable Toxins from Enterotoxigenic E. coli

    PubMed Central

    Andrade, Fernanda B.; Nepomuceno, Roberto; Silva, Anderson; Munhoz, Danielle D.; Yamamoto, Bruno B.; Luz, Daniela; Abreu, Patrícia A. E.; Horton, Denise S. P. Q.; Elias, Waldir P.; Ramos, Oscar H. P.; Piazza, Roxane M. F.

    2015-01-01

    Background Diarrhea is a prevalent pathological condition frequently associated to the colonization of the small intestine by enterotoxigenic Escherichia coli (ETEC) strains, known to be endemic in developing countries. These strains can produce two enterotoxins associated with the manifestation of clinical symptoms that can be used to detect these pathogens. Although several detection tests have been developed, minimally equipped laboratories are still in need of simple and cost-effective methods. With the aim to contribute to the development of such diagnostic approaches, we describe here two mouse hybridoma-derived single chain fragment variable (scFv) that were produced in E. coli against enterotoxins of ETEC strains. Methods and Findings Recombinant scFv were developed against ETEC heat-labile toxin (LT) and heat-stable toxin (ST), from previously isolated hybridoma clones. This work reports their design, construction, molecular and functional characterization against LT and ST toxins. Both antibody fragments were able to recognize the cell-interacting toxins by immunofluorescence, the purified toxins by ELISA and also LT-, ST- and LT/ST-producing ETEC strains. Conclusion The developed recombinant scFvs against LT and ST constitute promising starting point for simple and cost-effective ETEC diagnosis. PMID:26154103

  16. A mycobacterial lipoarabinomannan specific monoclonal antibody and its F(ab′)2 fragment prolong survival of mice infected with Mycobacterium tuberculosis

    PubMed Central

    HAMASUR, B; HAILE, M; PAWLOWSKI, A; SCHRÖDER, U; KÄLLENIUS, G; SVENSON, S B

    2004-01-01

    Lipoarabinomannan (LAM) is a major structural carbohydrate antigen of the outer surface of Mycobacterium tuberculosis. High antibody titres against LAM are often seen in active tuberculosis (TB). The role of such LAM-specific antibodies in the immune response against TB is unknown. Here we have investigated a monoclonal antibody (MoAb) SMITB14 of IgG1 subclass and its corresponding F(ab′)2 fragment directed against LAM from M. tuberculosis strain H37Rv. MoAb SMITB14 was shown by immunofluorescence to bind to whole cells of the clinical isolate M. tuberculosis strain Harlingen as well as to M. tuberculosis H37Rv. The binding of MoAb SMITB14 to LAM was inhibited by arabinomannan (AM) and oligosaccharides (5·2 kDa) derived from LAM, showing that the MoAb binds specifically to the AM carbohydrate portion of LAM. In passive protection experiments BALB/c mice were infected intravenously with M. tuberculosis Harlingen. MoAb SMITB14 was added intravenously either prior to, or together with, the bacteria. The antibody proved to be protective against the M. tuberculosis infection in terms of a dose-dependent reduction in bacterial load in spleens and lungs, reduced weight loss and, most importantly, increased long-term survival. PMID:15373902

  17. Crystallization and preliminary X-ray diffraction analysis of the Fab fragment of WO2, an antibody specific for the Aβ peptides associated with Alzheimer’s disease

    SciTech Connect

    Wun, Kwok S.; Miles, Luke A.; Crespi, Gabriela A. N.; Wycherley, Kaye; Ascher, David B.; Barnham, Kevin J.; Cappai, Roberto; Beyreuther, Konrad; Masters, Colin L.; Parker, Michael W.; McKinstry, William J.

    2008-05-01

    Crystallization and X-ray diffraction data collection of the Fab fragment of the monoclonal antibody WO2 in the absence or presence of amyloid β peptides associated with Alzheimer’s disease are reported. The murine monoclonal antibody WO2 specifically binds the N-terminal region of the amyloid β peptide (Aβ) associated with Alzheimer’s disease. This region of Aβ has been shown to be the immunodominant B-cell epitope of the peptide and hence is considered to be a basis for the development of immunotherapeutic strategies against this prevalent cause of dementia. Structural studies have been undertaken in order to characterize the molecular basis for antibody recognition of this important epitope. Here, details of the crystallization and X-ray analysis of the Fab fragment of the unliganded WO2 antibody in two crystal forms and of the complexes that it forms with the truncated Aβ peptides Aβ{sub 1–16} and Aβ{sub 1–28} are presented. These crystals were all obtained using the hanging-drop vapour-diffusion method at 295 K. Crystals of WO2 Fab were grown in polyethylene glycol solutions containing ZnSO{sub 4}; they belonged to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1} and diffracted to 1.6 Å resolution. The complexes of WO2 Fab with either Aβ{sub 1–@}@{sub 16} or Aβ{sub 1–28} were cocrystallized from polyethylene glycol solutions. These two complex crystals grew in the same space group, P2{sub 1}2{sub 1}2{sub 1}, and diffracted to 1.6 Å resolution. A second crystal form of WO2 Fab was grown in the presence of the sparingly soluble Aβ{sub 1–42} in PEG 550 MME. This second form belonged to space group P2{sub 1} and diffracted to 1.9 Å resolution.

  18. Antibody Fragments Directed against Different Portions of the Human Neural Cell Adhesion Molecule L1 Act as Inhibitors or Activators of L1 Function

    PubMed Central

    Wang, Yan; Loers, Gabriele; Pan, Hong-Chao; Gouveia, Ricardo; Zhao, Wei-Jiang; Shen, Yan-Qin; Kleene, Ralf; Costa, Julia; Schachner, Melitta

    2012-01-01

    The neural cell adhesion molecule L1 plays important roles in neuronal migration and survival, neuritogenesis and synaptogenesis. L1 has also been found in tumors of different origins, with levels of L1 expression correlating positively with the metastatic potential of tumors. To select antibodies targeting the varied functions of L1, we screened the Tomlinson library of recombinant human antibody fragments to identify antibodies binding to recombinant human L1 protein comprising the entire extracellular domain of human L1. We obtained four L1 binding single-chain variable fragment antibodies (scFvs), named I4, I6, I13, and I27 and showed by enzyme-linked immunosorbent assay (ELISA) that scFvs I4 and I6 have high affinity to the immunoglobulin-like (Ig) domains 1–4 of L1, while scFvs I13 and I27 bind strongly to the fibronectin type III homologous (Fn) domains 1–3 of L1. Application of scFvs I4 and I6 to human SK-N-SH neuroblastoma cells reduced proliferation and transmigration of these cells. Treatment of SK-N-SH cells with scFvs I13 and I27 enhanced cell proliferation and migration, neurite outgrowth, and protected against the toxic effects of H2O2 by increasing the ratio of Bcl-2/Bax. In addition, scFvs I4 and I6 inhibited and scFvs I13 and I27 promoted phosphorylation of src and Erk. Our findings indicate that scFvs reacting with the immunoglobulin-like domains 1–4 inhibit L1 functions, whereas scFvs interacting with the fibronectin type III domains 1–3 trigger L1 functions of cultured neuroblastoma cells. PMID:23272240

  19. Characterization of a Lipopolysaccharide-Targeted Monoclonal Antibody and Its Variable Fragments as Candidates for Prophylaxis against the Obligate Intracellular Bacterial Pathogen Coxiella burnetii

    PubMed Central

    Peng, Ying; Schoenlaub, Laura; Elliott, Alexandra; Mitchell, William J.

    2014-01-01

    Our previous study demonstrated that treatment of Coxiella burnetii with the phase I lipopolysaccharide (PI-LPS)-targeted monoclonal antibody (MAb) 1E4 significantly inhibited C. burnetii infection in mice, suggesting that 1E4 is a protective MAb. To determine whether passive transfer of antibodies (Abs) can provide protection against C. burnetii natural infection, we examined if passive transfer of 1E4 would protect SCID mice against C. burnetii aerosol infection. The results indicated that 1E4 conferred significant protection against aerosolized C. burnetii, suggesting that 1E4 may be useful for preventing C. burnetii natural infection. To further understand the mechanisms of 1E4-mediated protection and to test the possibility of using humanized 1E4 to prevent C. burnetii infection, we examined whether the Fab fragment of 1E4 (Fab1E4), a recombinant murine single-chain variable fragment (muscFv1E4), and a humanized single-chain variable fragment (huscFv1E4) retained the ability of 1E4 to inhibit C. burnetii infection. The results indicated that Fab1E4, muscFv1E4, and huscFv1E4 were able to inhibit C. burnetii infection in mice but that their ability to inhibit C. burnetii infection was lower than that of 1E4. In addition, treatment of C. burnetii with Fab1E4, muscFv1E4, or huscFv1E4 can block C. burnetii infection of macrophages. Interestingly, treatment of C. burnetii with huscFv1E4 can significantly reduce C. burnetii infectivity in human macrophages. This report provides the first evidence to demonstrate that the humanized variable fragments of an LPS-specific MAb can neutralize C. burnetii infection and appears to be a promising step toward the potential use of a humanized MAb as emergency prophylaxis against C. burnetii exposure. PMID:25114119

  20. An integrated top-down and bottom-up proteomic approach to characterize the antigen binding fragment of antibodies

    SciTech Connect

    Dekker, Leendert J.; Wu, Si; vanDuijn, Martijn M.; Tolic, Nikola; Stingl, Christoph; Zhao, Rui; Luider, Theo N.; Pasa-Tolic, Ljiljana

    2014-05-31

    We have previously shown that different individuals exposed to the same antigen produce antibodies with identical mutations in their complementarity determining regions (CDR), suggesting that CDR tryptic peptides can serve as biomarkers for disease diagnosis and prognosis. Complete Fabs derived from disease specific antibodies have even higher potential; they could potentially be used for disease treatment and are required to identify the antigens towards which the antibodies are directed. However, complete Fab sequence characterization via LC-MS analysis of tryptic peptides (i.e. bottom-up) has proven to be impractical for mixtures of antibodies. To tackle this challenge, we have developed an integrated bottom-up and top-down MS approach, employing 2D chromatography coupled with Fourier transform mass spectrometry (FTMS), and applied this approach for full characterization of the variable parts of two pharmaceutical monoclonal antibodies with sensitivity comparable to the bottom-up standard. These efforts represent an essential step towards the identification of disease specific antibodies in patient samples with potentially significant clinical impact.

  1. Characterization of a human antibody fragment Fab and its calcium phosphate nanoparticles that inhibit rabies virus infection with vaccine.

    PubMed

    Liu, Xinjian; Lin, Hong; Tang, Qi; Li, Chen; Yang, Songtao; Wang, Zhongcan; Wang, Changjun; He, Qing; Cao, Brian; Feng, Zhenqing; Guan, Xiaohong; Zhu, Jin

    2011-01-01

    Recombinant antibody phage display technology has been used to mimic many aspects of the processes that govern the generation and selection of high-affinity natural human antibodies in the human immune system, especially for infectious disease prophylaxis. An anti-rabies virus immunized phage-display Fab library was constructed from peripheral blood lymphocytes from vaccinated volunteers. The immunized antibody library, with a diversity of 6.7×10(8), was used to select and produce antibodies that bound to rabies virus glycoprotein. After five rounds of immobilized fixed rabies virion panning, four unique DNA sequences were found in the higher binding clones, and only one, Fab094, showed neutralization activity. Fab094 components were analyzed by ELISA, immunoprecipitation and immunofluorescent staining. ELISA and immunofluorescence showed that Fab094 bound specifically to rabies virions. Immunoprecipitation and mass spectrometry showed that Fab094 reacted with rabies virus glycoprotein. To improve the penetration power of Fab094 antibodies, we developed Fab094 calcium phosphate nanoparticles (Fab094-CPNPs) and tested their efficacy. The rapid fluorescent focus inhibition test indicated that the neutralizing antibody titers of Fab094 and Fab094-CPNPs were reached at 200.17 IU/Kg and 246.12 IU/Kg, respectively. These findings were confirmed in vivo in a Kunming mouse challenge model. Our results demonstrate that human Fab094 and Fab094-CPNPs are efficacious candidate drugs to replace rabies immunoglobulin in post-exposure prophylaxis (PEP). PMID:21573024

  2. Recent Developments in Antibody-Based Assays for the Detection of Bacterial Toxins

    PubMed Central

    Zhu, Kui; Dietrich, Richard; Didier, Andrea; Doyscher, Dominik; Märtlbauer, Erwin

    2014-01-01

    Considering the urgent demand for rapid and accurate determination of bacterial toxins and the recent promising developments in nanotechnology and microfluidics, this review summarizes new achievements of the past five years. Firstly, bacterial toxins will be categorized according to their antibody binding properties into low and high molecular weight compounds. Secondly, the types of antibodies and new techniques for producing antibodies are discussed, including poly- and mono-clonal antibodies, single-chain variable fragments (scFv), as well as heavy-chain and recombinant antibodies. Thirdly, the use of different nanomaterials, such as gold nanoparticles (AuNPs), magnetic nanoparticles (MNPs), quantum dots (QDs) and carbon nanomaterials (graphene and carbon nanotube), for labeling antibodies and toxins or for readout techniques will be summarized. Fourthly, microscale analysis or minimized devices, for example microfluidics or lab-on-a-chip (LOC), which have attracted increasing attention in combination with immunoassays for the robust detection or point-of-care testing (POCT), will be reviewed. Finally, some new materials and analytical strategies, which might be promising for analyzing toxins in the near future, will be shortly introduced. PMID:24732203

  3. Genetic fusion of single-chain variable fragments to partial spider silk improves target detection in micro- and nanoarrays.

    PubMed

    Thatikonda, Naresh; Delfani, Payam; Jansson, Ronnie; Petersson, Linn; Lindberg, Diana; Wingren, Christer; Hedhammar, My

    2016-03-01

    Immobilizing biomolecules with retained functionality and stability on solid supports is crucial for generation of sensitive immunoassays. However, upon use of conventional immobilization strategies, a major portion of the biomolecules (e.g. antibodies) frequently tends to lose their bioactivity. In this study, we describe a procedure to immobilize human single-chain variable fragment (scFv) via genetic fusion to partial spider silk, which have a high tendency to adhere to solid supports. Two scFvs, directed towards serum proteins, were genetically fused to partial spider silk proteins and expressed as silk fusion proteins in E. coli. Antigen binding ability of scFvs attached to a partial silk protein denoted RC was investigated using microarray analysis, whereas scFvs fused to the NC silk variant were examined using nanoarrays. Results from micro- and nanoarrays confirmed the functionality of scFvs attached to both RC and NC silk, and also for binding of targets in crude serum. Furthermore, the same amount of added scFv gives higher signal intensity when immobilized via partial spider silk compared to when immobilized alone. Together, the results suggest that usage of scFv-silk fusion proteins in immunoassays could improve target detection, in the long run enabling novel biomarkers to be detected in crude serum proteomes. PMID:26470853

  4. Development of Phage-Based Single Chain Fv Antibody Reagents for Detection of Yersinia pestis

    PubMed Central

    Shou, Yulin; Graves, Steven W.; Bradbury, Andrew R. M.

    2011-01-01

    Background Most Yersinia pestis strains are known to express a capsule-like antigen, fraction 1 (F1). F1 is encoded by the caf1 gene located on the large 100-kb pFra plasmid, which is found in Y. pestis but not in closely related species such as Yersinia enterocolytica and Yersinia pseudotuberculosis. In order to find antibodies specifically binding to Y. pestis we screened a large single chain Fv antibody fragment (scFv) phage display library using purified F1 antigen as a selection target. Different forms of the selected antibodies were used to establish assays for recombinant F1 antigen and Y. pestis detection. Methods Phage antibody panning was performed against F1 in an automated fashion using the Kingfisher magnetic bead system. Selected scFvs were screened for F1-binding specificity by one-step alkaline phosphatase enzyme linked immunosorbant assay (ELISA), and assayed for binding to recombinant antigen and/or Y. pestis by flow cytometry and whole-cell ELISA. Results Seven of the eight selected scFvs were shown to specifically bind both recombinant F1 and a panel of F1-positive Yersinia cells. The majority of the soluble scFvs were found to be difficult to purify, unstable and prone to cross-reactivity with F1-negative Yersinia strains, whereas phage displayed scFvs were found to be easy to purify/label and remarkably stable. Furthermore direct fluorescent labeling of phage displaying scFv allowed for an easy one-step flow cytometry assay. Slight cross-reactivity was observed when fixed cells were used in ELISA. Conclusions Our high throughput methods of selection and screening allowed for time and cost effective discovery of seven scFvs specifically binding Y. pestis F1 antigen. We describe implementation of different methods for phage-based immunoassay. Based on the success of these methods and the proven stability of phage, we indicate that the use of phage-displayed, rather than phage-free proteins, might generally overcome the shortcomings of scFv

  5. Neutralization of West Nile virus by cross-linking of its surface proteins with Fab fragments of the human monoclonal antibody CR4354

    SciTech Connect

    Kaufmann, Bärbel; Vogt, Matthew R.; Goudsmit, Jaap; Holdaway, Heather A.; Aksyuk, Anastasia A.; Chipman, Paul R.; Kuhn, Richard J.; Diamond, Michael S.; Rossmann, Michael G.

    2010-11-15

    Many flaviviruses are significant human pathogens, with the humoral immune response playing an essential role in restricting infection and disease. CR4354, a human monoclonal antibody isolated from a patient, neutralizes West Nile virus (WNV) infection at a postattachment stage in the viral life-cycle. Here, we determined the structure of WNV complexed with Fab fragments of CR4354 using cryoelectron microscopy. The outer glycoprotein shell of a mature WNV particle is formed by 30 rafts of three homodimers of the viral surface protein E. CR4354 binds to a discontinuous epitope formed by protein segments from two neighboring E molecules, but does not cause any detectable structural disturbance on the viral surface. The epitope occurs at two independent positions within an icosahedral asymmetric unit, resulting in 120 binding sites on the viral surface. The cross-linking of the six E monomers within one raft by four CR4354 Fab fragments suggests that the antibody neutralizes WNV by blocking the pH-induced rearrangement of the E protein required for virus fusion with the endosomal membrane.

  6. Imaging of human leukemic T-cell xenografts in nude mice by radiolabeled monoclonal antibodies and F(ab')2 fragments

    SciTech Connect

    Vacca, A.; Buchegger, F.; Carrel, S.; Mach, J.P.

    1988-01-01

    Monoclonal antibodies (MoAb) that react with the T-lymphocyte markers called cluster of differentiation CD5 and CD2 were labeled with iodine 131 (/sup 131/I) and were injected intravenously in nude mice bearing solid subcutaneous xenografts derived from the human T-cell leukemia line Ichikawa. Both MoAb anti-CD5 and anti-CD2 yielded favorable mean tumor to whole-body ratios of 3.8 and 5.1, respectively. These ratios were further increased up to 10.0 for MoAb anti-CD5 and 15.5 for MoAb anti-CD2 by using their F(ab')2 fragments. The tumors could be imaged clearly by external scanning after injection of F(ab')2 fragments from both MoAb. F(ab')2 fragments from MoAb anti-CD2 and of a third MoAb recognizing the clonotypic determinant (Ti) of the antigen receptor expressed by the human T-cell line Jurkat were injected in mice bearing intrasplenic Jurkat xenografts. A selective localization of both fragments in tumor tissue was demonstrated with mean tumor to whole-body ratios of 7.5 and 4.1 for MoAb anti-CD2 and anti-Ti, respectively. These in vivo experimental results may provide useful information for the potential use of radiolabeled MoAb and fragments in the diagnosis and treatment of patients with T-cell lymphoma and different other forms of T-cell malignancies.

  7. Functional domains on von Willebrand factor. Recognition of discrete tryptic fragments by monoclonal antibodies that inhibit interaction of von Willebrand factor with platelets and with collagen.

    PubMed Central

    Sixma, J J; Sakariassen, K S; Stel, H V; Houdijk, W P; In der Maur, D W; Hamer, R J; de Groot, P G; van Mourik, J A

    1984-01-01

    We have identified two functional domains on the von Willebrand factor (VWF) moiety of the Factor VIII-von Willebrand factor complex (FVIII-VWF), one interacting with blood platelets, and one interacting with vessel wall collagens, by means of two monoclonal antibodies directed against the VWF molecule, CLB-RAg 35 and CLB-RAg 201. The monoclonal antibody CLB-RAg 35 inhibited virtually all platelet adherence to artery subendothelium and to purified vessel wall collagen type III, at relatively high wall shear rates. CLB-RAg 35 also inhibited the ristocetin-induced platelet aggregation and the binding of FVIII-VWF to the platelet in the presence of ristocetin but did not affect the binding of FVIII-VWF to collagen. The monoclonal antibody CLB-RAg 201 inhibited the binding of FVIII-VWF to purified vessel wall collagen type I and III and all platelet adherence to collagen type III and the platelet adherence to subendothelium that was mediated by FVIII-VWF in plasma. The two functional domains on FVIII-VWF that were recognized by CLB-RAg 35 and CLB-RAg 201 were identified by means of immunoprecipitation studies of trypsin-digested FVIII-VWF. The domains resided on different polypeptide fragments, with a Mr of 48,000 for the collagen binding domain and a Mr of 116,000 for the platelet binding domain. The 116,000-mol wt fragment consisted of subunits of 52,000/56,000 mol wt and 14,000 mol wt after reduction. The 52,000/56,000-mol wt subunits possessed the epitope for CLB-RAg 35. Images PMID:6332119

  8. Investigation of Fragment Antibody Stability and Its Release Mechanism from Poly(Lactide-co-Glycolide)-Triacetin Depots for Sustained-Release Applications.

    PubMed

    Chang, Debby P; Garripelli, Vivek Kumar; Rea, Jennifer; Kelley, Robert; Rajagopal, Karthikan

    2015-10-01

    Achieving long-term drug release from polymer-based delivery systems continues to be a challenge particularly for the delivery of large hydrophilic molecules such as therapeutic antibodies and proteins. Here, we report on the utility of an in situ-forming and injectable polymer-solvent system for the long-term release of a model antibody fragment (Fab1). The delivery system was prepared by dispersing a spray-dried powder of Fab1 within poly(lactide-co-glycolide) (PLGA)-triacetin solution. The formulation viscosity was within the range 1.0 ± 0.3 Pa s but it was injectable through a 27G needle. The release profile of Fab1, measured in phosphate-buffered saline (PBS), showed a lag phase followed by sustained-release phase for close to 80 days. Antibody degradation during its residence within the depot was comparable to its degradation upon long-term incubation in PBS. On the basis of temporal changes in surface morphology, stiffness, and depot mass, a mechanism to account for the drug release profile has been proposed. The unprecedented release profile and retention of greater than 80% of antigen-binding capacity even after several weeks demonstrates that PLGA-triacetin solution could be a promising system for the long-term delivery of biologics. PMID:26099467

  9. Translational fusion and redirection to thylakoid lumen as strategies to improve the accumulation of a camelid antibody fragment in transplastomic tobacco.

    PubMed

    Lentz, Ezequiel M; Garaicoechea, Lorena; Alfano, E Federico; Parreño, Viviana; Wigdorovitz, Andrés; Bravo-Almonacid, Fernando F

    2012-08-01

    Fragments from camelid single-chain antibodies known as VHHs or nanobodies represent a valuable tool in diagnostics, investigation and passive immunity therapy. Here, we explored different strategies to improve the accumulation of a neutralizing VHH antibody against rotavirus in tobacco transplastomic plants. First, we attempted to express the VHH in the chloroplast stroma and then two alternative strategies were carried out to improve the expression levels: expression as a translational fusion to the β-glucuronidase enzyme (GUS-E-VHH), and redirection of the VHH into the thylakoid lumen (pep-VHH). Every attempt to produce transplastomic plants expressing the VHH in the stroma was futile. The transgene turned out to be unstable and the presence of the VHH protein was almost undetectable. Although pep-VHH plants also presented some of the aforementioned problems, higher accumulation of the nanobody was observed (2-3% of the total soluble proteins). The use of β-glucuronidase as a partner protein turned out to be a successful strategy and expression levels reached 3% of the total soluble proteins. The functionality of the VHHs produced by pep-VHH and GUS-E-VHH plants was studied and compared with that of the antibody produced in Escherichia coli. This work contributes to optimizing the expression of VHH in transplastomic plants. Recombinant proteins could be obtained either by accumulation in the thylakoid lumen or as a fusion protein with β-glucuronidase, and both strategies allow for further optimization. PMID:22526499

  10. Crystallization of the receptor-binding domain of parathyroid hormone-related protein in complex with a neutralizing monoclonal antibody Fab fragment

    SciTech Connect

    McKinstry, William J.; Polekhina, Galina; Diefenbach-Jagger, Hannelore; Sato, Koh; Onuma, Etsuro; Gillespie, Matthew T.; Martin, Thomas J.; Parker, Michael W.

    2009-04-01

    Parathyroid hormone-related protein (PTHrP) plays an important role in regulating embryonic skeletal development and is abnormally regulated in the pathogenesis of skeletal complications observed with many cancers and osteoporosis. It exerts its action through binding to a G-protein-coupled seven-transmembrane cell-surface receptor (GPCR). Structurally, GPCRs are very difficult to study by X-ray crystallography. In this study, a monoclonal antibody Fab fragment which recognizes the same region of PTHrP as its receptor, PTH1R, was used to aid in the crystallization of PTHrP. The resultant protein complex was crystallized using the hanging-drop vapour-diffusion method with polyethylene glycol as a precipitant. The crystals belonged to the orthorhombic space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 72.6, b = 96.3, c = 88.5 {angstrom}, and diffracted to 2.0 {angstrom} resolution using synchrotron radiation. The crystal structure will shed light on the nature of the key residues of PTHrP that interact with the antibody and will provide insights into how the antibody is able to discriminate between PTHrP and the related molecule parathyroid homone.

  11. Differential analyses of major allergen proteins in wild-type rice and rice producing a fragment of anti-rotavirus antibody.

    PubMed

    Yuki, Yoshikazu; Kurokawa, Shiho; Kozuka-Hata, Hiroko; Tokuhara, Daisuke; Mejima, Mio; Kuroda, Masaharu; Oyama, Masaaki; Nishimaki-Mogami, Tomoko; Teshima, Reiko; Kiyono, Hiroshi

    2016-04-01

    To develop oral antibody therapy against rotavirus infection, we previously produced a recombinant fragment of llama heavy-chain antibody to rotavirus (ARP1) in rice seeds (MucoRice-ARP1). We intend to use a purification-free rice powder for clinical application but needed to check whether MucoRice-ARP1 had increased levels of known allergen proteins. For this purpose, we used two-dimensional fluorescence difference gel electrophoresis to compare the allergen protein levels in MucoRice-ARP1 and wild-type rice. We detected no notable differences, except in the levels of α-amylase/trypsin inhibitor-like family proteins. Because by this approach we could not completely separate ARP1 from the proteins of this family, we confirmed the absence of changes in the levels of these allergens by using shotgun mass spectrometry as well as immunoblot. By using immunoelectron microscopy, we also showed that RAG2, a member of the α-amylase/trypsin inhibitor-like protein family, was relocated from protein bodies II to the plasma membrane or cell wall in MucoRice-ARP1 seed. The relocation did not affect the level of RAG2. We demonstrated that most of the known rice allergens were not considerably upregulated by the genetic modification in MucoRice-ARP1. Our data suggest that MucoRice-ARP1 is a potentially safe oral antibody for clinical application. PMID:26851506

  12. Pharmacokinetics of the monoclonal antibody B72. 3 and its fragments labeled with either /sup 125/I or /sup 111/In

    SciTech Connect

    Brown, B.A.; Comeau, R.D.; Jones, P.L.; Liberatore, F.A.; Neacy, W.P.; Sands, H.; Gallagher, B.M.

    1987-02-15

    A comparison of the pharmacokinetics of intact B72.3 (a murine monoclonal antibody specific for human breast and colon carcinoma) with F(ab')2 and Fab fragments labeled with /sup 111/In and /sup 125/I was done in athymic mice bearing target (LS174T) and non-target (HCT-15) tumors. IgG B72.3 labeled with either isotype imaged LS174T. Biodistributions of both labels were similar in all organs except liver. F(ab')2 also imaged the LS174T tumor, while Fab bearing either isotype did not. The blood clearance was Fab greater than F(ab')2 greater than immunoglobulin G B72.3 for both isotopes. /sup 111/In-labeled fragments yielded large accumulations in the kidneys which persisted for 2 days. The different patterns of biodistribution for the various forms of B72.3 labeled with the two isotopes suggest that the most desirable combination of fragment and isotope will depend on the intended use.

  13. A Cancer Specific Cell-Penetrating Peptide, BR2, for the Efficient Delivery of an scFv into Cancer Cells

    PubMed Central

    Lim, Ki Jung; Sung, Bong Hyun; Shin, Ju Ri; Lee, Young Woong; Kim, Da Jung; Yang, Kyung Seok; Kim, Sun Chang

    2013-01-01

    Cell-penetrating peptides (CPPs) have proven very effective as intracellular delivery vehicles for various therapeutics. However, there are some concerns about non-specific penetration and cytotoxicity of CPPs for effective cancer treatments. Herein, based on the cell-penetrating motif of an anticancer peptide, buforin IIb, we designed several CPP derivatives with cancer cell specificity. Among the derivatives, a 17-amino acid peptide (BR2) was found to have cancer-specificity without toxicity to normal cells. After specifically targeting cancer cells through interaction with gangliosides, BR2 entered cells via lipid-mediated macropinocytosis. Moreover, BR2 showed higher membrane translocation efficiency than the well-known CPP Tat (49–57). The capability of BR2 as a cancer-specific drug carrier was demonstrated by fusion of BR2 to a single-chain variable fragment (scFv) directed toward a mutated K-ras (G12V). BR2-fused scFv induced a higher degree of apoptosis than Tat-fused scFv in K-ras mutated HCT116 cells. These results suggest that the novel cell-penetrating peptide BR2 has great potential as a useful drug delivery carrier with cancer cell specificity. PMID:23776609

  14. Sustained in vivo inhibition of protein domains using single-chain Fv recombinant antibodies and its application to dissect RGMa activity on axonal outgrowth.

    PubMed

    Tassew, Nardos G; Charish, Jason; Chestopalova, Larisa; Monnier, Philippe P

    2009-01-28

    Antibodies are powerful tools for delineating the specific function of protein domains, yet several limitations restrict their in vivo applicability. Here we present a new method to obtain sustained in vivo inhibition of specific protein domains using recombinant antibodies. We show that long term in vivo expression of single-chain Fv (scFv) fragments in the developing CNS can be achieved through retroviral transduction. Moreover, specific scFvs generated against the N- and C-terminal domains of the repulsive guidance molecule, RGMa, prevent proper axon targeting in the visual system. This work reveals a previously unappreciated role for the RGMa N-terminal domain in axon guidance, and provides a novel, broadly applicable and rapid procedure to functionally antagonize any protein domain in vivo. PMID:19176821

  15. Development of conventional monoclonal antibody and recombinant antibody (scFv) against Candidatus Liberibacter asiaticus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A non-culturable member of the alpha-proteobacteria, ‘Ca. Liberibacter asiaticus’, is consistently associated with huanglongbing (HLB) disease. This bacterium is transmitted by citrus psyllids and grows systemically in infected citrus phloem tissues. Control of the disease requires effective, conven...

  16. A Cryo-Electron Microscopy Study Identifies the Complete H16.V5 Epitope and Reveals Global Conformational Changes Initiated by Binding of the Neutralizing Antibody Fragment

    PubMed Central

    Lee, Hyunwook; Brendle, Sarah A.; Bywaters, Stephanie M.; Guan, Jian; Ashley, Robert E.; Yoder, Joshua D.; Makhov, Alexander M.; Conway, James F.; Christensen, Neil D.

    2014-01-01

    ABSTRACT Human papillomavirus 16 (HPV16) is a worldwide health threat and an etiologic agent of cervical cancer. To understand the antigenic properties of HPV16, we pursued a structural study to elucidate HPV capsids and antibody interactions. The cryo-electron microscopy (cryo-EM) structures of a mature HPV16 particle and an altered capsid particle were solved individually and as complexes with fragment of antibody (Fab) from the neutralizing antibody H16.V5. Fitted crystal structures provided a pseudoatomic model of the virus-Fab complex, which identified a precise footprint of H16.V5, including previously unrecognized residues. The altered-capsid–Fab complex map showed that binding of the Fab induced significant conformational changes that were not seen in the altered-capsid structure alone. These changes included more ordered surface loops, consolidated so-called “invading-arm” structures, and tighter intercapsomeric connections at the capsid floor. The H16.V5 Fab preferentially bound hexavalent capsomers likely with a stabilizing effect that directly correlated with the number of bound Fabs. Additional cryo-EM reconstructions of the virus-Fab complex for different incubation times and structural analysis provide a model for a hyperstabilization of the capsomer by H16.V5 Fab and showed that the Fab distinguishes subtle differences between antigenic sites. IMPORTANCE Our analysis of the cryo-EM reconstructions of the HPV16 capsids and virus-Fab complexes has identified the entire HPV.V5 conformational epitope and demonstrated a detailed neutralization mechanism of this clinically important monoclonal antibody against HPV16. The Fab bound and ordered the apical loops of HPV16. This conformational change was transmitted to the lower region of the capsomer, resulting in enhanced intercapsomeric interactions evidenced by the more ordered capsid floor and “invading-arm” structures. This study advances the understanding of the neutralization mechanism used

  17. A dual-targeting PDGFRβ/VEGF-A molecule assembled from stable antibody fragments demonstrates anti-angiogenic activity in vitro and in vivo

    PubMed Central

    Gilbertson, Debra G; Frank, Amanda; Vu, Tuyen; Ardourel, Dan; Ostrander, Craig; Stevens, Brenda; Julien, Susan; Franke, Secil; Meengs, Brent; Brody, Jennifer; Presnell, Scott; Hamacher, Nels B; Lantry, Megan; Wolf, Anitra; Bukowski, Tom; Rosler, Robert; Yen, Cindy; Anderson-Haley, Monica; Brasel, Kenneth; Pan, Qi; Franklin, Hank; Thompson, Penny; Dodds, Mike; Underwood, Sara; Peterson, Scott; Sivakumar, Pallavur V; Snavely, Mark

    2010-01-01

    Targeting angiogenesis is a promising approach to the treatment of solid tumors and age-related macular degeneration (AMD). Inhibition of vascularization has been validated by the successful marketing of monoclonal antibodies (mAbs) that target specific growth factors or their receptors, but there is considerable room for improvement in existing therapies. Combination of mAbs targeting both the VeGF and pDGF pathways has the potential to increase the efficacy of anti-angiogenic therapy without the accompanying toxicities of tyrosine kinase inhibitors and the inability to combine efficiently with traditional chemotherapeutics. However, development costs and regulatory issues have limited the use of combinatorial approaches for the generation of more efficacious treatments. The concept of mediating disease pathology by targeting two antigens with one therapeutic was proposed over two decades ago. While mAbs are particularly suitable candidates for a dual-targeting approach, engineering bispecificity into one molecule can be difficult due to issues with expression and stability, which play a significant role in manufacturability. Here, we address these issues upstream in the process of developing a bispecific antibody (bsAb). Single-chain antibody fragments (scFvs) targeting pDGFRβ and VeGF-A were selected for superior stability. the scFvs were fused to both termini of human Fc to generate a bispecific, tetravalent molecule. resulting molecule displays potent activity, binds both targets simultaneously, and is stable in serum. assembly of a bsAb using stable monomeric units allowed development of an anti-pDGFRB/VeGF-A antibody capable of attenuating angiogenesis through two distinct pathways and represents an efficient method for rapid engineering of dual-targeting molecules. PMID:20065654

  18. Tetravalent anti-CD20/CD3 bispecific antibody for the treatment of B cell lymphoma.

    PubMed

    Lu, Chia-Yen; Chen, Gregory J; Tai, Pei-Han; Yang, Yu-Chen; Hsu, Yu-Shen; Chang, Mingi; Hsu, Chuan-Lung

    2016-05-13

    Bispecific antibodies (bsAbs) are second generation antibodies for therapeutic application in immunotherapy. One of the major strategies of the bsAb platform is the recruitment of immune effector T cells by incorporating an anti-CD3 domain. A bispecific T-cell engager (BiTE), with one end having an affinity for CD3 and the other end with affinity for CD19, has been approved in the US and Europe for the treatment of acute lymphoblastic leukemia. However, due to their small size and lack of Fc region, these single-chain variable fragment (scFv) bsAbs have short half-lives in vivo. Additionally, poor solubility, structural instability, and low production yields have also become major challenges in the bulk production process. To overcome these challenges, we have engineered a tetravalent bsAb with bivalent binding specificity for the CD20 and CD3 antigen in an immunoglobulin G (IgG) format. The fusion of the anti-CD3 scFvs to the CD20 antibody via a linker-hinge domain (LHD) results in improved antibody stabilization and properties. Here we demonstrate this antibody's highly efficient cancer cell elimination in a dose-dependent manner in a CD20-expressing B lymphoblastoid cell line in vitro. Our data suggest the potential clinical application of this bsAb for the treatment of CD20-expressing B cell malignancies. PMID:27040766

  19. Fragments of the V1/V2 domain of HIV-1 glycoprotein 120 engineered for improved binding to the broadly neutralizing PG9 antibody.

    PubMed

    Morales, Javier F; Yu, Bin; Perez, Gerardo; Mesa, Kathryn A; Alexander, David L; Berman, Phillip W

    2016-09-01

    The V1/V2 domain of the HIV-1 envelope protein gp120 possesses two important epitopes: a glycan-dependent epitope recognized by the prototypic broadly neutralizing monoclonal antibody (bN-mAb), PG9, as well as an epitope recognized by non-neutralizing antibodies that has been associated with protection from HIV infection in the RV144 HIV vaccine trial. Because both of these epitopes are poorly immunogenic in the context of full length envelope proteins, immunization with properly folded and glycosylated fragments (scaffolds) represents a potential way to enhance the immune response to these specific epitopes. Previous studies showed that V1/V2 domain scaffolds could be produced from a few selected isolates, but not from many of the isolates that would be advantageous in a multivalent vaccine. In this paper, we used a protein engineering approach to improve the conformational stability and antibody binding activity of V1/V2 domain scaffolds from multiple diverse isolates, including several that were initially unable to bind the prototypic PG9 bN-mAb. Significantly, this effort required replicating both the correct glycan structure as well as the β-sheet structure required for PG9 binding. Although scaffolds incorporating the glycans required for PG9 binding (e.g., mannose-5) can be produced using glycosylation inhibitors (e.g., swainsonine), or mutant cell lines (e.g. GnTI(-) 293 HEK), these are not practical for biopharmaceutical production of proteins intended for clinical trials. In this report, we describe engineered glycopeptide scaffolds from three different clades of HIV-1 that bind PG9 with high affinity when expressed in a wildtype cell line suitable for biopharmaceutical production. The mutations that improved PG9 binding to scaffolds produced in normal cells included amino acid positions outside of the antibody contact region designed to stabilize the β-sheet and turn structures. The scaffolds produced address three major problems in HIV vaccine

  20. Structure of a human monoclonal antibody Fab fragment against gp41 of human immunodeficiency virus type 1

    NASA Technical Reports Server (NTRS)

    He, Xiao M.; Rueker, Florian; Casale, Elena; Carter, Daniel C.

    1992-01-01

    The three-dimensional structure of a human monoclonal antibody (Fab), which binds specifically to a major epitope of the transmembrane protein gp41 of the human immunodeficiency virus type 1, has been determined by crystallographic methods to a resolution of 2.7 A. It has been previously determined that this antibody recognizes the epitope SGKLICTTAVPWNAS, belongs to the subclass IgG1 (kappa), and exhibits antibody-dependent cellular cytotoxicity. The quaternary structure of the Fab is in an extended conformation with an elbow bend angle between the constant and variable domains of 175 deg. Structurally, four of the hypervariable loops can be classified according to previously recognized canonical structures. The third hypervariable loops of the heavy (H3) and light chain (L3) are structurally distinct. Hypervariable loop H3, residues 102H-109H, is unusually extended from the surface. The complementarity-determining region forms a hydrophobic binding pocket that is created primarily from hypervariable loops L3, H3, and H2.

  1. An Exopolysaccharide-Deficient Mutant of Lactobacillus rhamnosus GG Efficiently Displays a Protective Llama Antibody Fragment against Rotavirus on Its Surface

    PubMed Central

    Álvarez, Beatriz; Krogh-Andersen, Kasper; Tellgren-Roth, Christian; Martínez, Noelia; Günaydın, Gökçe; Lin, Yin; Martín, M. Cruz; Álvarez, Miguel A.; Hammarström, Lennart

    2015-01-01

    Rotavirus is the leading cause of infantile diarrhea in developing countries, where it causes a high number of deaths among infants. Two vaccines are available, being highly effective in developed countries although markedly less efficient in developing countries. As a complementary treatment to the vaccines, a Lactobacillus strain producing an anti-rotavirus antibody fragment in the gastrointestinal tract could potentially be used. In order to develop such an alternative therapy, the effectiveness of Lactobacillus rhamnosus GG to produce and display a VHH antibody fragment (referred to as anti-rotavirus protein 1 [ARP1]) on the surface was investigated. L. rhamnosus GG is one of the best-characterized probiotic bacteria and has intrinsic antirotavirus activity. Among four L. rhamnosus GG strains [GG (CMC), GG (ATCC 53103), GG (NCC 3003), and GG (UT)] originating from different sources, only GG (UT) was able to display ARP1 on the bacterial surface. The genomic analysis of strain GG (UT) showed that the genes welE and welF of the EPS cluster are inactivated, which causes a defect in exopolysaccharide (EPS) production, allowing efficient display of ARP1 on its surface. Finally, GG (UT) seemed to confer a level of protection against rotavirus-induced diarrhea similar to that of wild-type GG (NCC 3003) in a mouse pup model, indicating that the EPS may not be involved in the intrinsic antirotavirus activity. Most important, GG (EM233), a derivative of GG (UT) producing ARP1, was significantly more protective than the control strain L. casei BL23. PMID:26092449

  2. An Exopolysaccharide-Deficient Mutant of Lactobacillus rhamnosus GG Efficiently Displays a Protective Llama Antibody Fragment against Rotavirus on Its Surface.

    PubMed

    Álvarez, Beatriz; Krogh-Andersen, Kasper; Tellgren-Roth, Christian; Martínez, Noelia; Günaydın, Gökçe; Lin, Yin; Martín, M Cruz; Álvarez, Miguel A; Hammarström, Lennart; Marcotte, Harold

    2015-09-01

    Rotavirus is the leading cause of infantile diarrhea in developing countries, where it causes a high number of deaths among infants. Two vaccines are available, being highly effective in developed countries although markedly less efficient in developing countries. As a complementary treatment to the vaccines, a Lactobacillus strain producing an anti-rotavirus antibody fragment in the gastrointestinal tract could potentially be used. In order to develop such an alternative therapy, the effectiveness of Lactobacillus rhamnosus GG to produce and display a VHH antibody fragment (referred to as anti-rotavirus protein 1 [ARP1]) on the surface was investigated. L. rhamnosus GG is one of the best-characterized probiotic bacteria and has intrinsic antirotavirus activity. Among four L. rhamnosus GG strains [GG (CMC), GG (ATCC 53103), GG (NCC 3003), and GG (UT)] originating from different sources, only GG (UT) was able to display ARP1 on the bacterial surface. The genomic analysis of strain GG (UT) showed that the genes welE and welF of the EPS cluster are inactivated, which causes a defect in exopolysaccharide (EPS) production, allowing efficient display of ARP1 on its surface. Finally, GG (UT) seemed to confer a level of protection against rotavirus-induced diarrhea similar to that of wild-type GG (NCC 3003) in a mouse pup model, indicating that the EPS may not be involved in the intrinsic antirotavirus activity. Most important, GG (EM233), a derivative of GG (UT) producing ARP1, was significantly more protective than the control strain L. casei BL23. PMID:26092449

  3. Positron Emission Tomography Imaging of Tumor Angiogenesis with a 61/64Cu-Labeled F(ab')2 Antibody Fragment

    PubMed Central

    Hong, Hao; Zhang, Yin; Orbay, Hakan; Valdovinos, Hector F.; Nayak, Tapas R.; Bean, Jero; Theuer, Charles P.; Barnhart, Todd E.; Cai, Weibo

    2013-01-01

    The objective of this study was to characterize the in vitro and in vivo properties of the F(ab')2 fragment of TRC105, a human/murine chimeric IgG1 monoclonal antibody that binds with high avidity to human and murine CD105 (i.e. endoglin), and investigate its potential for positron emission tomography (PET) imaging of tumor angiogenesis after 61/64Cu-labeling. TRC105-F(ab')2 of high purity was produced by pepsin digestion of TRC105, which was confirmed by SDS-PAGE, HPLC analysis, and mass spectrometry. 61/64Cu-labeling of NOTA-TRC105-F(ab')2 (NOTA denotes 1,4,7-triazacyclononane-1,4,7-triacetic acid) was achieved with yields of > 75% (specific activity: ~115 GBq/μmol). PET imaging revealed rapid tumor uptake of 64Cu-NOTA TRC105-F(ab')2 in the 4T1 murine breast cancer model (5.8 ± 0.8, 7.6 ± 0.6, 5.6 ± 0.4, 5.0 ± 0.6, and 3.8 ± 0.7 %ID/g at 0.5, 3, 16, 24, and 48 h post-injection respectively; n = 4). Since tumor uptake peaked at 3 h post-injection, 61Cu-NOTA-TRC105-F(ab')2 also gave good tumor contrast at 3 and 8 h post-injection. CD105 specificity of the tracers was confirmed by blocking studies and histopathology. In conclusion, the use of a F(ab')2 fragment led to more rapid tumor uptake (which peaked at 3 h post-injection) than radiolabeled intact antibody (which often peaked after 24 h post-injection), which may allow for same day immunoPET imaging in future clinical studies. PMID:23316869

  4. Positron emission tomography imaging of tumor angiogenesis with a (61/64)Cu-labeled F(ab')(2) antibody fragment.

    PubMed

    Hong, Hao; Zhang, Yin; Orbay, Hakan; Valdovinos, Hector F; Nayak, Tapas R; Bean, Jero; Theuer, Charles P; Barnhart, Todd E; Cai, Weibo

    2013-02-01

    The objective of this study was to characterize the in vitro and in vivo properties of the F(ab')(2) fragment of TRC105, a human/murine chimeric IgG1 monoclonal antibody that binds with high avidity to human and murine CD105 (i.e., endoglin), and investigate its potential for positron emission tomography (PET) imaging of tumor angiogenesis after (61/64)Cu-labeling. TRC105-F(ab')(2) of high purity was produced by pepsin digestion of TRC105, which was confirmed by SDS-PAGE, HPLC analysis, and mass spectrometry. (61/64)Cu-labeling of NOTA-TRC105-F(ab')(2) (NOTA denotes 1,4,7-triazacyclononane-1,4,7-triacetic acid) was achieved with yields of >75% (specific activity: ∼115 GBq/μmol). PET imaging revealed rapid tumor uptake of (64)Cu-NOTA-TRC105-F(ab')(2) in the 4T1 murine breast cancer model (5.8 ± 0.8, 7.6 ± 0.6, 5.6 ± 0.4, 5.0 ± 0.6, and 3.8 ± 0.7% ID/g at 0.5, 3, 16, 24, and 48 h postinjection respectively; n = 4). Since tumor uptake peaked at 3 h postinjection, (61)Cu-NOTA-TRC105-F(ab')(2) also gave good tumor contrast at 3 and 8 h postinjection. CD105 specificity of the tracers was confirmed by blocking studies and histopathology. In conclusion, the use of a F(ab')(2) fragment led to more rapid tumor uptake (which peaked at 3 h postinjection) than radiolabeled intact antibody (which often peaked after 24 h postinjection), which may allow for same day immunoPET imaging in future clinical studies. PMID:23316869

  5. Epitope Mapping of Antigenic MUC1 Peptides to Breast Cancer Antibody Fragment B27.29: A Heteronuclear NMR Study

    SciTech Connect

    Grinstead, Jeffrey S.; Schuman, Jason T.; Campbell, Ann P.

    2003-11-13

    MUC1 mucin is a breast cancer-associated transmembrane glycoprotein, of which the extracellular domain is formed by the repeating 20-amino acid sequence GVTSAPDTRPAPGSTAPPAH. In neoplastic breast tissue, the highly immunogenic sequence PDTRPAP (in bold above) is exposed. Antibodies raised directly against MUC1-expressing tumors offer unique access to this neoplastic state, as they represent immunologically relevant ''reverse templates'' of the tumor-associated mucin. In a previous study [Grinstead, J. S., et al. (2002) Biochemistry 41, 9946-9961], 1H NMR methods were used to correlate the effects of cryptic glycosylation outside of the PDTRPAP core epitope sequence on the recognition and binding of Mab B27.29, a monoclonal antibody raised against breast tumor cells. In the study presented here, isotope-edited NMR methods, including 15N and 13C relaxation measurements, were used to probe the recognition and binding of the PDTRPAP epitope sequence to Fab B27.29. Two peptides were studied: a one-repeat MUC1 16mer peptide of the sequence GVTSAPDTRPAPGSTA and a two-repeat MUC1 40mer peptide of the sequence (VTSAPDTRPAPGSTAPPAHG)2. 15N and 13C NMR relaxation parameters were measured for both peptides free in solution and bound to Fab B27.29. The 13CR T1 values best represent changes in the local correlation time of the peptide epitope upon binding antibody, and demonstrate that the PDTRPAP sequence is immobilized in the antibody-combining site. This result is also reflected in the appearance of the 15N- and 13C-edited HSQC spectra, where line broadening of the same peptide epitope resonances is observed. The PDTRPAP peptide epitope expands upon the peptide epitope identified previously in our group as PDTRP by homonuclear NMR experiments [Grinstead, J. S., et al. (2002) Biochemistry 41, 9946-9961], and illustrates the usefulness of the heteronuclear NMR experiments. The implications of these results are discussed within the context of MUC1 breast cancer vaccine design.

  6. Astatine-211 labeling of an anti-melanoma antibody and its Fab fragment using N-succinimidyl para[{sup 211} At]astatobenzoate : comparisons In Vivo with the para-[{sup 125}1]iodobenzoyl conjugate.

    SciTech Connect

    Hadley, S. W.; Wilbur, D. S.; Gray, M. A.; Atcher, R. W.; Chemistry; NeoRx Corp.; Univ. of Washington Medical Center

    1991-01-01

    Astatine-211 labeling of an anti-melanoma antibody, NR-ML-05, and its Fab fragment using N-succinimidyl para[{sup 211} At]astatobenzoate has been described. Preparation of the astatinated intermediate 2a was accomplished by distilling astatine-211 from an irradiated bismuth target directly into a reaction mixture containing an organometallic compound, N-succinimidyl p-(tri-n-butylstannyl)benzoate (1), and an oxidant, N-chlorosuccinimide, in 5% HOAc/MeOH. Trapping of distilled astatine as 2a was found to be efficient, resulting in 70-90% yields based on the amount of astatine-211 which ranged from 20% to 75%. Conjugation of 2a to NR-ML-05 and its Fab fragment was accomplished in 40-60% yields. The [{sup 211}At]astatobenzoyl-conjugated antibodies were found to be stable in vitro when challenged by strong denaturants and nucleophilic reagents. Coinjected dual-labeled studies of the 2a astatinated antibodies and the same antibodies labeled with N-succinimidyl p-[{sup 125}I]iodobenzoate (2b) in athymic mice bearing the human tumor xenograft A375 Met/Mix demonstrated that both radiolabeled antibodies had equivalent tumor localization. Data from the dual-labeled biodistribution of the intact antibody suggests that the astatine is stably attached. Data from the dual-labeled Fab fragment suggests that a portion of the astatine label is released as astatide, either from the astatinated Fab or from a catabolite.

  7. Selection of diethylstilbestrol-specific single-chain antibodies from a non-immunized mouse ribosome display library.

    PubMed

    Sun, Yanan; Ning, Baoan; Liu, Ming; Gao, Xianjun; Fan, Xianjun; Liu, Jianqing; Gao, Zhixian

    2012-01-01

    Single chain variable fragments (scFvs) against diethylstilbestrol (DES) were selected from the splenocytes of non-immunized mice by ribosome display technology. A naive library was constructed and engineered to allow in vitro transcription and translation using an E. coli lysate system. Alternating selection in solution and immobilization in microtiter wells was used to pan mRNA-ribosome-antibody (ARM) complexes. After seven rounds of ribosome display, the expression vector pTIG-TRX containing the selected specific scFv DNAs were transformed into Escherichia coli BL21 (DE3) for expression. Twenty-six positive clones were screened and five clones had high antibody affinity and specificity to DES as evidenced by indirect competitive ELISA. Sequence analysis showed that these five DES-specific scFvs had different amino acid sequences, but the CDRs were highly similar. Surface plasmon resonance (SPR) analysis was used to determine binding kinetics of one clone (30-1). The measured K(D) was 3.79 µM. These results indicate that ribosome display technology can be used to efficiently isolate hapten-specific antibody (Ab) fragments from a naive library; this study provides a methodological framework for the development of novel immunoassays for multiple environmental pollutants with low molecular weight detection using recombinant antibodies. PMID:22427984

  8. GFP-SCFV: expression and possible applications as a tool for experimental investigations of atherosclerosis.

    PubMed

    Faulin, Tanize do Espirito Santo; Guilherme, Daniel Ferreira; Silva, Aldacilene Souza; Abdalla, Dulcineia Saes Parra; Hering, Vitor Renaux; Politi, Mario José; Maranhão, Andrea Queiroz

    2014-01-01

    Experimental studies on atherosclerosis are crucial for investigating its pathophysiology, defining new therapeutic targets, and developing new drugs and diagnostic tools. Thus, many imaging markers have been developed and introduced in experimental studies. The main advantage of these new tools is that they allow the noninvasive diagnosis of atherosclerotic vascular disease. Here, we describe the cloning, expression, purification, and stabilization of a chimeric protein specifically designed to probe cells and tissues for the presence of LDL(-), a relevant marker of atherosclerosis. The DNA sequence that encodes the anti-LDL(-) scFv, previously obtained from a hybridoma secreting an anti-LDL(-) monoclonal antibody, was inserted into the bacterial vector pET-28a(+) in tandem with a DNA sequence encoding GFP. The recombinant protein was expressed in high yields in E. coli as inclusion bodies. The applicability of GFP-scFv was assessed by ELISA, which determined its affinity for LDL(-) and confocal microscopy, that showed macrophage uptake of the protein along with LDL(-). In conclusion, our data suggest that the anti-LDL(-) GFP-scFv chimeric protein could be useful in studies on atherogenesis as well as for developing diagnostic tools for atherosclerosis. PMID:24911875

  9. Haemagglutination induced by Bordetella pertussis filamentous haemagglutinin adhesin (FHA) is inhibited by antibodies produced against FHA(430-873) fragment expressed in Lactobacillus casei.

    PubMed

    Colombi, Débora; Oliveira, Maria L S; Campos, Ivana B; Monedero, Vicente; Pérez-Martinez, Gaspar; Ho, Paulo L

    2006-12-01

    Filamentous haemagglutinin adhesin (FHA) is an important virulence factor from Bordetella pertussis related to the adhesion and spread of the bacteria through the respiratory tract. Three distinct domains have been characterized in mature FHA, and among them, the FHA(442-863) fragment was suggested to be responsible for the heparin-binding activity. In this study, we cloned the gene encoding the HEP fragment (FHA(430-873)) in a Lactobacillus casei-inducible expression vector based on the lactose operon. The recombinant bacteria, transformed with the resulting construct (L. casei-HEP), were able to express the heterologous protein depending on the sugar added to the culture. Subcutaneous inoculation of L. casei-HEP in Balb/C mice, using the cholera toxin B subunit as adjuvant, induced systemic anti-HEP antibodies that were able to inhibit in vitro erythrocyte haemagglutination induced by FHA. This is the first example of a B. pertussis antigen produced in lactic acid bacteria and opens new perspectives for alternative vaccine strategies against whooping cough. PMID:17106803

  10. A recombinant mimetics of the HIV-1 gp41 prehairpin fusion intermediate fused with human IgG Fc fragment elicits neutralizing antibody response in the vaccinated mice

    SciTech Connect

    Qi, Zhi; Pan, Chungen; Lu, Hong; Shui, Yuan; Li, Lin; Li, Xiaojuan; Xu, Xueqing; Liu, Shuwen; Jiang, Shibo

    2010-07-30

    Research highlights: {yields} One recombinant mimetics of gp41 prehairpin fusion intermediate (PFI) consisting of gp41 N46 sequence, foldon and IgG Fc, designated N46FdFc, was expressed. {yields} N46FdFc-induced antibodies in mice that neutralized HIV-1 infection, inhibited PIE7 binding to PFI, blocked gp41 six-helix bundle formation, and suppressed HIV-1 mediated cell-cell fusion. {yields} These findings provide an important clue for developing recombinant gp41 PFI mimetics-based HIV vaccines. -- Abstract: HIV-1 gp41 prehairpin fusion intermediate (PFI) composed of three N-terminal heptad repeats (NHR) plays a crucial role in viral fusion and entry and represents an attractive target for anti-HIV therapeutics (e.g., enfuvirtide) and vaccines. In present study, we constructed and expressed two recombinant gp41 PFI mimetics, designated N46Fd and N46FdFc. N46Fd consists of N46 (residues 536-581) in gp41 NHR and foldon (Fd), a trimerization motif. N46FdFc is composed of N46Fd fused with human IgG Fc fragment as an immunoenhancer. We immunized mice with N46 peptide, N46Fd and N46FdFc, respectively, and found that only N46FdFc elicited neutralizing antibody response in mice against infection by HIV-1 strains IIIB (clade B, X4), 92US657 (clade B, R5), and 94UG103 (clade A, X4R5). Anti-N46FdFc antibodies inhibited PIE7 binding to PFI, blocked gp41 six-helix bundle formation, and suppressed HIV-1 mediated cell-cell fusion. These findings provide an important clue for developing recombinant gp41 PFI mimetics-based HIV vaccines.

  11. A Nanoparticle Platform To Evaluate Bioconjugation and Receptor-Mediated Cell Uptake Using Cross-Linked Polyion Complex Micelles Bearing Antibody Fragments.

    PubMed

    Florinas, Stelios; Liu, Marc; Fleming, Ryan; Van Vlerken-Ysla, Lilian; Ayriss, Joanne; Gilbreth, Ryan; Dimasi, Nazzareno; Gao, Changshou; Wu, Herren; Xu, Ze-Qi; Chen, Shaoyi; Dirisala, Anjaneyulu; Kataoka, Kazunori; Cabral, Horacio; Christie, R James

    2016-05-01

    Targeted nanomedicines are a promising technology for treatment of disease; however, preparation and characterization of well-defined protein-nanoparticle systems remain challenging. Here, we describe a platform technology to prepare antibody binding fragment (Fab)-bearing nanoparticles and an accompanying real-time cell-based assay to determine their cellular uptake compared to monoclonal antibodies (mAbs) and Fabs. The nanoparticle platform was composed of core-cross-linked polyion complex (PIC) micelles prepared from azide-functionalized PEG-b-poly(amino acids), that is, azido-PEG-b-poly(l-lysine) [N3-PEG-b-PLL] and azido-PEG-b-poly(aspartic acid) [N3-PEG-b-PAsp]. These PIC micelles were 30 nm in size and contained approximately 10 polymers per construct. Fabs were derived from an antibody binding the EphA2 receptor expressed on cancer cells and further engineered to contain a reactive cysteine for site-specific attachment and a cleavable His tag for purification from cell culture expression systems. Azide-functionalized micelles and thiol-containing Fab were linked using a heterobifunctional cross-linker (FPM-PEG4-DBCO) that contained a fluorophenyl-maleimide for stable conjugation to Fabs thiols and a strained alkyne (DBCO) group for coupling to micelle azide groups. Analysis of Fab-PIC micelle conjugates by fluorescence correlation spectroscopy, size exclusion chromatography, and UV-vis absorbance determined that each nanoparticle contained 2-3 Fabs. Evaluation of cellular uptake in receptor positive cancer cells by real-time fluorescence microscopy revealed that targeted Fab-PIC micelles achieved higher cell uptake than mAbs and Fabs, demonstrating the utility of this approach to identify targeted nanoparticle constructs with unique cellular internalization properties. PMID:27007881

  12. Crystallization and preliminary X-ray diffraction analysis of the Fab fragment of WO2, an antibody specific for the A[beta] peptides associated with Alzheimer's disease

    SciTech Connect

    Wun, Kwok S.; Miles, Luke A.; Crespi, Gabriela A.N.; Wycherley, Kaye; Ascher, David B.; Barnham, Kevin J.; Cappai, Roberto; Beyreuther, Konrad; Masters, Colin L.; Parker, Michael W.; McKinstry, William J.

    2008-05-28

    The murine monoclonal antibody WO2 specifically binds the N-terminal region of the amyloid {beta} peptide (A{beta}) associated with Alzheimer's disease. This region of A{beta} has been shown to be the immunodominant B-cell epitope of the peptide and hence is considered to be a basis for the development of immunotherapeutic strategies against this prevalent cause of dementia. Structural studies have been undertaken in order to characterize the molecular basis for antibody recognition of this important epitope. Here, details of the crystallization and X-ray analysis of the Fab fragment of the unliganded WO2 antibody in two crystal forms and of the complexes that it forms with the truncated Az{beta} peptides A{beta}{sub 1-16} and A{beta}{sub 1-28} are presented. These crystals were all obtained using the hanging-drop vapour-diffusion method at 295 K. Crystals of WO2 Fab were grown in polyethylene glycol solutions containing ZnSO{sub 4}; they belonged to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1} and diffracted to 1.6 {angstrom} resolution. The complexes of WO2 Fab with either A{beta}{sub 1-16} or A{beta}{sub 1-28} were cocrystallized from polyethylene glycol solutions. These two complex crystals grew in the same space group, P2{sub 1}2{sub 1}2{sub 1}, and diffracted to 1.6 {angstrom} resolution. A second crystal form of WO2 Fab was grown in the presence of the sparingly soluble A{beta}{sub 1-42} in PEG 550 MME. This second form belonged to space group P2{sub 1} and diffracted to 1.9 {angstrom} resolution.

  13. Comparative tissue distribution in mice of the alpha-emitter 211At and 131I as labels of a monoclonal antibody and F(ab')2 fragment.

    PubMed

    Garg, P K; Harrison, C L; Zalutsky, M R

    1990-06-15

    Because it decays by the emission of short-range, high-energy alpha-particles, the radiohalogen 211At might be a particularly useful nuclide for some types of radioimmunotherapy. However, no suitable gamma-emitting nuclide of astatine exists which would permit either imaging prior to therapy to obtain radiation dosimetry estimates or performing experiments in paired-label format. Since iodine is the halogen above astatine in the periodic table, we investigated whether the in vivo distribution of 131I could be used to mimic the biodistribution of 211At. In this study, the N-succinimidyl 3-(trialkylstannyl)benzoate method was used to label C110 IgG, an antibody directed against carcinoembryonic antigen, and its (Fab')2 fragment with 211At and 131I. Paired-label experiments were performed in normal mice comparing the tissue distribution of 211At- versus 131I-labeled C110 IgG and F(ab')2 as well as [211At]astatide versus [131I]iodide and m-[211At]astatobenzoic acid versus m-[131I]iodobenzoic acid, potential catabolites of proteins radiohalogenated via the N-succinimidyl 3-(trialkylstannyl)benzoate method. With the exception of thyroid, retention of astatide in tissues was higher than that of iodide; and, with the halobenzoic acids, uptake of 211At was higher than 135I in thyroid, stomach, and spleen. Use of the N-succinimidyl 3-(trialkylstannyl)benzoate method to label C110 IgG with 211At and 131I resulted in similar distributions of the two nuclides. In contrast, loss of 211At from the F(ab')2 fragment was considerably more rapid than 131I, suggesting that different astatination methods may be required for use with F(ab')2 fragments. PMID:2340501

  14. Novel Camelid Antibody Fragments Targeting Recombinant Nucleoprotein of Araucaria hantavirus: A Prototype for an Early Diagnosis of Hantavirus Pulmonary Syndrome

    PubMed Central

    Pereira, Soraya S.; Moreira-Dill, Leandro S.; Morais, Michelle S. S.; Prado, Nidiane D. R.; Barros, Marcos L.; Koishi, Andrea C.; Mazarrotto, Giovanny A. C. A.; Gonçalves, Giselle M.; Zuliani, Juliana P.; Calderon, Leonardo A.; Soares, Andreimar M.; Pereira da Silva, Luiz H.; Duarte dos Santos, Claudia N.; Fernandes, Carla F. C.; Stabeli, Rodrigo G.

    2014-01-01

    In addition to conventional antibodies, camelids produce immunoglobulins G composed exclusively of heavy chains in which the antigen binding site is formed only by single domains called VHH. Their particular characteristics make VHHs interesting tools for drug-delivery, passive immunotherapy and high-throughput diagnosis. Hantaviruses are rodent-borne viruses of the Bunyaviridae family. Two clinical forms of the infection are known. Hemorrhagic Fever with Renal Syndrome (HFRS) is present in the Old World, while Hantavirus Pulmonary Syndrome (HPS) is found on the American continent. There is no specific treatment for HPS and its diagnosis is carried out by molecular or serological techniques, using mainly monoclonal antibodies or hantavirus nucleoprotein (N) to detect IgM and IgG in patient serum. This study proposes the use of camelid VHHs to develop alternative methods for diagnosing and confirming HPS. Phage display technology was employed to obtain VHHs. After immunizing one Lama glama against the recombinant N protein (prNΔ85) of a Brazilian hantavirus strain, VHH regions were isolated to construct an immune library. VHHs were displayed fused to the M13KO7 phage coat protein III and the selection steps were performed on immobilized prNΔ85. After selection, eighty clones recognized specifically the N protein. These were sequenced, grouped based mainly on the CDRs, and five clones were analyzed by western blot (WB), surface plasmon resonance (SPR) device, and ELISA. Besides the ability to recognize prNΔ85 by WB, all selected clones showed affinity constants in the nanomolar range. Additionaly, the clone KC329705 is able to detect prNΔ85 in solution, as well as the native viral antigen. Findings support the hypothesis that selected VHHs could be a powerful tool in the development of rapid and accurate HPS diagnostic assays, which are essential to provide supportive care to patients and reduce the high mortality rate associated with hantavirus infections. PMID

  15. Construction of an anti-IL-1β scfv and TNFRI fusion protein and its therapeutic effect on RA mice model.

    PubMed

    Kan, Fangming; Ren, Guiping; Guo, Mo; Qi, Jianying; Zhang, Yu; Han, Yang; Zhang, Yakun; Li, Deshan

    2014-01-01

    IL-1β and TNF-α play key roles in the inflammatory response. Their abnormal expression may cause the occurrence of various diseases, such as RA. Recently, medicines of target TNF-α and IL-1β have become popular in the clinical practice. Although these biological agents can get mostly good results, they are not effective in all patients. The reason for this result may be that these biological agents could not fully inhibit a variety of inflammatory cytokines in the inflammatory response. In the present study, a fusion protein gene which encoded human interleukin-1β scfv and soluble TNF receptor I (sTNFRI) was cloned. A number of in vitro assays demonstrated that anti-IL-1β scfv/TNFRI simultaneously bound to both targets. The bioactivity assay showed that the fusion protein could inhibit both the cytotoxicity of hTNF-α on L929 cells and hIL-1β-induced proliferation of L929 cells, indicating that the fusion protein has the ability to neutralize both hTNF-α and hIL-1β. In this study, we established the chicken type II collagen-induced rheumatoid arthritis model in Kunming mice, and evaluated the pharmacological effect of the fusion protein in vivo. Model mice were randomly divided into 8 groups (n=8): CIA model control group, DEX treatment group (1 mg/kg), intraperitoneal treatment group (highdose: 5 mg/kg; medium-dose: 2 mg/kg; low-dose: 0.8 mg/kg), subcutaneous treatment group (high-dose: 5 mg/kg; medium- dose: 2 mg/kg; low-dose: 0.8 mg/kg), and healthy mice as control. The control group received the same volume of saline. The mice were administrated once every 2 days. Arthritis index, anti-CII antibody titers, cytokine levels, histopathological changes were examined. The results showed that anti-IL-1β scfv/TNFRI fusion protein could reduce the degree of joint swelling, inflammatory cell infiltration, synovial cell proliferation and the level of CII antibody in the sera. The Real-time PCR analysis showed that anti-IL-1β scfv/TNFRI had the ability to

  16. Single Chain Antibodies Against gp55 of Human Cytomegalovirus (HCMV) for Prophylaxis and Treatment of HCMV Infections

    PubMed Central

    Moazen, Bahareh; Ebrahimi, Elahe; Nejatollahi, Foroogh

    2016-01-01

    Background: Immunotherapy is a promising prospective new treatment for cytomegalovirus (CMV) infections. Neutralizing effects have been reported using monoclonal antibodies. Recombinant single chain antibodies (scFvs) due to their advantages over monoclonal antibodies are potential alternatives and provide valuable clinical agents. Objectives: The aim of this study was to select specific single chain antibodies against gp55 of CMV and to evaluate their neutralizing effects. In the present study, we selected specific single chain antibodies against glycoprotein 55 (gp55) of CMV for their use in treatment and diagnosis. Materials and Methods: Single chain antibodies specific against an epitope located in the C-terminal part of gp55 were selected from a phage antibody display library. After four rounds of panning, twenty clones were amplified by the polymerase chain reaction (PCR) and fingerprinted by MvaI restriction enzyme. The reactivities of the specific clones were tested by the enzyme-linked immunosorbent assay (ELISA) and the neutralizing effects were evaluated by the plaque reduction assay. Results: Fingerprinting of selected clones revealed three specific single chain antibodies (scFv1, scFv2 and scFv3) with frequencies 25%, 20 and 20%. The clones produced positive ELISA with the corresponding peptide. The percentages of plaque reduction for scFv1, scFv2 and scFv3 were 23.7, 68.8 and 11.6, respectively. Conclusions: Gp55 of human CMV is considered as an important candidate for immunotherapy. In this study, we selected three specific clones against gp55. The scFvs reacted only with the corresponding peptide in a positive ELISA. The scFv2 with 68.8% neutralizing effect showed the potential to be considered for prophylaxis and treatment of CMV infections, especially in solid organ transplant recipients, for whom treatment of CMV is urgently needed. The scFv2 with neutralizing effect of 68.8%, has the potential to be considered for treatment of these patients

  17. Uses of monoclonal antibody 8H9

    DOEpatents

    Cheung, Nai-Kong V.

    2013-04-09

    This invention provides a composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a suitable carrier. This invention provides a pharmaceutical composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a pharmaceutically acceptable carrier. This invention also provides an antibody other than the monoclonal antibody 8H9 comprising the complementary determining regions of monoclonal antibody 8H9 or a derivative thereof, capable of binding to the same antigen as the monoclonal antibody 8H9. This invention provides a substance capable of competitively inhibiting the binding of monoclonal antibody 8H9. This invention also provides an isolated scFv of monoclonal antibody 8H9 or a derivative thereof. This invention also provides the 8H9 antigen. This invention also provides different uses of the monoclonal antibody 8H9 or its derivative.

  18. Uses of monoclonal antibody 8H9

    DOEpatents

    Cheung, Nai-Kong V.

    2010-06-22

    This invention provides a composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a suitable carrier. This invention provides a pharmaceutical composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a pharmaceutically acceptable carrier. This invention also provides an antibody other than the monoclonal antibody 8H9 comprising the complementary determining regions of monoclonal antibody 8H9 or a derivative thereof, capable of binding to the same antigen as the monoclonal antibody 8H9. This invention provides a substance capable of competitively inhibiting the binding of monoclonal antibody 8H9. This invention also provides an isolated scFv of monoclonal antibody 8H9 or a derivative thereof. This invention also provides the 8H9 antigen. This invention also provides different uses of the monoclonal antibody 8H9 or its derivative.

  19. Anti-human CD138 monoclonal antibodies and their bispecific formats: generation and characterization.

    PubMed

    Chen, Dan; Zou, Jianxuan; Zong, Yunhui; Meng, Huimin; An, Gangli; Yang, Lin

    2016-06-01

    Syndecan-1 (CD138), a heparan sulfate proteoglycan, acts as a co-receptor for growth factors and chemokines and is a molecular marker associated with the epithelial-mesenchymal transition during development and carcinogenesis. In this study, we generated two specific mouse anti-human CD138 monoclonal antibodies (mAbs, clone ID: 480CT5.4.3, 587CT7.3.6.5) using hybridoma technology and identified their immunological characteristics. After hybridoma sequencing, the single-chain variable fragments (ScFvs) cloned from two hybridoma cells were combined with anti-CD3 OKT-3 ScFv to generate two recombinant bispecific antibodies (h-STL002, m-STL002) against CD138 and CD3 molecules, respectively. The bispecific antibodies were able to specifically target CD138 + multiple myeloma (MM) cells and CD3 + T cells, and showed the potent cytotoxicity against MM RPMI-8226 cell line through T cell activation. However, these bispecific antibodies without T cells did not cause toxic side effect on MM cells. Overall, the two hybridoma clones and their bispecific formats have great potential to promote diagnosis and immunotherapy of plasma cell malignancy. PMID:26954291

  20. Genetically engineered colorimetric single-chain antibody fusion protein for rapid diagnosis of rabies virus.

    PubMed

    Mousli, M; Turki, I; Kharmachi, H; Dellagi, K

    2008-01-01

    The most widely used test for rabies diagnostics is the fluorescent antibody test, which is recommended by both the World Health Organization and the World Organisation for Animal Health (OIE). This test may be used directly on a smear, and can also be used to confirm the presence of rabies antigen in cell culture or in brain tissue for diagnosis. The colorimetric enzymes are usually coupled to an antibody by chemical means using cross-linking reagents. However, such non-specific procedures lead to heterogeneous conjugates, sometimes with reduced activity and specificity. To bypass these problems, genetic engineering has provided a way to create chimeric bifunctional molecules in which the variable domains of an antibody are genetically linked to unrelated protein tracers. In this study, we describe the successful production of a bifunctional chimeric protein based on alkaline phosphatase-fused anti-rabies virus glycoprotein scFv antibody fragment. We also report the antigen binding properties and the alkaline phosphatase activity of the recombinant conjugate protein. We established its value as a novel in vitro tool for detecting the rabies virus in brain smear in a one-step procedure; it presents a similar sensitivity and specificity to that obtained using standard reagents. PMID:18634511

  1. A Camelid-derived Antibody Fragment Targeting the Active Site of a Serine Protease Balances between Inhibitor and Substrate Behavior.

    PubMed

    Kromann-Hansen, Tobias; Oldenburg, Emil; Yung, Kristen Wing Yu; Ghassabeh, Gholamreza H; Muyldermans, Serge; Declerck, Paul J; Huang, Mingdong; Andreasen, Peter A; Ngo, Jacky Chi Ki

    2016-07-15

    A peptide segment that binds the active site of a serine protease in a substrate-like manner may behave like an inhibitor or a substrate. However, there is sparse information on which factors determine the behavior a particular peptide segment will exhibit. Here, we describe the first x-ray crystal structure of a nanobody in complex with a serine protease. The nanobody displays a new type of interaction between an antibody and a serine protease as it inserts its complementary determining region-H3 loop into the active site of the protease in a substrate-like manner. The unique binding mechanism causes the nanobody to behave as a strong inhibitor as well as a poor substrate. Intriguingly, its substrate behavior is incomplete, as 30-40% of the nanobody remained intact and inhibitory after prolonged incubation with the protease. Biochemical analysis reveals that an intra-loop interaction network within the complementary determining region-H3 of the nanobody balances its inhibitor versus substrate behavior. Collectively, our results unveil molecular factors, which may be a general mechanism to determine the substrate versus inhibitor behavior of other protease inhibitors. PMID:27226628

  2. A single domain antibody fragment that recognizes the adaptor ASC defines the role of ASC domains in inflammasome assembly.

    PubMed

    Schmidt, Florian I; Lu, Alvin; Chen, Jeff W; Ruan, Jianbin; Tang, Catherine; Wu, Hao; Ploegh, Hidde L

    2016-05-01

    Myeloid cells assemble inflammasomes in response to infection or cell damage; cytosolic sensors activate pro-caspase-1, indirectly for the most part, via the adaptors ASC and NLRC4. This leads to secretion of proinflammatory cytokines and pyroptosis. To explore complex formation under physiological conditions, we generated an alpaca single domain antibody, VHHASC, which specifically recognizes the CARD of human ASC via its type II interface. VHHASC not only impairs ASC(CARD) interactions in vitro, but also inhibits inflammasome activation in response to NLRP3, AIM2, and NAIP triggers when expressed in living cells, highlighting a role of ASC in all three types of inflammasomes. VHHASC leaves the Pyrin domain of ASC functional and stabilizes a filamentous intermediate of inflammasome activation. Incorporation of VHHASC-EGFP into these structures allowed the visualization of endogenous ASC(PYD) filaments for the first time. These data revealed that cross-linking of ASC(PYD) filaments via ASC(CARD) mediates the assembly of ASC foci. PMID:27069117

  3. Identification of framework residues in a secreted recombinant antibody fragment that control production level and localization in Escherichia coli.

    PubMed

    Forsberg, G; Forsgren, M; Jaki, M; Norin, M; Sterky, C; Enhörning, A; Larsson, K; Ericsson, M; Björk, P

    1997-05-01

    The monoclonal antibody 5T4, directed against a human tumor-associated antigen, was expressed as a secreted Fab superantigen fusion protein in Escherichia coli. The product is a putative agent for immunotherapy of non-small cell lung cancer. During fermentation, most of the fusion protein leaked out from the periplasm to the growth medium at a level of approximately 40 mg/liter. This level was notably low compared with similar products containing identical CH1, CL, and superantigen moieties, and the Fv framework was therefore engineered. Using hybrid molecules, the light chain was found to limit high expression levels. Substituting five residues in VL increased the level almost 15 times, exceeding 500 mg/liter in the growth medium. Here, the substitutions Phe-10 --> Ser, Thr-45 --> Lys, Thr-77 --> Ser, and Leu-78 --> Val were most powerful. In addition, replacing four VH residues diminished cell lysis during fermentation. Thereby the product was preferentially located in the periplasm instead of the growth medium, and the total yield was more than 700 mg/liter. All engineered products retained a high affinity for the tumor-associated antigen. It is suggested that at least some of the identified framework residues generally have to be replaced to obtain high level production of recombinant Fab products in E. coli. PMID:9139690

  4. A Humanized Anti-CD22-Onconase Antibody-Drug Conjugate Mediates Highly Potent Destruction of Targeted Tumor Cells

    PubMed Central

    Weber, Tobias; Mavratzas, Athanasios; Kiesgen, Stefan; Haase, Stephanie; Bötticher, Benedikt; Exner, Evelyn; Mier, Walter; Grosse-Hovest, Ludger; Jäger, Dirk; Arndt, Michaela A. E.; Krauss, Jürgen

    2015-01-01

    Antibody-drug conjugates (ADCs) have evolved as a new class of potent cancer therapeutics. We here report on the development of ADCs with specificity for the B-cell lineage specific (surface) antigen CD22 being expressed in the majority of hematological malignancies. As targeting moiety a previously generated humanized anti-CD22 single-chain variable fragment (scFv) derivative from the monoclonal antibody RFB4 was reengineered into a humanized IgG1 antibody format (huRFB4). Onconase (ranpirnase), a clinically active pancreatic-type ribonuclease, was employed as cytotoxic payload moiety. Chemical conjugation via thiol-cleavable disulfide linkage retained full enzymatic activity and full binding affinity of the ADC. Development of sophisticated purification procedures using size exclusion and ion exchange chromatography allowed the separation of immunoconjugate species with stoichiometrically defined number of Onconase cargos. A minimum of two Onconase molecules per IgG was required for achieving significant in vitro cytotoxicity towards lymphoma and leukemia cell lines. Antibody-drug conjugates with an Onconase to antibody ratio of 3 : 1 exhibited an IC50 of 0.08 nM, corresponding to more than 18,400-fold increased cytotoxicity of the ADC when compared with unconjugated Onconase. These results justify further development of this ADC as a promising first-in-class compound for the treatment of CD22-positive malignancies. PMID:26605343

  5. Inhibitors and Antibody Fragments as Potential Anti-Inflammatory Therapeutics Targeting Neutrophil Proteinase 3 in Human Disease.

    PubMed

    Korkmaz, Brice; Lesner, Adam; Guarino, Carla; Wysocka, Magdalena; Kellenberger, Christine; Watier, Hervé; Specks, Ulrich; Gauthier, Francis; Jenne, Dieter E

    2016-07-01

    Proteinase 3 (PR3) has received great scientific attention after its identification as the essential antigenic target of antineutrophil cytoplasm antibodies in Wegener's granulomatosis (now called granulomatosis with polyangiitis). Despite many structural and functional similarities between neutrophil elastase (NE) and PR3 during biosynthesis, storage, and extracellular release, unique properties and pathobiological functions have emerged from detailed studies in recent years. The development of highly sensitive substrates and inhibitors of human PR3 and the creation of PR3-selective single knockout mice led to the identification of nonredundant roles of PR3 in cell death induction via procaspase-3 activation in cell cultures and in mouse models. According to a study in knockout mice, PR3 shortens the lifespan of infiltrating neutrophils in tissues and accelerates the clearance of aged neutrophils in mice. Membrane exposure of active human PR3 on apoptotic neutrophils reprograms the response of macrophages to phagocytosed neutrophils, triggers secretion of proinflammatory cytokines, and undermines immune silencing and tissue regeneration. PR3-induced disruption of the anti-inflammatory effect of efferocytosis may be relevant for not only granulomatosis with polyangiitis but also for other autoimmune diseases with high neutrophil turnover. Inhibition of membrane-bound PR3 by endogenous inhibitors such as the α-1-protease inhibitor is comparatively weaker than that of NE, suggesting that the adverse effects of unopposed PR3 activity resurface earlier than those of NE in individuals with α-1-protease inhibitor deficiency. Effective coverage of PR3 by anti-inflammatory tools and simultaneous inhibition of both PR3 and NE should be most promising in the future. PMID:27329045

  6. Optimization modeling of single-chain antibody against hepatoma based on similarity algorithm.

    PubMed

    Zhao, Zhi-Jun; Chen, Jing-Tao; Yuan, Jia-Ying; Yin, Xiao-Xiang; Song, Hua-Yong; Wang, Xin-Chun

    2015-01-01

    The purposes was to establish optimal modeling of single-chain antibody molecules based on similarity algorithm and seek the connecting peptides that had the minimal effect on the structure and bioactivity of the variable region of heavy chain (VH) and that of light chain (VL) in a single-chain antibody against liver cancer. After the Linker with different lengths (n=0~7) had been added into single chain fragment variable (ScFv), modeling of the overall sequences of VH, VL and ScFv were conducted r