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Sample records for melanoma antibody fragments

  1. 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

  2. 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.

  3. Indium-111 labeled anti-melanoma monoclonal antibodies

    DOEpatents

    Srivastava, S.C.; Fawwaz, R.A.; Ferrone, S.

    1984-04-30

    A monoclonal antibody to a high molecular weight melanoma-associated antigen was chelated and radiolabeled with indium-111. This material shows high affinity for melanoma and thus can be used in the detection, localization and imaging of melanoma. 1 figure.

  4. Palladium-109 labeled anti-melanoma monoclonal antibodies

    DOEpatents

    Srivastava, S.C.; Fawwaz, R.A.; Ferrone, S.

    1984-04-30

    The invention consists of new monoclonal antibodies labelled with Palladium 109, a beta-emitting radionuclide, the method of preparing this material, and its use in the radiotherapy of melanoma. The antibodies are chelate-conjugated and demonstrate a high uptake in melanomas. (ACR)

  5. Stabilization of antibody fragments in adverse environments.

    PubMed

    Dooley, H; Grant, S D; Harris, W J; Porter, A J

    1998-08-01

    Antibody fragments have the potential to be used as sensitive and specific binding agents in a broad range of industrial applications. Genetic manipulation has been used to design a series of antibody fragment configurations with a flexible linker and/or a disulphide bond between the heavy chain and light chain of an antibody fragment against the herbicide atrazine. The thermostability and stability to a range of denaturants, polar and non-polar solvents, surfactants and proteases have been compared. It has been found that a novel antibody fragment construct (STAB: stabilized antibody) containing both a flexible linker and a disulphide bond can be effectively produced and shows greatly improved stability in these diverse environments. These STABs should be useful in environmental diagnostics and remediation, and may provide a generic approach for stabilizing antibody fragments in formulations containing detergents and penetrants for topical application in the pharmaceutical and cosmetic industries.

  6. Baculovirus display of functional antibody Fab fragments.

    PubMed

    Takada, Shinya; Ogawa, Takafumi; Matsui, Kazusa; Suzuki, Tasuku; Katsuda, Tomohisa; Yamaji, Hideki

    2015-08-01

    The generation of a recombinant baculovirus that displays antibody Fab fragments on the surface was investigated. A recombinant baculovirus was engineered so that the heavy chain (Hc; Fd fragment) of a mouse Fab fragment was expressed as a fusion to the N-terminus of baculovirus gp64, while the light chain of the Fab fragment was simultaneously expressed as a secretory protein. Following infection of Sf9 insect cells with the recombinant baculovirus, the culture supernatant was analyzed by enzyme-linked immunosorbent assay using antigen-coated microplates and either an anti-mouse IgG or an anti-gp64 antibody. A relatively strong signal was obtained in each case, showing antigen-binding activity in the culture supernatant. In western blot analysis of the culture supernatant using the anti-gp64 antibody, specific protein bands were detected at an electrophoretic mobility that coincided with the molecular weight of the Hc-gp64 fusion protein as well as that of gp64. Flow cytometry using a fluorescein isothiocyanate-conjugated antibody specific to mouse IgG successfully detected the Fab fragments on the surface of the Sf9 cells. These results suggest that immunologically functional antibody Fab fragments can be displayed on the surface of baculovirus particles, and that a fluorescence-activated cell sorter with a fluorescence-labeled antigen can isolate baculoviruses displaying specific Fab fragments. This successful baculovirus display of antibody Fab fragments may offer a novel approach for the efficient selection of specific antibodies.

  7. Localization of malignant melanoma using monoclonal antibodies

    SciTech Connect

    Wasselle, J.; Becker, J.; Cruse, W.; Espinosa, C.; Cox, C.; Reintgen, D. )

    1991-04-01

    Finding a screening test to evaluate patients with cancer for occult metastatic disease, as well as imaging all known disease, is a goal of research efforts. Twenty-nine evaluable patients with deeply invasive (stage I), regional nodal (stage II), or systemic (stage III) melanoma underwent imaging by administration of a preparation of the antimelanoma antibody labeled with technetium 99m. Scan results indicated that 28 of 32 confirmed metastatic sites were imaged with this technique (88% sensitivity). Analysis of the individual positive sites revealed that nodal basins and visceral metastases accounted for the highest percentage of metastatic sites imaged, with 14 (88%) of 16 nodal basin metastases and all four visceral metastases being detected through imaging. Occult nodal disease was detected in the iliac nodal chain in two of the 29 patients. The imaging of benign tumors and nodal basins not containing disease accounted for a confirmed false-positive rate of 21%. Three (10%) of the 29 scan results were confirmed to be false-negative. In vivo tumor localization with monoclonal antibodies showed a sensitivity similar to that of other roentgenographic procedures for identifying metastatic disease and was useful in two of three patients in identifying occult iliac nodal disease, a region that is difficult to evaluate with physical examination and other imaging modalities.

  8. A technetium-labeled monoclonal antibody for imaging metastatic melanoma

    SciTech Connect

    Frytak, S.; Creagan, E.T.; Brown, M.L.; Salk, D.; Nelp, W. )

    1991-04-01

    Twenty patients with histologically proven metastatic melanoma were scanned with a 99mtechnetium ({sup 99}mTc)-labeled melanoma antibody to determine the detection rate of known malignant lesions and to evaluate the antibody's ability to discover occult metastases. Isotope localization in different organs was as follows: liver 100%, bone 100%, subcutaneous lesions 80%, lymph nodes 54%, and lung 33%. Four unsuspected bone lesions and 16 occult subcutaneous lesions were found. False positive lesions were noted in two instances--one benign thyroid adenoma, and one arthritic bone lesion. One patient developed an atypical serum sickness reaction with a rash and arthralgias that responded rapidly to treatment. The {sup 99}mTc antimelanoma antibody is a safe and effective method to detect metastatic melanoma. It has potential use for screening newly diagnosed melanomas that carry an increased risk of recurrence.

  9. Radioimaging of melanoma using sup 99m Tc-labeled Fab fragment reactive with a high molecular weight melanoma antigen

    SciTech Connect

    Lamki, L.M.; Zukiwski, A.A.; Shanken, L.J.; Legha, S.S.; Benjamin, R.S.; Plager, C.E.; Salk, D.F.; Schroff, R.W.; Murray, J.L. )

    1990-02-01

    Twenty patients with metastatic malignant melanoma were studied with 99mTc-labeled monoclonal antibody (MoAb) Fab fragment (NR-Ml-05) reactive with a high molecular weight (Mr 240,000) melanoma associated antigen. Patients received 40 mg unlabeled irrelevant MoAb (NR-2AD-IgG) and 7.5 mg unlabeled NR-Ml-05 (whole IgG) prior to infusion of 10 mg 99mTc-labeled (10-25 mCi) NR-Ml-05 Fab. Unlabeled MoAb were given to block nonspecific and specific binding sites. Gamma camera scans and single photon emission computed tomography were performed at 8 and 24 h postadministration. Of 172 preexisting lesions, 136 were imaged for a sensitivity of detection of 79%. Imaging was site and size dependent with the greatest sensitivity for liver lesions (100%) and the least for bowel (0%). Six sites (2 skin, 1 lung, 3 liver) were detected by single photon emission computed tomography that were missed on routine planar images. Forty-one additional unconfirmed sites were seen. Of these, 7 (17%) have been confirmed as tumor after a median follow-up time of 6 months. False positive scans included scar tissue, areas of chronic inflammation, an infected femoral aneurysm, and septic emboli. Nonspecific uptake of radioactivity occurred in kidney, gallbladder, bowel, thyroid, and myocardium. Human anti-mouse antibodies were detected in up to 69% of patients. In summary, radioimaging with {sup 99m}Tc-NR-Ml-05 is a sensitive test, especially for detecting liver lesions. It is safe, simple to administer, and convenient for the patient. Biodistribution and imaging sensitivity differ significantly from studies in which {sup 111}In-labeled anti-melanoma MoAb have been used.

  10. 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.

  11. Clinical deployment of antibodies for treatment of melanoma.

    PubMed

    Curti, Brendan D; Urba, Walter J

    2015-10-01

    The concept of using immunotherapy to treat melanoma has existed for decades. The rationale comes from the knowledge that many patients with melanoma have endogenous immune responses against their tumor cells and clinically meaningful tumor regression can be achieved in a minority of patients using cytokines such as interleukin-2 and adoptive cellular therapy. In the last 5 years there has been a revolution in the clinical management of melanoma in large measure based on the development of antibodies that influence T cell regulatory pathways by overcoming checkpoint inhibition and providing co-stimulation, either of which results in significantly more effective immune-mediated tumor destruction. This review will describe the pre-clinical and clinical application of antagonistic antibodies targeting the T-cell checkpoints cytotoxic T-lymphocyte antigen 4 (CTLA-4) and programmed death 1 (PD-1), and agonistic antibodies targeting the costimulatory pathways OX40 and 4-1BB. Recent progress and opportunities for future investigation of combination antibody therapy will be described.

  12. 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

  13. Radiolabeled monoclonal antibodies in the diagnosis and treatment of malignant melanoma

    SciTech Connect

    Divgi, C.R.; Larson, S.M. )

    1989-10-01

    The use of antibodies directed against tumors has found increasing usefulness after the discovery by Kohler and Milstein of hybridoma technology, which made it possible to obtain monoclonal antibody (MoAb) that reacted specifically against a particular epitope on a particular antigen site. Relative tumor specificity and a lack of significant toxicity, together with the ability to link radionuclides (both halogens and metals) without significant deterioration of biologic behavioral characteristics such as immunoreactivity, have enabled widespread use of radiolabeled MoAbs in several malignancies, including and especially malignant melanoma. There is a significant body of data indicating that radiolabeled MoAbs directed against melanoma-associated antigens have an important role in the detection and therapy of metastatic malignant melanoma. Detection of visceral disease, while currently suboptimal, will in the future improve with optimization of SPECT imaging using 99mTc-labeled MoAb Fab fragments. This may result in an attenuated or absent antimouse response, especially after one injection, unless of course coinfused with either specific and/or nonspecific intact immunoglobulin (Ig). Radiolabeled fragments play an important role in radioimmunotherapy in metastatic melanoma. This role may be enhanced by the development of newer chelating agents that will decrease nonspecific hepatic uptake of radionuclide, enabling the use of beta-emitting radiometals such as 90Y. The recent report demonstrating diminished hepatic uptake of 99mTc-labeled anti-high molecular weight antigen (HMWA) Fab shows promise, since the same labeling technique can be used to deliver radiotherapeutic agents such as 186Re, which may be labeled to MoAb with methods similar to those used for 99mTc.82 references.

  14. Technetium-99m labeled monoclonal antibodies in the detection of metastatic melanoma

    SciTech Connect

    Serafini, A.N.; Kotler, J.; Feun, L.; Dewanjee, M.; Robinson, D.; Salk, D.; Sfakianakis, G.; Abrams, P.; Savaraj, N.; Goodwin, D.; )

    1989-08-01

    Twenty-six stage II/III malignant melanoma patients with 321 measurable metastatic lesions were imaged using Fab fragments of an IgG murine monoclonal antibody labeled specifically with 10-30 mCi Tc-99m with a bi-functional chelating method (NeoRx, Seattle, WA). There were no side effects or adverse reactions. Immunoscintigraphy demonstrated 66.6% of lesions larger than 1 cm and 92.5% of lesions larger than 3 cm. Most frequently detected metastases were in lymph nodes, subcutaneous areas, and bone. Of lesions less than 1 cm, 23.6% were detected if superficial cutaneous lesions were excluded. The smallest detectable lesion was 4 mm. Twenty-one additional clinically unsuspected sites were visualized in 12 of the 26 patients studied. Of these, 56% were confirmed as metastasis by other tests. There were apparent nonspecific localizations owing to other causes, including fracture, varicosities, skin abscess and pneumonitis. Increased experience in image analysis facilitates correct interpretation of these localizations. This study demonstrates that imaging with Tc-99m labeled antibody fragments detects melanoma lesions in organs routinely surveyed and in other areas not routinely assessed by other imaging techniques. The procedure is readily performed and safe. The principal advantage of the test is its ability to survey the entire body and all organs with a single test. Its principal limitation, in common with other diagnostic imaging procedures, is its poor sensitivity for detecting lesions less than 1 cm.

  15. Cloning, bacterial expression and crystallization of Fv antibody fragments

    NASA Astrophysics Data System (ADS)

    E´, Jean-Luc; Boulot, Ginette; Chitarra, V´ronique; Riottot, Marie-Madeleine; Souchon, H´le`ne; Houdusse, Anne; Bentley, Graham A.; Narayana Bhat, T.; Spinelli, Silvia; Poljak, Roberto J.

    1992-08-01

    The variable Fv fragments of antibodies, cloned in recombinant plasmids, can be expressed in bacteria as functional proteins having immunochemical properties which are very similar or identical with those of the corresponding parts of the parent eukaryotic antibodies. They offer new possibilities for the study of antibody-antigen interactions since the crystals of Fv fragments and of their complexes with antigen reported here diffract X-rays to a higher resolution that those obtained with the cognate Fab fragments. The Fv approach should facilitate the structural study of the combining site of antibodies and the further characterization of antigen-antibody interactions by site-directed mutagenesis experiments.

  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. Development trends for generation of single-chain antibody fragments.

    PubMed

    Farajnia, Safar; Ahmadzadeh, Vahideh; Tanomand, Asghar; Veisi, Kamal; Khosroshahi, Shiva Ahdi; Rahbarnia, Leila

    2014-10-01

    Recombinant antibodies are increasingly being employed as therapeutic agents especially in combination with anti-cancer drugs. The single-chain antibody fragments are small antigen-binding proteins which provide the most commonly used antibody formats for diagnostic and therapeutic purposes. These antibody fragments have more rapid tumor penetration and clearance from the serum relative to full-length monoclonal antibodies. There are in vitro antibody-display technologies such as phage display, cell surface display, ribosome display and mRNA display that can be used to isolate high specificity and affinity single-chain antibodies against a wide variety of targets. We review these strategies for generation of stable and active antibody fragments in the present article.

  18. Monoclonal Antibody-Directed Effector Cells Selectively Lyse Human Melanoma Cells in vitro and in vivo

    NASA Astrophysics Data System (ADS)

    Schulz, Gregor; Bumol, Thomas F.; Reisfeld, Ralph A.

    1983-09-01

    Monoclonal antibody 9.2.27 (mAb 9.2.27) directed to a chondroitin sulfate proteoglycan on human melanoma cells was able to suppress tumor growth in athymic (nu/nu) mice more effectively when bound with polyethylene glycol to murine effector cells than when injected alone. These ``armed'' effector cells also proved more effective than the monoclonal antibody in eliciting antibody-dependent cellular cytotoxicity against human melanoma target cells in vitro.

  19. Serological survey of normal humans for natural antibody to cell surface antigens of melanoma.

    PubMed Central

    Houghton, A N; Taormina, M C; Ikeda, H; Watanabe, T; Oettgen, H F; Old, L J

    1980-01-01

    Sera of 106 normal adult men were tested for antibodies reacting with cell surface antigens of three established lines of cultured malignant melanoma. Positive reactions with a protein A assay for IgG antibodies were extremely rare (1-2%). The frequency of positive reactions with assays for IgM antibodies was higher: 5-15% in immune adherence assays and 55-82% in anti-C3 mixed hemadsorption assays. After low-titered sera and sera reacting with fetal calf serum components, conventional alloantigens, and widely distributed class 3 antigens were excluded, sera from seven individuals (one with IgG antibody and six with IgM antibodies) were selected for detailed analysis. The serum containing the IgG antibody came from a healthy 65-year-old Caucasian man; titers of antibody in his serum ranged from < 1/10 to 1/40,000 in tests with different melanoma cell lines. This IgG antibody identifies a differentiation antigen of melanocytes, provisionally designated Mel 1, that distinguishes two classes of melanomas: 22 melanoma cell lines typed Mel 1+ and 17 types Mel 1-. Mel 1 is expressed by fetal fibroblasts but not adult fibroblasts and can be found on a proportion of cultured epithelial cancer cell lines (5 out of 23) but not on glioma or B-cell lines. The melanoma antigens detected by the naturally occurring IgM antibodies are serologically unrelated to Mel 1 but, like Mel 1, appear to be differentiation antigens that distinguish subsets of melanoma. These IgM antibodies detect antigens that are identical or closely related to the AH antigen, a melanoma surface antigen that was initially defined by autologous antibody in a patient with melanoma. In view of the immunogenicity of both Mel 1 and the AH antigens in humans and their occurrence on more than 50% of melanomas, it remains to be seen whether antibody to these antigens can be elicited by specific vaccination of seronegative melanoma patients and whether this will have an influence on the clinical course of the disease

  20. Monoclonal antibody imaging of human melanoma. Radioimmunodetection by subcutaneous or systemic injection

    SciTech Connect

    Lotze, M.T.; Carrasquillo, J.A.; Weinstein, J.N.; Bryant, G.J.; Perentesis, P.; Reynolds, J.C.; Matis, L.A.; Eger, R.R.; Keenan, A.M.; Hellstroem, Ie.

    1986-09-01

    Fab fragments of monoclonal antibodies (MoAb) to melanoma, radiolabeled with /sup 131/I, were evaluated as diagnostic reagents to determine their ability to localize systemic--MoAb injected intravenously (IV)--or nodal metastatic disease--injected subcutaneously (SQ) at a site proximal to draining lymph nodes. Sixty-one scans were performed (40 IV, 21 SQ) in 59 patients who had injections of 0.2-50 mg of /sup 131/I coupled (0.2-12 mCi) antibody. These included 48.7, which identifies a high molecular weight antigen (HMW), or 96.5, which identifies a transferrin like molecule, p97. 125I coupled nonspecific Fab 1.4, reacting with murine leukemia virus, or the whole antibody BL3, reactive with a human B cell idiotypic determinant, was generally used in tandem with the patients injected SQ as a nonspecific control. All patients had immunohistochemical studies performed and demonstrated binding to the antibodies injected. Of the IV patients, 22/38 (58%) had (+) scans, 13 at SQ or nodal sites, four at visceral sites, and five at visceral and SQ sites. Patients with clinical stage II disease had SQ injection of MoAb, including 11 additional patients injected with the whole antibody 9.2.27 (anti-HMW) labeled with 111In (6 patients) or /sup 131/I (5 patients). Nodal dissection was performed 2-4 days later. All 111In coupled antibodies demonstrated excellent nodal delineation without specific identification of tumor deposits. Of the 21 patients injected SQ with MoAb, 17 had confirmed tumor in nodes. Of patients injected with Fab fragments, 50% had specific uptake of MoAb, although only two were successfully imaged. Increased uptake of antimelanoma antibodies was observed in some patients in lymph nodes not containing tumor. Clearance of labeled antibody from the injection site occurred with a half life of 16-50 hours. Toxicity was limited to local discomfort at the site of SQ injection.

  1. Engineered Antibody Fragments for Immunodiagnosis of Papaya ringspot virus.

    PubMed

    Maheshwari, Yogita; Verma, H N; Jain, R K; Mandal, Bikash

    2015-07-01

    The present study was undertaken to clone and express the genes encoding antibody to the recombinant coat protein (rCP) of Papaya ringspot virus (PRSV) and to assess the engineered antibody for the detection of PRSV. A 33-kDa rCP of PRSV, which was produced in Escherichia coli, generated PRSV specific antibody in immunized mouse. The heavy and light chain variable domain genes (VH and VL) of 351 and 360 nucleotides, respectively, were cloned from the mRNA isolated from the spleen of the immunized mouse with rCP of PRSV. The VH and VL belong to the family IgG1 and kappa chain, respectively, and contained the framework regions and complementarity determining regions. The VH and VL genes were individually used to develop the expression constructs in pET28a (+) vector and 14-kDa proteins were obtained in E. coli. The amount of purified VH and VL proteins was 3-4 mg/l of bacterial culture. Both the antibody fragments recognized PRSV in the crude sap; however, the VL antibody fragment showed higher affinity to PRSV. The mixture of VH and VL detected PRSV as effectively as polyclonal antibody. The recombinant antibody fragments mixture detected PRSV in the field samples with 100 % accuracy in dot immunobinding assay (DIBA) and enzyme-linked immunosorbent assay (ELISA). The sensitivity of the detection of PRSV using antibody fragments was 1.0 and 10.0 ng in DIBA and ELISA, respectively. The results showed successful isolation of functional single-domain antibody encoding genes to PRSV directly from the immunized spleen cells of mouse. This study for the first time demonstrates application of bacterial expressed recombinant antibody fragments in immunodiagnosis of PRSV.

  2. Generation of recombinant antibody fragments for membrane protein crystallization.

    PubMed

    Mir, Syed H; Escher, Claudia; Kao, Wei-Chun; Birth, Dominic; Wirth, Christophe; Hunte, Carola

    2015-01-01

    Membrane proteins are challenging targets for crystallization and structure determination by X-ray crystallography. Hurdles can be overcome by antibody-mediated crystallization. More than 25 unique structures of membrane protein:antibody complexes have already been determined. In the majority of cases, hybridoma-derived antibody fragments either in Fab or Fv fragment format were employed for these complexes. We will briefly introduce the background and current status of the strategy and describe in detail the current protocols of well-established methods for the immunization, the selection, and the characterization of antibodies, as well as the cloning, the production, and the purification of recombinant antibodies useful for structural analysis of membrane proteins.

  3. The Biochemical Properties of Antibodies and Their Fragments.

    PubMed

    Hnasko, Robert M

    2015-01-01

    Immunoglobulins (Ig) or antibodies are powerful molecular recognition tools that can be used to identify minute quantities of a given target analyte. Their antigen-binding properties define both the sensitivity and selectivity of an immunoassay. Understanding the biochemical properties of this class of protein will provide users with the knowledge necessary to select the appropriate antibody composition to maximize immunoassay results. Here we define the general biochemical properties of antibodies and their similarities and differences, explain how these properties influence their functional relationship to an antigen target, and describe a method for the enzymatic fragmentation of antibodies into smaller functional parts.

  4. 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

  5. Effect of anti-B16 melanoma monoclonal antibody on established murine B16 melanoma liver metastases

    SciTech Connect

    Eisenthal, A.; Lafreniere, R.; Lefor, A.T.; Rosenberg, S.A.

    1987-06-01

    The administration of anti-B16 monoclonal antibody of the IgG2b isotype to mice bearing established B16 melanoma liver metastases caused a significant and consistent reduction of up to 90% in the number of these metastases. No reduction in the number of metastases was noted when antigenically unrelated tumor or nonspecific immunoglobulin were employed. The antibody-mediated antitumor effect was completely abrogated by total body irradiation of the host. Treatment of the tumor-bearing host with antiserum directed against asialo GM1 prior to anti B16 antibody administration, abrogated the therapeutic effect indicating the involvement of a radiosensitive, ASGM1-positive cell in the tumor regression. The antitumor effect of the antibody treatment could be augmented by the concomitant administration of recombinant interleukin-2. The effect seen may have possible application in the treatment of liver metastases in humans by combined immunotherapy using recombinant interleukin-2 and specific antitumor monoclonal antibodies.

  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. Common antigenic determinants on human melanoma, glioma, neuroblastoma, and sarcoma cells defined with monoclonal antibodies.

    PubMed

    Seeger, R C; Rosenblatt, H M; Imai, K; Ferrone, S

    1981-07-01

    Antigenic determinants that are common to melanomas, gliomas, neuroblastomas, and sarcomas but that are minimally or not detectably expressed by adult tissues were defined with monoclonal antibodies. Quantitative absorption of monoclonal antibody (Ab 165) with adult tissues followed by testing on antigen-positive UCLA-SO-M14 melanoma cells did not demonstrate antigenic determinant (Ag 165) in brain, lung, liver, kidney, intestine, adrenal, and muscle, Absorption of Ab 376 demonstrated Ag 376 in adult lung but minimal or no antigen in other tissues. Both antigens were associated with a variety of fetal tissues. Assessment of 28 human tumor cell lines with the 131I-staphylococcal Protein A-binding test demonstrated that Ab 165 reacted strongly with melanomas and gliomas and weakly with sarcomas. Ab 376 reacted strongly with melanomas, gliomas, neuroblastomas, and sarcomas. Neither of these antibodies reacted appreciably with carcinoma or teratoma cell lines. Absorption of Ab 165 and Ab 376 with noncultured tumors demonstrated that melanomas, sarcomas, and neuroblastomas can have greater quantities of these antigens in vivo than do normal adult tissues. Qualitative and quantitative antigenic heterogeneity within positive classes of tumors was demonstrated for both cultured and noncultured tumors. The differences in antigen expression in vivo between normal and neoplastic cells suggest potential value for these antibodies in immunodiagnosis and possibly immunotherapy.

  8. In vivo Imaging with Antibodies and Engineered Fragments

    PubMed Central

    Freise, Amanda C.; Wu, Anna M.

    2015-01-01

    Antibodies have clearly demonstrated their utility as therapeutics, providing highly selective and effective drugs to treat diseases in oncology, hematology, cardiology, immunology and autoimmunity, and infectious diseases. More recently, a pressing need for equally specific and targeted imaging agents for assessing disease in vivo, in preclinical models and patients, has emerged. This review summarizes strategies for developing and optimizing antibodies as targeted probes for use in non-invasive imaging using radioactive, optical, magnetic resonance, and ultrasound approaches. Recent advances in engineered antibody fragments and scaffolds, conjugation and labeling methods, and multimodality probes are highlighted. Importantly, antibody-based imaging probes are seeing new applications in detection and quantitation of cell surface biomarkers, imaging specific responses to targeted therapies, and monitoring immune responses in oncology and other diseases. Antibody-based imaging will provide essential tools to facilitate the transition to truly precision medicine. PMID:25934435

  9. Paraneoplastic cerebellar degeneration with anti-Yo antibodies associated with metastatic uveal melanoma.

    PubMed

    Valpione, Sara; Zoccarato, Marco; Parrozzani, Raffaele; Pigozzo, Jacopo; Giometto, Bruno; Laveder, Francesco; Aliberti, Camillo; Chiarion-Sileni, Vanna

    2013-12-15

    Paraneoplastic cerebellar degeneration (PCD) is characterized by subacute development of pancerebellar dysfunction as a remote effect of a systemic cancer and usually develops in patients affected by gynecological tumors. Uveal melanoma is a very rare disease with a severe prognosis. A 58-year-old man affected by uveal melanoma developed anti-Yo positive paraneoplastic cerebellar degeneration (PCD) 42 months after the initial diagnosis. The onset and worsening of the neurological symptoms were parallel to the course of liver metastasis. To our knowledge this is the first case of PCD in a patient with uveal melanoma. We speculate that the cerebellar degeneration-related protein 2 (CDR2), to which the anti-Yo antibodies are directed, may have been expressed in melanoma cells and conferred proliferative advantage to the disease. PMID:24035275

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

    PubMed

    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 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.

  11. 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

  12. 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

  13. 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

  14. Influence of molecular size on tissue distribution of antibody fragments

    PubMed Central

    Li, Zhe; Krippendorff, Ben-Fillippo; Sharma, Sharad; Walz, Antje C.; Lavé, Thierry; Shah, Dhaval K.

    2016-01-01

    Biodistribution coefficients (BC) allow estimation of the tissue concentrations of proteins based on the plasma pharmacokinetics. We have previously established the BC values for monoclonal antibodies. Here, this concept is extended by development of a relationship between protein size and BC values. The relationship was built by deriving the BC values for various antibody fragments of known molecular weight from published biodistribution studies. We found that there exists a simple exponential relationship between molecular weight and BC values that allows the prediction of tissue distribution of proteins based on molecular weight alone. The relationship was validated by a priori predicting BC values of 4 antibody fragments that were not used in building the relationship. The relationship was also used to derive BC50 values for all the tissues, which is the molecular weight increase that would result in 50% reduction in tissue uptake of a protein. The BC50 values for most tissues were found to be ~35 kDa. An ability to estimate tissue distribution of antibody fragments based on the BC vs. molecular size relationship established here may allow better understanding of the biologics concentrations in tissues responsible for efficacy or toxicity. This relationship can also be applied for rational development of new biotherapeutic modalities with optimal biodistribution properties to target (or avoid) specific tissues. PMID:26496429

  15. Aglycosylated antibodies and antibody fragments produced in a scalable in vitro transcription-translation system.

    PubMed

    Yin, Gang; Garces, Eudean D; Yang, Junhao; Zhang, Juan; Tran, Cuong; Steiner, Alexander R; Roos, Christine; Bajad, Sunil; Hudak, Susan; Penta, Kalyani; Zawada, James; Pollitt, Sonia; Murray, Christopher J

    2012-01-01

    We describe protein synthesis, folding and assembly of antibody fragments and full-length aglycosylated antibodies using an Escherichia coli-based open cell-free synthesis (OCFS) system. We use DNA template design and high throughput screening at microliter scale to rapidly optimize production of single-chain Fv (scFv) and Fab antibody fragments that bind to human IL-23 and IL-13α1R, respectively. In addition we demonstrate production of aglycosylated immunoglobulin G (IgG 1) trastuzumab. These antibodies are produced rapidly over several hours in batch mode in standard bioreactors with linear scalable yields of hundreds of milligrams/L over a 1 million-fold change in scales up to pilot scale production. We demonstrate protein expression optimization of translation initiation region (TIR) libraries from gene synthesized linear DNA templates, optimization of the temporal assembly of a Fab from independent heavy chain and light chain plasmids and optimized expression of fully assembled trastuzumab that is equivalent to mammalian expressed material in biophysical and affinity based assays. These results illustrate how the open nature of the cell-free system can be used as a seamless antibody engineering platform from discovery to preclinical development of aglycosylated monoclonal antibodies and antibody fragments as potential therapeutics.

  16. 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

  17. Polyreactivity of natural antibodies: exchange by HL-fragments.

    PubMed

    Sedykh, M A; Buneva, V N; Nevinsky, G A

    2013-12-01

    The polyreactivity of binding (formation of antibody (AB) complexes not only with specific but also with foreign antigens) is a widespread phenomenon that in some cases can be caused by a conformational lability of the antigen-binding sites of antibodies (which increases upon treatment with various destabilizing agents) and leads to AB binding with very different antigens. Some ABs exist as dimers of the initial ABs and their idiotypes (or anti-idiotypes) capable of producing intramolecular cyclic complexes with features of polyreactants. Another mechanism of binding polyreactivity is an exchange in blood by halves of IgG4 molecules (HL-fragments) against various antigens. Also, for the first time catalytic polyfunctionality of human milk ABs has been detected, which is caused by an exchange by HL-fragments between molecules of λ- and κ-IgG (IgG1-IgG4) and also by λ- and κ-sIgA against different antigens with formation of very different chimeric antibodies. This review considers all possible pathways of formation of polyspecific immunoglobulins and their biological functions described in the literature, as well as mechanisms of binding polyreactivity and catalytic polyfunctionality of natural antibodies.

  18. Cell-surface antigens of melanoma recognized by human monoclonal antibodies.

    PubMed Central

    Yamaguchi, H; Furukawa, K; Fortunato, S R; Livingston, P O; Lloyd, K O; Oettgen, H F; Old, L J

    1987-01-01

    Human monoclonal antibodies (mAbs) were derived from lymph node lymphocytes and peripheral blood lymphocytes (PBL) from patients with melanoma. Four methods for generating human mAbs were compared: fusion with human [LICR-LON-HMy-2 (LICR-2)] or mouse (NS-1) cells; transformation by Epstein-Barr virus (EBV); and EBV transformation followed by NS-1 fusion. NS-1 fusion with lymph node lymphocytes resulted in a higher number of growing hybrids than LICR-2 fusion. Virtually no hybrids were obtained from NS-1 or LICR-2 fusions with PBL. EBV transformed lymphocytes from lymph node and peripheral blood with equal efficiency, and the yield of proliferating cultures for antibody screening was more than 10- to 30-fold greater than that obtained by fusion techniques. However, once antibody-producing cultures had been identified, stability and clonability of EBV-transformed cells were poorer than that of NS-1 hybrid cells. To combine the strengths of both methods, cultures of EBV-transformed cells were fused with NS-1; and hybrid clones were isolated that showed vigorous growth, clonability, and stable antibody secretion. Detailed specificity analysis of the mAbs produced by six of these clones indicated detection of a class 1 (unique) melanoma antigen, a class 3 melanoma antigen, and four ganglioside antigens (GD3, GM3, and two other, as yet uncharacterized, heterophile antigens). Images PMID:3031684

  19. Chemical macrocyclization of peptides fused to antibody Fc fragments.

    PubMed

    Angelini, Alessandro; Diderich, Philippe; Morales-Sanfrutos, Julia; Thurnheer, Sarah; Hacker, David; Menin, Laure; Heinis, Christian

    2012-09-19

    To extend the plasma half-life of a bicyclic peptide antagonist, we chose to link it to the Fc fragment of the long-lived serum protein IgG1. Instead of chemically conjugating the entire bicyclic peptide, we recombinantly expressed its peptide moiety as a fusion protein to an Fc fragment and subsequently cyclized the peptide by chemically reacting its three cysteine residues with tris-(bromomethyl)benzene. This reaction was efficient and selective, yielding completely modified peptide fusion protein and no side products. After optimization of the linker and the Fc fragment format, the bicyclic peptide was fully functional as an inhibitor (K(i) = 76 nM) and showed an extended terminal half-life of 1.5 days in mice. The unexpectedly clean reaction makes chemical macrocyclization of peptide-Fc fusion proteins an attractive synthetic approach. Its good compatibility with the Fc fragment may lend the bromomethylbenzene-based chemistry also for the generation of antibody-drug conjugates.

  20. PD-1 Antibody Monotherapy for Malignant Melanoma: A Systematic Review and Meta-Analysis

    PubMed Central

    Lin, Zhijuan; Chen, Xing; Li, Zhifeng; Luo, Yiming; Fang, Zhihong; Xu, Bing; Han, Mingzhe

    2016-01-01

    Antibodies targeting programmed death 1 (PD-1) help prevent tumor cells from escaping immune-mediated destruction. We conducted this systematic review and meta-analysis to gain insight into the efficacy of PD-1 antibodies for the treatment of melanoma. Five trials involving 2,828 adult patients were included in this meta-analysis. In patients with previously untreated or refractory melanoma, treatment with PD-1 antibodies significantly improved the six-month progression-free survival (PFS) (HR 0.55, 95% CI 0.50–0.60, P<0.00001) and the overall response rate (OR 3.89, 95% CI 3.12–4.83, P<0.00001). This meta-analysis indicated that anti-PD-1 treatment might provide a significant survival benefit in patients with melanoma. In addition, we found that patients treated with nivolumab reported significantly fewer treatment-related adverse events (OR 0.74, 95% CI 0.57–0.97, P = 0.03) than those treated with other agents, but there was a dose-dependent increase in the frequency of adverse events in patients treated with pembrolizumab. PMID:27483468

  1. Nivolumab and pembrolizumab as immune-modulating monoclonal antibodies targeting the PD-1 receptor to treat melanoma.

    PubMed

    Faghfuri, Elnaz; Faramarzi, Mohammad Ali; Nikfar, Shekoufeh; Abdollahi, Mohammad

    2015-01-01

    Malignant melanoma is an important issue in oncology due to its high incidence, high mortality, and resistance to systemic therapy; however, targeted immunotherapy has noticeably improved the survival rates of melanoma patients. Promising targeted immunotherapies for malignant melanoma include the blockade of immune checkpoints with antibodies targeting cytotoxic T lymphocyte-associated antigen 4 and the programmed cell death protein 1 pathway. The US FDA-approved antibody ipilimumab targets cytotoxic T lymphocyte-associated antigen 4; however, it was limited by toxicity and a low response. Nivolumab and pembrolizumab (formerly lambrolizumab), the two FDA-approved anti-programmed death-1 monoclonal antibodies, show highly durable response rates and long-term safety, validating the importance of the programmed cell death protein 1 pathway blockade for treatment of malignant melanoma.

  2. Hormone Conjugated with Antibody to CD3 Mediates Cytotoxic T Cell Lysis of Human Melanoma Cells

    NASA Astrophysics Data System (ADS)

    Liu, Margaret Ann; Nussbaum, Samuel R.; Eisen, Herman N.

    1988-01-01

    Cytotoxic T lymphocytes can be activated by antibodies to their antigen-specific receptor complex (TCR-CD3) to destroy target cells, regardless of the specificity of the cytotoxic T cells. A novel hormone-antibody conjugate, consisting of an analog of melanocyte-stimulating hormone chemically coupled to a monoclonal antibody to CD3, the invariant component of the T cell receptor complex, was used to target human melanoma cells for destruction by human cytotoxic T lymphocytes that bear no specificity for the tumor cells. As targeting components of such anti-CD3 conjugates, hormones or growth factors are expected to prove more effective than antibodies to tumor-associated antigens in focusing the destructive activity of cytotoxic T cells on tumor target cells.

  3. Antibody Therapy Targeting CD47 and CD271 Effectively Suppresses Melanoma Metastasis in Patient-Derived Xenografts.

    PubMed

    Ngo, Michael; Han, Arum; Lakatos, Anita; Sahoo, Debashis; Hachey, Stephanie J; Weiskopf, Kipp; Beck, Andrew H; Weissman, Irving L; Boiko, Alexander D

    2016-08-01

    The high rate of metastasis and recurrence among melanoma patients indicates the existence of cells within melanoma that have the ability to both initiate metastatic programs and bypass immune recognition. Here, we identify CD47 as a regulator of melanoma tumor metastasis and immune evasion. Protein and gene expression analysis of clinical melanoma samples reveals that CD47, an anti-phagocytic signal, correlates with melanoma metastasis. Antibody-mediated blockade of CD47 coupled with targeting of CD271(+) melanoma cells strongly inhibits tumor metastasis in patient-derived xenografts. This therapeutic effect is mediated by drastic changes in the tumor and metastatic site immune microenvironments, both of whichwhich exhibit greatly increased density of differentiated macrophages and significantly fewer inflammatory monocytes, pro-metastatic macrophages (CCR2(+)/VEGFR1(+)), and neutrophils, all of which are associated with disease progression. Thus, antibody therapy that activates the innate immune response in combination with selective targeting of CD271(+) melanoma cells represents a powerful therapeutic approach against metastatic melanoma. PMID:27477289

  4. The antibody response against MART-1 differs in patients with melanoma-associated leucoderma and vitiligo.

    PubMed

    Teulings, Hansje-Eva; Willemsen, Karin J; Glykofridis, Iris; Krebbers, Gabrielle; Komen, Lisa; Kroon, Marije W; Kemp, E Helen; Wolkerstorfer, Albert; van der Veen, J P Wietze; Luiten, Rosalie M; Tjin, Esther P M

    2014-11-01

    Patients with melanoma may develop skin depigmentation spontaneously or following therapy, referred to as melanoma-associated leucoderma (MAL). As clinical presentation of MAL may precede primary/metastatic melanoma detection, recognition of MAL is important to prevent its misdiagnosis as vitiligo and the subsequent application of immunosuppressive treatment. To reveal the immunity involved in MAL development, we investigated the presence of antibody and T-cell immune responses directed against the melanocyte-differentiation-antigens MART-1 (Melan-A), tyrosinase and gp100 in patients with MAL, as compared to patients with vitiligo. Autoantibodies to gp100 and tyrosinase were commonly found in both diseases. Interestingly, MART-1 antibodies were only present in patients with MAL. Melanocyte antigen-specific T cells were found in all patients, with relatively more specific T cells in patients with active vitiligo. Although MAL and vitiligo may appear clinically similar, our results indicate that the humoral immune responses against MART-1 differ between these diseases, which can help to differentiate MAL from vitiligo.

  5. Inhibition of gallium-67 uptake in melanoma by an anti-human transferrin receptor monoclonal antibody

    SciTech Connect

    Chan, S.M.; Hoffer, P.B.; Maric, N.; Duray, P.

    1987-08-01

    The effect of an anti-human transferrin receptor (anti-TFR) monoclonal antibody (MoAb), designated B3/25, and an anti-melanoma antibody, designated 96.5, on the uptake of gallium-67 (/sup 67/Ga) by tumor was studied. Three groups of six athymic mice bearing a human melanoma were injected via tail vein with (a) 0.55 mg human serum albumin (HSA) (control group), (b) 0.5 mg MoAb B3/25 + 0.55 mg HSA, and (c) 0.5 mg MoAb 96.5 + 0.55 mg HSA, respectively. Twenty-four hours later, each mouse was given an intravenous dose of 5 microCi (/sup 67/Ga) citrate. Biodistribution of activity (percent injected dose per gram) determined 48 hr after injection of /sup 67/Ga showed a 75% decrease in tumor uptake in the group of mice that received B3/25 (anti-TFR MoAb) compared with the control group. In contrast, MoAb 96.5 did not show any effect on melanoma uptake of /sup 67/Ga. Histologic findings suggest that the decreased uptake was not due to cellular damage resulting from binding of B3/25 to TFR. The results of this study strongly suggest the involvement of TFR in the in vivo tumor uptake of /sup 67/Ga.

  6. Monoclonal Antibody and an Antibody-Toxin Conjugate to a Cell Surface Proteoglycan of Melanoma Cells Suppress in vivo Tumor Growth

    NASA Astrophysics Data System (ADS)

    Bumol, T. F.; Wang, Q. C.; Reisfeld, R. A.; Kaplan, N. O.

    1983-01-01

    A monoclonal antibody directed against a cell surface chondroitin sulfate proteoglycan of human melanoma cells, 9.2.27, and its diphtheria toxin A chain (DTA) conjugate were investigated for their effects on in vitro protein synthesis and in vivo tumor growth of human melanoma cells. The 9.2.27 IgG and its DTA conjugate display similar serological activities against melanoma target cells but only the conjugate can induce consistent in vitro inhibition of protein synthesis and toxicity in M21 melanoma cells. However, both 9.2.27 IgG and its DTA conjugate effect significant suppression of M21 tumor growth in vivo in an immunotherapy model of a rapidly growing tumor in athymic nu/nu mice, suggesting that other host mechanisms may mediate monoclonal antibody-induced tumor suppression.

  7. Combined treatment with dabrafenib and trametinib with immune-stimulating antibodies for BRAF mutant melanoma.

    PubMed

    Homet Moreno, Blanca; Mok, Stephen; Comin-Anduix, Begonya; Hu-Lieskovan, Siwen; Ribas, Antoni

    2016-07-01

    The combination of targeted therapy with BRAF and MEK inhibitors has become the standard of care in patients with BRAF (V600E) mutant melanoma, but responses are not durable. In addition, the impressive clinical benefits with anti-PD-1 and anti-PD-L1 antibodies (Ab) in patients with heavily pretreated metastatic melanoma and the synergistic effect of dabrafenib, trametinib and anti-PD-1 compared with single therapy alone groups support the idea that combining dabrafenib, trametinib and immunotherapy based on PD-1 blockade could be an interesting approach in the treatment of metastatic melanoma. With our mouse model of syngeneic BRAF (V600E) driven melanoma (SM1), we tested whether the addition of an immunostimulatory Ab targeting CD137 (4-1BB) and/or CD134 (OX40) would enhance the antitumor effect of dabrafenib, trametinib and anti-PD-1 or anti-PD-L1 therapy. In vitro studies showed that the combination group of dabrafenib, trametinib and anti-PD-1 increases CD8(+) tumor infiltrating lymphocytes (TILs), as well as CD4(+) T cells and tumor-associated macrophages (TAMs). An upregulation of PD-L1 was observed in the combination of dabrafenib, trametinib and anti-PD-1 therapy. Combination of dabrafenib, trametinib and anti-PD-1, with either anti-CD137 or anti-CD134, showed a superior antitumor effect, but the five-agent combination was not superior to the four-agent combinations. In conclusion, the combination of dabrafenib, trametinib, anti-PD1 or anti-PD-L1 therapy results in robust antitumor activity, which is further improved by adding the immune-stimulating Ab anti-CD137 or anti-CD134. Our findings support the testing of these combinations in patients with BRAF (V600E) mutant metastatic melanoma.

  8. Combined treatment with dabrafenib and trametinib with immune-stimulating antibodies for BRAF mutant melanoma.

    PubMed

    Homet Moreno, Blanca; Mok, Stephen; Comin-Anduix, Begonya; Hu-Lieskovan, Siwen; Ribas, Antoni

    2016-07-01

    The combination of targeted therapy with BRAF and MEK inhibitors has become the standard of care in patients with BRAF (V600E) mutant melanoma, but responses are not durable. In addition, the impressive clinical benefits with anti-PD-1 and anti-PD-L1 antibodies (Ab) in patients with heavily pretreated metastatic melanoma and the synergistic effect of dabrafenib, trametinib and anti-PD-1 compared with single therapy alone groups support the idea that combining dabrafenib, trametinib and immunotherapy based on PD-1 blockade could be an interesting approach in the treatment of metastatic melanoma. With our mouse model of syngeneic BRAF (V600E) driven melanoma (SM1), we tested whether the addition of an immunostimulatory Ab targeting CD137 (4-1BB) and/or CD134 (OX40) would enhance the antitumor effect of dabrafenib, trametinib and anti-PD-1 or anti-PD-L1 therapy. In vitro studies showed that the combination group of dabrafenib, trametinib and anti-PD-1 increases CD8(+) tumor infiltrating lymphocytes (TILs), as well as CD4(+) T cells and tumor-associated macrophages (TAMs). An upregulation of PD-L1 was observed in the combination of dabrafenib, trametinib and anti-PD-1 therapy. Combination of dabrafenib, trametinib and anti-PD-1, with either anti-CD137 or anti-CD134, showed a superior antitumor effect, but the five-agent combination was not superior to the four-agent combinations. In conclusion, the combination of dabrafenib, trametinib, anti-PD1 or anti-PD-L1 therapy results in robust antitumor activity, which is further improved by adding the immune-stimulating Ab anti-CD137 or anti-CD134. Our findings support the testing of these combinations in patients with BRAF (V600E) mutant metastatic melanoma. PMID:27622011

  9. Combined treatment with dabrafenib and trametinib with immune-stimulating antibodies for BRAF mutant melanoma

    PubMed Central

    Homet Moreno, Blanca; Mok, Stephen; Comin-Anduix, Begonya; Hu-Lieskovan, Siwen; Ribas, Antoni

    2016-01-01

    The combination of targeted therapy with BRAF and MEK inhibitors has become the standard of care in patients with BRAFV600E mutant melanoma, but responses are not durable. In addition, the impressive clinical benefits with anti-PD-1 and anti-PD-L1 antibodies (Ab) in patients with heavily pretreated metastatic melanoma and the synergistic effect of dabrafenib, trametinib and anti-PD-1 compared with single therapy alone groups support the idea that combining dabrafenib, trametinib and immunotherapy based on PD-1 blockade could be an interesting approach in the treatment of metastatic melanoma. With our mouse model of syngeneic BRAFV600E driven melanoma (SM1), we tested whether the addition of an immunostimulatory Ab targeting CD137 (4-1BB) and/or CD134 (OX40) would enhance the antitumor effect of dabrafenib, trametinib and anti-PD-1 or anti-PD-L1 therapy. In vitro studies showed that the combination group of dabrafenib, trametinib and anti-PD-1 increases CD8+ tumor infiltrating lymphocytes (TILs), as well as CD4+ T cells and tumor-associated macrophages (TAMs). An upregulation of PD-L1 was observed in the combination of dabrafenib, trametinib and anti-PD-1 therapy. Combination of dabrafenib, trametinib and anti-PD-1, with either anti-CD137 or anti-CD134, showed a superior antitumor effect, but the five-agent combination was not superior to the four-agent combinations. In conclusion, the combination of dabrafenib, trametinib, anti-PD1 or anti-PD-L1 therapy results in robust antitumor activity, which is further improved by adding the immune-stimulating Ab anti-CD137 or anti-CD134. Our findings support the testing of these combinations in patients with BRAFV600E mutant metastatic melanoma. PMID:27622011

  10. Melanoma

    MedlinePlus

    ... have melanoma that has spread. Help the patient’s immune system fight the cancer Ipilimumab (Yervoy®), which was FDA ... How ipilimumab works : This drug helps the patient’s immune system to recognize, target, and attack cancer cells. Healthy ...

  11. Ganglioside GD2-specific trifunctional surrogate antibody Surek demonstrates therapeutic activity in a mouse melanoma model

    PubMed Central

    2012-01-01

    Background Trifunctional bispecific antibodies (trAb) are a special class of bispecific molecules recruiting and activating T cells and accessory immune cells simultaneously at the targeted tumor. The new trAb Ektomab that targets the melanoma-associated ganglioside antigen GD2 and the signaling molecule human CD3 (hCD3) on T cells demonstrated potent T-cell activation and tumor cell destruction in vitro. However, the relatively low affinity for the GD2 antigen raised the question of its therapeutic capability. To further evaluate its efficacy in vivo it was necessary to establish a mouse model. Methods We generated the surrogate trAb Surek, which possesses the identical anti-GD2 binding arm as Ektomab, but targets mouse CD3 (mCD3) instead of hCD3, and evaluated its chemical and functional quality as a therapeutic antibody homologue. The therapeutic and immunizing potential of Surek was investigated using B78-D14, a B16 melanoma transfected with GD2 and GD3 synthases and showing strong GD2 surface expression. The induction of tumor-associated and autoreactive antibodies was evaluated. Results Despite its low affinity of approximately 107 M-1 for GD2, Surek exerted efficient tumor cell destruction in vitro at an EC50 of 70ng/ml [0.47nM]. Furthermore, Surek showed strong therapeutic efficacy in a dose-dependent manner and is superior to the parental GD2 mono-specific antibody, while the use of a control trAb with irrelevant target specificity had no effect. The therapeutic activity of Surek was strictly dependent on CD4+ and CD8+ T cells, and cured mice developed a long-term memory response against a second challenge even with GD2-negative B16 melanoma cells. Moreover, tumor protection was associated with humoral immune responses dominated by IgG2a and IgG3 tumor-reactive antibodies indicating a Th1-biased immune response. Autoreactive antibodies against the GD2 target antigen were not induced. Conclusion Our data suggest that Surek revealed strong tumor elimination

  12. Monoclonal antibody recognizing human melanoma-carcinoma cross-reacting oncofetal antigen epitopically associated with carcinoembryonic antigen.

    PubMed

    Liao, S K; Kwong, P C; Clarke, B J; Dent, P B; Ryan, E D; Khosravi, M J; Laferte, S; Krantz, M J

    1985-05-01

    By fusion of mouse NS1 myeloma cells with splenocytes from a BALB/c mouse immunized with human melanoma cells, an IgG1 monoclonal antibody, designated as 140.72, was produced. By the mixed hemadsorption antibody binding assay, 140.72 was shown to react with 17 of 20 melanoma cell lines and with 5 of 14 carcinoma cell lines. This antibody also reacted with 3 of 3 normal melanocyte cultures in much lower titers. It did not react with any of 35 other normal and malignant lines, including neuroblastoma, glioblastoma, sarcoma, teratoma, fibroblast, and lymphoid cell lines. Absorption with fresh melanoma and carcinoma homogenates confirmed the results of direct tests. Fetal reactivity of antibody 140.72 was determined by positive absorption with 10 of 11 tissue homogenates derived from different fetuses of 10-16 weeks' gestation. The reactivity of this antibody was completely removed by absorption with a highly purified preparation of carcinoembryonic antigen (CEA) derived from a colon carcinoma. The antigenic activity was detected in the culture medium of reactive cell lines. Immunoprecipitation analyses of melanoma and carcinoma cells indicated that the antigenic determinant recognized by antibody 140.72 is on a glycoprotein with an apparent molecular weight of 95,000-150,000 common to both serologically reactive cell types. Additionally, a 200,000-molecular-weight glycoprotein corresponding to the CEA molecule was detected only on the reactive carcinoma cells. These data confirmed previous findings obtained with polyclonal anti-CEA antisera for the existence of shared CEA-related antigenic determinants on human carcinomas and melanomas and provided additional molecular characterization of these glycoproteins. Further characterization of the molecules bearing the antigenic determinant recognized by antibody 140.72 should be performed with a view to exploring its potential in the immunodiagnosis and immunotherapy of patients with melanoma.

  13. Boronated monoclonal antibody 225. 28S for potential use in neutron capture therapy of malignant melanoma

    SciTech Connect

    Tamat, S.R.; Moore, D.E.; Patwardhan, A.; Hersey, P. )

    1989-07-01

    The concept of conjugating boron cluster compounds to monoclonal antibodies has been examined by several groups of research workers in boron neutron capture therapy (BNCT). The procedures reported to date for boronation of monoclonal antibodies resulted in either an inadequate level of boron incorporation, the precipitation of the conjugates, or a loss of immunological activity. The present report describes the conjugation of dicesium-mercapto-undecahydrododecaborate (Cs2B12H11SH) to 225.28S monoclonal antibody directed against high molecular weight melanoma-associated antigens (HMW-MAA), using poly-L-ornithine as a bridge to increase the carrying capacity of the antibody and to minimize change in the conformational structure of antibody. The method produces a boron content of 1,300 to 1,700 B atoms per molecule 225.28S while retaining the immunoreactivity. Characterization in terms of the homogeneity of the conjugation of the boron-monoclonal antibody conjugates has been studied by gel electrophoresis and ion-exchange HPLC.

  14. 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

  15. Solution Equilibrium Titration for High-Throughput Affinity Estimation of Unpurified Antibodies and Antibody Fragments.

    PubMed

    Della Ducata, Daniela; Jaehrling, Jan; Hänel, Cornelia; Satzger, Marion; Wolber, Meike; Ostendorp, Ralf; Pabst, Stefan; Brocks, Bodo

    2015-12-01

    The generation of therapeutic antibodies with extremely high affinities down to the low picomolar range is today feasible with state-of-the art recombinant technologies. However, reliable and efficient identification of lead candidates with the desired affinity from a pool of thousands of antibody clones remains a challenge. Here, we describe a high-throughput procedure that allows reliable affinity screening of unpurified immunoglobulin G or antibody fragments. The method is based on the principle of solution equilibrium titration (SET) using highly sensitive electrochemiluminescence as a readout system. Because the binding partners are not labeled, the resulting KD represents a sound approximation of the real affinity. For screening, diluted bacterial lysates or cell culture supernatants are equilibrated with four different concentrations of a soluble target molecule, and unbound antibodies are subsequently quantified on 384-well Meso Scale Discovery (MSD) plates coated with the respective antigen. For determination of KD values from the resulting titration curves, fit models deduced from the law of mass action for 1:1 and 2:1 binding modes are applied to assess hundreds of interactions simultaneously. The accuracy of the method is demonstrated by comparing results from different screening campaigns from affinity optimization projects with results from detailed affinity characterization.

  16. Internalization and re-expression of antigens of human melanoma cells following exposure to monoclonal antibody

    SciTech Connect

    Wang, B.S.; Lumanglas, A.L.; Silva, J.; Ruszala-Mallon, V.; Durr, F.E.

    1987-04-15

    Modulation of the surface membrane of human Sk-Mel-28 melanoma cells by monoclonal antibody (MoAb) 96.5 recognizing p97 determinants was examined using direct radioimmunoassay and indirect fluorescent antibody-staining techniques. It was determined that the majority of /sup 111/In-labeled antibody that remained associated with cells after a 24-hr incubation at 37 degrees C had been internalized because MoAb 96.5 was no longer visible on the cell surface. A second treatment of these cells with the same antibody 24 hr later not only increased the cell-associated radioactivity, reflecting an increase of total antibody bound, but also rendered these cells membrane immunofluorescent again, indicating the re-expression of surface antigens. Autoradiographs of the electrophoretically analyzed membrane components of Sk-Mel-28 cells further demonstrated the appearance of newly synthesized 97-kDa proteins that were immunoprecipitable with MoAb 96.5. Taken together, the present findings suggest that p97 antigens undergo endocytosis in Sk-Mel-28 cells following exposure to MoAb 96.5. However, the same antigens were regenerated and expressed on the cell surface within a period of 24 hr. The re-expression of tumor cell surface antigen following initial internalization of the MoAb-antigen complex may have implications for diagnosis and therapy.

  17. A Sex Chromosomal Restriction-Fragment-Length Marker Linked to Melanoma-Determining Tu Loci in Xiphophorus

    PubMed Central

    Schartl, M.

    1988-01-01

    In Xiphophorus, the causative genetic information for melanoma formation has been assigned by classical genetics to chromosomal loci, which are located on the sex chromosomes. In our attempts to molecularly clone these melanoma-determining loci, named Tu, we have looked for restriction-fragment-length markers (RFLMs) linked to the Tu loci. These RFLMs should be useful in obtaining a physical map of a Tu locus, which will aid in the cloning of the corresponding sequences. DNA samples from various Xiphophorus strains and hybrids including those bearing different Tu wild-type, deletion and translocation chromosomes, were screened for the presence of random RFLMs using homologous or heterologous sequences as hybridization probes. We find an EcoRI restriction fragment which shows limited crosshybridization to the v-erb B gene--but not representing the authentic c-erb B gene of Xiphophorus--to be polymorphic with respect to different sex chromosomes. Linkage analysis revealed that a 5-kb fragment is linked to the Tu-Sd locus on the X chromosome, a 7-kb fragment is linked to the Tu-Sr locus on the Y chromosome, both of Xiphophorus maculatus, and that a 12-kb fragment is linked to the Tu-Li locus on the X chromosome of Xiphophorus variatus. Using different chromosomal mutants this RFLM has been mapped to a frequent deletion/translocation breakpoint of the X chromosome, less than 0.3 cM apart from the Tu locus. PMID:2841190

  18. A sex chromosomal restriction-fragment-length marker linked to melanoma-determining Tu loci in Xiphophorus.

    PubMed

    Schartl, M

    1988-07-01

    In Xiphophorus, the causative genetic information for melanoma formation has been assigned by classical genetics to chromosomal loci, which are located on the sex chromosomes. In our attempts to molecularly clone these melanoma-determining loci, named Tu, we have looked for restriction-fragment-length markers (RFLMs) linked to the Tu loci. These RFLMs should be useful in obtaining a physical map of a Tu locus, which will aid in the cloning of the corresponding sequences. DNA samples from various Xiphophorus strains and hybrids including those bearing different Tu wild-type, deletion and translocation chromosomes, were screened for the presence of random RFLMs using homologous or heterologous sequences as hybridization probes. We find an EcoRI restriction fragment which shows limited crosshybridization to the v-erb B gene--but not representing the authentic c-erb B gene of Xiphophorus--to be polymorphic with respect to different sex chromosomes. Linkage analysis revealed that a 5-kb fragment is linked to the Tu-Sd locus on the X chromosome, a 7-kb fragment is linked to the Tu-Sr locus on the Y chromosome, both of Xiphophorus maculatus, and that a 12-kb fragment is linked to the Tu-Li locus on the X chromosome of Xiphophorus variatus. Using different chromosomal mutants this RFLM has been mapped to a frequent deletion/translocation breakpoint of the X chromosome, less than 0.3 cM apart from the Tu locus. PMID:2841190

  19. Clinical evaluation of metastases of malignant melanoma imaging with 99Tcm-glutathione and 99Tcm-anti-melanoma antibody: a comparative study.

    PubMed

    Duman, Y; Burak, Z; Ercan, M T; Dirlik, A; Bilkay, B C; Akin, Y; Taner, M; Bekdik, C F

    1995-11-01

    The aim of this investigation was to test for the scintigraphic detection of metastases of malignant melanoma with a new radiopharmaceutical, 99Tcm-glutathione (99Tcm-GSH), in comparison with 99Tcm-anti-melanoma antibody (99Tcm-AMAb). Glutathione was labelled with 99Tcm by a Sn2+ reduction method with an efficiency of > 99% as determined by instant thin layer chromatography (ITLC). Anti-melanoma antibody was obtained as a kit from SORIN (Italy) and labelled with 99TcmO-4. Forty-three patients with a total of 55 biopsy-proven metastatic melanoma foci, 1 ocular melanoma and 20 benign pathologic foci, also confirmed by ultrasound, computed tomography and magnetic resonance imaging, were included in the study after giving their informed consent. Following the intravenous (i.v.) injection of 500 MBq 99Tcm-AMAb, scintigraphic images of the involved areas were obtained 6 h post-injection. Three days later, the same patients were given 500 MBq 99Tcm-GSH i.v. and images were obtained 6 and 24 h post-injection. The images were classified as positive (focal abnormal accumulation) or negative. Quantitative evaluation was also applied. Regions of interest were drawn over the involved areas and nearby soft tissues and the target-to-nontarget (T/NT) ratios obtained with 99Tcm-AMAb (T/NT: 1.92 +/- 0.2) and 99Tcm-GSH (T/NT: 1.84 +/- 0.2) were compared (0.1 < P < or = 0.3). The sensitivity (and specificity) of 99Tcm-AMAb and 99Tcm-GSH in the detection of malignant melanoma metastases were 91% (95%) and 84% (90%), respectively. Compared with 99Tcm-AMAb, the advantages of 99Tcm-GSH are lower levels of blood radioactivity, lower costs and easy in-house preparation. In conclusion, our results show that 99Tcm-GSH is a potentially useful radiopharmaceutical for the detection of metastases of malignant melanoma.

  20. Selective binding of anti-DNA antibodies to native dsDNA fragments of differing sequence.

    PubMed

    Uccellini, Melissa B; Busto, Patricia; Debatis, Michelle; Marshak-Rothstein, Ann; Viglianti, Gregory A

    2012-03-30

    Systemic autoimmune diseases are characterized by the development of autoantibodies directed against a limited subset of nuclear antigens, including DNA. DNA-specific B cells take up mammalian DNA through their B cell receptor, and this DNA is subsequently transported to an endosomal compartment where it can potentially engage TLR9. We have previously shown that ssDNA-specific B cells preferentially bind to particular DNA sequences, and antibody specificity for short synthetic oligodeoxynucleotides (ODNs). Since CpG-rich DNA, the ligand for TLR9 is found in low abundance in mammalian DNA, we sought to determine whether antibodies derived from DNA-reactive B cells showed binding preference for CpG-rich native dsDNA, and thereby select immunostimulatory DNA for delivery to TLR9. We examined a panel of anti-DNA antibodies for binding to CpG-rich and CpG-poor DNA fragments. We show that a number of anti-DNA antibodies do show preference for binding to certain native dsDNA fragments of differing sequence, but this does not correlate directly with the presence of CpG dinucleotides. An antibody with preference for binding to a fragment containing optimal CpG motifs was able to promote B cell proliferation to this fragment at 10-fold lower antibody concentrations than an antibody that did not selectively bind to this fragment, indicating that antibody binding preference can influence autoreactive B cell responses.

  1. Radioimmunodetection of melanoma utilizing In-111 96. 5 monoclonal antibody: A preliminary report

    SciTech Connect

    Halpern, S.E.; Dillman, R.O.; Witztum, K.F.; Shega, J.F.; Hagan, P.L.; Burrows, W.M.; Dillman, J.B.; Clutter, M.L.; Sobol, R.E.; Frincke, J.M.

    1985-05-01

    The murine 96.5 monoclonal antimelanoma antibody (MoAb) was labeled with In-111, and 1-20 mg were administered to 21 patients who had proved or suspected melanoma metastases. In four patients, unlabeled 96.5 MoAb was administered prior to the radiopharmaceutical. The scans were interpreted by two observers, one with full knowledge, the other with no knowledge of the cases. Increasing the MoAb mass or preinfusing unlabeled MoAb prior to the administration of In-111 MoAb resulted in a prolongation of In-111 MoAb of the serum half time, and appeared to improve tumor detection. In all patients who had metastatic disease, at least one tumor site was apparent. Fifty-six per cent of known lesions 1.5 cm or greater in size were detected by the physician who had knowledge of the cases when data from all doses were considered. Forty-nine per cent were detected by the other physician. The 96.5 In-111 MoAb appears to have utility for the detection of metastatic melanoma.

  2. 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

  3. Acetylcholine receptor binding antibody-associated myasthenia gravis and rhabdomyolysis induced by nivolumab in a patient with melanoma.

    PubMed

    Shirai, Takushi; Sano, Tasuku; Kamijo, Fuminao; Saito, Nana; Miyake, Tomomi; Kodaira, Minori; Katoh, Nagaaki; Nishie, Kenichi; Okuyama, Ryuhei; Uhara, Hisashi

    2016-01-01

    We reported an 81-year-old woman with metastatic melanoma, in whom myasthenia gravis and rhabdomyolysis developed after nivolumab monotherapy. The first symptom of myasthenia gravis was dyspnea. Ultrasonography detected hypokinesis of the bilateral diaphragm suggesting myasthenia gravis, although there was no abnormal finding of the lungs in computed tomography images. Acetylcholine receptor binding antibodies were low-titer positive in the preserved serum before administration of nivolumab, strongly suggesting that the myasthenia gravis was a nivolumab-related immune adverse event. Despite the remarkable clinical benefits of immune checkpoint inhibitors for patients with advanced melanoma, it is important to recognize unexpected immune-related adverse events.

  4. Expression of Human Immunodeficiency Virus Type 1 Neutralizing Antibody Fragments Using Human Vaginal Lactobacillus

    PubMed Central

    Marcobal, Angela; Liu, Xiaowen; Zhang, Wenlei; Dimitrov, Antony S.; Jia, Letong; Lee, Peter P.; Fouts, Timothy R.; Parks, Thomas P.

    2016-01-01

    Abstract Eradication of human immunodeficiency virus type 1 (HIV-1) by vaccination with epitopes that produce broadly neutralizing antibodies is the ultimate goal for HIV prevention. However, generating appropriate immune responses has proven difficult. Expression of broadly neutralizing antibodies by vaginal colonizing lactobacilli provides an approach to passively target these antibodies to the mucosa. We tested the feasibility of expressing single-chain and single-domain antibodies (dAbs) in Lactobacillus to be used as a topical microbicide/live biotherapeutic. Lactobacilli provide an excellent platform to express anti-HIV proteins. Broadly neutralizing antibodies have been identified against epitopes on the HIV-1 envelope and have been made into active antibody fragments. We tested single-chain variable fragment m9 and dAb-m36 and its derivative m36.4 as prototype antibodies. We cloned and expressed the antibody fragments m9, m36, and m36.4 in Lactobacillus jensenii-1153 and tested the expression levels and functionality. We made a recombinant L. jensenii 1153-1128 that expresses dAb-m36.4. All antibody fragments m9, m36, and m36.4 were expressed by lactobacilli. However, we noted the smaller m36/m36.4 were expressed to higher levels, ≥3 μg/ml. All L. jensenii-expressed antibody fragments bound to gp120/CD4 complex; Lactobacillus-produced m36.4 inhibited HIV-1BaL in a neutralization assay. Using a TZM-bl assay, we characterized the breadth of neutralization of the m36.4. Delivery of dAbs by Lactobacillus could provide passive transfer of these antibodies to the mucosa and longevity at the site of HIV-1 transmission. PMID:26950606

  5. Crystallization of the Fab fragments of anti-peptide monoclonal antibodies and a complex with peptide.

    PubMed

    Griest, R E; Jeffrey, P D; Taylor, G L; Rees, A R

    1992-01-01

    The antigen-binding fragments of four monoclonal antibodies that cross-react with both the "loop" peptide of hen egg-white lysozyme (residues 57 to 84) against which they were raised, and with the native protein (HEL) have been crystallized. One of these fragments also crystallizes as a complex with the peptide antigen. PMID:1731084

  6. Uptake of indium-111-labeled monoclonal antibody ZME-018 as a function of tumor size in a patient with melanoma

    SciTech Connect

    Macey, D.J.; Denardo, S.J.; Denardo, G.L.; Goodnight, J.K.; Unger, M.W.

    1988-01-01

    The accumulation of an Indium-111-labeled monoclonal antibody (MoAb), ZME-018, in melanoma tumors in a patient was determined by sequential, quantitative gamma camera imaging. The amount and concentration of In-111 in each tumor changed in a characteristic pattern with time, reaching a peak at day 3 followed by a steady clearance. The concentration of In-111 in the tumor and the ratios of tumor to whole-body or blood decreased as the size of the tumor increased. These results were interpreted to indicate that the fraction of active, perfused tumor decreased as the melanoma lesions increased in size. The maximum number of MoAb molecules bound per melanoma cell was calculated to be abut 35,000. The implications of these observations for radioimmunoimaging and therapy are significant.

  7. Antibody-drug conjugates: targeting melanoma with cisplatin encapsulated in protein-cage nanoparticles based on human ferritin

    NASA Astrophysics Data System (ADS)

    Falvo, Elisabetta; Tremante, Elisa; Fraioli, Rocco; Leonetti, Carlo; Zamparelli, Carlotta; Boffi, Alberto; Morea, Veronica; Ceci, Pierpaolo; Giacomini, Patrizio

    2013-11-01

    A novel antibody-drug conjugate (ADC) was synthesized incorporating ferritin-based nanoparticles. An average of three molecules of monoclonal antibody (mAb) Ep1 to the human melanoma-specific antigen CSPG4 were conjugated to a single ferritin cage encapsulating about 50 cisplatin molecules (HFt-Pt-Ep1). The HFt-Pt-Ep1 nanoparticle had an estimated molecular size of about 900 kD and 33 nm, and flow cytometry demonstrated specific binding to a CSPG4+ melanoma cell line, but not to a CSPG4- breast carcinoma cell line. As compared to the cisplatin-containing ferritin nanoparticle alone (HFt-Pt), which inhibited thymidine incorporation more efficiently in breast carcinoma than melanoma cells, the mAb-derivatized HFt-Pt-Ep1 nanoparticle had a 25-fold preference for the latter. A similar preference for melanoma was observed upon systemic intravenous administration of HFt-Pt-Ep1 to nude mice xenotransplanted with pre-established, palpable melanoma and breast carcinoma tumors. Thus, we have been able to determine precise combinations and stoichiometric relationships between mAbs and nanoparticle protein cages, whereby the latter lose their tropism for ubiquitously distributed cellular receptors, and acquire instead remarkably lineage-selective binding. HFt-Pt-Ep1 is therefore an interesting model to improve the therapeutic index of antiblastic therapy in a tumor such as melanoma, which at its advanced stages is totally refractory to mono- and combination-chemotherapy.A novel antibody-drug conjugate (ADC) was synthesized incorporating ferritin-based nanoparticles. An average of three molecules of monoclonal antibody (mAb) Ep1 to the human melanoma-specific antigen CSPG4 were conjugated to a single ferritin cage encapsulating about 50 cisplatin molecules (HFt-Pt-Ep1). The HFt-Pt-Ep1 nanoparticle had an estimated molecular size of about 900 kD and 33 nm, and flow cytometry demonstrated specific binding to a CSPG4+ melanoma cell line, but not to a CSPG4- breast carcinoma cell

  8. 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.

  9. Fully humanized neutralizing antibodies to interleukin-8 (ABX-IL8) inhibit angiogenesis, tumor growth, and metastasis of human melanoma.

    PubMed

    Huang, Suyun; Mills, Lisa; Mian, Badar; Tellez, Carmen; McCarty, Marya; Yang, X-D; Gudas, Jean M; Bar-Eli, Menashe

    2002-07-01

    Interleukin-8 (IL-8) has recently been shown to contribute to human melanoma progression by functioning as a mitogenic and angiogenic factor. In the present study, we investigated whether targeting IL-8 by a fully human anti-IL-8 antibody (ABX-IL8) could be a potential therapeutic strategy to control angiogenesis, growth, and metastasis of melanoma. The human melanoma cells A375SM (high IL-8 producer) and TXM-13 (intermediate IL-8 producer) were injected subcutaneously into nude mice, which were then treated with ABX-IL8 (1 mg/3 times weekly, i.p., for 3 weeks). Tumor growth of both melanomas in ABX-IL8-treated mice was significantly inhibited when compared with control IgG-treated animals. ABX-IL8 treatment also suppressed experimental metastasis when the melanoma cells were injected intravenously. IL-8 blockade by ABX-IL8 significantly inhibited the promoter activity and the collagenase activity of matrix metalloproteinase-2 in human melanoma cells, resulting in decreased invasion through reconstituted basement membrane in vitro. In vivo, ABX-IL8 treatment resulted in decreased expression of matrix metalloproteinase-2, and decreased vascularization (angiogenesis) of tumors concomitant with increased apoptosis of tumor cells. Moreover, in an in vitro vessel formation assay, ABX-IL8 directly interfered with the tubule formation by human umbilical vein endothelial cells. Taken together, these results point to the potential utility of ABX-IL8 as a modality to treat melanoma and other solid tumors either alone or in combination with conventional chemotherapy or other anti-tumor agents. PMID:12107097

  10. Development of human antibody fragments using antibody phage display for the detection and diagnosis of Venezuelan equine encephalitis virus (VEEV)

    PubMed Central

    Kirsch, Martina Inga; Hülseweh, Birgit; Nacke, Christoph; Rülker, Torsten; Schirrmann, Thomas; Marschall, Hans-Jürgen; Hust, Michael; Dübel, Stefan

    2008-01-01

    Background Venezuelan equine encephalitis virus (VEEV) belongs to the Alphavirus group. Several species of this family are also pathogenic to humans and are recognized as potential agents of biological warfare and terrorism. The objective of this work was the generation of recombinant antibodies for the detection of VEEV after a potential bioterrorism assault or an natural outbreak of VEEV. Results In this work, human anti-VEEV single chain Fragments variable (scFv) were isolated for the first time from a human naïve antibody gene library using optimized selection processes. In total eleven different scFvs were identified and their immunological specificity was assessed. The specific detection of the VEEV strains TC83, H12/93 and 230 by the selected antibody fragments was proved. Active as well as formalin inactivated virus particles were recognized by the selected antibody fragments which could be also used for Western blot analysis of VEEV proteins and immunohistochemistry of VEEV infected cells. The anti-VEEV scFv phage clones did not show any cross-reactivity with Alphavirus species of the Western equine encephalitis virus (WEEV) and Eastern equine encephalitis virus (EEEV) antigenic complex, nor did they react with Chikungunya virus (CHIKV), if they were used as detection reagent. Conclusion For the first time, this study describes the selection of antibodies against a human pathogenic virus from a human naïve scFv antibody gene library using complete, active virus particles as antigen. The broad and sensitive applicability of scFv-presenting phage for the immunological detection and diagnosis of Alphavirus species was demonstrated. The selected antibody fragments will improve the fast identification of VEEV in case of a biological warfare or terroristic attack or a natural outbreak. PMID:18764933

  11. Eradication of Human Hepatic and Pulmonary Melanoma Metastases in SCID Mice by Antibody--Interleukin 2 Fusion Proteins

    NASA Astrophysics Data System (ADS)

    Becker, Jurgen C.; Pancook, James D.; Gillies, Stephen D.; Mendelsohn, John; Reisfeld, Ralph A.

    1996-04-01

    Antibody--cytokine fusion proteins combine the unique targeting ability of antibodies with the multifunctional activity of cytokines. Here, we demonstrate the therapeutic efficacy of such constructs for the treatment of hepatic and pulmonary metastases of different melanoma cell lines. Two antibody--interleukin 2 (IL-2) fusion proteins, ch225-IL2 and ch14.18-IL2, constructed by fusion of a synthetic sequence coding for human IL-2 to the carboxyl end of the Cγ 1 gene of the corresponding antibodies, were tested for their therapeutic efficacy against xenografted human melanoma in vivo. Tumorspecific fusion proteins completely inhibited the growth of hepatic and pulmonary metastases in C.B-17 scid/scid mice previously reconstituted with human lymphokine-activated killer cells, whereas treatment with combinations of the corresponding antibodies plus recombinant IL-2 only reduced the tumor load. Even when treatment with fusion proteins was delayed up to 8 days after inoculation of tumor cells, it still resulted in complete eradication of micrometastases that were established at that time point. Selection of tumor cell lines expressing or lacking the targeted antigen of the administered fusion protein proved the specificity of the observed antitumor effect. Biodistribution analysis demonstrated that the tumorspecific fusion protein accumulated not only in subcutaneous tumors but also in lungs and livers affected with micrometastases. Survival times of animals treated with the fusion protein were more than doubled as compared to those treated with the combination of the corresponding antibody plus IL-2. Our data demonstrate that an immunotherapeutic approach using cytokines targeted by antibodies to tumor sites has potent effects against disseminated human melanoma.

  12. Novel amyloid-beta specific scFv and VH antibody fragments from human and mouse phage display antibody libraries.

    PubMed

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

    2010-06-01

    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 (Abeta1-42) recombinant monoclonal antibodies in a single chain fragment variable (scFv) and a single-domain (VH) format. We demonstrated that these antibody fragments recognize in a specific manner amyloid-beta deposits in APP/Tg mouse brains, inhibit toxicity of oligomeric Abeta1-42 in neuroblastoma cell cultures in a concentration-dependent manner and reduced amyloid deposits in APP/Tg2576 mice after intracranial administration. These antibody fragments recognize epitopes in the middle/C-terminus region of Abeta, which makes them strong therapeutic candidates due to the fact that most of the Abeta species found in the brains of AD patients display extensive N-terminus truncations/modifications.

  13. 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

  14. 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

  15. Assessment of the effect of triton X-114 on the physicochemical properties of an antibody fragment.

    PubMed

    Malpiedi, Luciana P; Nerli, Bibiana B; Abdalla, Dulcineia S P; Pessoa, Adalberto

    2014-01-01

    The effect of Triton X-114 on the physicochemical properties of a single-chain antibody fragment (scFv) has been studied. According to the far UV circular dichroism spectroscopy, the secondary structure of the recombinant antibody was not significantly affected by the presence of Triton. From the antibody tertiary structure analysis, it was found that the surfactant could be located around the tryptophan molecules accessible to the solvent, diminishing the polarity of its environment but maintaining most of the protein structure integrity. However, in certain conditions of high temperature and high concentration of denaturant molecules, the presence of TX could compromise the antibody fragment stability. These results represent a previous step in designing scFv purification protocols and should be considered prior to developing scFv liquid-liquid extraction procedures.

  16. 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.

  17. Stirred batch crystallization of a therapeutic antibody fragment.

    PubMed

    Hebel, Dirk; Huber, Sabine; Stanislawski, Bernd; Hekmat, Dariusch

    2013-07-20

    Technical-scale crystallization of therapeutic proteins may not only allow for a significant cost-reduction in downstream processing, but also enable new applications, e.g., the use of crystal suspensions for subcutaneous drug delivery. In this work, the crystallization of the antigen-binding fragment FabC225 was studied. First, vapor diffusion crystallization conditions from the literature were transferred to 10μL-scale microbatch experiments. A phase diagram was developed in order to identify the crystallization window. The conditions obtained from the microbatch experiments were subsequently transferred to parallelized 5mL-scale stirred-tank crystallizers. This scalable and reproducible agitated crystallization system allowed for an optimization of the crystallization process based on quantitative measurements. The optimized crystallization process resulted in an excellent yield of 99% in less than 2h by increasing the concentration of the crystallization agent ammonium sulfate during the process. The successful scalability of the Fab fragment crystallization process to 100mL-scale crystallizers based on geometric similarity was demonstrated. A favorable crystal size distribution was obtained. Furthermore, a wash step was introduced in order to remove unfavorable low-molecular substances from the crystals.

  18. 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

  19. 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.

  20. Quantitative Spatiotemporal Analysis of Antibody Fragment Diffusion and Endocytic Consumption in Tumor Spheroids

    PubMed Central

    Thurber, Greg M.; Wittrup, K. Dane

    2010-01-01

    Antibody-based cancer treatment depends upon distribution of the targeting macromolecule throughout tumor tissue, and spatial heterogeneity could significantly limit efficacy in many cases. Antibody distribution in tumor tissue is a function of drug dosage, antigen concentration, binding affinity, antigen internalization, drug extravasation from blood vessels, diffusion in the tumor extracellular matrix, and systemic clearance rates. We have isolated the effects of a subset of these variables by live-cell microscopic imaging of single-chain antibody fragments against carcinoembryonic antigen in LS174T tumor spheroids. The measured rates of scFv penetration and retention were compared with theoretical predictions based on simple scaling criteria. The theory predicts that antibody dose must be large enough to drive a sufficient diffusive flux of antibody to overcome cellular internalization, and exposure time must be long enough to allow penetration to the spheroid center. The experimental results in spheroids are quantitatively consistent with these predictions. Therefore, simple scaling criteria can be applied to accurately predict antibody and antibody fragment penetration distance in tumor tissue. PMID:18451160

  1. 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...

  2. Antibody fragments for stabilization and crystallization of G protein-coupled receptors and their signaling complexes.

    PubMed

    Shukla, Arun K; Gupta, Charu; Srivastava, Ashish; Jaiman, Deepika

    2015-01-01

    G protein-coupled receptors (GPCRs) are one of the key players in extracellular signal recognition and their subsequent communications with cellular signaling machinery. Crystallization and high-resolution structure determination of GPCRs has been one of the major advances in the area of GPCR biology over the last 7-8 years. There have primarily been three approaches to GPCR crystallization till date. These are fusion protein strategy, thermostabilization, and antibody fragment-mediated crystallization. Of these, antibody fragment-mediated crystallization has not only provided the first breakthrough in structure determination of a non-rhodopsin GPCR but it has also assisted in obtaining structures of fully active conformations of GPCRs. Antibody fragment approach has also been crucial in obtaining structural information on GPCR signaling complexes. Here, we highlight the specific examples of GPCR crystal structures that have utilized antibody fragments for promoting crystallogenesis and structure solution. We also discuss emerging powerful technologies such as the nanobody technology and the synthetic phage display libraries in the context of GPCR crystallization and underline how these tools are likely to propel key GPCR structural studies in future.

  3. 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.

  4. Covalent and oriented immobilization of scFv antibody fragments via an engineered glycan moiety.

    PubMed

    Hu, Xuejun; Hortigüela, María J; Robin, Sylvain; Lin, Heng; Li, Yajie; Moran, Anthony P; Wang, Wenxin; Wall, J Gerard

    2013-01-14

    Antibody-derived fragments have enormous potential application in solid-phase assays such as biomarker detection and protein purification. Controlled orientation of the immobilized antibody molecules is a critical requirement for the sensitivity and efficacy of such assays. We present an approach for covalent, correctly oriented attachment of scFv antibody fragments on solid supports. Glycosylated scFvs were expressed in Escherichia coli and the C-terminal, binding pocket-distal glycan tag was oxidized for covalent attachment to amine-functionalized beads. The glycosylated scFvs could be immobilized at salt concentrations that precluded nonspecific adsorption of unglycosylated molecules and the covalently attached antibody fragments exhibited 4-fold higher functional activity than ionically adsorbed scFvs. The glyco-tethered scFvs were stable in NaCl concentrations that removed greater than 90% of adsorbed scFvs and they exhibited improved stability of antigen binding over both adsorbed scFvs and soluble, nonimmobilized scFvs in accelerated degradation tests. The simple expression and immobilization approach reported is likely to find broad application in in vitro antibody tests.

  5. Selection of phage-displayed human antibody fragments on Dengue virus particles captured by a monoclonal antibody: application to the four serotypes.

    PubMed

    Cabezas, Sheila; Rojas, Gertrudis; Pavon, Alequis; Alvarez, Mayling; Pupo, Maritza; Guillen, Gerardo; Guzman, Maria G

    2008-02-01

    Antibody fragments to the four Dengue virus serotypes were isolated from a human universal naïve library using phage display technology. Phage-displayed antibody fragments were selected on Dengue virus particles directly captured from infected Vero cells supernatant by an anti-dengue monoclonal antibody, in order to avoid laborious virus concentration/purification procedures. A total of nine phage-displayed antibody fragments were obtained. Seven of them were highly specific for three of the selector serotypes (two for Dengue 1, four for Dengue 3 and one for Dengue 4). One clone (Dengue 3-selected) cross-reacted with Dengue 1, whereas another (selected with Dengue 2) cross-reacted with the three remaining serotypes. The soluble variants of six antibody fragments recognized their target viruses when used at nanomolar and even subnanomolar concentrations. All phage-displayed antibody fragments were cross-reactive against several strains of distinct genotypes within the corresponding serotype(s). These antibody fragments are potentially useful for the future development of tools for viral diagnosis and serotype identification. The simple phage selection method on captured virus could be applied in a high throughput way to obtain larger panels of antibody fragments to Dengue virus for multiple applications.

  6. 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.

  7. 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

  8. 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

  9. 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.

  10. Potential to involve multiple effector cells with human recombinant interleukin-2 and antiganglioside monoclonal antibodies in a canine malignant melanoma immunotherapy model.

    PubMed

    Helfand, S C; Soergel, S A; Donner, R L; Gan, J; Hank, J A; Lindstrom, M J; Sondel, P M

    1994-10-01

    Human tumors originating from neuroectodermal cells such as malignant melanoma and neuroblastoma express high levels of disialogangliosides GD2 and GD3, making these antigens ideal for targeting by monoclonal antibodies (Mabs). The purpose of this study was to investigate expression and targeting of gangliosides on canine melanoma. Using immunohistochemical methods, we analyzed the expression of disialogangliosides GD2 and GD3 on canine oral malignant melanomas with murine Mabs 14.G2a and R24 that recognize GD2 and GD3 disialogangliosides, respectively, on human tumors. We also assessed the ability of Mab 14.G2a (and its mouse-human chimera, ch 14.18) to mediate antibody-dependent cellular cytotoxicity (ADCC) in vitro against a canine malignant melanoma cell line with human recombinant interleukin-2 (IL-2) activated canine peripheral blood lymphocytes (PBL), or canine neutrophil effector cells. Our data show that Mabs 14.G2a and R24 recognized fresh frozen canine oral melanoma. Mabs 14.G2a or ch 14.18, or IL-2, potentiated lysis of the canine malignant melanoma cell line by canine PBL. The killing effect observed using the combination of either Mab with IL-2 was additive. Mab 14.G2a mediated potent ADCC of canine melanoma by canine neutrophils. These studies indicate that disialogangliosides are expressed on fresh canine melanoma cells. Mabs reactive with these antigens can target and trigger tumor killing by multiple canine effector populations and IL-2 can potentiate these effects by canine lymphocytes. Thus, canine oral malignant melanoma, a spontaneously occurring, metastatic cancer in the dog, may be a relevant animal model to investigate combination immunotherapy using antitumor Mab and IL-2.

  11. 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

  12. Anti-chondroitin sulfate proteoglycan 4-specific antibodies modify the effects of vemurafenib on melanoma cells differentially in normoxia and hypoxia

    PubMed Central

    PUCCIARELLI, DANIELA; LENGGER, NINA; TAKACOVA, MARTINA; CSADEROVA, LUCIA; BARTOSOVA, MARIA; BREITENEDER, HEIMO; PASTOREKOVA, SILVIA; HAFNER, CHRISTINE

    2015-01-01

    Chondroitin sulfate proteoglycan 4 (CSPG4), a highly immunogenic melanoma tumor antigen, is a potential target for antibody-based immunotherapy. The mechanism by which CSPG4 affects melanoma progression is only partly understood, in particular the involvement of other receptor tyrosine kinases and the tumor microenvironment. We have previously reported on a mimotope-based vaccine against CSPG4 in a human melanoma xenograft model that resulted in reduction of tumor growth. Herein we describe the influence of hypoxia on the response to polyclonal anti-CSPG4-antibodies induced by this vaccine in combination with the BRAF inhibitor vemurafenib to enhance therapeutic efficacy by simultaneously targeting multiple signaling pathways. Melanoma cells were treated with polyclonal anti-CSPG4-antibodies and vemurafenib. Proliferation, migration and invasion were evaluated in a real-time setting in the impedance-based x-CELLigence® system. Western blotting and quantitative PCR arrays were used to determine protein and mRNA expression of hypoxia inducible factor 1α (HIF1α), carbonic anhydrase IX (CAIX) and signaling pathway proteins. A melanoma xenograft model was used to detect HIF1α and CAIX expression in vivo. Hypoxia enhanced the antiproliferative response to vemurafenib. The migration and invasion capacities of vemurafenib-treated melanoma cells were increased, in spite of vemurafenib-decreased expression of HIF1α and CAIX. Polyclonal anti-CSPG4-antibodies reduced the Transwell migration of vemurafenib-treated, BRAF V600E-mutant and CSPG4-expressing melanoma cells in hypoxia. This was associated with the downregulation of phosphorylated AKT, a kinase contributing to tumor cell migration. Our results highlight CSPG4 as a potential target for modulating treatment resistance to vemurafenib induced by the hypoxic microenvironment. PMID:25997619

  13. Phase I trial of the murine monoclonal anti-GD2 antibody 14G2a in metastatic melanoma.

    PubMed

    Saleh, M N; Khazaeli, M B; Wheeler, R H; Dropcho, E; Liu, T; Urist, M; Miller, D M; Lawson, S; Dixon, P; Russell, C H

    1992-08-15

    In a phase I trial, 12 patients with GD2 antigen-positive metastatic melanoma received the murine anti-GD2 monoclonal antibody 14G2a. The monoclonal antibody was administered in four doses over an 8-day period with total dose ranging from 10 to 120 mg. All patients receiving greater than 10 mg of 14G2a experienced transient abdominal/pelvic pain during the antibody infusion. Five patients had a delayed extremity pain syndrome following the third and fourth antibody infusion. Four of the five patients developed neurological toxicity, including two patients with significant although reversible motor neuropathy. Two of the patients developed hyponatremia secondary to a syndrome of inappropriate antidiuretic hormone. All 12 patients developed high levels of human anti-14G2a antibody. The plasma half-life of 14G2a was 42 +/- 6 (SD) h. One patient each had a partial response, mixed response, and stable disease, respectively. The very modest antitumor activity accompanied by dose-limiting neurological toxicity at total doses greater than 80 mg may restrict the clinical utility of murine 14G2a. PMID:1643631

  14. 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

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

    PubMed

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

    2015-07-01

    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.

  16. Single-chain Fv antibody fragments retain binding properties of the monoclonal antibody raised against peptide P1 of the human prion protein.

    PubMed

    Skrlj, Nives; Serbec, Vladka Curin; Dolinar, Marko

    2010-03-01

    Prion diseases are incurable neurodegenerative diseases that affect both humans and animals. The infectious agent is a pathogenic form of the prion protein that accumulates in brain as amyloids. Currently, there is neither cure nor reliable preclinical diagnostics on the market available. The growing number of reports shows that passive immunisation is one of the most promising strategies for prion disease therapy, where antibodies against prions may prevent and even cure the infection. Since antibodies are large molecules and, thus, might not be suitable for the therapy, different antibody fragments are a good alternative. Therefore, we have designed and prepared single-chain antibody fragments (scFvs) derived from the PrP(Sc)-specific murine monoclonal antibody V5B2. Using a new expression vector pMD204, we produced scFvs in two opposing chain orientations in the periplasm of Escherichia coli. Both recombinant antibody fragments retained the specificity of the parent antibody and one of these exhibited binding properties comparable to the corresponding murine Fab fragments with the affinity in nM range. Our monovalent antibody fragments are of special interest in view of possible therapeutic reagents for prion diseases as well as for development of a new generation of diagnostics. PMID:19597999

  17. 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

  18. 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

  19. A Novel Single-Chain Antibody Fragment for Detection of Mannose 6-Phosphate-Containing Proteins

    PubMed Central

    Müller-Loennies, Sven; Galliciotti, Giovanna; Kollmann, Katrin; Glatzel, Markus; Braulke, Thomas

    2010-01-01

    Newly synthesized soluble lysosomal hydrolases require mannose 6-phosphate (Man6P) residues on their oligosaccharides for their transport to lysosomes. The formation of Man6P residues is catalyzed by the GlcNAc-1-phosphotransferase, which is defective in the lysosomal storage disorders mucolipidosis type II (ML II) and ML III. Both hypersecretion and reduced intracellular level of lysosomal enzymes as well as direct sequencing of GlcNAc-1-phosphotransferase genes are important diagnostic markers for ML II and ML III. A high-affinity Man6P-specific single-chain antibody fragment was generated, allowing the rapid indirect demonstration of defective GlcNAc-1-phosphotransferase. In media and extracts of cultured fibroblasts of healthy controls but not of ML II and ML III patients, several Man6P-containing proteins could be detected by anti-Man6P Western blotting. Immunoprecipitation of Man6P-containing proteins from conditioned media or mouse brain extracts followed by arylsulfatase A and cathepsin D Western blotting confirmed the specificity of the antibody fragment for lysosomal proteins. Application of the antibody fragment in immunohistochemistry of human brain slices from nonaffected patients showed strong neuronal immunoreactivity, which was not observed in cortical sections of an ML II patient. Finally, in brain extracts of a novel GlcNAc-1-phosphotransferase knock-in mouse no Man6P-containing proteins were detectable. Thus, the single-chain antibody fragment against Man6P was demonstrated to allow the specific, rapid, and convenient detection of Man6P-containing proteins and facilitates the diagnosis of ML II and ML III. PMID:20472886

  20. Fab(nimotuzumab)-HYNIC-99mTc: Antibody Fragmentation for Molecular Imaging Agents.

    PubMed

    Calzada, Victoria; García, María Fernanda; Alonso-Martínez, Luis Michel; Camachoc, Ximena; Goicochea, Enzo; Fernández, Marcelo; Castillo, Abmel Xiques; Díaz-Miqueli, Arlhee; Iznaga-Escobar, Normando; Montaña, René Leyva; Alonso, Omar; Gambini, Juan Pablo; Cabral, Pablo

    2016-01-01

    Finally, fast blood clearance nimotuzumab is a humanized monoclonal antibody that recognise, with high specific affinity, the epidermal growth factor receptor (EGF-R) which play an important role in the growth process associated with many solid tumors. In this work, the whole antibody was digested with papain in order to generate a Fab fragment, derivatized with NHS-HYNIC-Tfa and radiolabel with technetium-99m (99mTc) as a potential agent of molecular imaging of cancer. Both, whole and fragment radiolabels were in-vivo and in-vitro characterized. Radiolabeling conditions with Tricine as coligand and quality controls were assessed to confirm the integrity of the labeled fragment. Biodistribution and imaging studies in normal and spontaneous adenocarcinoma mice were performed at different times to determine the in-vivo characteristics of the radiolabel fragment. Tumor localization was visualized by conventional gamma camera imaging studies, and the results were compared with the whole antibody. Also, an immunoreactivity assay was carried out for both. The results showed clearly the integrity of the nimotuzumab fragment and the affinity by the receptor was verified. Fab(nimotuzumab)-HYNIC was obtained with high purity and a simple strategy of radiolabeling was performed. Finally, a fast blood clearance was observed in the biodistribution studies increasing the tumor uptake of Fab(nimotuzumab)- HYNIC-99mTc over time, with tumor/muscle ratios of 3.81 ± 0.50, 5.16 ± 1.97 and 6.32 ± 1.98 at 1 h, 4 h and 24 h post injection. Urinary excretion resulted in 32.89 ± 3.91 %ID eliminated at 24 h. Scintigraphy images showed uptake in the tumor and the activity in non-target organs was consistent with the biodistribution data at the same time points. Hence, these preliminary results showed important further characteristic of Fab(nimotuzumab)-HYNIC-99mTc as a molecular imaging agent of cancer. PMID:26961312

  1. Fab(nimotuzumab)-HYNIC-99mTc: Antibody Fragmentation for Molecular Imaging Agents.

    PubMed

    Calzada, Victoria; García, María Fernanda; Alonso-Martínez, Luis Michel; Camachoc, Ximena; Goicochea, Enzo; Fernández, Marcelo; Castillo, Abmel Xiques; Díaz-Miqueli, Arlhee; Iznaga-Escobar, Normando; Montaña, René Leyva; Alonso, Omar; Gambini, Juan Pablo; Cabral, Pablo

    2016-01-01

    Finally, fast blood clearance nimotuzumab is a humanized monoclonal antibody that recognise, with high specific affinity, the epidermal growth factor receptor (EGF-R) which play an important role in the growth process associated with many solid tumors. In this work, the whole antibody was digested with papain in order to generate a Fab fragment, derivatized with NHS-HYNIC-Tfa and radiolabel with technetium-99m (99mTc) as a potential agent of molecular imaging of cancer. Both, whole and fragment radiolabels were in-vivo and in-vitro characterized. Radiolabeling conditions with Tricine as coligand and quality controls were assessed to confirm the integrity of the labeled fragment. Biodistribution and imaging studies in normal and spontaneous adenocarcinoma mice were performed at different times to determine the in-vivo characteristics of the radiolabel fragment. Tumor localization was visualized by conventional gamma camera imaging studies, and the results were compared with the whole antibody. Also, an immunoreactivity assay was carried out for both. The results showed clearly the integrity of the nimotuzumab fragment and the affinity by the receptor was verified. Fab(nimotuzumab)-HYNIC was obtained with high purity and a simple strategy of radiolabeling was performed. Finally, a fast blood clearance was observed in the biodistribution studies increasing the tumor uptake of Fab(nimotuzumab)- HYNIC-99mTc over time, with tumor/muscle ratios of 3.81 ± 0.50, 5.16 ± 1.97 and 6.32 ± 1.98 at 1 h, 4 h and 24 h post injection. Urinary excretion resulted in 32.89 ± 3.91 %ID eliminated at 24 h. Scintigraphy images showed uptake in the tumor and the activity in non-target organs was consistent with the biodistribution data at the same time points. Hence, these preliminary results showed important further characteristic of Fab(nimotuzumab)-HYNIC-99mTc as a molecular imaging agent of cancer.

  2. 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.

  3. 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

  4. 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

  5. Cutaneous melanoma.

    PubMed

    Eggermont, Alexander M M; Spatz, Alan; Robert, Caroline

    2014-03-01

    In the past decade, major advances have been made in the understanding of melanoma. New predisposition genes have been reported and key somatic events, such as BRAF mutation, directly translated into therapeutic management. Surgery for localised melanoma and regional lymph node metastases is the standard of care. Sentinel-node biopsy provides precise staging, but has not been reported to affect survival. The effect of lymph-node dissection on survival is a topic of investigation. Two distinct approaches have emerged to try to extend survival in patients with metastatic melanoma: immunomodulation with anti-CTLA4 monoclonal antibodies, and targeted therapy with BRAF inhibitors or MEK inhibitors for BRAF-mutated melanoma. The combination of BRAF inhibitors and MEK inhibitors might improve progression-free survival further and, possibly, increase overall survival. Response patterns differ substantially-anti-CTLA4 immunotherapy can induce long-term responses, but only in a few patients, whereas targeted drugs induce responses in most patients, but nearly all of them relapse because of pre-existing or acquired resistance. Thus, the long-term prognosis of metastatic melanoma remains poor. Anti-PD1 and anti-PDL1 antibodies have emerged as breakthrough drugs for melanoma that have high response rates and long durability. Biomarkers that have predictive value remain elusive in melanoma, although emerging data for adjuvant therapy indicate that interferon sensitivity is associated with ulceration of the primary melanoma. Intense investigation continues for clinical and biological markers that predict clinical benefit of immunotherapeutic drugs, such as interferon alfa or anti-CTLA4 antibodies, and the mechanisms that lead to resistance of targeted drugs.

  6. 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

  7. Combination of antibodies directed against different ErbB3 surface epitopes prevents the establishment of resistance to BRAF/MEK inhibitors in melanoma.

    PubMed

    Fattore, Luigi; Malpicci, Debora; Marra, Emanuele; Belleudi, Francesca; Noto, Alessia; De Vitis, Claudia; Pisanu, Maria Elena; Coluccia, Pierpaolo; Camerlingo, Rosa; Roscilli, Giuseppe; Ribas, Antoni; Di Napoli, Arianna; Torrisi, Maria Rosaria; Aurisicchio, Luigi; Ascierto, Paolo Antonio; Mancini, Rita; Ciliberto, Gennaro

    2015-09-22

    Patients with metastatic melanoma bearing V600 mutations in BRAF oncogene clinically benefit from the treatment with BRAF inhibitors alone or in combination with MEK inhibitors. However, a limitation to such treatment is the occurrence of resistance. Tackling the adaptive changes helping cells survive from drug treatment may offer new therapeutic opportunities. Very recently the ErbB3 receptor has been shown to act as a central node promoting survival of BRAF mutated melanoma. In this paper we first demonstrate that ErbB3/AKT hyperphosphorylation occurs in BRAF mutated melanoma cell lines following exposure to BRAF and/or MEK inhibitors. This strongly correlates with increased transcriptional activation of its ligand neuregulin. Anti-ErbB3 antibodies impair the establishment of de novo cell resistance to BRAF inhibition in vitro. In order to more potently ablate ErbB3 activity we used a combination of two anti-ErbB3 antibodies directed against distinct epitopes of its extracellular domain. These two antibodies in combo with BRAF/MEK inhibitors potently inhibit in vitro cell growth and tumor regrowth after drug withdrawal in an in vivo xenograft model. Importantly, residual tumor masses from mice treated by the antibodies and BRAF/ERK inhibitors combo are characterized almost exclusively by large necrotic areas with limited residual areas of tumor growth. Taken together, our findings support the concept that triple therapy directed against BRAF/MEK/ErbB3 may be able to provide durable control of BRAF mutated metastatic melanoma.

  8. Combination of antibodies directed against different ErbB3 surface epitopes prevents the establishment of resistance to BRAF/MEK inhibitors in melanoma

    PubMed Central

    Fattore, Luigi; Malpicci, Debora; Marra, Emanuele; Belleudi, Francesca; Noto, Alessia; De Vitis, Claudia; Pisanu, Maria Elena; Coluccia, Pierpaolo; Camerlingo, Rosa; Roscilli, Giuseppe; Ribas, Antoni; Di Napoli, Arianna; Torrisi, Maria Rosaria; Aurisicchio, Luigi; Ascierto, Paolo Antonio; Mancini, Rita; Ciliberto, Gennaro

    2015-01-01

    Patients with metastatic melanoma bearing V600 mutations in BRAF oncogene clinically benefit from the treatment with BRAF inhibitors alone or in combination with MEK inhibitors. However, a limitation to such treatment is the occurrence of resistance. Tackling the adaptive changes helping cells survive from drug treatment may offer new therapeutic opportunities. Very recently the ErbB3 receptor has been shown to act as a central node promoting survival of BRAF mutated melanoma. In this paper we first demonstrate that ErbB3/AKT hyperphosphorylation occurs in BRAF mutated melanoma cell lines following exposure to BRAF and/or MEK inhibitors. This strongly correlates with increased transcriptional activation of its ligand neuregulin. Anti-ErbB3 antibodies impair the establishment of de novo cell resistance to BRAF inhibition in vitro. In order to more potently ablate ErbB3 activity we used a combination of two anti-ErbB3 antibodies directed against distinct epitopes of its extracellular domain. These two antibodies in combo with BRAF/MEK inhibitors potently inhibit in vitro cell growth and tumor regrowth after drug withdrawal in an in vivo xenograft model. Importantly, residual tumor masses from mice treated by the antibodies and BRAF/ERK inhibitors combo are characterized almost exclusively by large necrotic areas with limited residual areas of tumor growth. Taken together, our findings support the concept that triple therapy directed against BRAF/MEK/ErbB3 may be able to provide durable control of BRAF mutated metastatic melanoma. PMID:26208478

  9. 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

  10. 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.

  11. 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

  12. Cytotoxic activity against human neuroblastoma and melanoma cells mediated by IgM antibodies derived from peripheral blood of healthy donors.

    PubMed

    Devarapu, Satish Kumar; Mamidi, Srinivas; Plöger, Frank; Dill, Othmar; Blixt, Ola; Kirschfink, Michael; Schwartz-Albiez, Reinhard

    2016-06-15

    A small percentage of healthy donors identified in the Western population carry antibodies in their peripheral blood which convey cytotoxic activity against certain human melanoma and neuroblastoma cell lines. We measured the cytotoxic activity of sera and plasmas from healthy donors on the human neuroblastoma cell line Kelly and various melanoma cell lines. Antibodies of IgM isotype, presumably belonging to the class of naturally occurring antibodies, exerted cytotoxic activity in a complement-dependent fashion. Apart from complement-dependent tumor cell lysis, we observed C3 opsonization in all tumor cell lines upon treatment with cytotoxic plasmas. Cell lines tested primarily expressed membrane complement regulatory proteins (mCRP) CD46, CD55 and CD59 to various extents. Blocking of mCRPs by monoclonal antibodies enhanced cell lysis and opsonization, though some melanoma cells remained resistant to complement attack. Epitopes recognized by cytotoxic antibodies were represented by gangliosides such as GD2 and GD3, as evidenced by cellular sialidase pretreatment and enhanced expression of distinct gangliosides. It remains to be clarified why only a small fraction of healthy persons carry these antitumor cytotoxic antibodies. PMID:26830059

  13. Cytotoxic activity against human neuroblastoma and melanoma cells mediated by IgM antibodies derived from peripheral blood of healthy donors.

    PubMed

    Devarapu, Satish Kumar; Mamidi, Srinivas; Plöger, Frank; Dill, Othmar; Blixt, Ola; Kirschfink, Michael; Schwartz-Albiez, Reinhard

    2016-06-15

    A small percentage of healthy donors identified in the Western population carry antibodies in their peripheral blood which convey cytotoxic activity against certain human melanoma and neuroblastoma cell lines. We measured the cytotoxic activity of sera and plasmas from healthy donors on the human neuroblastoma cell line Kelly and various melanoma cell lines. Antibodies of IgM isotype, presumably belonging to the class of naturally occurring antibodies, exerted cytotoxic activity in a complement-dependent fashion. Apart from complement-dependent tumor cell lysis, we observed C3 opsonization in all tumor cell lines upon treatment with cytotoxic plasmas. Cell lines tested primarily expressed membrane complement regulatory proteins (mCRP) CD46, CD55 and CD59 to various extents. Blocking of mCRPs by monoclonal antibodies enhanced cell lysis and opsonization, though some melanoma cells remained resistant to complement attack. Epitopes recognized by cytotoxic antibodies were represented by gangliosides such as GD2 and GD3, as evidenced by cellular sialidase pretreatment and enhanced expression of distinct gangliosides. It remains to be clarified why only a small fraction of healthy persons carry these antitumor cytotoxic antibodies.

  14. The Intrinsic Dynamics and Unfolding Process of an Antibody Fab Fragment Revealed by Elastic Network Model.

    PubMed

    Su, Ji-Guo; Zhang, Xiao; Han, Xiao-Ming; Zhao, Shu-Xin; Li, Chun-Hua

    2015-01-01

    Antibodies have been increasingly used as pharmaceuticals in clinical treatment. Thermal stability and unfolding process are important properties that must be considered in antibody design. In this paper, the structure-encoded dynamical properties and the unfolding process of the Fab fragment of the phosphocholine-binding antibody McPC603 are investigated by use of the normal mode analysis of Gaussian network model (GNM). Firstly, the temperature factors for the residues of the protein were calculated with GNM and then compared with the experimental measurements. A good result was obtained, which provides the validity for the use of GNM to study the dynamical properties of the protein. Then, with this approach, the mean-square fluctuation (MSF) of the residues, as well as the MSF in the internal distance (MSFID) between all pairwise residues, was calculated to investigate the mobility and flexibility of the protein, respectively. It is found that the mobility and flexibility of the constant regions are higher than those of the variable regions, and the six complementarity-determining regions (CDRs) in the variable regions also exhibit relative large mobility and flexibility. The large amplitude motions of the CDRs are considered to be associated with the immune function of the antibody. In addition, the unfolding process of the protein was simulated by iterative use of the GNM. In our method, only the topology of protein native structure is taken into account, and the protein unfolding process is simulated through breaking the native contacts one by one according to the MSFID values between the residues. It is found that the flexible regions tend to unfold earlier. The sequence of the unfolding events obtained by our method is consistent with the hydrogen-deuterium exchange experimental results. Our studies imply that the unfolding behavior of the Fab fragment of antibody McPc603 is largely determined by the intrinsic dynamics of the protein.

  15. 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

  16. 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

  17. 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

  18. A Naturally Occurring Antibody Fragment Neutralizes Infectivity of Diverse Infectious Agents

    PubMed Central

    Polonelli, Luciano; Ciociola, Tecla; Elviri, Lisa; Zanello, Pier Paolo; Giovati, Laura; Arruda, Denise C.; Muñoz, Julián E.; Mortara, Renato A.; Morace, Giulia; Borghi, Elisa; Galati, Serena; Marin, Oriano; Casoli, Claudio; Pilotti, Elisabetta; Ronzi, Paola; Travassos, Luiz R.; Magliani, Walter; Conti, Stefania

    2016-01-01

    A phosphorylated peptide, named K40H, derived from the constant region of IgMs was detected in human serum by liquid chromatography coupled to high-resolution mass spectrometry. Synthetic K40H proved to exert a potent in vitro activity against fungal pathogens, and to inhibit HIV-1 replication in vitro and ex vivo. It also showed a therapeutic effect against an experimental infection by Candida albicans in the invertebrate model Galleria mellonella. K40H represents the proof of concept of the innate role that naturally occurring antibody fragments may exert against infectious agents, shedding a new light upon the posthumous role of antibodies and opening a new scenario on the multifaceted functionality of humoral immunity. PMID:27725769

  19. Blocking FGF2 with a new specific monoclonal antibody impairs angiogenesis and experimental metastatic melanoma, suggesting a potential role in adjuvant settings.

    PubMed

    de Aguiar, Rodrigo Barbosa; Parise, Carolina Bellini; Souza, Carolina Rosal Teixeira; Braggion, Camila; Quintilio, Wagner; Moro, Ana Maria; Navarro Marques, Fabio Luiz; Buchpiguel, Carlos Alberto; Chammas, Roger; de Moraes, Jane Zveiter

    2016-02-28

    Compelling evidence suggests that fibroblast growth factor 2 (FGF2), overexpressed in melanomas, plays an important role in tumor growth, angiogenesis and metastasis. In this study, we evaluated the therapeutic use of a new anti-FGF2 monoclonal antibody (mAb), 3F12E7, using for that the B16-F10 melanoma model. The FGF2 neutralizing effect of this antibody was certified by in vitro assays, which allowed the further track of its possible in vivo application. 3F12E7 mAb could be retained in B16-F10 tumors, as shown by antibody low-pH elution and nuclear medicine studies, and also led to reduction in number and size of metastatic foci in lungs, when treatment starts one day after intravenous injection of B16-F10 cells. Such data were accompanied by decreased CD34(+) tumor vascular density and impaired subcutaneous tumor outgrowth. Treatments starting one week after melanoma cell intravenous injection did not reduce tumor burden, remaining the therapeutic effectiveness restricted to early-adopted regimens. Altogether, the presented anti-FGF2 3F12E7 mAb stands as a promising agent to treat metastatic melanoma tumors in adjuvant settings. PMID:26655277

  20. Optimizing radiolabeled engineered anti-p185HER2 antibody fragments for in vivo imaging.

    PubMed

    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

    2005-07-01

    We have recently described the in vivo properties of an iodinated anti-p185HER2 engineered antibody fragment [minibody (scFv-C(H)3)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), radiometal 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-p185(HER2) 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-C(H)2-C(H)3)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(HER2) antibody fragments, the kidney activity was reduced and high or low expression of p185HER2 in xenografts could be distinguished by microPET imaging.

  1. 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

  2. Immunospecific saturable clearance mechanisms for indium-111-labeled anti-melanoma monoclonal antibody 96. 5 in humans

    SciTech Connect

    Murray, J.L.; Lamki, L.M.; Shanken, L.J.; Blake, M.E.; Plager, C.E.; Benjamin, R.S.; Schweighardt, S.; Unger, M.W.; Rosenblum, M.G.

    1988-08-01

    Liver uptake of 111In-labeled monoclonal antibodies (MoAb) remains a significant problem in radioimaging studies to date. To determine if the observed liver uptake of an 111In-labeled anti-melanoma antibody 96.5 (111In-96.5) was dependent on the presence of hepatic antigen or on recognition of circulating murine antibody, escalating doses of an unlabeled nonimmunoreactive MoAb (NIR-MoAb) were administered to 18 patients with metastatic malignant melanoma either 1 or 24 h prior to an infusion of 1 mg of 111In-96.5. The number of metastases imaged, pharmacokinetics, and the ratio of radioactivity (expressed as average counts/pixel) in liver (L), spleen (S), bone (B), and kidney (K) compared to blood pool (heart = H) were examined. Results were prospectively compared with data from six patients who received immunoreactive unlabeled 96.5 prior to 111In-96.5. Increasing dose or changes in the preinfusion time of NIR-MoAb had no significant effect on the biodistribution of 111In-96.5. In contrast, patients who received unlabeled, immunoreactive 96.5 prior to 111In-96.5 infusion demonstrated a significant drop (P less than 0.001) in the liver/heart ratio of radioactivity (2.81 +/- 0.35 (SEM)) compared to patients receiving the identical dose of NIR-MoAb (10.35 +/- 1.33). Significant decreases in spleen/heart and bone/heart ratios were also observed. Pharmacokinetic studies showed that the volume of distribution (Vd) and the plasma t1/2 both decreased when 96.5 was administered compared to NIR-MoAb. In addition, a 4-fold increase in concentration X time was obtained after 96.5 antibody was administered compared to NIR-MoAb. More metastases were imaged in patients receiving preinfusions of 96.5 (23 of 28) than in patients receiving NIR-MoAb (10 of 18; P less than 0.05).

  3. An Autopsy Case of Anti-melanoma Differentiation-associated Gene-5 Antibody-positive Clinical Amyopathic Dermatomyositis Complicated by Rapidly Progressive Interstitial Lung Disease.

    PubMed

    Yoshida, Naomi; Kaieda, Shinjiro; Tomozoe, Kumi; Tajiri, Morihiro; Wakasugi, Daisuke; Okamoto, Masaki; Tominaga, Masaki; Ida, Hiroaki; Hoshino, Tomoaki

    2016-01-01

    A 62-year-old man presented with heliotrope rash, Gottron's sign, and mild muscle weakness. Both of his lung fields showed interstitial changes that worsened rapidly. He was diagnosed with clinical amyopathic dermatomyositis with rapidly progressive interstitial lung disease. The patient died of respiratory failure, despite the administration of immunosuppressive therapy. Autopsy revealed diffuse alveolar damage. An antibody analysis, which was performed postmortem, detected the presence of anti-melanoma differentiation-associated gene (MDA)-5 antibodies. Clinicians should note the clinical, radiologic, and serologic findings to predict anti-MDA-5 antibody-associated rapidly progressive interstitial lung disease. PMID:27301523

  4. Immobilization and functional reconstitution of antibody Fab fragment by solid-phase refolding.

    PubMed

    Kumada, Yoichi; Hamasaki, Kyoto; Nakagawa, Aya; Sasaki, Eiju; Shirai, Tatsunori; Okumura, Masahiro; Inoue, Manami; Kishimoto, Michimasa

    2013-12-31

    In this study, we demonstrated the successful preparation of a Fab antibody-immobilized hydrophilic polystyrene (phi-PS) plate via one- and two-step solid-phase refolding methods. Both polystyrene-binding peptide (PS-tag)-fused Fd fragment of heavy chain (Fab H-PS) and full-length of light-chain (Fab L-PS) were individually produced in insoluble fractions of Escherichia coli cells, and they were highly purified in the presence of 8M of urea. Antigen-binding activities of Fab antibody immobilized were correctly recovered by the one-step solid-phase refolding method that a mixture of Fab H-PS and Fab L-PS was immobilized in the presence of 0.5-2M urea, followed by surface washing of the phi-PS plate with PBST. These results indicate that by genetic fusion of a PS-tag, a complex between Fab H and Fab L was efficiently immobilized on the surface of a phi-PS plate even in the presence of a low concentration of urea, and was then correctly refolded to retain its high antigen-binding activity via removal of the urea. A two-step solid-phase refolding method whereby Fab H-PS and Fab L-PS were successively refolded on the surface of a phi-PS plate also resulted in Fab antibody formation on the plate. Furthermore, both the binding affinity and the specificity of the Fab antibody produced by the two-step method were highly maintained, according to the results of sandwich ELISA and competitive ELISA using Fab antibody-immobilized plate via two-step solid-phase refolding. Thus, the solid-phase refolding method demonstrated in this study should be quite useful for the preparation of a Fab antibody-immobilized PS surface with high efficiency from individually produced Fab H-PS and Fab L-PS. This method will be applicable to the preparation of a large Fab antibody library on the surface of a PS plate for use in antibody screening.

  5. The novel panel assay to define tumor-associated antigen-binding antibodies in patients with metastatic melanomas may have diagnostic value.

    PubMed

    Kotlan, Beatrix; Liszkay, Gabriella; Blank, Miri; Csuka, Orsolya; Balatoni, Timea; Toth, Laszlo; Eles, Klara; Horvath, Szabolcs; Naszados, Gyorgy; Olasz, Judit; Banky, Balazs; Toth, Jozsef; Godeny, Maria; Marincola, Francesco M; Kasler, Miklos; Shoenfeld, Yehuda

    2015-02-01

    We aim to harness the natural humoral immune response by various technologies to get novel biomarkers. A complex antibody analysis in sera and in the tumor microenvironment leads to reveal tumor-specific antibodies. More strategies were introduced to select the most effective one to identify potential tumor antigen-binding capacity of the host. Epstein-Barr virus transformation and cloning with limiting dilution assay, magnetic cell sorting and antibody phage display with further methodological improvements were used in epithelial and neuroectodermal cancers. Column-purified sera of patient with melanoma were tested by immunofluorescence assay, while sera of further melanoma patients were processed for membrane-binding enzyme-linked immunosorbent assay. Some supernatants of selected B cell clones and purified antibodies showed considerable cancer cell binding capacity by immunofluorescence FACS analysis and confocal laser microscopy. Our native tumor cell membrane preparations helped to test soluble scFv and patients' sera for tumor binder antibodies. A complex tumor immunological study was introduced for patients with melanoma (ethical permission: ETT TUKEB 16462-02/2010); peripheral blood (n = 57) and surgically removed primary or metastatic tumors (n = 44) were gathered and processed at cellular immunological level. The technological developments proved to be important steps forward to the next antibody profile analyses at DNA sequence level. Cancer cell binding of patient-derived antibodies and natural immunoglobulin preparations of pooled plasma product intravenous immunoglobulins support the importance of natural human antibodies. Important cancer diagnostics and novel anticancer strategies are going to be built on these tools.

  6. Germline humanization of a murine Abeta antibody and crystal structure of the humanized recombinant Fab fragment.

    PubMed

    Robert, Remy; Streltsov, Victor A; Newman, Janet; Pearce, Lesley A; Wark, Kim L; Dolezal, Olan

    2010-02-01

    Alzheimer's disease is the most common form of dementia, affecting 26 million people worldwide. The Abeta peptide (39-43 amino acids) derived from the proteolytic cleavage of the amyloid precursor protein is one of the main constituents of amyloid plaques associated with disease pathogenesis and therefore a validated target for therapy. Recently, we characterized antibody fragments (Fab and scFvs) derived from the murine monoclonal antibody WO-2, which bind the immunodominant epitope ((3)EFRH(6)) in the Abeta peptide at the N-terminus. In vitro, these fragments are able to inhibit fibril formation, disaggregate preformed amyloid fibrils, and protect neuroblastoma cells against oligomer-mediated toxicity. In this study, we describe the humanization of WO-2 using complementary determining region loop grafting onto the human germline gene and the determination of the three-dimensional structure by X-ray crystallography. This humanized version retains a high affinity for the Abeta peptide and therefore is a potential candidate for passive immunotherapy of Alzheimer's disease.

  7. 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

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

    PubMed Central

    Sommeregger, Wolfgang; Mayrhofer, Patrick; Steinfellner, Willibald; Reinhart, David; Henry, Michael; Clynes, Martin

    2016-01-01

    ABSTRACT 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

  9. 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

  10. 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.

  11. Immunoscintigraphy of colorectal carcinoma with F (ab')2 fragments of anti-CEA monoclonal antibody

    SciTech Connect

    Buraggi, G.; Callegaro, L.; Turrin, A.; Gennari, L.; Bombardieri, E.; Mariani, G.; Deleide, G.; Dovis, M.; Gasparini, M.; Doci, R.

    1987-01-01

    A monoclonal antibody to carcinoembryonic antigen (CEA) (F023C5), belonging to IgG1 class, was obtained by cell fusion technique. Preliminary screening on different tissues was performed with immunoperoxidase staining, which showed good specificity of the antibody for gastric and colorectal carcinomas. F(ab')2 fragments were subsequently prepared and labeled with /sup 131/I and /sup 111/In. After immunoreactivity check the radiopharmaceuticals were injected intravenously. Sixteen patients with 22 primary or secondary localizations of colorectal carcinoma were studied following the recommendations of the ethical Committee of the Istituto Nazionale Tumori, Milan, Italy. Serial scans were performed after injection of the two radioactive reagents. In vivo pharmacokinetics of the compound was studied. Radioactivity level in surgical specimens was measured, and immunostaining was performed. All tumors were found to express the antigen. Eleven out of 12 tumor localizations of the gastrointestinal tract and three out of ten liver metastases were imaged. Specificity of tumor uptake was assessed by simultaneous injection of an irrelevant antibody.

  12. Reaction of systemic lupus erythematosus antinative DNA antibodies with native DNA fragments from 20 to 1,200 base pairs.

    PubMed Central

    Papalian, M; Lafer, E; Wong, R; Stollar, B D

    1980-01-01

    Double-stranded DNA fragments of varying sizes were isolated and tested for binding to systemic lupus erythematosus (SLE) antinative DNA antibodies. Fragments of 20-25, 40-50, 90-110, and 160-180 base pairs (bp), along with intermediate-size pieces were isolated by preparative gel electrophoresis of a limited micrococcal nuclease digest of calf thymus DNA. Larger helical polynucleotides of 160-200, 380, 600-1,000, and 1,200 bp were isolated by preparative gel electrophoresis of DNA from chicken erythrocyte nucleosomes and oligonucleosomes. The fragments behaved as base-paired structures as tested by thermal denaturation, resistance to S1 nuclease, and serological assays with antibodies to native or denatured DNA. At a concentration of 0.27 muM, fragments of 20-25 bp were able to react with two SLE sera in competition with native DNA. With these and two other sera, DNA of 40-50 bp was a much more effective competitor. One serum required DNA greater than 180 bp for competition in the concentration range tested. Denatured fragments were much less effective than native fragments. The results emphasize the heterogeneity of SLE antinative DNA antibodies, confirm that secondary structure of the antigen is important for specific binding to these antibodies, and support the suggestion that bivalent binding to one molecule may be important for high functional affinity. Images PMID:6153184

  13. Anti-coagulation effect of Fc fragment against anti-β2-GP1 antibodies in mouse models with APS.

    PubMed

    Xie, Weidong; Zhang, Yaou; Bu, Cunya; Sun, Shijing; Hu, Shaoliang; Cai, Guoping

    2011-01-01

    Anti-beta (2)-glycoprotein I (anti-β2-GP1) is one of the important pathogenesis factors responsible for thrombosis formation in patients with antiphospholipid syndrome (APS). Administration of intravenous immunoglobulin (IVIg) is a common method used to inhibit the abnormal antibody levels and decrease the mortality of APS in emergency situations. We hypothesize that the Fc fragment of IgG is the molecular structure responsible for these effects. The present study investigates the beneficial effects of both recombinant and natural human Fc fragments of heterogeneous IgG against human anti-β2-GP1 antibodies in mouse models with APS. Results showed that both recombinant and natural human Fc fragments moderately but significantly decreased the levels of serum anti-β2-GP1 antibodies and had anti-coagulation effects in human β2-GP1-immunized mice. Furthermore, both recombinant and natural human Fc fragments inhibited thrombosis formation and decreased mortality in mouse models infused intravenously with human anti-β2GP1 antibodies from patients with APS. Findings suggest that the Fc fragment might be one of the active structural units of heterogeneous IgG. Thus, recombinant human Fc fragment administration may be a useful treatment for individuals with APS.

  14. A Natural Bacterial-Derived Product, the Metalloprotease Arazyme, Inhibits Metastatic Murine Melanoma by Inducing MMP-8 Cross-Reactive Antibodies

    PubMed Central

    Pereira, Felipe V.; Ferreira-Guimarães, Carla A.; Paschoalin, Thaysa; Scutti, Jorge A. B.; Melo, Filipe M.; Silva, Luis S.; Melo, Amanda C. L.; Silva, Priscila; Tiago, Manoela; Matsuo, Alisson L.; Juliano, Luiz; Juliano, Maria A.; Carmona, Adriana K.; Travassos, Luiz R.; Rodrigues, Elaine G.

    2014-01-01

    The increased incidence, high rates of mortality and few effective means of treatment of malignant melanoma, stimulate the search for new anti-tumor agents and therapeutic targets to control this deadly metastatic disease. In the present work the antitumor effect of arazyme, a natural bacterial-derived metalloprotease secreted by Serratia proteomaculans, was investigated. Arazyme significantly reduced the number of pulmonary metastatic nodules after intravenous inoculation of B16F10 melanoma cells in syngeneic mice. In vitro, the enzyme showed a dose-dependent cytostatic effect in human and murine tumor cells, and this effect was associated to the proteolytic activity of arazyme, reducing the CD44 expression at the cell surface, and also reducing in vitro adhesion and in vitro/in vivo invasion of these cells. Arazyme treatment or immunization induced the production of protease-specific IgG that cross-reacted with melanoma MMP-8. In vitro, this antibody was cytotoxic to tumor cells, an effect increased by complement. In vivo, arazyme-specific IgG inhibited melanoma lung metastasis. We suggest that the antitumor activity of arazyme in a preclinical model may be due to a direct cytostatic activity of the protease in combination with the elicited anti-protease antibody, which cross-reacts with MMP-8 produced by tumor cells. Our results show that the bacterial metalloprotease arazyme is a promising novel antitumor chemotherapeutic agent. PMID:24788523

  15. β-Actin-binding Complementarity-determining Region 2 of Variable Heavy Chain from Monoclonal Antibody C7 Induces Apoptosis in Several Human Tumor Cells and Is Protective against Metastatic Melanoma*

    PubMed Central

    Arruda, Denise C.; Santos, Luana C. P.; Melo, Filipe M.; Pereira, Felipe V.; Figueiredo, Carlos R.; Matsuo, Alisson L.; Mortara, Renato A.; Juliano, Maria A.; Rodrigues, Elaine G.; Dobroff, Andrey S.; Polonelli, Luciano; Travassos, Luiz R.

    2012-01-01

    Complementarity-determining regions (CDRs) from monoclonal antibodies tested as synthetic peptides display anti-infective and antitumor activities, independent of the specificity of the native antibody. Previously, we have shown that the synthetic peptide C7H2, based on the heavy chain CDR 2 from monoclonal antibody C7, a mAb directed to a mannoprotein of Candida albicans, significantly reduced B16F10 melanoma growth and lung colony formation by triggering tumor apoptosis. The mechanism, however, by which C7H2 induced apoptosis in tumor cells remained unknown. Here, we demonstrate that C7H2 interacts with components of the tumor cells cytoskeleton, being rapidly internalized after binding to the tumor cell surface. Mass spectrometry analysis and in vitro validation revealed that β-actin is the receptor of C7H2 in the tumor cells. C7H2 induces β-actin polymerization and F-actin stabilization, linked with abundant generation of superoxide anions and apoptosis. Major phenotypes following peptide binding were chromatin condensation, DNA fragmentation, annexin V binding, lamin disruption, caspase 8 and 3 activation, and organelle alterations. Finally, we evaluated the cytotoxic efficacy of C7H2 in a panel of human tumor cell lines. All tumor cell lines studied were equally susceptible to C7H2 in vitro. The C7H2 amide without further derivatization significantly reduced lung metastasis of mice endovenously challenged with B16F10-Nex2 melanoma cells. No significant cytotoxicity was observed toward nontumorigenic cell lines on short incubation in vitro or in naïve mice injected with a high dose of the peptide. We believe that C7H2 is a promising peptide to be developed as an anticancer drug. PMID:22334655

  16. Immunomodulating antibodies in the treatment of metastatic melanoma: the experience with anti-CTLA-4, anti-CD137, and anti-PD1.

    PubMed

    Simeone, Ester; Ascierto, Paolo A

    2012-01-01

    Clinical activity of anti-CTLA-4 (cytotoxic T-lymphocyte antigen-4) monoclonal antibodies (mAb) has changed the approaches for the treatment of cancer in terms of patterns of response, duration of response, and adverse event profiles. In fact, antibodies that block the interaction of CTLA-4 with its ligands B7.1 and B7.2 can enhance immune responses, including anti-tumor immunity. Two recent studies using ipilimumab (an anti-CTLA-4 mAb) demonstrated improvements in overall survival in the treatment of advanced melanoma. These studies utilized two different schedules of treatment in different patient categories (first and second line of treatment). However, the results were quite similar despite the different dosage used and the combination with dacarbazine in the first line treatment. Ongoing clinical studies will establish the efficacy of ipilimumab as monotherapy or in combination with other drugs for the treatment of metastatic melanoma and a variety of other cancers. Other antibodies, such as CD137 agonists and PD-1 antagonists, are currently in various stages of pre-clinical and clinical development. Agonist antibodies directed against CD137 (4-1BB) on the surface of antigen-primed T-lymphocytes increase tumor immunity that is curative against some transplantable murine tumors. Programmed death-1 (PD1) is a surface molecule delivering inhibitory signals important to maintain T-cell functional silence against their cognate antigens. Interference with PD1 or its ligand PD-L1 (B7-H1) increases anti-tumor immunity. As a result, human mAbs anti-PD1 and anti-PD-L1 are under clinical development. This paper reviews recent studies in the treatment of advanced melanoma with these types of monoclonal antibodies. Ipilimumab can be considered a cornerstone of a new era in melanoma treatment. However, the aim is to optimize the therapy with anti-CTLA-4 antibodies to define the best schedule for next combination regimens (other immunomodulatory antibodies, BRAF

  17. Imaging findings and pharmacokinetics of 111-indium ZME-018 monoclonal antibody (MoAb) in malignant melanoma

    SciTech Connect

    Murray, J.L.; Rosenblum, M.; Lamki, L.; Haynie, T.P.; Glenn, H.; Jahns, M.; Plager, C.; Hersh, E.M.; Unger, M.; Carlo, D.L.

    1985-05-01

    13 patients with metastatic melanoma were studied using 5 mCi of In-111 labeled MoAb ZME-018 which reacts with GP 240 melanoma-associated antigen. The MoAb was infused over 2 h at doses of 2.5 mg (5 pts), 5 mg (5 pts), and 10 mg (3 pts). Total body tomograms and planar spot views with region of interest analysis were performed at 4, 24 and 72 hours post infusion. No adverse side effects were noted. There was rapid distribution to spleen, bone, bone marrow, liver, and testes. Tumor sites could be visualized as early as 24 hours but were more easily seen at 72 hours when the background activity was less. 20 of 46 (43%) previously documented metastases were identified. More sites imaged with increasing concentrations of MoAB, I.E., 25% at 2.5 mg; 67% at 5 mg; 70% at 10 mg. Tumor localization occurred in a significant number of patients especially at MoAb doses above 2.5 mg. In two instances, uptake of 111-In occurred in previously undiagnosed sites. The pharmacokinetics of MoAb were analyzed at each dose level. At the 5 mg dose, the terminal phase half-life for 111-In in plasma was 24.5 +- 2.7 hours. The apparent volume of distribution (Vd) was 4.03 +- 5iota similar to the plasma value, and the calculated clearance rate for 111-In label was 0.0259 + 0.002 ml/kg/min. Mean urinary excretion of 111-In label was 8.7 +- 0.6% of the administered dose over 48 hours after administration. The calculated pharmacokinetic parameters were independent of antibody dose. ZME 018 was cleared more rapidly from plasma, compared to previous studies with P97 antimelanoma MoAb.

  18. [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.

  19. Serrumab: a novel human single chain-fragment antibody with multiple scorpion toxin-neutralizing capacities.

    PubMed

    Pucca, Manuela Berto; Cerni, Felipe Augusto; Peigneur, Steve; Arantes, Eliane Candiani; Tytgat, Jan; Barbosa, José Elpidio

    2014-01-01

    In Brazil, scorpion envenomation is an important public health problem. The yellow scorpion, Tityus serrulatus (Ts), is considered the most dangerous species in the country, being responsible for the most severe clinical cases of envenomation. Currently, the administration of serum produced in horses is recognized and used as a treatment for accidents with scorpions. However, horse herds' maintenance is costly and the antibodies are heterologous, which can cause anaphylaxis and Serum Sickness. In the present work, a human monoclonal fragment antibody, Serrumab, has been analysed. Toxin neutralizing effects of Serrumab were evaluated using a two-electrode voltage-clamp technique. The results show that Serrumab presented a high neutralizing effect against Ts β-toxins (Ts1, 43.2% and Ts2, 68.8%) and none or low neutralizing effect against α-toxins (Ts3, 0% and Ts5, 10%). Additional experiments demonstrated that Serrumab was also able to neutralize the action of toxins from other scorpion genus (Css II, 45.96% and Lqh III, 100%/β- and α-toxins, respectively). This work indicated that Serrumab is able to neutralize many toxins in Ts venom, and could being considered as a neutralizing antibody for formulating a human anti-scorpion serum in Brazil. Additionally, this work demonstrated that Serrumab could neutralize different toxins from distinct scorpion genus. All these results reinforce the idea that Serrumab is a scFv antibody with multiple neutralizing capacities and a promising candidate for inclusion in scorpion anti-venoms against different genera. PMID:24001307

  20. 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.

  1. Arthritis and tenosynovitis associated with the anti-PD1 antibody pembrolizumab in metastatic melanoma.

    PubMed

    Chan, Matthew M K; Kefford, Richard F; Carlino, Matteo; Clements, Arthur; Manolios, Nicholas

    2015-01-01

    We report the acute onset of polyarticular inflammatory arthritis in 2 patients receiving the immune check-point inhibitor, pembrolizumab (MK-3475), anti-PD1 drug for metastatic melanoma after 14 and 11 months therapy, respectively. The first patient had severe tenosynovitis, synovitis, bone marrow edema, and myositis, whereas the second patient had predominantly synovitis and tenosynovitis. Good symptomatic control was obtained with bisphosphonates and salazopyrin, avoiding the use of T-cell immunosuppressants. These cases raise important questions on whether anti-PD1 therapy allows preexisting autoimmune T-cell clones to escape tolerance by suppressing regulatory T cells or whether they allow autoimmunity to develop de novo. These conditions heighten our awareness of complications associated with the clinical use of these agents, and provide a prototypical model for future research into the understanding of autoimmunity.

  2. Fine-mapping naturally occurring NY-ESO-1 antibody epitopes in melanoma patients' sera using short overlapping peptides and full-length recombinant protein.

    PubMed

    Komatsu, Nobukazu; Jackson, Heather M; Chan, Kok-fei; Oveissi, Sara; Cebon, Jonathan; Itoh, Kyogo; Chen, Weisan

    2013-07-01

    The tumor antigen NY-ESO-1 is one of the most antigenic cancer-testis antigens, first identified by serologic analysis of a recombinant cDNA expression library (SEREX). NY-ESO-1 is expressed in different types of cancers including melanoma. NY-ESO-1-specific spontaneous humoral and cellular immune responses are detected in a large proportion of patients with advanced NY-ESO-1-expressing cancers. Therefore NY-ESO-1 is a good candidate antigen for immunotherapy. Although cellular immune responses to NY-ESO-1 are well characterized, much less is known about the humoral immune responses. In this study, we finely mapped linear antibody epitopes using sera from melanoma patients and shorter overlapping peptide sets. We have shown that melanoma patients' humoral immune systems responded to NY-ESO-1 differently in each individual with widely differing antibody specificity, intensity and antibody subtypes. This knowledge will help us further understand anti-tumor immunity and may also help us to monitor cancer progress and cancer vaccine efficacy in the future.

  3. Radioiodination and biodistribution of the monoclonal antibody TU-20 and its scFv fragment

    NASA Astrophysics Data System (ADS)

    Kubaštová, H.; Kleinova, V.; Seifert, D.; Fišer, M.; Kranda, K.

    2006-01-01

    The ability of the monoclonal antibody TU-20 and its scFv fragment to specifically bind to the C-end of the class III beta-tubulin makes these preparations useful as potential diagnostics for in vivo determination of neurodegenerative diseases that entail degradation of neuronal cytoskeleton. To examine this hypothesis, TU-20 and its scFv were labelled with 125I and their properties were extensively investigated. TU-20 and its scFv were labelled via chloramine-T with the yield 90 95% and 64 78%, respectively. Their quality control, performed by an ELISA and gel electrophoresis, determined adequate properties for further studies. The in vitro experiment, involving autoradiography and immunohistochemistry of mice’ brain slices, enabled confirmation of preserved immunospecificity of the radiolabelled substances. Finally, the in vivo biodistribution proved differences in elimination of either TU-20, scFv TU-20, or iodide from the mice.

  4. An ELISA for detection of trout antibodies to viral haemorrhagic septicemia virus using recombinant fragments of their viral G protein.

    PubMed

    Encinas, P; Gomez-Casado, E; Estepa, A; Coll, J M

    2011-09-01

    An enzyme linked immunosorbent assay (ELISA) method to study serum antibodies to viral haemorrhagic septicemia virus (VHSV) was designed by using recombinant fragments of their G protein. By using this fragment-ELISA, we describe the binding of antibodies against recombinant G fragments of 45-445 amino acids present in VHSV-hyperimmunized trout sera. Fragments were designed by taking into account their tridimensional pH-dependent structure and functional domains. Sera were obtained from hyperimmunized trout following 4-5 intraperitoneal injections of VHSV antigens by using Freund's or saponin adjuvants. Sera from different hyperimmunized trout differed quantitatively rather than qualitatively in their recognition of solid-phase frg11 (56-110), frg12 (65-109), frg13 (97-167), frg14 (141-214), frg15 (65-250), frg16 (252-450) and G (G21-465) by Western blot and ELISA. However, titres were higher when using frg11, frg15 or frg16, rather than G21-465, suggesting higher accessibility to G epitopes. Further knowledge of the antigenicity of the G protein of rhabdoviruses by using fragments might be used to improve current vaccines. On the other hand, they might be used to dissect the trout antibody response to VHSV infections, to complement in vitro neutralizing assays, and/or to quantitate anti-VHSV antibodies in VHSV-infected/vaccinated trout, other fish and/or other body fluids such as mucus.

  5. Establishment of hapten-specific monoclonal avian IgY by conversion of antibody fragments obtained from combinatorial libraries.

    PubMed

    Deckers, Susanne; Braren, Ingke; Greunke, Kerstin; Meyer, Nadine; Rühl, Dana; Bredehorst, Reinhard; Spillner, Edzard

    2009-01-01

    Nowadays, recombinant antibody and phage display technology enable the efficient generation of immunotools and a subsequent manipulation for optimized affinity, specificity or overall performance. Such advantages are of particular interest for haptenic target structures, such as TNT (2,4,6-trinitrotoluene). The toxicity of TNT and its breakdown products makes a reliable and fast detection of low levels in aqueous samples highly important. In the present study, we aimed for the generation of scFvs (single-chain antibody fragments) specific for the TNT-surrogate TNP (2,4,6-trinitrophenyl) and their subsequent production as monoclonal avian IgY immunoglobulins providing improved assay performance. Therefore we subjected a human synthetic scFv library to selection following different strategies. TNP-specific human antibody fragments could be identified, characterized for their primary structure and evaluated for production as soluble scFv in Escherichia coli. Additionally, a murine TNP-specific antibody fragment was obtained from the hybridoma 11B3; however, the prokaryotic expression level was found to be limited. To generate and evaluate immunoglobulin formats with superior characteristics, all recombinant antibody fragments then were converted into two different chimaeric bivalent IgY antibody formats. After expression in mammalian cells, the IgY antibodies were assessed for their reactivity towards TNT. The IgY antibodies generated on the basis of the combinatorial library proved to be useful for detection of TNT, thereby emphasizing the high potential of this approach for the development of detection devices for immunoassay-based techniques.

  6. 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

  7. 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

  8. 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

  9. Melanoma immunotherapy.

    PubMed

    Sivendran, Shanthi; Glodny, Bradley; Pan, Michael; Merad, Miriam; Saenger, Yvonne

    2010-01-01

    Melanoma immunotherapy has been an area of intense research for decades, and this work is now yielding more tangible results for patients. Work has focused on 4 main areas: cytokine therapy, administration of immune-modulating antibodies, adoptive T-cell therapy, and vaccines. Cytokine therapy is an established treatment for advanced melanoma, and immune-modulating antibodies have recently emerged as an exciting new area of drug development with efficacy now established in a phase III trial. Adoptive T-cell therapy provides the proof of principle that T cells can attack and eliminate tumors. It has been challenging, however, to adapt this treatment for widespread use. Vaccines have generally yielded poor results, but intratumor pathogen-based strategies have shown encouraging results in recent trials, perhaps due to stronger immune stimulation. A review of the field of melanoma immunotherapy is provided here, with emphasis on those agents that have reached clinical testing. Novel strategies to induce the immune system to attack melanomas are reviewed. In the future, it is envisioned that immunotherapy will have further application in combination with cytotoxic and targeted therapies.

  10. Rice-based oral antibody fragment prophylaxis and therapy against rotavirus infection

    PubMed Central

    Tokuhara, Daisuke; ρlvarez, Beatriz; Mejima, Mio; Hiroiwa, Tomoko; Takahashi, Yuko; Kurokawa, Shiho; Kuroda, Masaharu; Oyama, Masaaki; Kozuka-Hata, Hiroko; Nochi, Tomonori; Sagara, Hiroshi; Aladin, Farah; Marcotte, Harold; Frenken, Leon G.J.; Iturriza-Gómara, Miren; Kiyono, Hiroshi; Hammarström, Lennart; Yuki, Yoshikazu

    2013-01-01

    Rotavirus-induced diarrhea is a life-threatening disease in immunocompromised individuals and in children in developing countries. We have developed a system for prophylaxis and therapy against rotavirus disease using transgenic rice expressing the neutralizing variable domain of a rotavirus-specific llama heavy-chain antibody fragment (MucoRice-ARP1). MucoRice-ARP1 was produced at high levels in rice seeds using an overexpression system and RNAi technology to suppress the production of major rice endogenous storage proteins. Orally administered MucoRice-ARP1 markedly decreased the viral load in immunocompetent and immunodeficient mice. The antibody retained in vitro neutralizing activity after long-term storage (>1 yr) and boiling and conferred protection in mice even after heat treatment at 94°C for 30 minutes. High-yield, water-soluble, and purification-free MucoRice-ARP1 thus forms the basis for orally administered prophylaxis and therapy against rotavirus infections. PMID:23925294

  11. Identification and molecular characterization of human antibody fragments specific for dengue NS5 protein.

    PubMed

    Zhao, Yongqian; Moreland, Nicole J; Tay, Moon Y F; Lee, Chin Chin; Swaminathan, Kunchithapadam; Vasudevan, Subhash G

    2014-01-22

    The multifunctional dengue nonstructural (NS) protein 5 from the four serotypes of dengue virus (DENV1-4) is essential for viral replication and harbors a methyl transferase (MTase) and a RNA-dependent RNA-polymerase domain (RdRp). There are limited comparative studies of NS5 from the four DENV serotypes and this is further hampered by a lack of cross-reactive NS5 antibodies. In this study, recombinant NS5 proteins were expressed, purified, enzymatically characterized, and used strategically as bait in biopanning experiments with a naïve human Fab phage-display library to identify serotype specific or cross-reactive Fab fragments. Using a combination of peptide competition ELISA and peptide phage display the epitopes of the cross-reactive Fabs were mapped to the first alpha helix of the MTase domain (5M1) and the priming loop of the RdRp domain (5R3). The epitope of a third, serotype-specific Fab (5M3) was mapped to aa19-30 of the DENV3 MTase domain. Together the recombinant proteins and specific antibodies will facilitate further mechanistic studies of the DENV replication complex.

  12. Structural analysis of bacterial ABC transporter inhibition by an antibody fragment.

    PubMed

    Ahuja, Shivani; Rougé, Lionel; Swem, Danielle L; Sudhamsu, Jawahar; Wu, Ping; Russell, Stephen J; Alexander, Mary Kate; Tam, Christine; Nishiyama, Mireille; Starovasnik, Melissa A; Koth, Christopher M

    2015-04-01

    Bacterial ATP-binding cassette (ABC) importers play critical roles in nutrient acquisition and are potential antibacterial targets. However, structural bases for their inhibition are poorly defined. These pathways typically rely on substrate binding proteins (SBPs), which are essential for substrate recognition, delivery, and transporter function. We report the crystal structure of a Staphylococcus aureus SBP for Mn(II), termed MntC, in complex with FabC1, a potent antibody inhibitor of the MntABC pathway. This pathway is essential and highly expressed during S. aureus infection and facilitates the import of Mn(II), a critical cofactor for enzymes that detoxify reactive oxygen species (ROS). Structure-based functional studies indicate that FabC1 sterically blocks a structurally conserved surface of MntC, preventing its interaction with the MntB membrane importer and increasing wild-type S. aureus sensitivity to oxidative stress by more than 10-fold. The results define an SBP blocking mechanism as the basis for ABC importer inhibition by an engineered antibody fragment.

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

    PubMed

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

    2016-01-01

    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

  14. Anti-neuropilin 1 antibody Fab' fragment conjugated liposomal docetaxel for active targeting of tumours.

    PubMed

    Manjappa, Arehalli S; Goel, Peeyush N; Gude, Rajiv P; Ramachandra Murthy, Rayasa S

    2014-09-01

    Neuropilin-1, a transmembrane receptor entailed in wide range of human tumour cell lines and diverse neoplasms, mediates the effects of VEGF and Semaphorins during the processes of cellular proliferation, survival and migration. In view of this, we had developed and evaluated in vitro and in vivo efficacy of anti-neuropilin-1 immunoliposomes against neuropilin-1 receptor expressing tumours. The PEGylated liposomes loaded with docetaxel were prepared using thin film hydration method. Functionalised PEGylated liposomes were prepared using post-insertion technique. Anti-neuropilin-1 immunoliposomes were prepared by covalently conjugating Fab' fragments of neuropilin-1 antibody to functionalised PEGylated liposomes via thioether linkage. In vivo evaluation of Taxotere and liposomal formulations was performed using intradermal tumour model to demonstrate anti-angiogenic and tumour regression ability. The modified Fab' fragments and immunoliposomes were found to be immunoreactive against A549 cells. Further, docetaxel loaded PEGylated liposomes and PEGylated immunoliposomes demonstrated higher in vitro cytotoxicity than Taxotere formulation at the same drug concentration and exposure time. The live imaging showed distinctive cellular uptake of functional immunoliposomes. Further, significant decrease in micro-blood vessel density and tumour volumes was observed using bio-engineered liposomes. The results clearly highlight the need to seek neuropilin-1 as one of the prime targets in developing an anti-angiogenic therapy.

  15. A collagen-binding EGFR single-chain Fv antibody fragment for the targeted cancer therapy.

    PubMed

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

    2015-07-10

    Collagen, a primary component of the extracellular matrix (ECM), is highly expressed in a variety of cancers and influences the tumor microenvironment by increasing the recruitment of macrophages and endothelial cells. Therefore, collagen is a highly promising target for cancer therapy. The collagen-binding domain (CBD) can dynamically bind to collagen and achieve the sustained release of CBD-fused protein in the collagen network. Here, we developed a collagen-binding epidermal growth factor receptor (EGFR) antibody fragment for targeting the collagen-rich ECM in tumors. The single chain fragment variable (scFv) of cetuximab was fused to CBD (CBD-scFv) and expressed in Pichia pastoris. CBD-scFv preserved the antigen binding domain and anti-tumor activity of cetuximab in vitro. Moreover, CBD-scFv displayed a collagen binding ability due to the function of CBD. In vivo experiments revealed that CBD-scFv bound to collagen and achieved sustained release in tumors. Furthermore, CBD-scFv significantly suppressed the growth of tumors in A431 xenografts. Therefore, CBD-scFv had a potential therapeutic value for the collagen-rich carcinomas. The specific target and sustained release of CBD-scFv in tumors could be a new approach for targeted drug delivery in cancer therapy.

  16. Identification of human antibody fragment clones specific for tetanus toxoid in a bacteriophage. lambda. immunoexpression library

    SciTech Connect

    Mullinax, R.L.; Gross, E.A.; Amberg, J.R.; Hogrefe, H.H.; Kubitz, M.M.; Greener, A.; Alting-Mees, M.; Ardourel, D.; Short, J.M.; Sorge, J.A. ); Hay, B.N.; Shopes, B. )

    1990-10-01

    The authors have applied a molecular biology approach to the identification of human monoclonal antibodies. Human peripheral blood lymphocyte mRNA was converted to cDNA and a select subset was amplified by the polymerase chain reaction. These products, containing coding sequences for numerous immunoglobulin heavy- and {kappa} light-chain variable and constant region domains, were inserted into modified bacteriophase {lambda} expression vectors and introduced into Escherichia coli by infection to yield a combinatorial immunoexpression library. Clones with binding activity to tetanus toxoid were identified by filter hybridization with radiolabeled antigen and appeared at a frequency of 0.2{percent} in the library. These human antigen binding fragments, consisting of a heavy-chain fragment covalently linked to a light chain, displayed high affinity of binding to tetanus toxoid with equilibrium constants in the nanomolar range but did not cross-react with other proteins tested. They estimate that this human immunoexpression library contains 20,000 clones with high affinity and specificity to our chosen antigen.

  17. Evaluation of radioiodinated and radiocopper labeled monovalent fragments of monoclonal antibody chCE7 for targeting of neuroblastoma.

    PubMed

    Carrel, F; Amstutz, H; Novak-Hofer, I; Schubiger, P A

    1997-08-01

    Monovalent fragments of antineuroblastoma antibody mAb chCE7 were evaluated for their in vitro and in vivo tumor cell binding properties. Single chain fragments were constructed from the variable region genes cloned from hybridoma cells, expressed in E.coli and purified by metal chelate affinity chromatography. Radioiodinated CE7-scFv fragments were found to bind with high affinity (Kd approximately 10(-9) M) to target cells in vitro but formed aggregates at 37 degrees C, and bound to serum proteins in vitro and in vivo. Circular Dichroism spectra revealed the protein to be in a conformationally altered form and no permanent "refolding" could be achieved. In contrast, chCE7- Fab fragments were found to bind to target tumor cells with similar affinity than the parent mAb chCE7 (Kd approximately 10(-10) M), showed no tendency to aggregate and were stable in serum both in vitro and in vivo. Kinetics of association and dissociation of radioiodinated scFv and Fab fragments were found to be rapid. Radioiodination with the Iodogen method led to impaired immunoreactivity which was found to further increase the off- rates of radioiodinated fragments from tumor cells. Radioiodination with the Bolton-Hunter reagent as well as labeling of chCE7-Fab fragments with 67Cu via the macrocyclic CPTA ligand led to fully immunoreactive Fab fragments. Radioiodinated and radiocopper labeled monovalent CE7 fragments did not internalize into target tumor cells as the parent mAb and its F(ab')2 fragment. A comparison of the biodistribution in tumor bearing nude mice of the radiocopper labeled monovalent, non internalizing Fab fragments with the internalizing divalent F(ab')2 fragments showed in both cases high levels of radioactivity in the kidneys. Concerning tumor uptake, radioactivity from both internalizing and non internalizing fragments remained associated with tumor tissue for longer times than in case of the corresponding radioiodinated fragments. When compared with the

  18. Production of an anti-idiotypic antibody single chain variable fragment vaccine against Edwardsiella tarda.

    PubMed

    Qin, Hong; Jin, Xiaohang; Huang, Weiquan; Liu, Yulin

    2010-02-01

    Edwardsiella tarda is the pathogen responsible for edwardsiellosis, a serious infectious disease of freshwater and marine fish species, and currently recognized to be the species pathogenic for human. An anti-idiotypic monoclonal antibody (mAb), 1E11, has been developed. It mimics the protective epitope of E. tarda and can prevent fish from infection of E. tarda. In this study, the correct variable heavy (VH) and variable light (VL) genes were obtained from 1E11 by using bioinformatics methods, and a 15 amino acid (Gly4Ser)3 linker was used to hold the two V domains together for the construction of VL-linker-VH form of single chain variable fragment (scFv) gene. Then, the scFv was subcloned into the vector pET-28a, expressed in the Escherichia coli BL21 cells, and identified by SDS-PAGE and western blotting. Red drum (Sciaenops ocellatus L.) weighing about 50 g was subjected to challenge with different E. tarda strains after 4 weeks followed by vaccination, the mortality rates and relative percentage survival were recorded and calculated, and the survival rate of fish in the scFv subgroups was obviously higher than that of control subgroups (P<0.01). Enzyme-linked immunosorbent assay results show that after 4 weeks of post-vaccination, the level of specific antibody in fish sera of scFv groups was significantly higher than control groups. This study indicates that the recombinant antibody scFv was successfully developed, and it may serve as an effective vaccine candidate against E. tarda. PMID:20119624

  19. 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

  20. Lung histopathological pattern in a survivor with rapidly progressive interstitial lung disease and anti-melanoma differentiation-associated gene 5 antibody-positive clinically amyopathic dermatomyositis.

    PubMed

    Suzuki, Atsushi; Kondoh, Yasuhiro; Taniguchi, Hiroyuki; Tabata, Kazuhiko; Kimura, Tomoki; Kataoka, Kensuke; Ono, Kenzo; Hashisako, Mikiko; Fukuoka, Junya

    2016-01-01

    Anti-melanoma differentiation-associated gene 5 (MDA5) antibodies are specific indicators of patients with dermatomyositis, particularly clinically amyopathic dermatomyositis (CADM). CADM is occasionally accompanied by fatal, treatment-resistant, rapidly-progressive interstitial lung disease (RP-ILD). All previous reports showed that histopathological findings in RP-ILD with anti-MDA5 antibody-positive CADM indicated diffuse alveolar damage (DAD). This is the first report describing a non-DAD pattern in RP-ILD with anti-MDA5 antibody-positive CADM, which was improved by immunosuppressive therapy. This case may be a milder clinical phenotype than a typical DAD pattern in RP-ILD with anti-MDA5 antibody-positive CADM. PMID:27354955

  1. Immunoliposome co-delivery of bufalin and anti-CD40 antibody adjuvant induces synergetic therapeutic efficacy against melanoma

    PubMed Central

    Li, Ying; Yuan, Jiani; Yang, Qian; Cao, Wei; Zhou, Xuanxuan; Xie, Yanhua; Tu, Honghai; Zhang, Ya; Wang, Siwang

    2014-01-01

    Liposomes constitute one of the most popular nanocarriers for improving the delivery and efficacy of agents in cancer patients. The purpose of this study was to design and evaluate immunoliposome co-delivery of bufalin and anti-CD40 to induce synergetic therapeutic efficacy while eliminating systemic side effects. Bufalin liposomes (BFL) conjugated with anti-CD40 antibody (anti-CD40-BFL) showed enhanced cytotoxicity compared with bufalin alone. In a mouse B16 melanoma model, intravenous injection of anti-CD40-BFL achieved smaller tumor volume than did treatment with BFL (average: 117 mm3 versus 270 mm3, respectively); the enhanced therapeutic efficacy through a caspase-dependent pathway induced apoptosis, which was confirmed using terminal deoxynucleotidyl transferase-mediated dUTP-Fluorescein nick end labeling and Western blot assay. Meanwhile, anti-CD40-BFL elicited unapparent body-weight changes and a significant reduction in serum levels of tumor necrosis factor-α, interleukin-1β, interleukin-6, interferon-γ, and hepatic enzyme alanine transaminase, suggesting minimized systemic side effects. This may be attributed to the mechanism by which liposomes are retained within the tumor site for an extended period of time, which is supported by the following biodistribution and flow cytometric analyses. Taken together, the results demonstrated a highly promising strategy for liposomal vehicle transport of anti-CD40 plus bufalin that can be used to enhance antitumor effects via synergetic systemic immunity while blocking systemic toxicity. PMID:25506218

  2. Intracellular interference of tick-borne flavivirus infection by using a single-chain antibody fragment delivered by recombinant Sindbis virus.

    PubMed Central

    Jiang, W; Venugopal, K; Gould, E A

    1995-01-01

    A single-chain antibody fragment that identifies a neutralizing epitope on the envelope protein of louping ill and some other tick-borne flaviviruses was previously expressed in soluble form from bacteria and shown to be functionally active in vitro. To see whether or not the single-chain antibody could bind and inactivate infectious virus in vivo, we have used recombinant Sindbis virus as a delivery vehicle for intracellular expression of the antibody fragment. The variable genes and interchain linker encoding the single-chain antibody were cloned into a double subgenomic Sindbis virus expression vector to generate recombinant Sindbis virus. Infection with this recombinant Sindbis virus provided high-level cytoplasmic expression of the antibody fragment in mammalian cells. We demonstrate (i) that the antibody fragment was antigen binding and (ii) that louping ill virus infectivity was significantly reduced in the presence of intracellular antibody expressed by the superinfecting recombinant Sindbis virus. PMID:7815482

  3. Method for the preparation of bispecific F(ab')2mu fragments from mouse monoclonal antibodies of the immunoglobulin M class and characterization of the fragments.

    PubMed

    Morimoto, K; Inouye, K

    1999-04-22

    Bispecific F(ab')2mu fragments (Bs F(ab')2mu) binding simultaneously both sialyl Lewis A antigen (SLA) and human carcinoembryonic antigen (CEA) were prepared by disulfide bond exchange between F(ab')2mu fragments derived from IgM monoclonal antibodies (mAbs) against SLA and CEA, and were purified to homogeneity in a one-step procedure of hydrophobic interaction HPLC. The final yield of Bs F(ab')2mu from F(ab')2mu fragments was 70-78%, and the purity was higher than 98%. The immunoreactivities of the Bs F(ab')2mu fragments against SLA and CEA were almost the same as those of the respective parental F(ab')2mu fragments. The dissociation constant (0.17 microM) of the Bs F(ab')2mu for CEA was in good agreement with that of the parental F(ab')2mu fragments. Although the number of applications of IgM mAbs is restricted because of the large molecular mass and low solubility, Bs F(ab')2mu might, nevertheless, be a useful tool for immunotherapy and immunodiagnosis.

  4. 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.

  5. 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...

  6. 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).

  7. Monoclonal antibody OKM5 inhibits the in vitro binding of Plasmodium falciparum-infected erythrocytes to monocytes, endothelial, and C32 melanoma cells

    SciTech Connect

    Barnwell, J.W.; Ockenhouse, C.F.; Knowles, D.M. II

    1985-11-01

    Plasmodium falciparum-infected erythrocytes bind in vitro to human endothelial cells, monocytes, and a certain melanoma cell line. Evidence suggests that this interaction is mediated by similar mechanisms which lead to the sequestration of parasitized erythrocytes in vivo through their attachment to endothelial cells of small blood vessels. They show here the monoclonal antibody OKM5, previously shown to react with the membranes of endothelial cells, monocyte,s and platelets, also reacts with the C32 melanoma cell line which also binds P. falciparum-infected erythrocytes. At relatively low concentrations, OKM5 inhibits and reverses the in vitro adherence of infected erythrocytes to target cells. As with monocytes, OKM5 antibody recognizes an /sup 125/I-labeled protein of approximately 88 Kd on the surface of C32 melanoma cells. It seems likely, therefore, that the 88 Kd polypeptide plays a role in cytoadherence, possibly as the receptor or part of a receptor for a ligand on the surface of infected erythrocytes.

  8. Reduced toxicity of expression, in Escherichia coli, of antipollutant antibody fragments and their use as sensitive diagnostic molecules.

    PubMed

    Strachan, G; Williams, S; Moyle, S P; Harris, W J; Porter, A J

    1999-09-01

    Single-chain antibody fragments (scAb), specific for the chlorophenoxy acid herbicide mecoprop, have been expressed and purified from the bacterium Escherichia coli. Co-expression with the colE1-compatible, arabinose-inducible, skp expression vector pHELP1 prevented bacterial lysis and significantly increased both total and functional expression yield. The periplasmic protein, SKP, may have a role as a generic detoxification protein. Surface plasmon resonance (BIAcore 2000) analysis confirmed that the purified scAb retained similar binding kinetics to the monoclonal antibody (Mab) from which it was cloned. In competition ELISA, the bacterial scAb showed the same specificity for mecoprop and a related herbicide, MCPA, as the Mab but an increase in sensitivity for free antigen in all ELISA formats. Bacterially expressed antibody fragments provide a simple, sensitive and cost-effective alternative to the traditional production of diagnostic Mabs via tissue culture.

  9. 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

  10. Management of side effects of immune checkpoint blockade by anti-CTLA-4 and anti-PD-1 antibodies in metastatic melanoma.

    PubMed

    Kähler, Katharina C; Hassel, Jessica C; Heinzerling, Lucie; Loquai, Carmen; Mössner, Rotraut; Ugurel, Selma; Zimmer, Lisa; Gutzmer, Ralf

    2016-07-01

    CTLA-4 and PD-1 are potential targets for tumor-induced downregulation of lymphocytic immune responses. Immune checkpoint-modifying monoclonal antibodies oppose these effects, inducing T cell-mediated immune responses to various tumors including melanoma. Both anti-CTLA-4 and anti-PD-1 antibodies modify the interaction between tumor, antigen-presenting cells, and T lymphocytes. With respect to overall survival, clinical studies have shown a major benefit for the anti-CTLA-4 antibody ipilimumab as well as the two anti-PD-1 antibodies nivolumab and pembrolizumab. Following approval of ipilimumab in 2011, the latter two achieved market authorization in the summer of 2015. Immune responses thus induced and enhanced inevitably entail autoimmune phenomena, affecting various organs to varying degrees. Knowledge of these side effects is crucial with regard to prevention and management by treating physicians. Typically occurring early on and presenting with pronounced and persistent diarrhea, colitis represents a major and severe side effect. Other immune-mediated disorders include dermatitis, hypophysitis, thyroiditis, hepatitis, iridocyclitis as well as other less common autoimmune phenomena. Early recognition and initiation of treatment can reduce risks and sequelae for patients. This review describes the mechanisms of action of immune checkpoint blockade as well as its clinical effects in metastatic melanoma, with a detailed focus on the spectrum of adverse events and their therapeutic management. PMID:27373241

  11. Integrated NY-ESO-1 antibody and CD8+ T-cell responses correlate with clinical benefit in advanced melanoma patients treated with ipilimumab.

    PubMed

    Yuan, Jianda; Adamow, Matthew; Ginsberg, Brian A; Rasalan, Teresa S; Ritter, Erika; Gallardo, Humilidad F; Xu, Yinyan; Pogoriler, Evelina; Terzulli, Stephanie L; Kuk, Deborah; Panageas, Katherine S; Ritter, Gerd; Sznol, Mario; Halaban, Ruth; Jungbluth, Achim A; Allison, James P; Old, Lloyd J; Wolchok, Jedd D; Gnjatic, Sacha

    2011-10-01

    Ipilimumab, a monoclonal antibody against cytotoxic T lymphocyte antigen 4 (CTLA-4), has been shown to improve survival in patients with advanced metastatic melanoma. It also enhances immunity to NY-ESO-1, a cancer/testis antigen expressed in a subset of patients with melanoma. To characterize the association between immune response and clinical outcome, we first analyzed NY-ESO-1 serum antibody by ELISA in 144 ipilimumab-treated patients with melanoma and found 22 of 140 (16%) seropositive at baseline and 31 of 144 (22%) seropositive following treatment. These NY-ESO-1-seropositive patients had a greater likelihood of experiencing clinical benefit 24 wk after ipilimumab treatment than NY-ESO-1-seronegative patients (P = 0.02, relative risk = 1.8, two-tailed Fisher test). To understand why some patients with NY-ESO-1 antibody failed to experience clinical benefit, we analyzed NY-ESO-1-specific CD4(+) and CD8(+) T-cell responses by intracellular multicytokine staining in 20 NY-ESO-1-seropositive patients and found a surprising dissociation between NY-ESO-1 antibody and CD8 responses in some patients. NY-ESO-1-seropositive patients with associated CD8(+) T cells experienced more frequent clinical benefit (10 of 13; 77%) than those with undetectable CD8(+) T-cell response (one of seven; 14%; P = 0.02; relative risk = 5.4, two-tailed Fisher test), as well as a significant survival advantage (P = 0.01; hazard ratio = 0.2, time-dependent Cox model). Together, our data suggest that integrated NY-ESO-1 immune responses may have predictive value for ipilimumab treatment and argue for prospective studies in patients with established NY-ESO-1 immunity. The current findings provide a strong rationale for the clinical use of modulators of immunosuppression with concurrent approaches to favor tumor antigen-specific immune responses, such as vaccines or adoptive transfer, in patients with cancer.

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

    PubMed

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

    2016-06-01

    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 Ca(2+) 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.

  13. 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

  14. Exploiting Cross-reactivity to Neutralize Two Different Scorpion Venoms with One Single Chain Antibody Fragment*

    PubMed Central

    Riaño-Umbarila, Lidia; Contreras-Ferrat, Gabriel; Olamendi-Portugal, Timoteo; Morelos-Juárez, Citlalli; Corzo, Gerardo; Possani, Lourival D.; Becerril, Baltazar

    2011-01-01

    We report the optimization of a family of human single chain antibody fragments (scFv) for neutralizing two scorpion venoms. The parental scFv 3F recognizes the main toxins of Centruroides noxius Hoffmann (Cn2) and Centruroides suffusus suffusus (Css2), albeit with low affinity. This scFv was subjected to independent processes of directed evolution to improve its recognition toward Cn2 (Riaño-Umbarila, L., Juárez-González, V. R., Olamendi-Portugal, T., Ortíz-León, M., Possani, L. D., and Becerril, B. (2005) FEBS J. 272, 2591–2601) and Css2 (this work). Each evolved variant showed strong cross-reactivity against several toxins, and was capable of neutralizing Cn2 and Css2. Furthermore, each variant neutralized the whole venoms of the above species. As far as we know, this is the first report of antibodies with such characteristics. Maturation processes revealed key residue changes to attain expression, stability, and affinity improvements as compared with the parental scFv. Combination of these changes resulted in the scFv LR, which is capable of rescuing mice from severe envenomation by 3 LD50 of freshly prepared whole venom of C. noxius (7.5 μg/20 g of mouse) and C. suffusus (26.25 μg/20 g of mouse), with surviving rates between 90 and 100%. Our research is leading to the formulation of an antivenom consisting of a discrete number of human scFvs endowed with strong cross-reactivity and low immunogenicity. PMID:21156801

  15. The development of antibody-IL-2 based immunotherapy with hu14.18-IL2 (EMD-273063) in melanoma and neuroblastoma.

    PubMed

    Yamane, Brett H; Hank, Jacquelyn A; Albertini, Mark R; Sondel, Paul M

    2009-07-01

    Patients with high risk melanoma and neuroblastoma frequently experience recurrence despite surgical resection and appropriate adjuvant therapies. Immunotherapy with the immunocytokine hu14.18-IL2 (EMD-273063) was developed by means of fusion of two molecules of IL-2 to the monoclonal antibody, 14.18, that recognizes GD2, expressed on the earlier mentioned malignancies. This article will discuss the results of preclinical work using EMD-273063 therapy, including data suggesting that intratumoral therapy may have enhanced antitumor benefit compared with intravenous therapy. Initial clinical trials in adult melanoma and pediatric neuroblastoma have demonstrated acceptable toxicity profiles in dosing that induces immune activation. Preclinical and initial clinical data suggest greater efficacy in the setting of minimal residual disease; therefore, future clinical testing is planned to test the benefit of EMD-273063 in this setting.

  16. The development of antibody –based immunotherapy with (EMD-273063) Hu14.18-IL2 in melanoma and neuroblastoma

    PubMed Central

    Yamane, Brett H.; Hank, Jacquelyn A.; Albertini, Mark R.; Sondel, Paul M.

    2010-01-01

    Patients with high risk melanoma and neuroblastoma frequently recur despite surgical resection and appropriate adjuvant therapies. Immunotherapy with the immunocytokine, hu14.18-IL2, was developed via fusion of two molecules of IL2 to the monoclonal antibody, 14.18, that recognizes GD2, expressed on the above malignancies. This article will discuss the results of preclinical work utilizing hu14.18-IL2 therapy, including data suggesting that intratumoral therapy may have enhanced antitumor benefit compared with IV therapy. Initial clinical trials in adult melanoma and pediatric neuroblastoma have demonstrated acceptable toxicity profiles in dosing that induces immune activation. Preclinical and initial clinical data suggest greater efficacy in the setting of minimal residual disease, therefore future clinical testing is planned to test the benefit of hu14.18-IL2 in this setting. PMID:19548853

  17. Effects of protein engineering and rational mutagenesis on crystal lattice of single chain antibody fragments

    PubMed Central

    Kalyoncu, Sibel; Hyun, Jeongmin; Pai, Jennifer C.; Johnson, Jennifer L.; Entzminger, Kevin; Jain, Avni; Heaner, David P.; Morales, Ivan A.; Truskett, Thomas M.; Maynard, Jennifer A.; Lieberman, Raquel L.

    2014-01-01

    Protein crystallization is dependent upon, and sensitive to, the intermolecular contacts that assist in ordering proteins into a three dimensional lattice. Here we used protein engineering and mutagenesis to affect the crystallization of single chain antibody fragments (scFvs) that recognize the EE epitope (EYMPME) with high affinity. These hypercrystallizable scFvs are under development to assist difficult proteins, such as membrane proteins, in forming crystals, by acting as crystallization chaperones. Guided by analyses of intermolecular crystal lattice contacts, two second-generation anti-EE scFvs were produced, which bind to proteins with installed EE tags. Surprisingly, although non-complementarity determining region (CDR) lattice residues from the parent scFv framework remained unchanged through the processes of protein engineering and rational design, crystal lattices of the derivative scFvs differ. Comparison of energy calculations and the experimentally-determined lattice interactions for this basis set provides insight into the complexity of the forces driving crystal lattice choice and demonstrates the availability of multiple well-ordered surface features in our scFvs capable of forming versatile crystal contacts. PMID:24615866

  18. 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.

  19. 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

  20. Primary malignant melanoma

    PubMed Central

    Mısır, A. Ferhat; Durmuşlar, Mustafa C.; Zerener, Tamer; Gün, Banu D.

    2016-01-01

    Malignant melanomas (MM) of the oral cavity are extremely rare, accounting for 0.2% to 8.0% of all malignant melanomas. Malignant melanomas is more frequently seen at the level of the hard palate and gingiva. Early diagnosis and treatment are important for reducing morbidity. Malignant melanoma cells stain positively with antibodies to human melanoma black 45, S-100 protein, and vimentin; therefore, immunohistochemistry can play an important role in evaluating the depth of invasion and the location of metastases. A 76-year-old man developed an oral malignant melanoma, which was originally diagnosed as a bluish reactive denture hyperplasia caused by an ill-fitting lower denture. The tumor was removed surgically, and histopathological examination revealed a nodular-type MM. There was no evidence of recurrence over a 4-year follow-up period. PMID:27052289

  1. Cell-free eukaryotic systems for the production, engineering, and modification of scFv antibody fragments

    PubMed Central

    Stech, Marlitt; Hust, Michael; Schulze, Corina; Dübel, Stefan; Kubick, Stefan

    2014-01-01

    Open cell-free translation systems based on Escherichia coli cell lysates have successfully been used to produce antibodies and antibody fragments. In this study, we demonstrate the cell-free expression of functional single-chain antibody variable fragments (scFvs) in a eukaryotic and endotoxin-free in vitro translation system based on Spodoptera frugiperda (Sf21) insect cell extracts. Three scFv candidates with different specificities were chosen as models. The first scFv candidate SH527-IIA4 specifically discriminates between its phosphorylated (SMAD2-P) and nonphosphorylated antigens (SMAD2) (where SMAD is mothers against decapentaplegic homolog 2), whereas the second scFv candidate SH527-IIC10 recognizes both, SMAD2-P and SMAD2. The third scFv candidate SH855-C11 binds specifically to a linear epitope of the CXC chemokine receptor type 5. The translocation of antibody fragments into the lumen of endogenous microsomal vesicles, which are contained in the lysate, was facilitated by fusion of scFv genes to the insect cell specific signal sequence of honeybee melittin. We compared the binding capabilities of scFv fragments with and without melittin signal peptide and detected that translocated scFv fragments were highly functional, whereas scFvs synthesized in the cytosol of the cell extract showed strongly decreased binding capabilities. Additionally, we describe a cell-free protein synthesis method for the incorporation of noncanonical amino acids into scFv molecules in eukaryotic cell lysates. We demonstrate the successful cotranslational labeling of de novo synthesized scFv molecules with fluorescent amino acids, using residue-specific as well as site-specific labeling. PMID:25821419

  2. Detailed analysis of immunologic effects of the cytotoxic T lymphocyte-associated antigen 4-blocking monoclonal antibody tremelimumab in peripheral blood of patients with melanoma

    PubMed Central

    Comin-Anduix, Begoña; Lee, Yohan; Jalil, Jason; Algazi, Alain; de la Rocha, Pilar; Camacho, Luis H; Bozon, Viviana A; Bulanhagui, Cecile A; Seja, Elisabeth; Villanueva, Arturo; Straatsma, Bradley R; Gualberto, Antonio; Economou, James S; Glaspy, John A; Gomez-Navarro, Jesus; Ribas, Antoni

    2008-01-01

    Background CTLA4-blocking antibodies induce tumor regression in a subset of patients with melanoma. Analysis of immune parameters in peripheral blood may help define how responses are mediated. Methods Peripheral blood from HLA-A*0201-positive patients with advanced melanoma receiving tremelimumab (formerly CP-675,206) at 10 mg/kg monthly was repeatedly sampled during the first 4 cycles. Samples were analyzed by 1) tetramer and ELISPOT assays for reactivity to CMV, EBV, MART1, gp100, and tyrosinase; 2) activation HLA-DR and memory CD45RO markers on CD4+/CD8+ cells; and 3) real-time quantitative PCR of mRNA for FoxP3 transcription factor, preferentially expressed by T regulatory cells. The primary endpoint was difference in MART1-specific T cells by tetramer assay. Immunological data were explored for significant trends using clustering analysis. Results Three of 12 patients eligible for immune monitoring had tumor regression lasting > 2 years without relapse. There was no significant change in percent of MART1-specific T cells by tetramer assay. Additionally, there was no generalized trend toward postdosing changes in other antigen-specific CD8+ cell populations, FoxP3 transcripts, or overall changes in surface expression of T-cell activation or memory markers. Unsupervised hierarchical clustering based on immune monitoring data segregated patients randomly. However, clustering according to T-cell activation or memory markers separated patients with clinical response and most patients with inflammatory toxicity into a common subgroup. Conclusion Administration of CTLA4-blocking antibody tremelimumab to patients with advanced melanoma results in a subset of patients with long-lived tumor responses. T-cell activation and memory markers served as the only readout of the pharmacodynamic effects of this antibody in peripheral blood. Clinical trial registration number NCT00086489 PMID:18452610

  3. Generation and characterization of a human single-chain fragment variable (scFv) antibody against cytosine deaminase from Yeast

    PubMed Central

    Mallano, Alessandra; Zamboni, Silvia; Carpinelli, Giulia; Santoro, Filippo; Flego, Michela; Ascione, Alessandro; Gellini, Mara; Tombesi, Marina; Podo, Franca; Cianfriglia, Maurizio

    2008-01-01

    Background The ability of cytosine deaminase (CD) to convert the antifungal agent 5-fluorocytosine (5-FC) into one of the most potent and largely used anticancer compound such as 5-fluorouracil (5-FU) raised considerable interest in this enzyme to model gene or antibody – directed enzyme-prodrug therapy (GDEPT/ADEPT) aiming to improve the therapeutic ratio (benefit versus toxic side-effects) of cancer chemotherapy. The selection and characterization of a human monoclonal antibody in single chain fragment (scFv) format represents a powerful reagent to allow in in vitro and in vivo detection of CD expression in GDEPT/ADEPT studies. Results An enzymatic active recombinant CD from yeast (yCD) was expressed in E. coli system and used as antigen for biopanning approach of the large semi-synthetic ETH-2 antibody phage library. Several scFvs were isolated and specificity towards yCD was confirmed by Western blot and ELISA. Further, biochemical and functional investigations demonstrated that the binding of specific scFv with yCD did not interfere with the activity of the enzyme in converting 5-FC into 5-FU. Conclusion The construction of libraries of recombinant antibody fragments that are displayed on the surface of filamentous phage, and the selection of phage antibodies against target antigens, have become an important biotechnological tool in generating new monoclonal antibodies for research and clinical applications. The scFvH5 generated by this method is the first human antibody which is able to detect yCD in routinary laboratory techniques without interfering with its enzymatic function. PMID:18783590

  4. Functional Production and Characterization of a Fibrin-Specific Single-Chain Antibody Fragment from Bacillus subtilis: Effects of Molecular Chaperones and a Wall-Bound Protease on Antibody Fragment Production

    PubMed Central

    Wu, Sau-Ching; Yeung, Jonathan C.; Duan, Yanjun; Ye, Ruiqiong; Szarka, Steven J.; Habibi, Hamid R.; Wong, Sui-Lam

    2002-01-01

    To develop an ideal blood clot imaging and targeting agent, a single-chain antibody (SCA) fragment based on a fibrin-specific monoclonal antibody, MH-1, was constructed and produced via secretion from Bacillus subtilis. Through a systematic study involving a series of B. subtilis strains, insufficient intracellular and extracytoplasmic molecular chaperones and high sensitivity to wall-bound protease (WprA) were believed to be the major factors that lead to poor production of MH-1 SCA. Intracellular and extracytoplasmic molecular chaperones apparently act in a sequential manner. The combination of enhanced coproduction of both molecular chaperones and wprA inactivation leads to the development of an engineered B. subtilis strain, WB800HM[pEPP]. This strain allows secretory production of MH-1 SCA at a level of 10 to 15 mg/liter. In contrast, with WB700N (a seven-extracellular-protease-deficient strain) as the host, no MH-1 SCA could be detected in both secreted and cellular fractions. Secreted MH-1 SCA from WB800HM[pMH1, pEPP] could be affinity purified using a protein L matrix. It retains comparable affinity and specificity as the parental MH-1 monoclonal antibody. This expression system can potentially be applied to produce other single-chain antibody fragments, especially those with folding and protease sensitivity problems. PMID:12089002

  5. Obstruction of dengue virus maturation by Fab fragments of the 2H2 antibody.

    PubMed

    Wang, Zhiqing; Li, Long; Pennington, Janice G; Sheng, Ju; Yap, Moh Lan; Plevka, Pavel; Meng, Geng; Sun, Lei; Jiang, Wen; Rossmann, Michael G

    2013-08-01

    The 2H2 monoclonal antibody recognizes the precursor peptide on immature dengue virus and might therefore be a useful tool for investigating the conformational change that occurs when the immature virus enters an acidic environment. During dengue virus maturation, spiky, immature, noninfectious virions change their structure to form smooth-surfaced particles in the slightly acidic environment of the trans-Golgi network, thereby allowing cellular furin to cleave the precursor-membrane proteins. The dengue virions become fully infectious when they release the cleaved precursor peptide upon reaching the neutral-pH environment of the extracellular space. Here we report on the cryo-electron microscopy structures of the immature virus complexed with the 2H2 antigen binding fragments (Fab) at different concentrations and under various pH conditions. At neutral pH and a high concentration of Fab molecules, three Fab molecules bind to three precursor-membrane proteins on each spike of the immature virus. However, at a low concentration of Fab molecules and pH 7.0, only two Fab molecules bind to each spike. Changing to a slightly acidic pH caused no detectable change of structure for the sample with a high Fab concentration but caused severe structural damage to the low-concentration sample. Therefore, the 2H2 Fab inhibits the maturation process of immature dengue virus when Fab molecules are present at a high concentration, because the three Fab molecules on each spike hold the precursor-membrane molecules together, thereby inhibiting the normal conformational change that occurs during maturation.

  6. Redistribution of flexibility in stabilizing antibody fragment mutants follows Le Châtelier's principle.

    PubMed

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

    2014-01-01

    Le Châtelier's principle is the cornerstone of our understanding of chemical equilibria. When a system at equilibrium undergoes a change in concentration or thermodynamic state (i.e., temperature, pressure, etc.), La Châtelier's principle states that an equilibrium shift will occur to offset the perturbation and a new equilibrium is established. We demonstrate that the effects of stabilizing mutations on the rigidity ⇔ flexibility equilibrium within the native state ensemble manifest themselves through enthalpy-entropy compensation as the protein structure adjusts to restore the global balance between the two. Specifically, we characterize the effects of mutation to single chain fragments of the anti-lymphotoxin-β receptor antibody using a computational Distance Constraint Model. Statistically significant changes in the distribution of both rigidity and flexibility within the molecular structure is typically observed, where the local perturbations often lead to distal shifts in flexibility and rigidity profiles. Nevertheless, the net gain or loss in flexibility of individual mutants can be skewed. Despite all mutants being exclusively stabilizing in this dataset, increased flexibility is slightly more common than increased rigidity. Mechanistically the redistribution of flexibility is largely controlled by changes in the H-bond network. For example, a stabilizing mutation can induce an increase in rigidity locally due to the formation of new H-bonds, and simultaneously break H-bonds elsewhere leading to increased flexibility distant from the mutation site via Le Châtelier. Increased flexibility within the VH β4/β5 loop is a noteworthy illustration of this long-range effect.

  7. Redistribution of flexibility in stabilizing antibody fragment mutants follows Le Châtelier's principle.

    PubMed

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

    2014-01-01

    Le Châtelier's principle is the cornerstone of our understanding of chemical equilibria. When a system at equilibrium undergoes a change in concentration or thermodynamic state (i.e., temperature, pressure, etc.), La Châtelier's principle states that an equilibrium shift will occur to offset the perturbation and a new equilibrium is established. We demonstrate that the effects of stabilizing mutations on the rigidity ⇔ flexibility equilibrium within the native state ensemble manifest themselves through enthalpy-entropy compensation as the protein structure adjusts to restore the global balance between the two. Specifically, we characterize the effects of mutation to single chain fragments of the anti-lymphotoxin-β receptor antibody using a computational Distance Constraint Model. Statistically significant changes in the distribution of both rigidity and flexibility within the molecular structure is typically observed, where the local perturbations often lead to distal shifts in flexibility and rigidity profiles. Nevertheless, the net gain or loss in flexibility of individual mutants can be skewed. Despite all mutants being exclusively stabilizing in this dataset, increased flexibility is slightly more common than increased rigidity. Mechanistically the redistribution of flexibility is largely controlled by changes in the H-bond network. For example, a stabilizing mutation can induce an increase in rigidity locally due to the formation of new H-bonds, and simultaneously break H-bonds elsewhere leading to increased flexibility distant from the mutation site via Le Châtelier. Increased flexibility within the VH β4/β5 loop is a noteworthy illustration of this long-range effect. PMID:24671209

  8. Redistribution of Flexibility in Stabilizing Antibody Fragment Mutants Follows Le Châtelier’s Principle

    PubMed Central

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

    2014-01-01

    Le Châtelier’s principle is the cornerstone of our understanding of chemical equilibria. When a system at equilibrium undergoes a change in concentration or thermodynamic state (i.e., temperature, pressure, etc.), La Châtelier’s principle states that an equilibrium shift will occur to offset the perturbation and a new equilibrium is established. We demonstrate that the effects of stabilizing mutations on the rigidity ⇔ flexibility equilibrium within the native state ensemble manifest themselves through enthalpy-entropy compensation as the protein structure adjusts to restore the global balance between the two. Specifically, we characterize the effects of mutation to single chain fragments of the anti-lymphotoxin-β receptor antibody using a computational Distance Constraint Model. Statistically significant changes in the distribution of both rigidity and flexibility within the molecular structure is typically observed, where the local perturbations often lead to distal shifts in flexibility and rigidity profiles. Nevertheless, the net gain or loss in flexibility of individual mutants can be skewed. Despite all mutants being exclusively stabilizing in this dataset, increased flexibility is slightly more common than increased rigidity. Mechanistically the redistribution of flexibility is largely controlled by changes in the H-bond network. For example, a stabilizing mutation can induce an increase in rigidity locally due to the formation of new H-bonds, and simultaneously break H-bonds elsewhere leading to increased flexibility distant from the mutation site via Le Châtelier. Increased flexibility within the VH β4/β5 loop is a noteworthy illustration of this long-range effect. PMID:24671209

  9. 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.

  10. Phase I trial of the chimeric anti-GD2 monoclonal antibody ch14.18 in patients with malignant melanoma.

    PubMed

    Saleh, M N; Khazaeli, M B; Wheeler, R H; Allen, L; Tilden, A B; Grizzle, W; Reisfeld, R A; Yu, A L; Gillies, S D; LoBuglio, A F

    1992-01-01

    The chimeric monoclonal anti-GD2 antibody ch14.18 is made up of the variable region of the murine anti-GD2 antibody 14.18 (or its IgG2a switch variant 14G2a) and the constant region of human IgG1k. Ch14.18 mediates antibody dependent cytotoxicity and complement dependent lysis in vitro. In a phase I trial, 13 patients with metastatic melanoma received ch14.18 as a single dose of 5-100 mg. Therapy was associated with an infusion-related abdominal/pelvic pain syndrome, which required intravenous morphine for control. The pharmacokinetics of ch14.18 best fit a two-compartment model with a T1/2 alpha of 24 +/- 1 hr and a T1/2 beta of 181 +/- 73 hr. Eight of 13 patients developed a weak-modest antibody response directed at the variable region of ch14.18. Clinical antitumor responses were not observed at the doses employed in this study. However, patients receiving greater than 45 mg of ch14.18 had antibody detectable on tumor cells analyzed by fluorescent activated cell sorter. Further modification of the therapeutic regime employing larger doses and frequent administration of ch14.18 are planned. PMID:1576319

  11. The cytotoxicity and microdosimetry of astatine-211-labeled chimeric monoclonal antibodies in human glioma and melanoma cells in vitro.

    PubMed

    Larsen, R H; Akabani, G; Welsh, P; Zalutsky, M R

    1998-02-01

    The cytotoxicity of alpha-particle-emitting endoradiotherapeutic compounds is of increasing interest because clinical evaluation of these potential therapeutic agents is commencing. Astatine-211 is a radionuclide with a 7.2-h half-life that emits 5.87 and 7.45 MeV alpha particles. In the present work, we have investigated the in vitro cytotoxicity of 211At-labeled chimeric monoclonal antibodies (mAbs) in monolayers of D-247 MG human glioma cells and SK-MEL-28 human melanoma cells. The mAbs studied were 81C6, reactive with the extracellular matrix antigen tenascin, Mel-14, directed against the cell membrane antigen proteoglycan chondroitin sulfate, and a nonspecific control mAb, TPS3.2. Cell uptake increased as a function of activity concentration after a 1-h exposure to the 211At-labeled mAbs. The retention of activity was also measured to calculate cumulative activity associated with the cells and the medium. The clonogenic survival as a function of activity concentration was linear in all cases with no detectable shoulder. Microdosimetric analyses were performed based on measured cell geometry, cumulative activity and Monte Carlo transport of alpha particles. Using 18 kBq/ml activity concentration and 1 h of incubation, a two to five times higher activity bound to the microcolonies was found for the specific mAbs compared to the nonspecific mAb. These calculations indicated that a survival fraction of 0.37 was achieved with 0.24-0.28 Gy for D-247 MG cells and 0.27-0.29 Gy for SK-MEL-28 cells. The microdosimetric cell sensitivity, z0, for D-247 MG cells was significantly lower than for SK-MEL-28 cells (0.08 compared to 0.15 Gy). For both cell lines, reduction in survival to 0.37 required an average of only 1-2 alpha-particle hits to the cell nucleus.

  12. 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

  13. Construction and characterization of DNA vaccines encoding the single-chain variable fragment of the anti-idiotype antibody 1A7 mimicking the tumor-associated antigen disialoganglioside GD2.

    PubMed

    Zeytin, H E; Tripathi, P K; Bhattacharya-Chatterjee, M; Foon, K A; Chatterjee, S K

    2000-11-01

    Anti-idiotype antibody, 1A7, functionally mimics the tumor-associated antigen disialoganglioside GD2, which is overexpressed on the surface of a number of neuroectodermal tumors such as melanoma, neuroblastoma, soft tissue sarcoma, and small cell carcinoma of the lung. Immunization of mice with 1A7 generated the production of anti-GD2 antibodies. In a phase I clinical trial, immunization of patients with 1A7, mixed with the adjuvant QS21, demonstrated that 1A7 could act as a surrogate antigen for GD2 and induce strong humoral immune responses in advanced stage melanoma patients. DNA vaccines have recently been shown to invoke humoral as well as cellular responses in injected hosts against the transgene product. To evaluate the efficiency of DNA vaccines encoding anti-idiotype antibodies, we constructed expression plasmids encoding the variable heavy (VH) and variable light (VL) chains of 1A7. The plasmids were made in two configurations, expressing either the VH (pc1A7VHLnVL) or the VL (pc1A7VLLnVH) chain of 1A7 at the amino terminus, linked together by a 15-amino acid linker (Ln). In vitro transcription/translation assays and transfection of CHO-K1 cells with the plasmids demonstrated that a approximately 30-kDa protein was expressed by both configurations of the single-chain variable fragment. This protein can be specifically precipitated by monoclonal anti-GD2 antibody, 14G2a. Following intramuscular injection in mice, the plasmids were detectable in the injected tissues for at least 3 months and the injected plasmids actively transcribed the single-chain variable fragment 1A7 gene at the injected site. A single, intramuscular immunization of a group of C57BL/6 mice with pc1A7VLLnVH in phosphate-buffered saline induced humoral immune responses against 1A7 as well as GD2, the nominal antigen. Multiple immunizations, however, were required to elicit stronger immune responses. PMID:11129285

  14. 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.

  15. 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

  16. Peptide linkers lead to modification of liver metabolism and improved tumor targeting of copper-67-labeled antibody fragments.

    PubMed

    Novak-Hofer, I; Zimmermann, K; Schubiger, P A

    2001-12-01

    In order to determine if tumor/nontarget tissue ratios of 67Cu-labeled antibody fragments can be improved, modifying the DO3A copper chelate with tripeptide linkers was investigated. The peptide-linked chelates 1,4,7,10-tetraazacyclodecane-N,N',N",N"'-tetraacetate (DOTA)-triglycyl-L-p-isothiocyanato-phenylalanine (DOTA-R1-NCS), DOTA-glycyl-phenylalanyl-glycyl-L-p-isothiocyanato-phenylalanine (DOTA-R2-NCS), DOTA-glycyl-prolyl-glycyl-L-p-isothiocyanato-phenylalanine (DOTA-R3-NCS) and DOTA-glycyl-L-p-isothiocyanato-phenylalanine (DOTA-R4-NCS) were synthesized and coupled to F(ab')2 fragments of anti-colon carcinoma mAb35. In vitro, the 67Cu-labeled antibody fragments were fully immunoreactive and stable in human serum. In vivo in nude mice bearing human colon carcinoma xenografts the conjugates R1 and R3 showed improved tumor uptake and lower levels of radioactivity in the liver compared with the other conjugates. Biodistributions of the DOTA-R2-F(ab')2 showed at early time points after injection higher levels of radioactivity in the liver, lower levels of activity persisting in the blood and lower accumulation of activity in the tumor. When liver homogenates were analyzed 30 min post injection by SDS-PAGE or FPLC gel chromatography, it was found that radioactivity was released more slowly from the triglycine (R1)-F(ab')2 than from the immunoconjugates with the R2 or the R4 linker. The main radioactive metabolites were protein bands at 66 kD, 31 kD and low molecular weight fragments. The results show that the rate of cleavage of the copper complex from F(ab')2 fragments in vivo can be influenced by the amino acid sequence close to the complex, with significant consequences on biodistributions.

  17. High-dose intravenous immunoglobulin therapy for rapidly progressive interstitial pneumonitis accompanied by anti-melanoma differentiation-associated gene 5 antibody-positive amyopathic dermatomyositis

    PubMed Central

    Hamada-Ode, Kazu; Taniguchi, Yoshinori; Kimata, Takahito; Kawaguchi, Yasushi; Shimamura, Yoshiko; Kuwana, Masataka; Fujimoto, Shimpei; Terada, Yoshio

    2015-01-01

    Anti-melanoma differentiation-associated gene 5 (MDA5) antibody-positive amyopathic dermatomyositis (ADM) associated with rapidly progressive interstitial pneumonitis (RPIP) frequently has a poor prognosis and optimal treatment is not well defined. Here, we report a 62-year-old Japanese man with anti-MDA5 antibody-positive ADM associated with RPIP presented with progressive shortness of breath, Heliotrope rash, Gottron’s papules, arthralgia, and fatigue but no sign of muscle weakness. Laboratory investigation revealed serum levels of the following biomarkers: ferritin, 1393 ng/mL; Krebs von der Lungen-6, 1880 U/mL; and creatine kinase, 85 U/L. Computed tomography (CT) images showed diffuse ground-glass opacity in both lung fields. Because anti-MDA5 was positive, we made a diagnosis of ADM associated with RPIP and initiated treatment. Following five courses of combination therapy with prednisolone, cyclosporine A, and intravenous cyclophosphamide (IVCY), IVCY treatment was switched to high-dose intravenous immunoglobulin therapy (IVIg) because of the reactivation of interstitial pneumonia with an increased serum ferritin level. Additional treatment with IVIg improved RPIP, with normalization of anti-ADM antibody levels. Therefore, IVIg mayt be a new candidate treatment for anti-MDA5 antibody-positive ADM associated with RPIP. PMID:27708934

  18. 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

  19. 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...

  20. 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

  1. 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

  2. Preclinical evaluation of 67Cu-labeled intact and fragmented anti-colon carcinoma monoclonal antibody MAb35.

    PubMed

    Smith, A; Alberto, R; Blaeuenstein, P; Novak-Hofer, I; Maecke, H R; Schubiger, P A

    1993-12-01

    The anti-carcinoembryonic antigen murine monoclonal antibody MAb35 and its F(ab')2 fragment were labeled with 131I or the potential therapeutic nuclide 67Cu. In vivo distribution patterns were compared in nude mice bearing human tumor xenografts by coinjection of the 131I- and 67Cu-labeled materials, thereby minimizing variations due to xenograft and host animal. The results showed that the 67Cu-labeled intact MAb35 achieved twice the percentage of injected dose/g tumor when compared to its 131I-labeled counterpart, without significant impairment of the wholebody distribution pattern. However, this effect was not evident in the case of F(ab')2, where high uptake of 67Cu was found in the kidney without any enhancement of accumulation in the target xenografts. To investigate the underlying causes of the different distribution patterns observed, iodine labeling was also performed using a more stable linkage, and the results indicated that the observed differences cannot be explained by simple deiodination of conventionally labeled preparations. We conclude that the intact form of the 67Cu-labeled antibody may be superior to the F(ab')2 fragment for use in our intended clinical studies. Our continuing work on the processing of radiometal-labeled F(ab')2 fragments, at the systemic and cellular level, will hopefully lead to a strategy to circumvent the problem of high kidney accumulation.

  3. [Issues around melanoma].

    PubMed

    Haanen, John

    2011-01-01

    Over the past 30 years little progress has been made in the treatment of patients with a metastatic melanoma. Recently there have been two new developments. One of these is ipilimumab, a monoclonal antibody that blocks the function of the protein cytotoxic T lymphocyte-associated antigen 4 (CTLA4) which inhibits activated T lymphocytes. This gives the immune system a chance to build up an immune response to the melanoma. The other development is vemurafenib, a small molecule that inhibits a mutated protein (BRAF) that occurs in many melanomas. The BRAF mutation leads to uninhibited proliferation.

  4. Biologic Effects of Anti-Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) Antibody Formation in Patients Treated with GM-CSF (Sargramostim) as Adjuvant Therapy of Melanoma

    PubMed Central

    Spitler, Lynn E.; Cao, Huynh; Piironen, Timo; Whiteside, Theresa L.; Weber, Robert W.; Cruickshank, Scott

    2014-01-01

    OBJECTIVES We investigated the development of binding and neutralizing antibodies to GM-CSF in patients receiving prolonged therapy with GM-CSF as adjuvant therapy of melanoma and the impact of these antibodies on biologic effects. METHODS Fifty-three patients with high-risk melanoma which had been surgically excised were treated with GM-CSF, 125 µg/m2 daily for 14 days every 28 days for 1 year following surgical resection of disease. Serum samples for antibodies to GM-CSF were measured before treatment and on Study Days 155 and 351. Blood draws for testing biologic effects were keyed to GM-CSF administration: Days 0 (before), 15 (after 14 days on GM-CSF), 29 (after 14 days off GM-CSF), 155, and 351 (after 14 days on GM-CSF in the 6th and 13th cycle of treatment). RESULTS Of 53 patients enrolled, 43 were evaluable for the development of anti-GM-CSF antibodies. Of these, 93% developed binding antibodies and 42% developed both binding and neutralizing antibodies. The increase in the white blood cell (WBC) count, percent eosinophils, or neopterin levels engendered by GM-CSF administration, was abrogated or markedly decreased in patients with neutralizing antibodies but not in patients who developed only binding antibodies. CONCLUSIONS Ninety-three percent of patients with melanoma treated with GM-CSF as adjuvant therapy develop antibodies to GM-CSF. In those with neutralizing antibodies, a diminution of the biologic effects of GM-CSF was observed. The development of neutralizing antibodies might also abrogate the potential clinical benefit of this treatment and should be considered in the design of future clinical trials. PMID:25286079

  5. Nephritogenic Lupus Antibodies Recognize Glomerular Basement Membrane-Associated Chromatin Fragments Released from Apoptotic Intraglomerular Cells

    PubMed Central

    Kalaaji, Manar; Mortensen, Elin; Jørgensen, Leif; Olsen, Randi; Rekvig, Ole Petter

    2006-01-01

    Antibodies to dsDNA represent a classification criterion for systemic lupus erythematosus. Subpopulations of these antibodies are involved in lupus nephritis. No known marker separates nephritogenic from non-nephritogenic anti-dsDNA antibodies. It is not clear whether specificity for glomerular target antigens or intrinsic antibody-affinity for dsDNA or nucleosomes is a critical parameter. Furthermore, it is still controversial whether glomerular target antigen(s) is constituted by nucleosomes or by non-nucleosomal glomerular structures. Previously, we have demonstrated that antibodies eluted from murine nephritic kidneys recognize nucleosomes, but not other glomerular antigens. In this study, we determined the structures that bind nephritogenic autoantibodies in vivo by transmission electron microscopy, immune electron microscopy, and colocalization immune electron microscopy using experimental antibodies to dsDNA, to histones and transcription factors, or to laminin. The data obtained are consistent and point at glomerular basement membrane-associated nucleosomes as target structures for the nephritogenic autoantibodies. Terminal deoxynucleotidyl-transferase-mediated dUTP nick end-labeling or caspase-3 assays demonstrate that lupus nephritis is linked to intraglomerular cell apoptosis. The data suggest that nucleosomes are released by apoptosis and associate with glomerulus basement membranes, which may then be targeted by pathogenic anti-nucleosome antibodies. Thus, apoptotic nucleosomes may represent both inducer and target structures for nephritogenic autoantibodies in systemic lupus erythematosus. PMID:16723695

  6. Nephritogenic lupus antibodies recognize glomerular basement membrane-associated chromatin fragments released from apoptotic intraglomerular cells.

    PubMed

    Kalaaji, Manar; Mortensen, Elin; Jørgensen, Leif; Olsen, Randi; Rekvig, Ole Petter

    2006-06-01

    Antibodies to dsDNA represent a classification criterion for systemic lupus erythematosus. Subpopulations of these antibodies are involved in lupus nephritis. No known marker separates nephritogenic from non-nephritogenic anti-dsDNA antibodies. It is not clear whether specificity for glomerular target antigens or intrinsic antibody-affinity for dsDNA or nucleosomes is a critical parameter. Furthermore, it is still controversial whether glomerular target antigen(s) is constituted by nucleosomes or by non-nucleosomal glomerular structures. Previously, we have demonstrated that antibodies eluted from murine nephritic kidneys recognize nucleosomes, but not other glomerular antigens. In this study, we determined the structures that bind nephritogenic autoantibodies in vivo by transmission electron microscopy, immune electron microscopy, and colocalization immune electron microscopy using experimental antibodies to dsDNA, to histones and transcription factors, or to laminin. The data obtained are consistent and point at glomerular basement membrane-associated nucleosomes as target structures for the nephritogenic autoantibodies. Terminal deoxynucleotidyl-transferase-mediated dUTP nick end-labeling or caspase-3 assays demonstrate that lupus nephritis is linked to intraglomerular cell apoptosis. The data suggest that nucleosomes are released by apoptosis and associate with glomerulus basement membranes, which may then be targeted by pathogenic anti-nucleosome antibodies. Thus, apoptotic nucleosomes may represent both inducer and target structures for nephritogenic autoantibodies in systemic lupus erythematosus.

  7. 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

  8. 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

  9. Anti-proliferative and pro-apoptotic activity of GD2 ganglioside-specific monoclonal antibody 3F8 in human melanoma cells

    PubMed Central

    Tsao, Chun-Yen; Sabbatino, Francesco; Cheung, Nai-Kong V; Hsu, Jeff Chi-feng; Villani, Vincenzo; Wang, Xinhui; Ferrone, Soldano

    2015-01-01

    The beneficial clinical effects of immunotherapy with GD2-specific monoclonal antibodies (mAbs) in melanoma and neuroblastoma patients have stimulated interest in characterizing the mechanisms underlying their antitumor effects. Previous studies have shown that GD2-specific mAbs mediate complement- and cell-dependent cytotoxicity and induce caspase-dependent apoptosis of tumor cells. In this study, we showed that GD2-specific mAb 3F8, which is undergoing clinical evaluation, inhibited the in vitro growth and induced apoptosis of melanoma cells. This effect was dose- and time-dependent, mediated by the interaction of mAb 3F8 combining site with GD2 ganglioside, associated with GD2 expression level on the cell surface, mAb internalization and increase of GD2 containing endosomes triggered by mAb 3F8. The induction of apoptosis by mAb 3F8 was mediated by caspase 3-, 7-, and 8-dependent pathways, downregulation of the anti-apoptotic molecules survivin and cytochrome c, and caspase 9 independent-AIF release from mitochondria. In addition, analyses of signaling pathway components demonstrated that mAb 3F8 strongly inhibited AKT and FAK activation and increased cleaved PARP expression. These results indicated that multiple mechanisms played a role in the antitumor activity of mAb 3F8 in melanoma cells. This information should provide a mechanistic basis for the optimization of the rational design of immunotherapeutic strategies in the mAb-based treatment of GD2 positive tumors. PMID:26405581

  10. Recombinant single-chain Fv antibody fragment-alkaline phosphatase conjugate for one-step immunodetection in molecular hybridization.

    PubMed

    Muller, B H; Chevrier, D; Boulain, J C; Guesdon, J L

    1999-07-30

    Using phage-display technology, a recombinant single-chain Fv antibody fragment (scFv) was rapidly generated from the K16-16 hybridoma secreting mouse monoclonal antibody (MAb) that binds to acetylaminofluorene-labeled DNA (AAF-DNA). The selected A4 phage-scFv specifically bound to AAF-DNA. The anti-AAF scFv gene was then recloned into a fusion vector for the production of a hybrid protein comprising the antibody fragment fused to a potent bacterial alkaline phosphatase variant (PhoAv). The anti-AAF scFv-PhoAv hybrid protein was bifunctional and possessed both antigen binding capacity and PhoA activity. The recombinant conjugate was directly used, without further purification, for one-step immunodetection in dot-blot hybridization. The detection limit was identical and the test was quicker than the conventional two-step procedure with the purified anti-AAF MAb revealed with a secondary enzyme-labeled antibody. To assess the value of this new reagent for the immunodetection of genomic nucleic acids, genomic DNAs of Campylobacter jejuni and Campylobacter coli were then one-step immunodetected with non-purified recombinant scFv-PhoAv conjugate in a Southern-blot hybridization experiment. The present study shows that the genetic fusion with PhoAv provides a new tool for immunodetection which presents easier and quicker production and use with the same sensitivity and specificity as classical reagents. The recombinant anti-AAF scFv-PhoAv conjugate is a promising alternative reagent for applications involving the immunodetection of specific DNA or RNA sequences, such as the detection and characterization of microorganisms.

  11. Activation of cellular cytotoxicity and complement-mediated lysis of melanoma and neuroblastoma cells in vitro by murine antiganglioside antibodies MB 3.6 and 14.G2a.

    PubMed

    Mayer, P; Handgretinger, R; Bruchelt, G; Schaber, B; Rassner, G; Fierlbeck, G

    1994-04-01

    Mouse monoclonal antibodies against tumour-associated gangliosides GD2 (14.G2a) and GD3 (MB 3.6) were tested to mediate antibody-dependent cellular cytotoxicity (ADCC) with various effector cells or complement-dependent cytolysis (CDC). We also evaluated the immunomodulating potential of interferons in combination with cellular cytotoxicity. Using effector:target (E/T) ratios of 40:1, ADCC with effector cells such as granulocytes or mononuclear blood cells was not detectable against melanoma cell lines GR, SK-MEL-28 and G-361 which preferentially express GD3 and bind antibody MB 3.6. Neuroblastoma cell line SK-N-LO, which was used for comparative purposes, mainly expressed GD2 and the tumour cells were killed effectively after labelling with antibody 14.G2a. Granulocytes did not show significant killing of melanoma cells by ADCC, but neuroblastoma cells were killed very efficiently. Peripheral blood mononuclear cells (PBMC) also failed to kill melanoma cells. Interferon-beta slightly stimulated PBMC and increased killing of neuroblastoma cells, but no additive effects with ADCC were detectable. Incubation of target cells with interferons produced no significant differences in susceptibility of the target cells to interferon-activated PBMC cytotoxicity. Despite the lack of effectiveness in mediating cellular cytotoxicity, GD3 antibody MB 3.6 showed strong complement-dependent cytolysis in the presence of human plasma. There were remarkable differences in individual activity and different susceptibility of the melanoma cell lines. We assume that CDC may have more activity against melanoma cells than cytotoxicity associated with various effector cells.

  12. 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

  13. Comparison of 2 monoclonal antibodies for immunohistochemical detection of BRAF V600E mutation in malignant melanoma, pulmonary carcinoma, gastrointestinal carcinoma, thyroid carcinoma, and gliomas.

    PubMed

    Routhier, Caitlin Ann; Mochel, Mark C; Lynch, Kerry; Dias-Santagata, Dora; Louis, David N; Hoang, Mai P

    2013-11-01

    BRAF mutation is seen in a variety of human neoplasms including cutaneous malignant melanoma, papillary thyroid carcinoma, colorectal carcinoma, non-small cell lung carcinoma, pleomorphic xanthoastrocytoma, and others. Currently, there are 2 commercially available monoclonal antibodies for the detection of BRAF V600E mutation; however, a full and practical comparison of their performance in various tumor types on an automated staining platform has not been done. We investigated their sensitivity and specificity in detecting the BRAF V600E mutation in a series of 152 tumors including 31 malignant melanomas, 25 lung carcinomas, 32 gastrointestinal carcinomas, 23 thyroid carcinomas, 35 gliomas, and 6 other malignancies. In this series, the concordance rate between immunohistochemistry (IHC) and mutational analyses was 97% (148/152) for VE1 and 88% (131/149) for anti-B-Raf. The sensitivity and specificity were 98% (60/61) and 97% (88/91) for monoclonal VE1 and 95% (58/61) and 83% (73/88) for anti-B-Raf, respectively. There were 4 cases with discordant IHC and mutational results for monoclonal VE1 in contrast to 18 cases for anti-B-Raf. Our studies showed that IHC with monoclonal VE1 has a better performance compared with anti-B-Raf in an automated staining platform and confirmed that clone VE1 provides excellent sensitivity and specificity for detecting the BRAF V600E mutation in a variety of tumor types in a clinical setting.

  14. Studies of nontarget-mediated distribution of human full-length IgG1 antibody and its FAb fragment in cardiovascular and metabolic-related tissues.

    PubMed

    Davidsson, Pia; Söderling, Ann-Sofi; Svensson, Lena; Ahnmark, Andrea; Flodin, Christine; Wanag, Ewa; Screpanti-Sundqvist, Valentina; Gennemark, Peter

    2015-05-01

    Tissue distribution and pharmacokinetics (PK) of full-length nontargeted antibody and its antigen-binding fragment (FAb) were evaluated for a range of tissues primarily of interest for cardiovascular and metabolic diseases. Mice were intravenously injected with a dose of 10 mg/kg of either human IgG1or its FAb fragment; perfused tissues were collected at a range of time points over 3 weeks for the human IgG1 antibody and 1 week for the human FAb antibody. Tissues were homogenized and antibody concentrations were measured by specific immunoassays on the Gyros system. Exposure in terms of maximum concentration (Cmax ) and area under the curve was assessed for all nine tissues. Tissue exposure of full-length antibody relative to plasma exposure was found to be between 1% and 10%, except for brain (0.2%). Relative concentrations of FAb antibody were the same, except for kidney tissue, where the antibody concentration was found to be ten times higher than in plasma. However, the absolute tissue uptake of full-length IgG was significantly higher than the absolute tissue uptake of the FAb antibody. This study provides a reference PK state for full-length whole and FAb antibodies in tissues related to cardiovascular and metabolic diseases that do not include antigen or antibody binding.

  15. Enhancement of toxin- and virus-neutralizing capacity of single-domain antibody fragments by N-glycosylation.

    PubMed

    Harmsen, M M; van Solt, C B; Fijten, H P D

    2009-10-01

    Single-domain antibody fragments (VHHs) have several beneficial properties as compared to conventional antibody fragments. However, their small size complicates their toxin- and virus-neutralizing capacity. We isolated 27 VHHs binding Escherichia coli heat-labile toxin and expressed these in Saccharomyces cerevisiae. The most potent neutralizing VHH (LT109) was N-glycosylated, resulting in a large increase in molecular mass. This suggests that N-glycosylation of LT109 improves its neutralizing capacity. Indeed, deglycosylation of LT109 decreased its neutralizing capacity three- to fivefold. We also studied the effect of glycosylation of two previously isolated VHHs on their ability to neutralize foot-and-mouth disease virus. For this purpose, these VHHs that lacked potential N-glycosylation sites were genetically fused to another VHH that was known to be glycosylated. The resulting fusion proteins were also N-glycosylated. They neutralized the virus at at least fourfold-lower VHH concentrations as compared to the single, non-glycosylated VHHs and at at least 50-fold-lower VHH concentrations as compared to their deglycosylated counterparts. Thus, we have shown that N-glycosylation of VHHs contributes to toxin- and virus-neutralizing capacity.

  16. 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.

  17. Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity.

    PubMed

    Boder, E T; Midelfort, K S; Wittrup, K D

    2000-09-26

    Single-chain antibody mutants have been evolved in vitro with antigen-binding equilibrium dissociation constant K(d) = 48 fM and slower dissociation kinetics (half-time > 5 days) than those for the streptavidin-biotin complex. These mutants possess the highest monovalent ligand-binding affinity yet reported for an engineered protein by over two orders of magnitude. Optimal kinetic screening of randomly mutagenized libraries of 10(5)-10(7) yeast surface-displayed antibodies enabled a >1,000-fold decrease in the rate of dissociation after four cycles of affinity mutagenesis and screening. The consensus mutations are generally nonconservative by comparison with naturally occurring mouse Fv sequences and with residues that do not contact the fluorescein antigen in the wild-type complex. The existence of these mutants demonstrates that the antibody Fv architecture is not intrinsically responsible for an antigen-binding affinity ceiling during in vivo affinity maturation.

  18. 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

  19. Single chain variable fragment antibodies block aggregation and toxicity induced by familial ALS-linked mutant forms of SOD1

    PubMed Central

    Ghadge, Ghanashyam D.; Pavlovic, John; Koduvayur, Sujatha P.; Kay, Brian K.; Roos, Raymond P.

    2013-01-01

    Approximately 10% of amyotrophic lateral sclerosis (ALS) cases are familial (known as FALS) with an autosomal dominant inheritance pattern, and ~25% of FALS cases are caused by mutations in Cu/Zn superoxide dismutase (SOD1). There is convincing evidence that mutant SOD1 (mtSOD1) kills motor neurons (MNs) because of a gain-of-function toxicity, most likely related to aggregation of mtSOD1. A number of recent reports have suggested that antibodies can be used to treat mtSOD1-induced FALS. To follow up on the use of antibodies as potential therapeutics, we generated single chain fragments of variable region antibodies (scFvs) against SOD1, and then expressed them as ‘intrabodies’ within a motor neuron cell line. In the present study, we describe isolation of human scFvs that interfere with mtSOD1 in vitro aggregation and toxicity. These scFvs may have therapeutic potential in sporadic ALS, as well as FALS, given that sporadic ALS may also involve abnormalities in the SOD1 protein or activity. PMID:23607939

  20. Feasibility study of the Fab fragment of a monoclonal antibody against tissue factor as a diagnostic tool.

    PubMed

    Tsumura, Ryo; Sato, Ryuta; Furuya, Fumiaki; Koga, Yoshikatsu; Yamamoto, Yoshiyuki; Fujiwara, Yuki; Yasunaga, Masahiro; Matsumura, Yasuhiro

    2015-12-01

    Tissue factor (TF) is expressed strongly in various types of cancer, especially cancers that are often refractory to treatment, such as pancreatic cancer. In this study, we compared the differences in the biophysical and pharmacological properties of whole IgG and the Fab fragment of anti-human TF monoclonal antibody (1849 antibodies), in order to determine their suitability for application in the diagnosis and treatment of cancers. In the biophysical examination, we investigated the characteristics of 1849-whole IgG and 1849-Fab by SPR sensing and confocal fluorescence microscopy analysis using recombinant human TF antigen and TF-overexpressing human pancreatic cancer cell line, BxPC3, respectively. After conjugation with Alexa-Flour-647, in vivo imaging was conducted in mice bearing BxPC3 xenograft tumors. Furthermore, the distribution of the conjugates in tumors and major organs was evaluated by ex vivo study. The in vitro experiments showed that 1849 antibodies had high affinity against TF antigen. In addition, 1849-Fab showed a faster dissociation rate from the antigen than 1849-whole IgG. In mice, 1849-Fab-Alexa-Flour-647 showed rapid renal clearance and faster tumor accumulation, achieving a high contrast signal over nearby normal tissues in the early phase and enhanced tumor penetration after administration. On the other hand, 1849-whole IgG-Alexa-Flour-647 showed slow clearance from the blood and sustained high tumor accumulation. These results suggest that 1849-Fab may be a useful tool for pancreatic cancer diagnosis.

  1. 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.

  2. 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

  3. Metastatic colorectal cancer: Radioimmunoscintigraphy with a stabilized In-111-labeled F(ab')2 fragment of an anti-CEA monoclonal antibody

    SciTech Connect

    Lamki, L.M.; Patt, Y.Z.; Rosenblum, M.G.; Shanken, L.J.; Thompson, L.B.; Schweighardt, S.A.; Frincke, J.M.; Murray, J.L. )

    1990-01-01

    Metastatic colorectal cancer was detected with stabilized F(ab')2 fragments of ZCE-025, an anti-carcino-embryonic antigen (CEA) monoclonal antibody (MoAb). The fragments were prepared by cross-linking Fab' with a bifunctional cross-linking agent, bis-(maleimido)methyl ether. The authors labeled 2 mg of ZCE-025 with 5 mCi (185 MBq) of indium-111 and injected the material intravenously, either alone or with unlabeled F(ab')2, into 16 patients. Lesion detection, pharmacokinetics, and relative body distribution were evaluated and compared with those of the intact immunoglobulin (IgG1) antibody. Stabilized F(ab')2 fragments were more useful than the intact antibody in detection of lesions: Overall sensitivity of F(ab')2 fragments for all the patients was 79.4%, whereas overall sensitivity of the intact IgG1 antibody was 32%. This anti-CEA-stabilized F(ab')2 fragment may be a powerful diagnostic tool that can achieve higher sensitivity at smaller protein doses than the intact IgG1 antibody.

  4. The protective effects and underlying mechanism of an anti-oligomeric Aβ42 single-chain variable fragment antibody.

    PubMed

    Zhang, Yuan; Chen, Xu; Liu, Jinyu; Zhang, Yingjiu

    2015-12-01

    Oligomeric Aβ42 aggregates have been identified as one of the major neurotoxic components of Alzheimer's disease (AD). Immunotherapy targeted against these Aβ42 aggregates has been proposed as an appropriate therapeutic approach for the treatment of AD. Here, we report an anti-oligomeric Aβ42 single-chain variable fragment (scFv) antibody, named MO6, obtained from the human antibody library of a healthy donor. ScFv MO6 specifically recognized and bound to the oligomeric Aβ42 (Aβ42 oligomers and immature protofibrils; 18-37 kDa), and reduced their levels mainly by blocking their formation, although scFv MO6 also induced disaggregation of Aβ42 aggregates. More importantly, scFv MO6 ameliorated or attenuated Aβ42-induced cytotoxicity and increased cell viability by up to 33%. Furthermore, scFv MO6 efficiently passed through an in vitro blood-brain barrier (BBB) model with a delivery efficiency of 66% after 60 min post-administration. ScFv MO6 is a monovalent antibody with an affinity constant (KD) of 5.2×10(-6) M for Aβ42 oligomers. Molecular docking simulations of Aβ42 to scFv MO6 revealed that the approach and specific binding of scFv MO6 to oligomeric Aβ42 aggregates was achieved by conformational recognition and directed induction, which resulted in a more dynamic adaptation of Aβ42 to scFv MO6, occurring mainly in the N-terminal (3-4), middle (12-19) and C-terminal (34-42) regions of Aβ42. This binding mode of scFv MO6 to Aβ42 explains its protective effects against oligomeric Aβ42. Our findings may be applied for the design of a smaller antibody specific for Aβ42 oligermers. PMID:26256421

  5. 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.

  6. 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

  7. 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

  8. 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

  9. 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.

  10. Phage-displayed antibody fragments recognizing dengue 3 and dengue 4 viruses as tools for viral serotyping in sera from infected individuals.

    PubMed

    Cabezas, Sheila; Rojas, Gertrudis; Pavon, Alequis; Bernardo, Lidice; Castellanos, Yinet; Alvarez, Mayling; Pupo, Maritza; Guillen, Gerardo; Guzman, Maria G

    2009-01-01

    The current study shows the usefulness of dengue-3- and dengue-4-specific phage-displayed antibody fragments as tools for viral detection and serotyping in sera from infected individuals. C6/36 HT cells were inoculated with acute-phase sera from patients, and supernatants were collected daily and analyzed by ELISA using phage-displayed antibody fragments as serotype-specific detector reagents. Serotyping of most samples was possible as early as two to three days postinoculation. Results were comparable with those obtained by indirect immunofluorescence assay but were obtained in a shorter period of time (<1 week). Phage-displayed antibody fragments were better tools for diagnosis and serotyping than their soluble counterparts. Our approach combines the advantages of viral isolation and ELISA techniques. These results could be the basis for the development of a high-throughput method for identifying dengue virus serotypes, which is crucial for the management and control of the disease.

  11. 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.

  12. 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

  13. 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

  14. Glycoprotein nonmetastatic melanoma protein B extracellular fragment shows neuroprotective effects and activates the PI3K/Akt and MEK/ERK pathways via the Na+/K+-ATPase

    PubMed Central

    Ono, Yoko; Tsuruma, Kazuhiro; Takata, Masafumi; Shimazawa, Masamitsu; Hara, Hideaki

    2016-01-01

    Glycoprotein nonmetastatic melanoma protein B (GPNMB) plays important roles in various types of cancer and amyotrophic lateral sclerosis (ALS). The details of GPNMB function and its interacting protein have not been clarified. Therefore, to identify GPNMB binding partners on the cell membrane, we used membrane protein library/BLOTCHIP-MS technology, which enables us to analyze all cell membrane proteins as binding partners of the GPNMB extracellular fragment. As a result of a comprehensive search, we identified the alpha subunits of Na+/K+-ATPase (NKA) as a possible binding partner. We confirmed the interaction between the GPNMB extracellular fragment and NKA by immunoprecipitation and immunostaining in NSC-34 cells. Indeed, endogenous GPNMB extracellular fragment bound to and colocalized with NKA alpha subunits. Furthermore, exogenous GPNMB extracellular fragment, i.e., human recombinant GPNMB, also bound to and colocalized with NKA alpha subunits. Additionally, we found that the GPNMB extracellular fragment had neuroprotective effects and activated the phosphoinositide 3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK)-extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK pathways via NKA. These findings indicated that NKA may act as a novel “receptor” for the GPNMB extracellular fragment, offering additional molecular targets for the treatment of GPNMB-related diseases, including various types of cancer and ALS. PMID:26988030

  15. 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.

  16. 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.

  17. Generation and characterization of functional recombinant antibody fragments against tomato yellow leaf curl virus replication-associated protein.

    PubMed

    Safarnejad, M R; Fischer, R; Commandeur, U

    2008-01-01

    Tomato yellow leaf curl virus (TYLCV) is a complex of geminivirus species prevalent in the tropics and sub-tropics, which causes severe diseases in economically important crops such as tomato. Conventional strategies for disease management have shown little success and new approaches based on genetic engineering need to be considered. We generated two single-chain variable fragment antibodies (scFv-ScRep1 and scFv-ScRep2) that bound strongly to continuous epitopes within the TYLCV replication-associated protein (Rep). The TYLCV-Ir C1 gene (encoding Rep) was expressed as glutathione-S-transferase (GST) and maltose-binding protein (MBP) fusions. Purified MBP-Rep was used to immunize mice allowing the construction of naïve and pre-immunized scFv phage display libraries. Immunoassays showed that scFv-ScRep1 recognized an N-terminal epitope of Rep, whereas scFv-ScRep2 recognized a more central epitope. This is the first successful production of scFv antibodies against a geminivirus Rep, the initial step in the production of transgenic plants with resistance to TYLCV. PMID:19226769

  18. 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

  19. Intratracheal exposure to Fab fragments of an allergen-specific monoclonal antibody regulates asthmatic responses in mice

    PubMed Central

    Yoshino, Shin; Mizutani, Nobuaki; Matsuoka, Daiko; Sae-Wong, Chutha

    2014-01-01

    Fab fragments (Fabs) maintain the ability to bind to specific antigens but lack effector functions due to the absence of the Fc portion. In the present study, we tested whether Fabs of an allergen-specific monoclonal antibody (mAb) were able to regulate asthmatic responses in mice. Asthmatic responses were induced in BALB/c mice by passive sensitization with anti-ovalbumin (OVA) polyclonal antibodies (pAbs) (day 0) and by active sensitization with OVA (days 0 and 14), followed by intratracheal (i.t.) challenge with OVA on day 1 and days 28, 29, 30 and 35. Fabs prepared by the digestion of an anti-OVA IgG1 (O1-10) mAb with papain were i.t. administered only once 30 min before antigenic challenge on day 1 or day 35. The results showed that i.t. administration of O1-10 Fabs with OVA markedly suppressed the early and/or late phases of asthmatic responses caused by passive and active sensitization. Similar results were obtained when Fabs of anti-OVA IgG2b mAb (O2B-3) were i.t. administered. In contrast, neither i.t. injection of intact 01-10/O2B-3 nor systemic injection of O1-10 Fabs suppressed the asthmatic responses. In vitro studies revealed that the capture of OVA by O1-10 Fabs prevented the subsequent binding of intact anti-OVA pAbs to the captured OVA. These results suggest that asthmatic responses may be down-regulated by the i.t. exposure to Fabs of an allergen-specific mAb via a mechanism involving the capture of allergen by Fabs in the respiratory tract before the interaction of intact antibody and allergen essential for the induction of asthmatic responses. PMID:24303921

  20. 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

  1. The single-chain immunotoxin MCSP-ETA’, targeting melanoma-associated chondroitin sulfate proteoglycan, is a potent inducer of apoptosis in cultured human melanoma cells

    PubMed Central

    Schwenkert, Michael; Birkholz, Katrin; Schwemmlein, Michael; Kellner, Christian; Peipp, Matthias; Nettelbeck, Dirk M.; Schuler-Thurner, Beatrice; Schaft, Niels; Dörrie, Jan; Ferrone, Soldano; Kämpgen, Eckhart; Fey, Georg H.

    2009-01-01

    A recombinant immunotoxin was constructed by fusing a single-chain Fv (scFv) antibody fragment, specific for the melanoma-associated chondroitin sulfate proteoglycan (MCSP), to a truncated variant of Pseudomonas Exotoxin A (ETA’), carrying a C-terminal KDEL peptide for improved intracellular transport. The resulting immunotoxin, MCSP-ETA’, induced antigen-specific, potent apoptosis in the cultured human melanoma-derived cell lines A2058 and A375M, and treatment with a single dose of the agent eliminated up to 80 % of these cells within 72 h. The dose needed for half-maximum killing (EC50) was approximately 1 nM for both cell lines. MCSP-ETA’ also displayed cytotoxic activity against cultured primary melanoma cells from patients with advanced disease, with net cell death reaching up to 70 % within 96 h after treatment with a single dose of 14 nM. MCSP-ETA’ induced cell death synergistically with Cyclosporin A (CsA), both in established human melanoma cell lines and cultured primary melanoma cells. The distinctive antigen-restricted induction of apoptosis and the synergy with CsA justify further evaluation of this novel agent with regard to its potential applications for the treatment of melanoma and other MCSP-positive malignancies. PMID:18337643

  2. 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.

  3. High-resolution characterization of antibody fragment/antigen interactions using Biacore T100.

    PubMed

    Papalia, Giuseppe A; Baer, Mark; Luehrsen, Kenneth; Nordin, Helena; Flynn, Peter; Myszka, David G

    2006-12-01

    A Biacore T100 optical biosensor was used to characterize the binding kinetics of a panel of antigen binding fragments (Fabs) directed against the PcrV protein from Pseudomonas aeruginosa. PcrV protein forms part of the type III secretion system complex of this opportunistic pathogen. We demonstrate that the biosensor response data for each Fab collected from three different surface densities of the antigen could be fit globally to a simple 1:1 interaction model. Importantly, we found that the Fabs with the slowest dissociation rate provided the best protection in cell cytotoxicity studies. To further characterize the Fab interactions, binding data were automatically acquired at different temperatures and under different buffer conditions. The comprehensive characterization of these Fabs shows how Biacore T100 can be used to complement protein therapeutic discovery programs from basic research to the selection of therapeutic candidates.

  4. Analysis of BRAF and NRAS Mutation Status in Advanced Melanoma Patients Treated with Anti-CTLA-4 Antibodies: Association with Overall Survival?

    PubMed

    Mangana, Joanna; Cheng, Phil F; Schindler, Katja; Weide, Benjamin; Held, Ulrike; Frauchiger, Anna L; Romano, Emanuella; Kähler, Katharina C; Rozati, Sima; Rechsteiner, Markus; Moch, Holger; Michielin, Olivier; Garbe, Claus; Hauschild, Axel; Hoeller, Christoph; Dummer, Reinhard; Goldinger, Simone M

    2015-01-01

    Ipilimumab and tremelimumab are human monoclonal antibodies (Abs) against cytotoxic T-lymphocyte antigen-4 (CTLA-4). Ipilimumab was the first agent to show a statistically significant benefit in overall survival in advanced melanoma patients. Currently, there is no proven association between the BRAFV600 mutation and the disease control rate in response to ipilimumab. This analysis was carried out to assess if BRAFV600 and NRAS mutation status affects the clinical outcome of anti-CTLA-4-treated melanoma patients. This is a retrospective multi-center analysis of 101 patients, with confirmed BRAF and NRAS mutation status, treated with anti-CTLA-4 antibodies from December 2006 until August 2012. The median overall survival, defined from the treatment start date with the anti-CTLA-4. Abs-treatment to death or till last follow up, of BRAFV600 or NRAS mutant patients (n = 62) was 10.12 months (95% CI 6.78-13.2) compared to 8.26 months (95% CI 6.02-19.9) in BRAFV600/NRASwt subpopulation (n = 39) (p = 0.67). The median OS of NRAS mutated patients (n = 24) was 12.1 months and although was prolonged compared to the median OS of BRAF mutated patients (n = 38, mOS = 8.03 months) or BRAFV600/NRASwt patients (n = 39, mOS = 8.26 months) the difference didn't reach statistical significance (p = 0.56). 69 patients were able to complete 4 cycles of anti-CTLA-4 treatment. Of the 24 patients treated with selected BRAF- or MEK-inhibitors, 16 patients received anti-CTLA 4 Abs following either a BRAF or MEK inhibitor with only 8 of them being able to finish 4 cycles of treatment. Based on our results, there is no difference in the median OS in patients treated with anti-CTLA-4 Abs implying that the BRAF/NRAS mutation status alone is not sufficient to predict the outcome of patients treated with anti-CTLA-4 Abs.

  5. 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 ...

  6. 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

  7. Differential Antitumor Effects of IgG and IgM Monoclonal Antibodies and Their Synthetic Complementarity-Determining Regions Directed to New Targets of B16F10-Nex2 Melanoma Cells.

    PubMed

    Dobroff, Andrey S; Rodrigues, Elaine G; Juliano, Maria A; Friaça, Dayson M; Nakayasu, Ernesto S; Almeida, Igor C; Mortara, Renato A; Jacysyn, Jacqueline F; Amarante-Mendes, Gustavo P; Magliani, Walter; Conti, Stefania; Polonelli, Luciano; Travassos, Luiz R

    2010-01-01

    Malignant melanoma has increased incidence worldwide and causes most skin cancer-related deaths. A few cell surface antigens that can be targets of antitumor immunotherapy have been characterized in melanoma. This is an expanding field because of the ineffectiveness of conventional cancer therapy for the metastatic form of melanoma. In the present work, antimelanoma monoclonal antibodies (mAbs) were raised against B16F10 cells (subclone Nex4, grown in murine serum), with novel specificities and antitumor effects in vitro and in vivo. MAb A4 (IgG2ak) recognizes a surface antigen on B16F10-Nex2 cells identified as protocadherin beta(13). It is cytotoxic in vitro and in vivo to B16F10-Nex2 cells as well as in vitro to human melanoma cell lines. MAb A4M (IgM) strongly reacted with nuclei of permeabilized murine tumor cells, recognizing histone 1. Although it is not cytotoxic in vitro, similarly with mAb A4, mAb A4M significantly reduced the number of lung nodules in mice challenged intravenously with B16F10-Nex2 cells. The V(H) CDR3 peptide from mAb A4 and V(L) CDR1 and CDR2 from mAb A4M showed significant cytotoxic activities in vitro, leading tumor cells to apoptosis. A cyclic peptide representing A4 CDR H3 competed with mAb A4 for binding to melanoma cells. MAb A4M CDRs L1 and L2 in addition to the antitumor effect also inhibited angiogenesis of human umbilical vein endothelial cells in vitro. As shown in the present work, mAbs A4 and A4M and selected CDR peptides are strong candidates to be developed as drugs for antitumor therapy for invasive melanoma.

  8. Cancer Immunology Miniatures: Immune activation and a 9-year ongoing complete remission following CD40 antibody therapy and metastasectomy in a patient with metastatic melanoma

    PubMed Central

    Bajor, David L.; Xu, Xiaowei; Torigian, Drew A.; Mick, Rosemarie; Garcia, Laura R.; Richman, Lee P.; Desmarais, Cindy; Nathanson, Katherine L.; Schuchter, Lynn M.; Kalos, Michael; Vonderheide, Robert H.

    2014-01-01

    Direct immune activation via agonistic monoclonal antibodies (mAb) is a potentially complementary approach to therapeutic blockade of inhibitory immune receptors in cancer. Here, we provide genetic analysis of the immunological consequences associated with the use of an agonistic CD40 mAb in a patient with metastatic melanoma who responded, underwent a single metastasectomy, and then achieved a complete remission ongoing for more than 9 years after starting therapy. Tumor microenvironment after immunotherapy was associated with pro-inflammatory modulations and emergence of a de novo T-cell repertoire as detected by next-generation sequencing of T-cell receptors (TCR) in the tumor and blood. The de-novo T-cell repertoire identified in the post-treatment metastasectomy sample was also present – and in some cases expanded – in the circulation years after completion of therapy. Comprehensive study of this “exceptional responder” highlights the emerging potential of direct immune agonists in the next wave of cancer immunotherapies and a potential role for TCR deep sequencing in cancer immune assessment. PMID:25252722

  9. Zebrafish Melanoma.

    PubMed

    Kaufman, Charles K

    2016-01-01

    Melanoma skin cancer is a potentially deadly disease in humans and has remained extremely difficult to treat once it has metastasized. In just the last 10 years, a number of models of melanoma have been developed in the zebrafish that are biologically faithful to the human disease and have already yielded important insights into the fundamental biology of melanoma and offered new potential avenues for treatment. With the diversity and breadth of the molecular genetic tools available in the zebrafish, these melanoma models will continue to be refined and expanded upon to keep pace with the rapidly evolving field of melanoma biology. PMID:27165365

  10. Complement-coated antibody-transfer (CCAT); serum IgA1 antibodies intercept and transport C4 and C3 fragments and preserve IgG1 deployment (PGD).

    PubMed

    Boackle, Robert J; Nguyen, Quang L; Leite, Renata S; Yang, Xiaofeng; Vesely, Jana

    2006-02-01

    in which complement-coated IgA1 antibodies transferred to non-complement-coated antigens is termed complement-coated antibody-transfer/transport (CCAT). In this way, IgA1 antibodies extended the efficiency of the complement system by insuring the specific IgA1 antibody-mediated transport of the captured biologically active complement fragments to those antigens stimulating the IgA1 antibody response but not yet neutralized (completely coated) with complement. Simultaneously by impeding the rate of C1 consumption and by intercepting C4b and C3b, IgA1 antibodies slowed C4b and C3b deposition on the antigenic surface and on the co-deposited IgG1 antibodies. Thus, in the presence of ongoing complement activation, the deposition of serum IgA1 antibodies enabled the co-deposited IgG1 antibodies to better maintain their ability to interact with antigens. We termed this latter phenomenon, preservation of IgG antibody deployment (PGD). In summary, co-deposited IgA1 antibodies maximized the efficiency of the complement system, transported their covalently bound complement fragments to specific antigens and sustained the effective deployment of IgG1 antibodies directed to those same antigens.

  11. 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.

  12. 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

  13. 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.

  14. Passive immunization of guinea pigs with llama single-domain antibody fragments against foot-and-mouth disease.

    PubMed

    Harmsen, M M; van Solt, C B; Fijten, H P D; van Keulen, L; Rosalia, R A; Weerdmeester, K; Cornelissen, A H M; De Bruin, M G M; Eblé, P L; Dekker, A

    2007-03-10

    Foot-and-mouth disease (FMD) is a highly contagious disease that occasionally causes outbreaks in Europe. There is a need for therapies that provide rapid protection against FMD in outbreak situations. We aim to provide such rapid protection by passive immunization with llama single-domain antibody fragments (VHHs). Twenty-four VHHs binding serotype O FMDV in vitro were isolated from immunized llamas by phage display and expressed in bakers yeast for further characterization. They recognized four functionally independent antigenic sites. Six strongly FMDV neutralizing VHHs bound to a peptide representing the GH-loop of viral protein 1 known to be involved in binding to the cellular receptor of FMDV. Clone M8, recognizing this antigenic site, and clone M23, recognizing another antigenic site, showed synergistic in vitro virus neutralization. Three FMDV specific VHHs were PEGylated in order to decrease their rapid blood clearance and thus enable in vivo guinea pig protection experiments. Passive immunization with individual VHHs showed no protection, but a mixture of M8 and M23 showed partial transient protection. The protection afforded by these VHHs was however low as compared to the complete protection afforded by convalescent guinea pig serum. In contrast, these VHHs showed far more efficient in vitro FMDV neutralization than convalescent guinea pig serum. This lack of correlation between in vitro neutralization and in vivo protection lends further credence to the notion that opsonophagocytosis of FMDV is important for protection in vivo.

  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).

  16. Effect of polyethylene glycol conjugation on conformational and colloidal stability of a monoclonal antibody antigen-binding fragment (Fab').

    PubMed

    Roque, Cristopher; Sheung, Anthony; Rahman, Nausheen; Ausar, S Fernando

    2015-02-01

    We have investigated the effects of site specific "hinge" polyethylene glycol conjugation (PEGylation) on thermal, pH, and colloidal stability of a monoclonal antibody antigen-binding fragment (Fab') using a variety of biophysical techniques. The results obtained by circular dichroism (CD), ultraviolet (UV) absorbance, and fluorescence spectroscopy suggested that the physical stability of the Fab' is maximized at pH 6-7 with no apparent differences due to PEGylation. Temperature-induced aggregation experiments revealed that PEGylation was able to increase the transition temperature, as well as prevent the formation of visible and subvisible aggregates. Statistical comparison of the three-index empirical phase diagram (EPD) revealed significant differences in thermal and pH stability signatures between Fab' and PEG-Fab'. Upon mechanical stress, micro-flow imaging (MFI) and measurement of the optical density at 360 nm showed that the PEG-Fab' had significantly higher resistance to surface-induced aggregation compared to the Fab'. Analysis of the interaction parameter, kD, indicated repulsive intermolecular forces for PEG-Fab' and attractive forces for Fab'. In conclusion, PEGylation appears to protect Fab' against thermal and mechanical stress-induced aggregation, likely due to a steric hindrance mechanism.

  17. Optimized extraction of a single-chain variable fragment of antibody by using aqueous micellar two-phase systems.

    PubMed

    Malpiedi, Luciana P; Nerli, Bibiana B; Taqueda, Maria E S; Abdalla, Dulcineia S P; Pessoa, Adalberto

    2015-07-01

    In this work, the purification of a single-chain variable fragment (scFv) of an antibody by using liquid-liquid extraction in aqueous micellar two-phase systems was optimized by means of central composite design. Protein partitioning assays were performed by using the selected system composition in previous works: Triton X-114 at 4% wt/wt, yeast fermentation supernatant at 60% wt/wt, McIlvaine buffer pH 7.00. The other system component concentrations, Cibacron Blue F3GA (CB), Fabsorbent™ F1P HF (HF) and NaCl, were selected as independent variables. ScFv recovery percentage (%R) and purification factor (PF) were selected as the responses. According to the optimization process both, scFv recovery percentage and purification factor were favored with the addition of HF and NaCl in a range of concentrations around the central point of the second central composite design (HF 0.0120% w/w, CB 0.0200% w/w, NaCl 0.200% w/w). These experimental conditions allowed the concentration and pre-purification of scFv in the micelle-rich bottom phase of the systems with a recovery percentage superior to 88% and a purification factor of approximately 3.5. These results improved the previously presented works and demonstrated the convenience of using aqueous micellar two-phase systems as a first step in the purification of scFv molecules.

  18. Passive immunization of guinea pigs with llama single-domain antibody fragments against foot-and-mouth disease.

    PubMed

    Harmsen, M M; van Solt, C B; Fijten, H P D; van Keulen, L; Rosalia, R A; Weerdmeester, K; Cornelissen, A H M; De Bruin, M G M; Eblé, P L; Dekker, A

    2007-03-10

    Foot-and-mouth disease (FMD) is a highly contagious disease that occasionally causes outbreaks in Europe. There is a need for therapies that provide rapid protection against FMD in outbreak situations. We aim to provide such rapid protection by passive immunization with llama single-domain antibody fragments (VHHs). Twenty-four VHHs binding serotype O FMDV in vitro were isolated from immunized llamas by phage display and expressed in bakers yeast for further characterization. They recognized four functionally independent antigenic sites. Six strongly FMDV neutralizing VHHs bound to a peptide representing the GH-loop of viral protein 1 known to be involved in binding to the cellular receptor of FMDV. Clone M8, recognizing this antigenic site, and clone M23, recognizing another antigenic site, showed synergistic in vitro virus neutralization. Three FMDV specific VHHs were PEGylated in order to decrease their rapid blood clearance and thus enable in vivo guinea pig protection experiments. Passive immunization with individual VHHs showed no protection, but a mixture of M8 and M23 showed partial transient protection. The protection afforded by these VHHs was however low as compared to the complete protection afforded by convalescent guinea pig serum. In contrast, these VHHs showed far more efficient in vitro FMDV neutralization than convalescent guinea pig serum. This lack of correlation between in vitro neutralization and in vivo protection lends further credence to the notion that opsonophagocytosis of FMDV is important for protection in vivo. PMID:17127019

  19. 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

  20. 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

  1. 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

    Zalutsky, M R; Garg, P K; Friedman, H S; Bigner, D D

    1989-09-01

    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.

  2. 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

  3. High contrast tumor imaging with radio-labeled antibody Fab fragments tailored for optimized pharmacokinetics via PASylation.

    PubMed

    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.

  4. Combination of alphavirus replicon particle-based vaccination with immunomodulatory antibodies: therapeutic activity in the B16 melanoma mouse model and immune correlates.

    PubMed

    Avogadri, Francesca; Zappasodi, Roberta; Yang, Arvin; Budhu, Sadna; Malandro, Nicole; Hirschhorn-Cymerman, Daniel; Tiwari, Shakuntala; Maughan, Maureen F; Olmsted, Robert; Wolchok, Jedd D; Merghoub, Taha

    2014-05-01

    Induction of potent immune responses to self-antigens remains a major challenge in tumor immunology. We have shown that a vaccine based on alphavirus replicon particles (VRP) activates strong cellular and humoral immunity to tyrosinase-related protein-2 (TRP2) melanoma antigen, providing prophylactic and therapeutic effects in stringent mouse models. Here, we report that the immunogenicity and efficacy of this vaccine is increased in combination with either antagonist anti-CTL antigen-4 (CTLA-4) or agonist anti-glucocorticoid-induced TNF family-related gene (GITR) immunomodulatory monoclonal antibodies (mAb). In the challenging therapeutic setting, VRP-TRP2 plus anti-GITR or anti-CTLA-4 mAb induced complete tumor regression in 90% and 50% of mice, respectively. These mAbs had similar adjuvant effects in priming an adaptive immune response against the vaccine-encoded antigen, augmenting, respectively, approximately 4- and 2-fold the TRP2-specific CD8(+) T-cell response and circulating Abs, compared with the vaccine alone. Furthermore, while both mAbs increased the frequency of tumor-infiltrating CD8(+) T cells, anti-CTLA-4 mAb also increased the quantity of intratumor CD4(+)Foxp3(-) T cells expressing the negative costimulatory molecule programmed death-1 (PD-1). Concurrent GITR expression on these cells suggests that they might be controlled by anti-GITR mAbs, thus potentially explaining their differential accumulation under the two treatment conditions. These findings indicate that combining immunomodulatory mAbs with alphavirus-based anticancer vaccines can provide therapeutic antitumor immune responses in a stringent mouse model, suggesting potential utility in clinical trials. They also indicate that tumor-infiltrating CD4(+)Foxp3(-)PD-1(+) T cells may affect the outcome of immunomodulatory treatments.

  5. Targeted Radionuclide Therapy of Melanoma.

    PubMed

    Norain, Abdullah; Dadachova, Ekaterina

    2016-05-01

    An estimated 60,000 individuals in the United States and 132,000 worldwide are yearly diagnosed with melanoma. Until recently, treatment options for patients with stages III-IV metastatic disease were limited and offered marginal, if any, improvement in overall survival. The situation changed with the introduction of B-RAF inhibitors and anti-cytotoxic T-lymphocyte antigen 4 and anti-programmed cell death protein 1 immunotherapies into the clinical practice. With only some patients responding well to the immune therapies and with very serious side effects and high costs of immunotherapy, there is still room for other approaches for the treatment of metastatic melanoma. Targeted radionuclide therapy of melanoma could be divided into the domains of radioimmunotherapy (RIT), radiolabeled peptides, and radiolabeled small molecules. RIT of melanoma is currently experiencing a renaissance with the clinical trials of alpha-emitter (213)Bi-labeled and beta-emitter (188)Rhenium-labeled monoclonal antibodies in patients with metastatic melanoma producing encouraging results. The investigation of the mechanism of efficacy of melanoma RIT points at killing of melanoma stem cells by RIT and involvement of immune system such as complement-dependent cytotoxicity. The domain of radiolabeled peptides for targeted melanoma therapy has been preclinical so far, with work concentrated on radiolabeled peptide analogues of melanocyte-stimulating hormone receptor and on melanin-binding peptides. The field of radiolabeled small molecule produced radioiodinated benzamides that cross the cellular membrane and bind to the intracellular melanin. The recent clinical trial demonstrated measurable antitumor effects and no acute or midterm toxicities. We are hopeful that the targeted radionuclide therapy of metastatic melanoma would become a clinical reality as a stand-alone therapy or in combination with the immunotherapies such as anti-PD1 programmed cell death protein 1 monoclonal antibodies

  6. Development and Characterization of Recombinant Antibody Fragments That Recognize and Neutralize In Vitro Stx2 Toxin from Shiga Toxin-Producing Escherichia coli

    PubMed Central

    Luz, Daniela; Chen, Gang; Maranhão, Andrea Q.; Rocha, Leticia B.; Sidhu, Sachdev; Piazza, Roxane M. F.

    2015-01-01

    Background Stx toxin is a member of the AB5 family of bacterial toxins: the active A subunit has N-glycosidase activity against 28S rRNA, resulting in inhibition of protein synthesis in eukaryotic cells, and the pentamer ligand B subunits (StxB) bind to globotria(tetra)osylceramide receptors (Gb3/Gb4) on the cell membrane. Shiga toxin-producing Escherichia coli strains (STEC) may produce Stx1 and/or Stx2 and variants. Strains carrying Stx2 are considered more virulent and related to the majority of outbreaks, besides being usually associated with hemolytic uremic syndrome in humans. The development of tools for the detection and/or neutralization of these toxins is a turning point for early diagnosis and therapeutics. Antibodies are an excellent paradigm for the design of high-affinity, protein-based binding reagents used for these purposes. Methods and Findings In this work, we developed two recombinant antibodies; scFv fragments from mouse hybridomas and Fab fragments by phage display technology using a human synthetic antibody library. Both fragments showed high binding affinity to Stx2, and they were able to bind specifically to the GKIEFSKYNEDDTF region of the Stx2 B subunit and to neutralize in vitro the cytotoxicity of the toxin up to 80%. Furthermore, the scFv fragments showed 79% sensitivity and 100% specificity in detecting STEC strains by ELISA. Conclusion In this work, we developed and characterized two recombinant antibodies against Stx2, as promising tools to be used in diagnosis or therapeutic approaches against STEC, and for the first time, we showed a human monovalent molecule, produced in bacteria, able to neutralize the cytotoxicity of Stx2 in vitro. PMID:25790467

  7. 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.

  8. Rainbow trout surviving infections of viral haemorrhagic septicemia virus (VHSV) show lasting antibodies to recombinant G protein fragments.

    PubMed

    Encinas, P; Gomez-Casado, E; Fregeneda-Grandes; Olesen, N J; Lorenzen, N; Estepa, A; Coll, J M

    2011-03-01

    Rainbow trout antibodies (Abs) binding to recombinant fragments (frgs) derived from the protein G of the viral haemorrhagic septicemia virus (VHSV)-07.71 strain, could be detected by ELISA (frg-ELISA) in sera from trout surviving laboratory-controlled infections. Abs were detected not only by using sera from trout infected with the homologous VHSV isolate but also with the VHSV-DK-201433 heterologous isolate, which had 13 amino acid changes. Sera from healthy trout and/or from trout surviving infectious haematopoietic necrosis virus (IHNV) infection, were used to calculate cut-off absorbances to differentiate negative from positive sera. Specific anti-VHSV Abs could then be detected by using any of the following frgs: frg11 (56-110), frg15 (65-250), frg16 (252-450) or G21-465. While high correlations were found among the ELISA values obtained with the different frgs, no correlations between any frg-ELISA and complement-dependent 50% plaque neutralization test (PNT) titres could be demonstrated. Between 4 and 10 weeks after VHSV infection, more trout sera were detected as positives by using heterologous frg-ELISA rather than homologous PNT. Furthermore, the percentage of positive sera detected by frg11-ELISA increased with time after infection to reach 100%, while those detected by complement-dependent PNT decreased to 29.4%, thus confirming that the lack of neutralizing Abs does not mean the lack of any anti-VHSV Abs in survivor trout sera. Preliminary results with sera from field samples suggest that further refinements of the frg-ELISA could allow detection of anti-VHSV trout Abs in natural outbreaks caused by different heterologous VHSV isolates. The homologous frg-ELISA method could be useful to follow G immunization attempts during vaccine development and/or to best understand the fish Ab response during VHSV infections. The viral frgs approach might also be used with other fish species and/or viruses.

  9. 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

  10. Immunotherapy in a rare case of primary pelvic retroperitoneal melanoma.

    PubMed

    Talag, Maria Monica; Alsharedi, Mohamed; Bou Zgheib, Nadim; Lebowicz, Yehuda

    2016-01-01

    Recent advances in novel immunotherapeutic and targeted therapeutic agents have increased treatment options in patients with advanced metastatic melanoma. However, evidence in the literature on whether or not extracutaneous melanoma will acquire an equivalent advantage from these therapies is very scarce. In general, extracutaneous melanomas are rare and aggressive melanomas, which are clinically and biologically unique, with higher incidence of metastatic disease and poor prognosis. In this case report, we present a very rare case of a 54-year-old woman with primary pelvic retroperitoneal melanoma treated with an anti-PD-1 antibody, pembrolizumab. Furthermore, we explore the role of novel immunotherapies in the treatment of advanced melanoma. PMID:27624447

  11. A stable cytosolic expression of VH antibody fragment directed against PVY NIa protein in transgenic potato plant confers partial protection against the virus.

    PubMed

    Bouaziz, Donia; Ayadi, Malika; Bidani, Amira; Rouis, Souad; Nouri-Ellouz, Oumèma; Jellouli, Raïda; Drira, Noureddine; Gargouri-Bouzid, Radhia

    2009-04-01

    The expression of recombinant antibodies in transgenic plants has been proved to be an efficient approach for large-scale production. However, the stability of these molecules and their accumulation level depend on their molecular properties and cellular targeting. The expression of single-domain antibody fragment (VH) can be advantageous since it offers small length, high expression, solubility and stability. It can therefore be preferred to other antibody derivatives avoiding the expression difficulties related to immunoglobulin domain folding via the formation of disulfide bridge. This report describes the production of transgenic potato plants expressing a VH antibody directed against the NIa protease of potato virus Y. The antibody was driven by the constitutive CaMV 35S RNA promoter. The expression cassette was transferred into potato plants via Agrobacterium tumefaciens mediated transformation. All transgenic lines showed detectable levels of VH protein confirming the efficient translation and stability of this protein. The cellular localisation of the VH antibody was investigated. Transgenic and control plants were transferred in the greenhouse and mechanically inoculated by PVY(o) suspension. Some of the transgenic lines showed delayed symptoms at the first period post inoculation and then displayed a recovery phenomenon while the virions were still detected in the leaves. PMID:26493138

  12. Melanoma therapy: Check the checkpoints.

    PubMed

    Furue, Masutaka; Kadono, Takafumi

    2016-02-01

    Recent mutational and translational studies have revealed that the Ras/Raf/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway plays a key role in melanomagenesis. Mutations in NRAS and BRAF are found in the majority of melanomas resulting in the formation of constitutively active NRAS and BRAF molecules, which leads to the proliferation and survival of melanoma cells through the activation of MEK/ERK signals. Inhibitors of BRAF or MEK significantly extend the progression-free survival and overall survival of melanoma patients compared with conventional chemotherapies. Combining BRAF and MEK inhibitors further enhances the clinical effectiveness. Cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) is an immune checkpoint molecule that downregulates T-cell activation by binding to B7 (CD80/CD86) molecules on antigen-presenting cells. Programmed death receptor ligand 1 on melanoma cells negatively regulates T-cell function by binding to the programmed death-1 (PD-1) receptor on T cells. Antibodies against CTLA-4 and PD-1 also enhance the survival of melanoma patients. In this review, we summarize the clinical effectiveness and adverse events of the BRAF inhibitors, MEK inhibitors and anti-immune checkpoint antibodies in melanoma treatment.

  13. 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.

  14. Targeting a homogeneously glycosylated antibody Fc to bind cancer cells using a synthetic receptor ligand.

    PubMed

    Xiao, Junpeng; Chen, Rui; Pawlicki, Mark A; Tolbert, Thomas J

    2009-09-30

    The targeting of a glycosylated antibody Fc fragment to bind to cancer cells by site-selective incorporation of a synthetic ligand is described. Homogeneously glycosylated immunoglobulin G subclass 1 fragment crystallizable (IgG1 Fc) was produced by expression in a glycosylation-deficient yeast strain and subsequent treatment with mannosidase IA. A N-terminal cysteine was generated on the expressed IgG1 Fc by utilizing proteolytic processing enzymes in the yeast secretory pathway. A cyclic RGD peptide thioester 2 was synthesized and then site-selectively attached to the N-terminus of the IgG1 Fc glycoprotein using native chemical ligation. The resulting chemically modified antibody fragment, RGD-Man(5)-IgG1 Fc (5), retained biological activity similar to that of the free cyclic RGD peptide 1 when assayed for its ability to both promote and inhibit the adhesion of alpha(v)beta(3) integrin receptor-expressing WM-115 melanoma cells. In addition, fluorescent microscopy experiments were conducted using FITC-labeled 5 and confirmed binding of 5 to WM-115 melanoma cells. Site-selectively modified antibody fragments such as the one described here may be used to combine the beneficial properties of synthetic receptor ligands with antibody fragments to develop useful biochemical tools and improved therapeutics. The methods described here can also be used to produce glycoprotein fragments for the chemoenzymatic synthesis of homogeneous glycoproteins.

  15. Detection of infectious myonecrosis virus using monoclonal antibody specific to N and C fragments of the capsid protein expressed heterologously.

    PubMed

    Kunanopparat, Areerat; Chaivisuthangkura, Parin; Senapin, Saengchan; Longyant, Siwaporn; Rukpratanporn, Sombat; Flegel, Timothy W; Sithigorngul, Paisarn

    2011-01-01

    The gene encoding the capsid protein in ORF1 of the genome of infectious myonecrosis virus (IMNV) (GenBank AY570982) was amplified into three parts named CP-N (nucleotides 2248-3045), CP-I (nucleotides 3046-3954) and CP-C (nucleotides 3955-4953). The CP-N fragment was inserted into expression vector pTYB1 while CP-I and CP-C were each inserted into expression vector pGEX-6P-1 for transformation of BL21 E. coli strain. After induction, intein-CP-N (84 kDa), glutathione-S-transferase (GST)-CP-I (60 kDa) and GST-CP-C (62 kDa) fusion proteins were produced. They were separated by SDS-PAGE and electroeluted before immunization of Swiss mice for monoclonal antibody (MAb) production. Two MAbs specific to CP-N and one MAb specific to CP-C were selected for use for detection of natural IMNV infections in Penaeus vannamei by dot blotting, Western blotting and immunohistochemistry. There was no cross-reaction with shrimp tissues or common shrimp viruses including white spot syndrome virus (WSSV), yellow head virus (YHV), Taura syndrome virus (TSV), Penaeus monodon nucleopolyhedrovirus (PemoNPV), Penaeus stylirostris densovirus (PstDNV) and Penaeus monodon densovirus (PmDNV). The detection sensitivities of the MAbs were approximately 6 fmol/spot of purified recombinant intein-CP-N protein and 8 fmol/spot of GST-CP-C as determined by dot blotting. A combination of all three MAbs resulted in a twofold increase in sensitivity over use of any single MAb. However, this sensitivity was approximately 10 times lower than that of one-step RT-PCR using the same sample. Immunohistochemical analysis using MAbs specific to CP-N and CP-C in IMNV-infected shrimp revealed intense staining patterns in muscles, the lymphoid organ, gills, the heart, hemocytes and connective tissue.

  16. Rapid optimization of antibotulinum toxin antibody fragment production by an integral approach utilizing RC-SELDI mass spectrometry and statistical design.

    PubMed

    Park, Jun T; Bradbury, Lisa; Kragl, Frank J; Lukens, Dennis C; Valdes, James J

    2006-01-01

    A process for the rapid development and optimization of the fermentation process for an antibotulinum neurotoxin antibody fragment (bt-Fab) production expressed in Escherichia coli was achieved via a high-throughput process proteomics and statistical experimental design. This process, using retentate chromatography-surface enhanced laser desorption/ionization mass spectrometry (RC-SELDI MS), was employed for identifying and quantifying bt-Fab antibody in complex biological samples for the optimization of microbial fermentation conditions. Five variables (type of culture media, glycerol concentration, post-induction temperature, IPTG concentration, and incubation time after induction) were statistically combined using an experimental 2(5)(-1) fractional factorial design and tested for their effects on maximal bt-Fab antibody production. When the effects of individual variables and their interactions were assessed, type of media and post-induction temperature showed statistically significant increase in yield of the fermentation process for the maximal bt-Fab antibody production. This study establishes an integral approach as a valuable tool for the rapid development of manufacturing processes for producing various biological materials. To verify the RC-SELDI MS method, a Fab-specific immuno-affinity HPLC assay developed here was also employed for the quantification of the bt-Fab antibody in crude lysate samples obtained during the fermentation optimization process. Similar results were obtained.

  17. Improved method for the direct labeling of antibodies with Tc-99m

    SciTech Connect

    Rhodes, B.A.; Hawkins, E.; Budd, P. ); Deleide, G.; Seccamani, E.; Bonino, C. )

    1990-01-01

    Antibodies and antibody fragments have been treated with stannous chloride or organic reducing agents to reduce disulfide bonds, providing sulfhydryl groups for binding reduced Tc-99m. The reduced antibody, additives and stannous salts are lyophilized. To radiolabel, sodium pertechnetate solution is added, which dissolves the protein and other reagents. The pertechnetate is reduced by the stannous ions and becomes bound to the antibody. After radiolabeling the shelf-life of the product exceeds the half-life of the radionuclide. One of the more effective additives is human serum albumin, which serves as a carrier protein, agent to protect against autoradiolysis and possibly as a transfer ligand. Many different antibodies have been labeled using this method. The most widely studied antibody is an anti-melanoma fragment which has now been used clinically in more than 1000 cases and has been proven effective for the diagnostic localization of melanoma. In summary, a single vial, one step procedure for the direct labeling of antibodies in the presence of human serum albumin has been developed, extensively characterized, and clinically validated. The method is used with murine monoclonal IgG fragments, IgM and human gamma globulin. 4 refs., 10 figs.

  18. [Choroidal melanoma].

    PubMed

    Desjardins, Laurence

    2016-03-01

    Choroidal melanoma is the most common form of eye cancer in adults. Treatments enabling the tumour to be destroyed or removed while preserving the eye socket are mainly based on surgery, proton therapy and brachytherapy. PMID:26944641

  19. Monoclonal Antibody Therapy in Treating Patients With Ovarian Epithelial Cancer, Melanoma, Acute Myeloid Leukemia, Myelodysplastic Syndrome, or Non-Small Cell Lung Cancer

    ClinicalTrials.gov

    2013-01-09

    Adult Acute Myeloid Leukemia With 11q23 (MLL) Abnormalities; Adult Acute Myeloid Leukemia With Inv(16)(p13;q22); Adult Acute Myeloid Leukemia With t(15;17)(q22;q12); Adult Acute Myeloid Leukemia With t(16;16)(p13;q22); Adult Acute Myeloid Leukemia With t(8;21)(q22;q22); Atypical Chronic Myeloid Leukemia, BCR-ABL1 Negative; Myelodysplastic/Myeloproliferative Neoplasm, Unclassifiable; Previously Treated Myelodysplastic Syndromes; Recurrent Adult Acute Myeloid Leukemia; Recurrent Melanoma; Recurrent Non-small Cell Lung Cancer; Recurrent Ovarian Epithelial Cancer; Stage IV Melanoma; Stage IV Non-small Cell Lung Cancer

  20. Hybridization of an Aβ-specific antibody fragment with aminopyrazole-based β-sheet ligands displays striking enhancement of target affinity.

    PubMed

    Hellmert, Marco; Müller-Schiffmann, Andreas; Peters, Max Sena; Korth, Carsten; Schrader, Thomas

    2015-03-14

    Determining Aβ levels in body fluids remains a powerful tool in the diagnostics of Alzheimer's disease. This report delineates a new supramolecular strategy which increases the affinity of antibodies towards Aβ to make diagnostic procedures more sensitive. A monoclonal antibody IC16 was generated to an N-terminal epitope of Aβ and the variable regions of the heavy and light chains were cloned as a recombinant protein (scFv). A 6 × histidine tag was fused to the C-terminus of IC16-scFv allowing hybridization with a small organic β-sheet binder via Ni-NTA complexation. On the other hand, a multivalent nitrilotriacetic acid (NTA)-equipped trimeric aminopyrazole (AP) derivative was synthesized based on a cyclam platform; and experimental evidence was obtained for efficient Ni(2+)-mediated complex formation with the histidine-tagged antibody species. In a proof of principle experiment the hybrid molecule showed a strong increase in affinity towards Aβ. Thus, the specific binding power of recombinant antibody fragments to their β-sheet rich targets can be conveniently enhanced by non-covalent hybridization with small organic β-sheet binders.

  1. 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

  2. Generation of human single-chain variable fragment antibodies specific to dengue virus non-structural protein 1 that interfere with the virus infectious cycle.

    PubMed

    Poungpair, Ornnuthchar; Bangphoomi, Kunan; Chaowalit, Prapaipit; Sawasdee, Nunghathai; Saokaew, Nichapatr; Choowongkomon, Kiattawee; Chaicumpa, Wanpen; Yenchitsomanus, Pa-thai

    2014-01-01

    Severe forms of dengue virus (DENV) infection frequently cause high case fatality rate. Currently, there is no effective vaccine against the infection. Clinical cases are given only palliative treatment as specific anti-DENV immunotherapy is not available and it is urgently required. In this study, human single-chain variable fragment (HuScFv) antibodies that bound specifically to the conserved non-structural protein-1 (NS1) of DENV and interfered with the virus replication cycle were produced by using phage display technology. Recombinant NS1 (rNS1) of DENV serotype 2 (DENV2) was used as antigen in phage bio-panning to select phage clones that displayed HuScFv from antibody phage display library. HuScFv from two phagemid transformed E. coli clones, i.e., clones 11 and 13, bound to the rNS1 as well as native NS1 in both secreted and intracellular forms. Culture fluids of the HuScFv11/HuScFv13 exposed DENV2 infected cells had significant reduction of the infectious viral particles, implying that the antibody fragments affected the virus morphogenesis or release. HuScFv epitope mapping by phage mimotope searching revealed that HuScFv11 bound to amino acids 1-14 of NS1, while the HuScFv13 bound to conformational epitope at the C-terminal portion of the NS1. Although the functions of the epitopes and the molecular mechanism of the HuScFv11 and HuScFv13 require further investigations, these small antibodies have high potential for development as anti-DENV biomolecules. PMID:24492300

  3. Design of an Escherichia coli Expressed HIV-1 gp120 Fragment Immunogen That Binds to b12 and Induces Broad and Potent Neutralizing Antibodies

    PubMed Central

    Bhattacharyya, Sanchari; Singh, Pranveer; Rathore, Ujjwal; Purwar, Mansi; Wagner, Denise; Arendt, Heather; DeStefano, Joanne; LaBranche, Celia C.; Montefiori, David C.; Phogat, Sanjay; Varadarajan, Raghavan

    2013-01-01

    b12, one of the few broadly neutralizing antibodies against HIV-1, binds to the CD4 binding site (CD4bs) on the gp120 subunit of HIV-1 Env. Two small fragments of HIV-1 gp120, b121a and b122a, which display about 70% of the b12 epitope and include solubility-enhancing mutations, were designed. Bacterially expressed b121a/b122a were partially folded and could bind b12 but not the CD4bs-directed non-neutralizing antibody b6. Sera from rabbits primed with b121a or b122a protein fragments and boosted with full-length gp120 showed broad neutralizing activity in a TZM-bl assay against a 16-virus panel that included nine Tier 2 and 3 viruses as well as in a five-virus panel previously designed to screen for broad neutralization. Using a mean IC50 cut-off of 50, sera from control rabbits immunized with gp120 alone neutralized only one virus of the 14 non-Tier 1 viruses tested (7%), whereas sera from b121a- and b122a-immunized rabbits neutralized seven (50%) and twelve (86%) viruses, respectively. Serum depletion studies confirmed that neutralization was gp120-directed and that sera from animals immunized with gp120 contained lower amounts of CD4bs-directed antibodies than corresponding sera from animals immunized with b121a/b122a. Competition binding assays with b12 also showed that b121a/2a sera contained significantly higher amounts of antibodies directed toward the CD4 binding site than the gp120 sera. The data demonstrate that it is possible to elicit broadly neutralizing sera against HIV-1 in small animals. PMID:23430741

  4. 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.

  5. 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.

  6. Expression of Tissue Factor by Melanoma Cells Promotes Efficient Hematogenous Metastasis

    NASA Astrophysics Data System (ADS)

    Mueller, Barbara M.; Reisfeld, Ralph A.; Edgington, Thomas S.; Ruf, Wolfram

    1992-12-01

    Metastasis is a multistep process which requires highly adapted interactions of tumor cells with host target organs. Compared with nonmetastatic cells, metastatic human melanoma cells express 1000-fold higher levels of tissue factor (TF), the major cellular initiator of the plasma coagulation protease cascades. To explore whether TF may contribute to metastatic tumor dissemination, we analyzed the effect of specific inhibition of TF function on human melanoma metastasis in severe combined immunodeficient (SCID) mice. Using species-specific antibodies to TF, we demonstrate that initial adherence is insufficient for successful tumor cell implantation in a target organ. Rapid arrest of human tumor cells in the lungs of mice was not diminished by inhibition of TF. However, inhibition of TF receptor function and consequent reduction in local protease generation abolished prolonged adherence of tumor cells, resulting in significantly reduced numbers of tumor cells retained in the vasculature of the lungs. The growth of pulmonary metastases was also significantly inhibited by a blocking anti-TF monoclonal antibody and Fab fragments thereof, whereas a noninhibitory antibody lacked antimetastatic effects. Cell surface expression of functional TF thus contributes to melanoma progression by allowing metastatic cells to provide requisite signals for prolonged adhesive interactions and/or transmigration of tumor cells across the endothelium, resulting in successful metastatic tumor implantation.

  7. Choroidal melanoma

    PubMed Central

    Singh, Parul; Singh, Abhishek

    2012-01-01

    Choroidal melanoma is the most common primary intra-ocular malignant tumor and second most common site of ten malignant melanoma sites in the body. Current diagnosis of choroidal melanoma is based on both the clinical experience of the specialist and modern diagnostic techniques such as indirect ophthalmoscopy, A- and B-ultrasonography scans, fundus fluorescein angiography, and transillumination. Invasive studies such as fine needle aspiration cytology can have significant morbidity and should only be considered if therapeutic intervention is indicated and diagnosis cannot be established by any other means. Several modes of treatment are available for choroidal melanoma. Multiple factors are taken into account when deciding one approach over other approaches, such as visual acuity of the affected eye, visual acuity of the contralateral eye, tumor size, location, ocular structures involved and presence of metastases. A comprehensive review of literature available in books and indexed journals was done. This article discusses in detail epidemiology, diagnosis, current available treatment options, and prognosis and survival of choroidal melanoma. PMID:22557869

  8. Melanoma Diagnosis

    NASA Astrophysics Data System (ADS)

    Horsch, Alexander

    The chapter deals with the diagnosis of the malignant melanoma of the skin. This aggressive type of cancer with steadily growing incidence in white populations can hundred percent be cured if it is detected in an early stage. Imaging techniques, in particular dermoscopy, have contributed significantly to improvement of diagnostic accuracy in clinical settings, achieving sensitivities for melanoma experts of beyond 95% at specificities of 90% and more. Automatic computer analysis of dermoscopy images has, in preliminary studies, achieved classification rates comparable to those of experts. However, the diagnosis of melanoma requires a lot of training and experience, and at the time being, average numbers of lesions excised per histology-proven melanoma are around 30, a number which clearly is too high. Further improvements in computer dermoscopy systems and their competent use in clinical settings certainly have the potential to support efforts of improving this situation. In the chapter, medical basics, current state of melanoma diagnosis, image analysis methods, commercial dermoscopy systems, evaluation of systems, and methods and future directions are presented.

  9. 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

  10. 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

  11. 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

  12. 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.

  13. 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.

  14. Neutralization of West Nile virus by cross-linking of its surface proteins with Fab fragments of the human monoclonal antibody CR4354.

    PubMed

    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

    2010-11-01

    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.

  15. 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.

  16. Platelet surface IgG in patients receiving infusions of Fab fragments of a chimaeric monoclonal antibody to glycoprotein IIb-IIIa.

    PubMed Central

    Christopoulos, C

    1994-01-01

    Platelet surface immunoglobulin G (PSIgG) was measured ex vivo in nine patients with stable angina pectoris receiving continuous (48-96 h) infusions of Fab fragments of a chimaeric MoAb (human IgG with murine variable regions) to platelet glycoprotein IIb-IIIa. PSIgG was measured using flow cytometry (FC) and an Fc-specific anti-IgG polyclonal antibody, which did not cross-react with the chimaeric Fab fragment (c7E3-Fab). A variable but statistically significant (P < 0.05) elevation of PSIgG was present within 24 h after the onset of the infusion, and was more marked (P < 0.01) several days after the end of the infusion despite an exponential fall in platelet surface c7E3-Fab post-infusion. PSIgG returned to normal within 2 weeks after the end of the infusion. The timing of IgG recruitment to the platelet surface suggested the pre-existence in the patients' plasma of IgG binding to c7E3-Fab-bearing platelets. None of the patients developed thrombocytopenia. In order to assess the incidence of IgG bindable to c7E3-Fab-bearing platelets in controls clinically comparable to the c7E3-Fab infusion patients, normal platelets coated with either chimaeric (c) or murine (m) 7E3-Fab were incubated with plasmas from 21 patients with ischaemic heart disease, and recruitment of IgG to the platelet surface was measured by FC. Fourteen of the 21 plasmas contained IgG bindable to c7E3-Fab-coated platelets, whereas only one of the 21 plasmas contained IgG bindable to m7E3-Fab-coated platelets (a highly significant difference, P < 0.001). These findings indicate that infusions of Fab fragments of the chimaeric anti-platelet antibody 7E3 are often associated with elevations in PSIgG, which are probably due to pre-existing 'naturally occurring' antibodies to the Fab fragments of chimaeric (but not murine) 7E3, and most probably other chimaeric MoAbs. The possible clinical significance of such ex vivo measured activities is at present a matter for speculation, and requires further

  17. Uveal Melanoma

    PubMed Central

    Papastefanou, Vasilios P.; Cohen, Victoria M. L.

    2011-01-01

    Uveal melanoma is the most common primary intraocular malignancy and the leading primary intraocular disease which can be fatal in adults. In this paper epidemiologic, pathogenetic, and clinical aspects of uveal melanoma are discussed. Despite the advance in local ocular treatments, there has been no change in patient survival for three decades. Development of metastases affects prognosis significantly. Current survival rates, factors predictive of metastatic potential and metastatic screening algorithms are discussed. Proposed and emerging treatments for uveal melanoma metastases are also overviewed. Current advances in genetics and cytogenetics have provided a significant insight in tumours with high metastatic potential and the molecular mechanisms that underlie their development. Biopsy of those lesions may prove to be important for prognostication and to allow further research into genetic mutations and potential new therapeutic targets in the future. PMID:21773036

  18. Expression of a Functional zipFv Antibody Fragment and Its Fusions with Alkaline Phosphatase in the Cytoplasm of an Escherichia coli

    PubMed Central

    Hur, Byung-ung; Choi, Hyo-jung; Yoon, Jae-bong

    2010-01-01

    Background Expression of recombinant antibodies and their derivatives fused with other functional molecules such as alkaline phosphatase in Escherichia coli is important in the development of molecular diagnostic reagents for biomedical research. Methods We investigated the possibility of applying a well-known Fos-Jun zipper to dimerize VH and VL fragments originated from the Fab clone (SP 112) that recognizes pyruvate dehydrogenase complex-E2 (PDC-E2), and demonstrated that the functional zipFv-112 and its alkaline phosphatase fusion molecules (zipFv-AP) can be produced in the cytoplasm of Origami(DE3) trxB gor mutant E. coli strain. Results The zipFv-AP fusion molecules exhibited higher antigen-binding signals than the zipFv up to a 10-fold under the same experimental conditions. However, conformation of the zipFv-AP seemed to be influenced by the location of an AP domain at the C-terminus of VH or VL domain [zipFv-112(H-AP) or zipFv-112(L-AP)], and inclusion of an AraC DNA binding domain at the C-terminus of VH of the zipFv-112(L-AP), termed zipFv-112(H-AD/L-AP), was also beneficial. Cytoplasmic co-expression of disulfide-binding isomerase C (DsbC) helped proper folding of the zipFv-112(H-AD/L-AP) but not significantly. Conclusion We believe that our zipFv constructs may serve as an excellent antibody format bi-functional antibody fragments that can be produced stably in the cytoplasm of E. coli. PMID:20532123

  19. 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.

  20. 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

  1. Crystallization and preliminary X-ray diffraction analysis of the complex between a human anti-alpha toxin antibody fragment and alpha toxin

    PubMed Central

    Oganesyan, Vaheh; Barnes, Arnita; Tkaczyk, Christine; Ferguson, Andrew; Wu, Herren; Dall’Acqua, William F.

    2013-01-01

    Staphylococcus aureus alpha toxin (AT) has been crystallized in complex with the Fab fragment of a human antibody (MEDI4893). This constitutes the first reported crystals of AT bound to an antibody. The monoclinic crystals belonged to space group P21, with unit-cell parameters a = 85.52, b = 148.50, c = 93.82 Å, β = 99.82°. The diffraction of the crystals extended to 2.56 Å resolution. The asymmetric unit contained two MEDI4893 Fab–AT complexes. This corresponds to a crystal volume per protein weight (V M) of 2.3 Å3 Da−1 and a solvent content of 47%. The three-dimensional structure of this complex will contribute to an understanding of the molecular basis of the interaction of MEDI4893 with AT. It will also shed light on the mechanism of action of this antibody, the current evaluation of which in the field of S. aureus-mediated diseases makes it a particularly interesting case study. Finally, this study will provide the three-dimensional structure of AT in a monomeric state for the first time. PMID:23519809

  2. 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

  3. 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.

  4. 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.

  5. 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

  6. A novel IFN-gamma regulated human melanoma associated antigen gp33-38 defined by monoclonal antibody Me14/D12. I. Identification and immunochemical characterization.

    PubMed

    Giuffré, L; Isler, P; Mach, J P; Carrel, S

    1988-09-15

    A novel melanoma-associated differentiation Ag whose surface expression can be enhanced or induced by IFN-gamma was identified by mAb Me14/D12. Testing of numerous tumor cell lines and tumor tissue sections showed that Me14/D12-defined Ag was present not only on melanoma but also on other tumor lines of neuroectodermal origin such as gliomas and neuroblastomas and on some lymphoblastic B cell lines, on monocytes and macrophages. Immunoprecipitation by mAb Me14/D12 of lysates from [35S]methionine-labeled melanoma cells analyzed by SDS-PAGE revealed two polypeptide chains of 33 and 38 KDa, both under reducing and nonreducing conditions. Cross-linking experiments indicated that the two chains were present at the cell surface as a dimeric structure. Two-dimensional gel electrophoresis showed that the two chains of 33 and 38 KDa had isoelectric points of 6.2 and 5.7, respectively. Treatment of the melanoma cells with tunicamycin, an inhibitor of N-linked glycosylation, resulted in a reduction of the Mr from 33 to 24 KDa and from 38 to 26 KDa. Peptide maps obtained after Staphylococcus aureus V8 protease digestion showed no shared peptides between the two chains. Although biochemical data indicate that Me14/D12 molecules do not correspond to any known MHC class II Ag, their dimeric structure, tissue distribution, and regulation of IFN-gamma suggest that they could represent a new member of the MHC class II family. PMID:3139751

  7. 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

  8. The Fab Fragment of a Humanized Anti-Toll Like Receptor 4 (TLR4) Monoclonal Antibody Reduces the Lipopolysaccharide Response via TLR4 in Mouse Macrophage.

    PubMed

    Cai, Binggang; Wang, Maorong; Zhu, Xuhui; Xu, Jing; Zheng, Wenkai; Zhang, Yiqing; Zheng, Feng; Feng, Zhenqing; Zhu, Jin

    2015-01-01

    Lipopolysaccharides (LPS) can induce acute inflammation, sepsis, or chronic inflammatory disorders through the Toll receptor 4 (TLR4) signaling pathway. The TLR4/MD2 (myeloid differentiation protein 2) complex plays a major role in the immune response to LPS. However, there is not a good method to suppress the immune response induced by LPS via this complex in macrophages. In this article, we aimed to evaluate the effects of humanized anti-TLR4 monoclonal antibodies on LPS-induced responses in mouse macrophages. The peritoneal macrophages of mice were incubated with anti-TLR4 monoclonal antibodies and stimulated with LPS. The expression levels of cytokines were analyzed by quantitative polymerase chain reaction and enzyme-linked immunosorbent assays. Additionally, activation of various signaling pathways was evaluated by Western blotting. The results showed that the humanized anti-TLR4 monoclonal antibody blocked the inflammatory cytokines expression at both the mRNA and protein level. We also found that the Fab fragment significantly inhibited the nuclear factor kappaB signaling pathway by reducing the phosphorylation of the inhibitor of kappaBalpha and decreasing the translocation of p65, resulting in the suppression of p38, extracellular signal-regulated kinase 1/2, c-Jun N-terminal kinase 1/2, and IFN-β regulatory factor 3 phosphorylation. Therefore, our study showed that this humanized anti-TLR4 monoclonal antibody could effectively protect against LPS-induced responses by blocking the TLR4 signaling pathway in mouse peritoneal macrophages.

  9. 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

  10. 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.

  11. Myeloid Cells' Evasion of Melanoma Immunity

    PubMed Central

    Wang, Jun; Chen, Lieping

    2015-01-01

    An immune-suppressive role of myeloid-derived suppressor cells (MDSCs) in melanoma has long been speculated, whereas molecular mechanisms underlying this role are not well understood. Here, Chung and colleagues show that dendritic cell-associated, heparan sulfate proteoglycans-dependent integrin ligand (DC-HIL), a cell surface immune-modulatory molecule, is highly expressed on tumor-associated MDSCs. Genetic ablation or antibody blockade of DC-HIL delays the growth of transplantable B16 melanoma in syngeneic mice, which is accompanied by enhanced antitumor T-cell activities. These findings support a role for DC-HIL in immune evasion within the melanoma microenvironment. PMID:25318429

  12. Diffusion and binding of monoclonal antibody TNT-1 in multicellular tumor spheroids

    SciTech Connect

    Cheng, F.M.; Hansen, E.B.; Taylor, C.R.; Epstein, A.L. )

    1991-02-06

    Tumor spheroids of HT-29 human colon adenocarcinoma and A375 melanoma were established to investigate the uptake and clearance kinetics of TNT-1, a monoclonal antibody that targets necrotic cells of tumors. Our data reveal that there was rapid uptake of TNT-1 and its F(ab')2 fragment in both spheroid models, whereas an antibody of irrelevant specificity, Lym-1, and its F(ab')2 fragment bound poorly to the spheroids. Unlike previously reported monoclonal antibodies to tumor cell-surface antigens, TNT-1 showed (1) a linear uptake that increased over time without saturation in tumor spheroids and (2) an unexpected uptake by a subpopulation of cells in the viable outer rim of the spheroids. These preclinical studies provide important information concerning the therapeutic potential of TNT monoclonal antibodies for the treatment of cancer and micrometastases.

  13. Malignant Melanoma of the Foot

    MedlinePlus

    ... Javascript in your browser. Malignant Melanoma of the Foot What is Malignant Melanoma? Melanoma is a cancer ... age groups, even the young. Melanoma in the Foot Melanoma that occurs in the foot or ankle ...

  14. Cell surface phosphatidylinositol-anchored heparan sulfate proteoglycan initiates mouse melanoma cell adhesion to a fibronectin-derived, heparin-binding synthetic peptide

    PubMed Central

    1992-01-01

    Cell surface heparan sulfate proteoglycan (HSPG) from metastatic mouse melanoma cells initiates cell adhesion to the synthetic peptide FN-C/H II, a heparin-binding peptide from the 33-kD A chain-derived fragment of fibronectin. Mouse melanoma cell adhesion to FN-C/H II was sensitive to soluble heparin and pretreatment of mouse melanoma cells with heparitinase. In contrast, cell adhesion to the fibronectin synthetic peptide CS1 is mediated through an alpha 4 beta 1 integrin and was resistant to heparin or heparitinase treatment. Mouse melanoma cell HSPG was metabolically labeled with [35S]sulfate and extracted with detergent. After HPLC-DEAE purification, 35S-HSPG eluted from a dissociative CL-4B column with a Kav approximately 0.45, while 35S- heparan sulfate (HS) chains eluted with a Kav approximately 0.62. The HSPG contained a major 63-kD core protein after heparitinase digestion. Polyclonal antibodies generated against HSPG purified from mouse melanoma cells grown in vivo also identified a 63-kD core protein. This HSPG is an integral plasma membrane component by virtue of its binding to Octyl Sepharose affinity columns and that anti-HSPG antibody staining exhibited a cell surface localization. The HSPG is anchored to the cell surface through phosphatidylinositol (PI) linkages, as evidenced in part by the ability of PI-specific phospholipase C to eliminate binding of the detergent-extracted HSPG to Octyl Sepharose. Furthermore, the mouse melanoma HSPG core protein could be metabolically labeled with 3H-ethanolamine. The involvement of mouse melanoma cell surface HSPG in cell adhesion to fibronectin was also demonstrated by the ability of anti-HSPG antibodies and anti-HSPG IgG Fab monomers to inhibit mouse melanoma cell adhesion to FN-C/H II. 35S- HSPG and 35S-HS bind to FN-C/H II affinity columns and require 0.25 M NaCl for elution. However, heparitinase-treated 125I-labeled HSPG failed to bind FN-C/H II, suggesting that HS, and not HSPG core protein, binds FN

  15. 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

  16. 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.

  17. Differential response of human thymus cells to CD2 antibodies: fragmentation of DNA of CD45RO+ and proliferation of CD45RO- subsets.

    PubMed Central

    Li, J; Campbell, D; Hayward, A R

    1992-01-01

    Human thymocytes bearing the CD45RO 'memory' cell phenotype do not proliferate in concanavalin A (Con A)-stimulated cultures and may be destined for intrathymic death. To determine whether this subset would exhibit characteristics of programmed cell death (apoptosis), we examined the integrity of the nuclear DNA by gel electrophoresis. DNA fragmentation was restricted to the CD45RO+ subset of human thymocytes following exposure to stimulating concentrations of anti-CD2 antibodies. Both CD45RO- and CD45RO+ subsets mobilized cytoplasmic Ca2+ following cell-surface CD2 ligation, but entry into the cell cycle and vigorous thymidine uptake were restricted to the CD45RO- subset. Our results provide a mechanism which may account for the failure of thymic CD45RO+ cells to respond to stimuli which elicit proliferation by the reciprocal CD45RA+ subset. Images Figure 1 Figure 2 PMID:1348052

  18. A case of Mobitz type II atrioventricular block due to Nerium oleander poisoning successfully managed with digoxin-specific Fab antibody fragments.

    PubMed

    Tatlısu, Mustafa Adem; Çekirdekçi, Elif İjlal; Akyüz, Şükrü; Nurkalem, Zekeriya

    2015-10-01

    Nerium oleander is a popular ornamental plant grown in many tropical and subtropical countries and in the Mediterranean region. It is dangerous because it has been shown to contain several types of cardiac glycosides, and hence can cause cardiac arrhythmias resembling digoxin in their toxicologic manifestations. We report a patient presenting to our hospital with Mobitz type II atrioventricular block after drinking herbal tea prepared from oleander leaves. Three hours after admission, a 200-mg empiric dose of digoxin-specific Fab antibody fragments was administered intravenously over 30 minutes. A 12-lead electrocardiogram (ECG) revealed sinus rhythm at the end of infusion. After 72 hours, the patient was discharged without any symptoms. PMID:26536992

  19. Epitope mapping of antigenic MUC1 peptides to breast cancer antibody fragment B27.29: a heteronuclear NMR study.

    PubMed

    Grinstead, Jeffrey S; Schuman, Jason T; Campbell, A Patricia

    2003-12-01

    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], (1)H 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 (15)N and (13)C 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). (15)N and (13)C NMR relaxation parameters were measured for both peptides free in solution and bound to Fab B27.29. The (13)C(alpha) T(1) 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 (15)N- and (13)C-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

  20. 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.

  1. 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

  2. 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

  3. 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

  4. [Focus on digitalis intoxication in the elderly. Report of a case treated with digoxin-specific Fab antibody fragments].

    PubMed

    Pautas, Éric; Lopez, Clara; Gouronnec, Adeline; Gravelaine, Sophie; Peyron, Isabelle; Lapostolle, Frédéric

    2012-12-01

    The indications for digoxin are currently limited to rare cases of heart failure and/or atrial fibrillation. Its use should be even more rare in geriatrics its pharmacological characteristics, associated with age-related changes and comorbidities, particularly increase the risk of digoxin poisoning in the elderly. However, at least a third of aged patients suffering from heart failure and/or atrial fibrillation is treated by digitalis. Digoxin intoxication can provoke gastrointestinal troubles, neurological disturbances and, above all, cardiac conduction impairment and dysrythmias, which explain its severity and high mortality rate. Presently, first-line therapy is the administration of digoxin specific antibodies. Poor prognosis factors, frequently found in digoxin intoxications in the elderly, have been established for guiding the prescription of antibodies and their dosage. It is important for geriatricians to be able to recognize poisoning signs and the conditions in which an antidote treatment is necessary. This will permit a more effective management of the case, with the support of a poison control center and possible referral of the patient to an intensive care unit.

  5. Chimpanzee Fab fragments and a derived humanized immunoglobulin G1 antibody that efficiently cross-neutralize dengue type 1 and type 2 viruses.

    PubMed

    Goncalvez, Ana P; Men, Ruhe; Wernly, Claire; Purcell, Robert H; Lai, Ching-Juh

    2004-12-01

    Passive immunization with monoclonal antibodies from humans or nonhuman primates represents an attractive alternative to vaccines for prevention of illness caused by dengue viruses (DENV) and other flaviviruses, including the West Nile virus. In a previous study, repertoire cloning to recover Fab fragments from bone marrow mRNA of chimpanzees infected with all four DENV serotypes (dengue virus serotype 1 [DENV-1] to DENV-4) was described. In that study, a humanized immunoglobulin G1 (IgG1) antibody that efficiently neutralized DENV-4 was recovered and characterized. In this study, the phage library constructed from the chimpanzees was used to recover Fab antibodies against the other three DENV serotypes. Serotype-specific neutralizing Fabs were not identified. Instead, we recovered DENV-neutralizing Fabs that specifically precipitated the envelope protein and were cross-reactive with all four DENV serotypes. Three of the Fabs competed with each other for binding to DENV-1 and DENV-2, although each of these Fabs contained a distinct complementarity determining region 3 (CDR3)-H sequence. Fabs that shared an identical or nearly identical CDR3-H sequences cross-neutralized DENV-1 and DENV-2 at a similar high 50% plaque reduction neutralization test (PRNT(50)) titer, ranging from 0.26 to 1.33 microg/ml, and neutralized DENV-3 and DENV-4 but at a titer 10- to 20-fold lower. One of these Fabs, 1A5, also neutralized the West Nile virus most efficiently among other flaviviruses tested. Fab 1A5 was converted to a full-length antibody in combination with human sequences for production in mammalian CHO cells. Humanized IgG1 1A5 proved to be as efficient as Fab 1A5 for cross-neutralization of DENV-1 and DENV-2 at a titer of 0.48 and 0.95 microg/ml, respectively. IgG1 1A5 also neutralized DENV-3, DENV-4, and the West Nile virus at a PRNT(50) titer of approximately 3.2 to 4.2 microg/ml. This humanized antibody represents an attractive candidate for further development of

  6. Plasma levels of plasminogen activator inhibitor type 1, factor VIII, prothrombin activation fragment 1+2, anticardiolipin, and antiprothrombin antibodies are risk factors for thrombosis in hemodialysis patients.

    PubMed

    Molino, Daniela; De Santo, Natale G; Marotta, Rosa; Anastasio, Pietro; Mosavat, Mahrokh; De Lucia, Domenico

    2004-09-01

    Patients with end-stage renal disease are prone to hemorrhagic complications and simultaneously are at risk for a variety of thrombotic complications such as thrombosis of dialysis blood access, the subclavian vein, coronary arteries, cerebral vessel, and retinal veins, as well as priapism. The study was devised for the following purposes: (1) to identify the markers of thrombophilia in hemodialyzed patients, (2) to establish a role for antiphospholipid antibodies in thrombosis of the vascular access, (3) to characterize phospholipid antibodies in hemodialysis patients, and (4) to study the effects of dialysis on coagulation cascade. A group of 20 hemodialysis patients with no thrombotic complications (NTC) and 20 hemodialysis patients with thrombotic complications (TC) were studied along with 400 volunteer blood donors. Patients with systemic lupus erythematosus and those with nephrotic syndrome were excluded. All patients underwent a screening prothrombin time, activated partial thromboplastin time, fibrinogen (Fg), coagulation factors of the intrinsic and extrinsic pathways, antithrombin III (AT-III), protein C (PC), protein S (PS), resistance to activated protein C, prothrombin activation fragment 1+2 (F1+2), plasminogen, tissue type plasminogen activator (t-PA), plasminogen tissue activator inhibitor type-1 (PAI-1), anticardiolipin antibodies type M and G (ACA-IgM and ACA-IgG), lupus anticoagulant antibodies, and antiprothrombin antibodies type M and G (aPT-IgM and aPT-IgG). The study showed that PAI-1, F 1+2, factor VIII, ACA-IgM, and aPT-IgM levels were increased significantly over controls both in TC and NTC, however, they could distinguish patients with thrombotic complications from those without, being increased maximally in the former group. The novelty of the study is represented by the significant aPT increase that was observed in non-systemic lupus erythematosus hemodialysis patients, and particularly in those with thrombotic events. In addition

  7. 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.

  8. 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).

  9. Multinuclear NMR study of the structure of the Fv fragment of anti-dansyl mouse IgG2a antibody

    SciTech Connect

    Takahashi, Hideo; Odaka, Asano; Matsunaga, Chigusa; Kato, Koichi; Shimada, Ichio; Arata, Yoji ); Kawaminami, Shunro )

    1991-07-02

    A multinuclear NMR study is reported of Fv, which is a minimum antigen-binding unit of immunoglobulin. Fv has been prepared by clostripain digestion of a mouse anti-dansyl IgG2a monoclonal antibody that lacks the entire C{sub H}1 domain. A variety of Fv analogues labeled with {sup 2}H in the aromatic rings and with {sup 13}C and/or {sup 15}N in the peptide bonds have been prepared and used for multinuclear NMR analyses of Fv spectra of Fv sensitively reflect the antigen binding and can be used along with {sup 1}H and {sup 13}C spectral data for the structural analyses of antigen-antibody interactions. Hydrogen-deuterium exchange of the amide protons has been folowed in the absence and presence of DNS-Lys by using the {sup 1}H-{sup 15}N shift correlation spectra. Use of the {beta}-shift observed for the carbonyl carbon resonances has also been helpful in following the hydrogen-deuterium exchange. On the basis of the NMR data obtained, the static and dynamic structure of the Fv fragment in the absence and presence of DNS-Lys has been discussed.

  10. 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

  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 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

  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

    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.

  13. 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.

  14. 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

  15. Cross-Neutralization Activity of Single-Chain Variable Fragment (scFv) Derived from Anti-V3 Monoclonal Antibodies Mediated by Post-Attachment Binding.

    PubMed

    Maruta, Yasuhiro; Kuwata, Takeo; Tanaka, Kazuki; Alam, Muntasir; Valdez, Kristel Paola Ramirez; Egami, Yoshika; Suwa, Yoshiaki; Morioka, Hiroshi; Matsushita, Shuzo

    2016-09-21

    The V3 loop in the envelope (Env) of HIV-1 is one of the major targets of neutralizing antibodies. However, this antigen is hidden inside the Env trimer in most isolates and is fully exposed only during CD4-gp120 interaction. Thus, primary HIV-1 isolates are relatively resistant to anti-V3 antibodies because IgG is too large to access the V3 loop. To overcome this obstacle, we constructed single-chain variable fragments (scFvs) from anti-V3 monoclonal antibodies 0.5γ, 5G2, and 16G6. Enhanced neutralization by 0.5γ and 5G2 scFvs was observed in strains resistant to their IgG counterparts. Neutralization coverage by 0.5γ scFv reached up to 90% of the tested viruses (tier 2 and 3 classes). The temperature-regulated neutralization assay revealed that extensive cross-neutralization of 0.5γ scFv can be explained by post-attachment neutralization. Neutralization assay involving viruses carrying an inter-subunit disulfide bond (SOS virus) showed that the neutralization-susceptible timeframe after attachment was 60 to 120 min. These results indicate that the scFvs efficiently access the V3 loop and subsequently neutralize HIV-1, even after virus attachment to the target cells. Based on its broad and potent neutralizing activity, further development of anti-V3 scFv for therapeutic and preventive strategies is warranted.

  16. 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.

  17. 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

  18. 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

  19. 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

  20. 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

    2015-07-10

    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.

  1. Novel camelid antibody fragments targeting recombinant nucleoprotein of Araucaria hantavirus: a prototype for an early diagnosis of Hantavirus Pulmonary Syndrome.

    PubMed

    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

  2. Structures of synthetic O-antigen fragments from serotype 2a Shigella flexneri in complex with a protective monoclonal antibody.

    PubMed

    Vulliez-Le Normand, B; Saul, F A; Phalipon, A; Bélot, F; Guerreiro, C; Mulard, L A; Bentley, G A

    2008-07-22

    The anti-LPS IgG mAb F22-4, raised against Shigella flexneri serotype 2a bacteria, protects against homologous, but not heterologous, challenge in an experimental animal model. We report the crystal structures of complexes formed between Fab F22-4 and two synthetic oligosaccharides, a decasaccharide and a pentadecasaccharide that were previously shown to be both immunogenic and antigenic mimics of the S. flexneri serotype 2a O-antigen. F22-4 binds to an epitope contained within two consecutive 2a serotype pentasaccharide repeat units (RU). Six sugar residues from a contiguous nine-residue segment make direct contacts with the antibody, including the nonreducing rhamnose and both branching glucosyl residues from the two RUs. The glucosyl residue, whose position of attachment to the tetrasaccharide backbone of the RU defines the serotype 2a O-antigen, is critical for recognition by F22-4. Although the complete decasaccharide is visible in the electron density maps, the last four pentadecasaccharide residues from the reducing end, which do not contact the antibody, could not be traced. Although considerable mobility in the free oligosaccharides can thus be expected, the conformational similarity between the individual RUs, both within and between the two complexes, suggests that short-range transient ordering to a helical conformation might occur in solution. Although the observed epitope includes the terminal nonreducing residue, binding to internal epitopes within the polysaccharide chain is not precluded. Our results have implications for vaccine development because they suggest that a minimum of two RUs of synthetic serotype 2a oligosaccharide is required for optimal mimicry of O-Ag epitopes.

  3. Novel approaches in melanoma prevention and therapy.

    PubMed

    Grimaldi, Antonio M; Cassidy, Pamela B; Leachmann, Sancy; Ascierto, Paolo A

    2014-01-01

    second BRAF inhibitor, in phase I and II trial obtained similar results to vemurafenib. A phase III trial is now ongoing. Taken together, the early clinical development of vemurafenib and dabrafenib clearly confirms that BRAF inhibitors can halt or reverse disease in patients with melanomas carrying this mutation, improving survival times compared with historically standard treatments (chemotherapy and interleukin-2). The clinical development of other new BRAF inhibitors such as RAF265 and LGX818 is now ongoing. Combination strategies of BRAF inhibitors with ipilimumab, an anti-CTLA-4 antibody, and/or MEK inhibitors or metformin are now under investigation in clinical trials.

  4. 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.

  5. Antibody fragment-armed mesoporous silica nanoparticles for the targeted delivery of bevacizumab in ovarian cancer cells.

    PubMed

    Zhang, Ying; Guo, Jing; Zhang, Xiao-Ling; Li, Da-Peng; Zhang, Ting-Ting; Gao, Fu-Feng; Liu, Nai-Fu; Sheng, Xiu-Gui

    2015-12-30

    In order to enhance the therapeutic efficacy and intracellular concentration of bevacizumab (BVC), we have designed a novel tumor endothelial marker 1 (TEM1)/endosialin (Ab-/scFv)-conjugated mesoporous silica nanoparticles (MSN) to target ovarian cancer cell. The Ab-/scFv-conjugated MSN were prepared by the conjugation of amine functional group of antibody of the carboxyl group of MSN. The resultant MSN was nanosized, spherical shaped, and exhibited a controlled release phenomenon in pH 7.4 conditions. Furthermore, BMSN/Ab was found to increase the cellular uptake and intracellular distribution of BVC in OVCAR-5 cancer cells. The Ab- conjugated MSN exhibited a superior anticancer effect with profound apoptosis in cancer cells in a time- and concentration dependent manner. Consistently, BMSN/Ab effectively inhibited the colony formation in transwell plate. Finally, BMSN/Ab showed a notable increase in the proportion of cells in G2/M phase of cell cycle indicating promising anticancer efficacy profile. Overall, Ab-/scFv-conjugated MSN might provide an effective strategy for the therapeutic management of ovarian cancers.

  6. 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

  7. Single chain fragment variable antibodies developed by using as target the 3rd fibronectin type III homologous repeat fragment of human neural cell adhesion molecule L1 promote cell migration and neuritogenesis.

    PubMed

    Tang, Dan-Yang; Yu, Yang; Zhao, Xuan-Jun; Schachner, Melitta; Zhao, Wei-Jiang

    2015-01-15

    L1CAM plays important roles during ontogeny, including promotion of neuronal cell migration and neuritogenesis, and stimulation of axonal outgrowth, fasciculation and myelination. These functions are at least partially exerted through a 16-mer amino acid sequence in the third fibronectin type III-like repeat of L1, which associates with several interaction partners, including integrins, other adhesion molecules and growth factor receptors. Here, using the Tomlinson I library for phage display, we obtained two single-chain variable fragment antibodies (scFvs) against this peptide sequence of human L1, hereafter called H3 peptide. Both scFvs recognize the H3 peptide and the extracellular domain of L1, as tested by enzyme-linked immunosorbent assay (ELISA), Western blot analysis and immunofluorescence staining of L1 expresssing cells. Furthermore, both scFvs reduce U-87 MG cell adhesion to fibronectin, while stimulating cell migration. Application of scFvs to human neuroblastoma SK-N-SH cells promote process outgrowth. Similar to triggering of endogenous L1 functions at the cell surface, both scFvs activate the signal transducers Erk and Src in these cells. Our results indicate that scFvs against a functionally pivotal domain in L1 trigger its regeneration-beneficial functions in vitro, encouraging thoughts on therapy of neurodegenerative diseases in the hope to ameliorate human nervous system diseases. PMID:25447207

  8. Fully Regressive Melanoma

    PubMed Central

    Ehrsam, Eric; Kallini, Joseph R.; Lebas, Damien; Modiano, Philippe; Cotten, Hervé

    2016-01-01

    Fully regressive melanoma is a phenomenon in which the primary cutaneous melanoma becomes completely replaced by fibrotic components as a result of host immune response. Although 10 to 35 percent of cases of cutaneous melanomas may partially regress, fully regressive melanoma is very rare; only 47 cases have been reported in the literature to date. AH of the cases of fully regressive melanoma reported in the literature were diagnosed in conjunction with metastasis on a patient. The authors describe a case of fully regressive melanoma without any metastases at the time of its diagnosis. Characteristic findings on dermoscopy, as well as the absence of melanoma on final biopsy, confirmed the diagnosis.

  9. Fully Regressive Melanoma

    PubMed Central

    Ehrsam, Eric; Kallini, Joseph R.; Lebas, Damien; Modiano, Philippe; Cotten, Hervé

    2016-01-01

    Fully regressive melanoma is a phenomenon in which the primary cutaneous melanoma becomes completely replaced by fibrotic components as a result of host immune response. Although 10 to 35 percent of cases of cutaneous melanomas may partially regress, fully regressive melanoma is very rare; only 47 cases have been reported in the literature to date. AH of the cases of fully regressive melanoma reported in the literature were diagnosed in conjunction with metastasis on a patient. The authors describe a case of fully regressive melanoma without any metastases at the time of its diagnosis. Characteristic findings on dermoscopy, as well as the absence of melanoma on final biopsy, confirmed the diagnosis. PMID:27672418

  10. Cutaneous Melanoma in Asians

    PubMed Central

    Kim, Sang Yub

    2016-01-01

    Malignant melanoma is a rare disease in Asians but potentially the most aggressive form of skin cancer worldwide. It can occur in any melanocyte-containing anatomic site. Four main cutaneous melanoma subtypes are recognized: lentigo maligna melanoma, superficial spreading melanoma, acral lentiginous melanoma (ALM), and nodular melanoma. Generally, excessive exposure to ultraviolet (UV) radiation increases the risk of melanoma. The exception is ALM, which is the most common melanoma subtype in Asians and is not associated with UV radiation. ALM presents as dark brownish to black, irregular maculopatches, nodules, or ulcers on the palms, soles, and nails. The lesions may be misdiagnosed as more benign lesions, such as warts, ulcers, hematomas, foreign bodies, or fungal infections, especially in amelanotic acral melanomas where black pigments are absent. The aim of this brief review is to improve understanding and the rate of early detection thereby reducing mortality, especially regarding cutaneous melanoma in Asians. PMID:27689028

  11. Nutrition and melanoma prevention.

    PubMed

    Jensen, J Daniel; Wing, Gregory J; Dellavalle, Robert P

    2010-01-01

    Melanoma has continued to rise in incidence despite public efforts to promote sun protection behaviors. Because sunscreen use does not completely prevent skin cancer induced by ultraviolet radiation, additional chemopreventive methods for protecting against and reversing the effects of ultraviolet photodamage need evaluation. Recent years have brought increased interest in dietary factors, such as natural botanicals and vitamins, for the prevention of melanoma. This contribution provides a narrative review of the relevant, nutrition-related literature found by searching the keywords "melanoma chemoprevention," "nutrition and melanoma," "dietary botanicals and melanoma prevention," "green tea and melanoma," "vitamin D and melanoma," and "vitamin E and melanoma" in the PubMed database. Although randomized controlled trials of humans are lacking, basic science and epidemiologic studies show promising benefits of many natural products in chemoprevention for melanoma. Future studies, hopefully, will yield concrete answers and clarify the role of commonly available dietary nutrients in melanoma chemoprevention.

  12. Cutaneous Melanoma in Asians.

    PubMed

    Kim, Sang Yub; Yun, Sook Jung

    2016-09-01

    Malignant melanoma is a rare disease in Asians but potentially the most aggressive form of skin cancer worldwide. It can occur in any melanocyte-containing anatomic site. Four main cutaneous melanoma subtypes are recognized: lentigo maligna melanoma, superficial spreading melanoma, acral lentiginous melanoma (ALM), and nodular melanoma. Generally, excessive exposure to ultraviolet (UV) radiation increases the risk of melanoma. The exception is ALM, which is the most common melanoma subtype in Asians and is not associated with UV radiation. ALM presents as dark brownish to black, irregular maculopatches, nodules, or ulcers on the palms, soles, and nails. The lesions may be misdiagnosed as more benign lesions, such as warts, ulcers, hematomas, foreign bodies, or fungal infections, especially in amelanotic acral melanomas where black pigments are absent. The aim of this brief review is to improve understanding and the rate of early detection thereby reducing mortality, especially regarding cutaneous melanoma in Asians. PMID:27689028

  13. Cutaneous Melanoma in Asians

    PubMed Central

    Kim, Sang Yub

    2016-01-01

    Malignant melanoma is a rare disease in Asians but potentially the most aggressive form of skin cancer worldwide. It can occur in any melanocyte-containing anatomic site. Four main cutaneous melanoma subtypes are recognized: lentigo maligna melanoma, superficial spreading melanoma, acral lentiginous melanoma (ALM), and nodular melanoma. Generally, excessive exposure to ultraviolet (UV) radiation increases the risk of melanoma. The exception is ALM, which is the most common melanoma subtype in Asians and is not associated with UV radiation. ALM presents as dark brownish to black, irregular maculopatches, nodules, or ulcers on the palms, soles, and nails. The lesions may be misdiagnosed as more benign lesions, such as warts, ulcers, hematomas, foreign bodies, or fungal infections, especially in amelanotic acral melanomas where black pigments are absent. The aim of this brief review is to improve understanding and the rate of early detection thereby reducing mortality, especially regarding cutaneous melanoma in Asians.

  14. 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

  15. 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.

  16. Cloning and in vitro expression of a melanoma-associated antigen immunogenic in patients with melanoma.

    PubMed

    Hayashibe, K; Mishima, Y; Ferrone, S

    1991-08-01

    The purpose of this study was to identify human melanoma-associated Ag (MAA) that are immunogenic in patients, because these molecules may be useful immunogens to implement active specific immunotherapy. To this end, an expression cDNA library constructed from the human melanoma cell line A375 was screened with sera from patients with melanoma. A 1029-bp cDNA (designated D-1) was isolated. Its nucleotide sequence showed no significant homology with viral and mammalian sequences stored in GE-NETYX. cDNA D-1 hybridized to a 2.0-kb mRNA species from human melanoma, neuroblastoma, erythroleukemia, B lymphoid, and T lymphoid cell lines but not from a renal carcinoma cell line, PBL, and cultured skin fibroblasts. The D-1 clone produced a fusion protein that displayed a significantly higher reactivity with sera from patients with melanoma than from healthy controls. Furthermore, D-1 fusion protein induced in mice antibodies that immunoprecipitated a 50-kDa component from cultured human melanoma cells. The structural properties of D-1 MAA are different from those of previously described MAA. These results suggest that the approach we have applied may be useful to identify novel MAA expressed by melanoma cells. Furthermore, the immunogenicity of recombinant D-1 protein suggests that it may be a valuable immunogen to implement active specific immunotherapy in patients with melanoma, if additional experiments show that it has the appropriate tissue distribution.

  17. Recombinant Interferon Alfa-2b in Treating Patients With Melanoma

    ClinicalTrials.gov

    2016-05-17

    Stage IA Skin Melanoma; Stage IB Skin Melanoma; Stage IIA Skin Melanoma; Stage IIB Skin Melanoma; Stage IIC Skin Melanoma; Stage IIIA Skin Melanoma; Stage IIIB Skin Melanoma; Stage IIIC Skin Melanoma; Stage IV Skin Melanoma

  18. Allergen-specific regulation of allergic rhinitis in mice by intranasal exposure to IgG1 monoclonal antibody Fab fragments against pathogenic allergen.

    PubMed

    Matsuoka, Daiko; Mizutani, Nobuaki; Sae-Wong, Chutha; Yoshino, Shin

    2014-09-01

    Fab fragments (Fabs) have the ability to bind to specific antigens but lack the Fc portion for binding to receptors on immune and inflammatory cells that play a critical role in allergic diseases. In the present study, we investigated whether Fabs of an allergen-specific IgG1 monoclonal antibody (mAb) inhibited allergic rhinitis in mice. BALB/c mice sensitized by intraperitoneal injections of ovalbumin (OVA) plus alum on days 0 and 14 were intranasally challenged with OVA on days 28-30, and 35. Fabs prepared by the digestion of an anti-OVA IgG1 mAb (O1-10) with papain were also intranasally administered 15min before each OVA challenge. The results showed that treatment with O1-10 Fabs significantly suppressed the sneezing frequency, associated with decrease of OVA-specific IgE in the serum and infiltration by mast cells in the nasal mucosa seen following the fourth antigenic challenge; additionally, the level of mouse mast cell protease-1, a marker of mast cell activation, in serum was decreased. Furthermore, infiltration of eosinophils and goblet cell hyperplasia in the nasal mucosa at the fourth challenge were inhibited by treatment with O1-10 Fabs. In conclusion, these results suggest that intranasal exposure to Fabs of a pathogenic antigen-specific IgG1 mAb may be effective in regulating allergic rhinitis through allergen capture by Fabs in the nasal mucosa before the interaction of the intact antibody and allergen.

  19. 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.

  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. Immunoscintigraphy of adenocarcinomas by means of radiolabeled F(ab')2 fragments of an anti-carcinoembryonic antigen monoclonal antibody: A multicenter study

    SciTech Connect

    Siccardi, A.G.; Buraggi, G.L.; Callegaro, L.; Colella, A.C.; De Filippi, P.G.; Galli, G.; Mariani, G.; Masi, R.; Palumbo, R.; Riva, P. )

    1989-06-01

    F(ab')2 fragments of anti-carcinoembryonic antigen (CEA) monoclonal antibody F023C5, determined to be more suitable than intact IgG and Fab fragments for immunoscintigraphy, were labeled with 131I or conjugated to DTPA for instant 111In-labeling, and administered i.v. (2-3 mCi/0.5 mg) to 509 patients in 11 nuclear medicine departments: 284 patients had gastrointestinal adenocarcinomas, 204 had nongastrointestinal adenocarcinomas and 21 were control; serum CEA was elevated in 169 patients, normal in 115, and not determined in 225. The following results were obtained: (a) no adverse reactions; (b) tumor imaging in 324 patients (in particular, in 81.5% CEA-seropositive and in 69.0% CEA-seronegative patients); (c) no significant difference in sensitivity among the results of the 11 departments; (d) no significant difference in overall sensitivity between 131I-and 111In-labeled immunoradiopharmaceuticals; (e) the fraction of documented lesions imaged was 73.3% in CEA-seropositive and 53.7% in CEA-seronegative patients; (f) the detection of liver metastases was hampered, particularly when using the 111In-labeled reagent, by nonspecific radioactivity uptake; (g) the major cause of negative immunoscintigraphy results was a lack of CEA in tumor lesions, as documented by immunohistochemistry; (h) lesion size is also important since the sensitivity was 64% for lesions up to 2 cm in diameter and 84% for larger lesions; (i) many unexpected radiolocalizations were recorded. Most were identified as occult tumor lesions. In 35 patients, this finding contributed to the early detection of tumor recurrences.

  2. Axitinib in Treating Patients With Melanoma That is Metastatic or Cannot Be Removed by Surgery

    ClinicalTrials.gov

    2016-05-31

    Extraocular Extension Melanoma; Metastatic Intraocular Melanoma; Recurrent Intraocular Melanoma; Recurrent Melanoma; Stage IIIA Intraocular Melanoma; Stage IIIA Melanoma; Stage IIIB Intraocular Melanoma; Stage IIIB Melanoma; Stage IIIC Intraocular Melanoma; Stage IIIC Melanoma; Stage IV Intraocular Melanoma; Stage IV Melanoma

  3. Engineering anti-GD2 monoclonal antibodies for cancer immunotherapy.

    PubMed

    Ahmed, Mahiuddin; Cheung, Nai-Kong V

    2014-01-21

    Ganglioside GD2 is highly expressed on neuroectoderm-derived tumors and sarcomas, including neuroblastoma, retinoblastoma, melanoma, small cell lung cancer, brain tumors, osteosarcoma, rhabdomyosarcoma, Ewing's sarcoma in children and adolescents, as well as liposarcoma, fibrosarcoma, leiomyosarcoma and other soft tissue sarcomas in adults. Since GD2 expression in normal tissues is restricted to the brain, which is inaccessible to circulating antibodies, and in selected peripheral nerves and melanocytes, it was deemed a suitable target for systemic tumor immunotherapy. Anti-GD2 antibodies have been actively tested in clinical trials for neuroblastoma for over the past two decades, with proven safety and efficacy. The main limitations have been acute pain toxicity associated with GD2 expression on peripheral nerve fibers and the inability of antibodies to treat bulky tumor. Several strategies have been developed to reduce pain toxicity, including bypassing complement activation, using blocking antibodies, or targeting of O-acetyl-GD2 derivative that is not expressed on peripheral nerves. To enhance anti-tumor efficacy, anti-GD2 monoclonal antibodies and fragments have been engineered into immunocytokines, immunotoxins, antibody drug conjugates, radiolabeled antibodies, targeted nanoparticles, T-cell engaging bispecific antibodies, and chimeric antigen receptors. The challenges of these approaches will be reviewed to build a perspective for next generation anti-GD2 therapeutics in cancer therapy.

  4. Plasma levels of plasminogen activator inhibitor type 1, factor VIII, prothrombin activation fragment 1+2, anticardiolipin, and antiprothrombin antibodies are risk factors for thrombosis in hemodialysis patients.

    PubMed

    Molino, Daniela; De Santo, Natale G; Marotta, Rosa; Anastasio, Pietro; Mosavat, Mahrokh; De Lucia, Domenico

    2004-09-01

    Patients with end-stage renal disease are prone to hemorrhagic complications and simultaneously are at risk for a variety of thrombotic complications such as thrombosis of dialysis blood access, the subclavian vein, coronary arteries, cerebral vessel, and retinal veins, as well as priapism. The study was devised for the following purposes: (1) to identify the markers of thrombophilia in hemodialyzed patients, (2) to establish a role for antiphospholipid antibodies in thrombosis of the vascular access, (3) to characterize phospholipid antibodies in hemodialysis patients, and (4) to study the effects of dialysis on coagulation cascade. A group of 20 hemodialysis patients with no thrombotic complications (NTC) and 20 hemodialysis patients with thrombotic complications (TC) were studied along with 400 volunteer blood donors. Patients with systemic lupus erythematosus and those with nephrotic syndrome were excluded. All patients underwent a screening prothrombin time, activated partial thromboplastin time, fibrinogen (Fg), coagulation factors of the intrinsic and extrinsic pathways, antithrombin III (AT-III), protein C (PC), protein S (PS), resistance to activated protein C, prothrombin activation fragment 1+2 (F1+2), plasminogen, tissue type plasminogen activator (t-PA), plasminogen tissue activator inhibitor type-1 (PAI-1), anticardiolipin antibodies type M and G (ACA-IgM and ACA-IgG), lupus anticoagulant antibodies, and antiprothrombin antibodies type M and G (aPT-IgM and aPT-IgG). The study showed that PAI-1, F 1+2, factor VIII, ACA-IgM, and aPT-IgM levels were increased significantly over controls both in TC and NTC, however, they could distinguish patients with thrombotic complications from those without, being increased maximally in the former group. The novelty of the study is represented by the significant aPT increase that was observed in non-systemic lupus erythematosus hemodialysis patients, and particularly in those with thrombotic events. In addition

  5. Mapping of antigenic determinants on a SAT2 foot-and-mouth disease virus using chicken single-chain antibody fragments.

    PubMed

    Opperman, Pamela A; Maree, Francois F; Van Wyngaardt, Wouter; Vosloo, Wilna; Theron, Jacques

    2012-08-01

    Recombinant single-chain variable fragments (scFvs) of antibodies make it possible to localize antigenic and immunogenic determinants, identify protective epitopes and can be exploited for the design of improved diagnostic tests and vaccines. A neutralizing epitope, as well as other potential antigenic sites of a SAT2 foot-and-mouth disease virus (FMDV) were identified using phage-displayed scFvs. Three unique ZIM/7/83-specific scFvs, designated scFv1, scFv2 and scFv3, were isolated. Further characterization of these scFvs revealed that only scFv2 was capable of neutralizing the ZIM/7/83 virus and was used to generate neutralization-resistant virus variants. Sequence analysis of the P1 region of virus escaping neutralization revealed a residue change from His to Arg at position 159 of the VP1 protein. Residue 159 is not only surface exposed but is also located at the C-terminal base of the G-H loop, a known immunogenic region of FMDV. A synthetic peptide, of which the sequence corresponded to the predicted antigenic site of the VP1 G-H loop of ZIM/7/83, inhibited binding of scFv2 to ZIM/7/83 in a concentration-dependent manner. This region can therefore be considered in the design of SAT2 vaccine seed viruses for the regional control of FMD in Africa.

  6. An ultra scale-down approach to study the interaction of fermentation, homogenization, and centrifugation for antibody fragment recovery from rec E. coli.

    PubMed

    Li, Qiang; Mannall, Gareth J; Ali, Shaukat; Hoare, Mike

    2013-08-01

    Escherichia coli is frequently used as a microbial host to express recombinant proteins but it lacks the ability to secrete proteins into medium. One option for protein release is to use high-pressure homogenization followed by a centrifugation step to remove cell debris. While this does not give selective release of proteins in the periplasmic space, it does provide a robust process. An ultra scale-down (USD) approach based on focused acoustics is described to study rec E. coli cell disruption by high-pressure homogenization for recovery of an antibody fragment (Fab') and the impact of fermentation harvest time. This approach is followed by microwell-based USD centrifugation to study the removal of the resultant cell debris. Successful verification of this USD approach is achieved using pilot scale high-pressure homogenization and pilot scale, continuous flow, disc stack centrifugation comparing performance parameters such as the fraction of Fab' release, cell debris size distribution and the carryover of cell debris fine particles in the supernatant. The integration of fermentation and primary recovery stages is examined using USD monitoring of different phases of cell growth. Increasing susceptibility of the cells to disruption is observed with time following induction. For a given recovery process this results in a higher fraction of product release and a greater proportion of fine cell debris particles that are difficult to remove by centrifugation. Such observations are confirmed at pilot scale.

  7. Hybrid metabolic flux analysis and recombinant protein prediction in Pichia pastoris X-33 cultures expressing a single-chain antibody fragment.

    PubMed

    Isidro, Inês A; Portela, Rui M; Clemente, João J; Cunha, António E; Oliveira, Rui

    2016-09-01

    Despite the growing importance of the Pichia pastoris expression system as industrial workhorse, the literature is almost absent in systematic studies on how culture medium composition affects central carbon fluxes and heterologous protein expression. In this study we investigate how 26 variations of the BSM+PTM1 medium impact central carbon fluxes and protein expression in a P. pastoris X-33 strain expressing a single-chain antibody fragment. To achieve this goal, we adopted a hybrid metabolic flux analysis (MFA) methodology, which is a modification of standard MFA to predict the rate of synthesis of recombinant proteins. Hybrid MFA combines the traditional parametric estimation of central carbon fluxes with non-parametric statistical modeling of product-related quantitative or qualitative measurements as a function of central carbon fluxes. It was observed that protein yield variability was 53.6 % (relative standard deviation) among the different experiments. Protein yield is much more sensitive to medium composition than biomass growth, which is mainly determined by the carbon source availability and main salts. Hybrid MFA was able to describe accurately the protein yield with normalized RMSE of 6.3 % over 5 independent experiments. The metabolic state that promotes high protein yields is characterized by high overall metabolic rates through main central carbon pathways concomitantly with a relative shift of carbon flux from biosynthetic towards energy generating pathways. PMID:27129458

  8. Crystal structure of the antigen-binding fragment of a monoclonal antibody specific for the multidrug-resistance-linked ABC transporter human P-glycoprotein.

    PubMed

    Esser, Lothar; Shukla, Suneet; Zhou, Fei; Ambudkar, Suresh V; Xia, Di

    2016-08-01

    P-glycoprotein (P-gp) is a polyspecific ATP-dependent transporter linked to multidrug resistance in cancers that plays important roles in the pharmacokinetics of a large number of drugs. The drug-resistance phenotype of P-gp can be modulated by the monoclonal antibody UIC2, which specifically recognizes human P-gp in a conformation-dependent manner. Here, the purification, sequence determination and high-resolution structure of the Fab fragment of UIC2 (UIC2/Fab) are reported. Purified UIC2/Fab binds human P-gp with a 1:1 stoichiometry. Crystals of UIC2/Fab are triclinic (space group P1), with unit-cell parameters a = 40.67, b = 44.91, c = 58.09 Å, α = 97.62, β = 99.10, γ = 94.09°, and diffracted X-rays to 1.6 Å resolution. The structure was determined by molecular replacement and refined to 1.65 Å resolution. The asymmetric unit contains one molecule of UIC2/Fab, which exhibits a positively charged antigen-binding surface, suggesting that it might recognize an oppositely charged extracellular epitope of P-gp.

  9. The sub-nanomolar binding of DNA-RNA hybrids by the single-chain Fv fragment of antibody S9.6.

    PubMed

    Phillips, Damilola D; Garboczi, David N; Singh, Kavita; Hu, Zonglin; Leppla, Stephen H; Leysath, Clinton E

    2013-08-01

    The monoclonal antibody S9.6 binds DNA-RNA hybrids with high affinity, making it useful in research and diagnostic applications, such as in microarrays and in the detection of R-loops. A single-chain variable fragment (scFv) of S9.6 was produced, and its affinities for various synthetic nucleic acid hybrids were measured by surface plasmon resonance (SPR). S9.6 exhibits dissociation constants of approximately 0.6 nM for DNA-RNA and, surprisingly, 2.7 nM for RNA-RNA hybrids that are AU-rich. The affinity of the S9.6 scFv did not appear to be strongly influenced by various buffer conditions or by ionic strength below 500 mM NaCl. The smallest epitope that was strongly bound by the S9.6 scFv contained six base pairs of DNA-RNA hybrid. Published 2013. This article is a U.S. Government work and is in the public domain in the USA.

  10. Construction, expression, and characterization of a single-chain variable fragment antibody against 2,4-dichlorophenoxyacetic acid in the hemolymph of silkworm larvae.

    PubMed

    Sakamoto, Seiichi; Pongkitwitoon, Benyakan; Nakamura, Seiko; Sasaki-Tabata, Kaori; Tanizaki, Yusuke; Maenaka, Katsumi; Tanaka, Hiroyuki; Morimoto, Satoshi

    2011-07-01

    A single-chain variable fragment antibody against herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D-scFv) has been successfully expressed in the hemolymph of silkworm larvae using a rapid Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system. Variable heavy- and light-chain domains were cloned directly from the cDNA of the hybridoma cell line 2C4 and assembled together with flexible peptide linker (Gly(4)Ser)(3) between two domains. The yield of functional 2,4-D-scFv after purification was 640 μg per 30 ml of hemolymph, which is equivalent to 21.3 mg per liter of hemolymph. The characterization of 2,4-D-scFv using an indirect competitive enzyme-linked immunosorbent assay (icELISA) revealed that it has wide cross-reactivities against 2,4,5-trichlorophenoxyacetic acid (65.5%), 2,4-dichlorophenol (47.9%), and 2,4-dichlorobenzoic acid (26.0%), making it possible to apply 2,4-D-scFv to icELISA for detecting/determining 2,4-D and its metabolites. Judging from its cost and time requirements and its ease of handling, this BmNPV bacmid DNA expression system is more useful for expressing functional scFv than bacterial systems, which frequently require costly and time-consuming refolding.

  11. Production and characterization of monoclonal antibody and its recombinant single chain variable fragment specific for a food-born mycotoxin, fumonisin B1.

    PubMed

    Min, Won-Ki; Cho, Young-Jin; Park, Jun-Bock; Bae, Yi-Hyun; Kim, Eun-Jeong; Park, Kyungmoon; Park, Yong-Cheol; Seo, Jin-Ho

    2010-01-01

    Fumonisin B(1) (FMB(1)) is a food-born mycotoxin produced by Fusarium moniliforme. Monoclonal antibody against FMB(1) (anti-FMB(1) mAb) was produced in the hybridoma DV9, which was established from a BALB/c mouse immunized with bovine serum albumin conjugated FMB(1) (FMB(1)-BSA). A competitive direct enzyme-linked immunosorbent assay (ELISA) showed that anti-FMB(1) mAb has about 10 ppb of minimum FMB(1) detection concentration and 220 ppb of 50% inhibition concentration (IC(50)). Much lower cross-reactivity of anti-FMB(1) mAb on ochratoxin A, aflatoxin B(1) and deoxynivalenol provided that anti-FMB(1) mAb was specific for FMB(1). The gene coding single chain variable fragment against FMB(1) (anti-FMB(1) scFv) was cloned from the hybridoma DV9 and was expressed in recombinant Escherichia coli. Insoluble anti-FMB(1) scFv required optimization of its refolding condition, and hence functional scFv was obtained. By using indirect ELISA, about 12-fold lower binding activity of anti-FMB(1) scFv on FMB(1)-BSA was obtained in comparison with that of the parental mAb. PMID:19597742

  12. A VL-linker-VH Orientation Dependent Single Chain Variable Antibody Fragment Against Rabies Virus G Protein with Enhanced Neutralizing Potency in vivo.

    PubMed

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

    2016-01-01

    Lethal rabies can be prevented effectively by post-exposure prophylactic (PEP) with rabies immunoglobulin (RIG). Single-chain variable fragment (scFv), which is composed of a variable heavy chain (VH) and variable light chain (VL) connected by a peptide linker, may be developed as alternative to RIG for neutralizing rabies virus (RV). However, our previously constructed scFv (FV57S) with the (NH2) VH-linker-VL (COOH) orientation showed a lower neutralizing potency than its parent RIG. This orientation may inhibit FV57S from refolding into an intact and correct conformation. Therefore, the RFV57S protein with a VL-linker-VH orientation was constructed based on FV57S. A HIS tag was incorporated to aid in purification and detection of RFV57S and FV57S. However, abilities of RFV57S and FV57S to bind with the anti-HIS tag mAb were different. Therefore, a novel direct ELISA was established by utilizing a biotin-labeled truncated glycoprotein of RV. Although with similar stability and in vitro neutralizing potency as FV57S, RFV57S showed enhanced binding ability, affinity and in vivo protective efficacy against lethal dose of RV. Our studies support the feasibility of developing a scFv with reversed orientation and provide a novel method for evaluating the binding ability, stability and affinity of engineered antibodies recognizing linear epitope.

  13. NY-ESO-1 specific antibody and cellular responses in melanoma patients primed with NY-ESO-1 protein in ISCOMATRIX and boosted with recombinant NY-ESO-1 fowlpox virus.

    PubMed

    Chen, Ji-Li; Dawoodji, Amina; Tarlton, Andrea; Gnjatic, Sacha; Tajar, Abdelouahid; Karydis, Ioannis; Browning, Judy; Pratap, Sarah; Verfaille, Christian; Venhaus, Ralph R; Pan, Linda; Altman, Douglas G; Cebon, Jonathan S; Old, Lloyd L; Nathan, Paul; Ottensmeier, Christian; Middleton, Mark; Cerundolo, Vincenzo

    2015-03-15

    Vaccination strategies based on repeated injections of NY-ESO-1 protein formulated in ISCOMATRIX particles (NY-ESO-1 ISCOMATRIX) have shown to elicit combined NY-ESO-1 specific antibody and T cell responses. However, it remains unclear whether heterologous prime-boost strategies based on the combination with NY-ESO-1 ISCOMATRIX with different NY-ESO-1 boosting reagents could be used to increase NY-ESO-1 CD8(+) or CD4(+) T cell responses. To address this question, we carried out a randomized clinical trial in 39 high-risk, resected melanoma patients vaccinated with NY-ESO-1 ISCOMATRIX, and then boosted with repeated injections of either recombinant fowlpox virus encoding full length NY-ESO-1 (rF-NY-ESO-1) (Arm A) or NY-ESO-1 ISCOMATRIX alone (Arm B). We have comprehensively analyzed NY-ESO-1 specific T cells and B cells response in all patients before and after vaccination for a total of seven time points per patient. NY-ESO-1 ISCOMATRIX alone elicited a strong NY-ESO-1 specific CD4(+) T cell and antibody response, which was maintained by both regiments at similar levels. However, CD8(+) T cell responses were significantly boosted in 3 out of 18 patients in Arm A after the first rF-NY-ESO-1 injection and such responses were maintained until the end of the trial, while no patients in Arm B showed similar CD8(+) T cell responses. In addition, our results clearly identified immunodominant regions in the NY-ESO-1 protein: NY-ESO-179-102 and NY-ESO-1115-138 for CD4+ T cells and NY-ESO-185-108 for CD8+ T cells in a large proportion of vaccinated patients. These regions of NY-ESO-1 protein should be considered in future clinical trials as immunodominant epitopes.

  14. Cixutumumab in Treating Patients With Metastatic Melanoma of the Eye

    ClinicalTrials.gov

    2015-06-25

    Ciliary Body and Choroid Melanoma, Medium/Large Size; Ciliary Body and Choroid Melanoma, Small Size; Iris Melanoma; Metastatic Intraocular Melanoma; Recurrent Intraocular Melanoma; Stage IV Intraocular Melanoma

  15. Astatine-211 labeling of an antimelanoma antibody and its Fab fragment using N-succinimidyl p-astatobenzoate: comparisons in vivo with the p-[125I]iodobenzoyl conjugate.

    PubMed

    Hadley, S W; Wilbur, D S; Gray, M A; Atcher, R W

    1991-01-01

    Astatine-211 labeling of an antimelanoma antibody, NR-ML-05, and its Fab fragment with N-succinimidyl p-[211At]astatobenzoate (2a) 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 in the reaction mixture. The dry distillation technique employed gave recoveries 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 [211At]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-[125I]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.

  16. Ga-67 vs In-111-DTPA-anti-p97 monoclonal antibody (MoAb) for scintigraphic detection of metastatic melanoma

    SciTech Connect

    Neumann, R.D.; Kirkwood, J.M.; Zoghbi, S.S.; Ernstoff, M.S.; Cornelius, E.A.; Hoffer, P.B.; Gottschalk, A.

    1985-05-01

    The authors have initiated a prospective comparative evaluation of tomographic (Pho/Con) Ga-67 (10 mCi) scintigraphy versus computerized gamma camera radioimmunoscintigraphy using anti-p97 murine MoAb (5 mCi In-111-DTPA conjugated to 1 mg MoAb + 19 mgs ''cold'' MoAb) for detection of metastatic melanoma. To date 9 patients have had both a Ga-67 scan and an anti-p97 MoAb study within a 1 month interval. The 9 patients had 45 known sites of disease greater than 1 cm minimum diameter, located in skin, soft tissue, and/or viscea. In-111-anti-p97 scans were done 72 hrs after i.v. administration of the radiolabeled MoAb mixed together with the ''cold'' carrier MoAb. Ga-67 scans were done 48-72 hrs after injection. MoAb scans detected 30/45 known sites (sensitivity=67%) while the Ga-67 scans found 29/45 of these known sites (sensitivity=64%). Heterogeneity of metastases was evident with the detection of partly overlapping subsets of metastases by the two techniques, i.e. some mets were localized with both techniques while other mets were detected by only one of the two methods. Combined detection with both techniques improved sensitivity from 64-67% to 91%. These preliminary data suggest: 1) comparable sensitivities for Ga-67 and In-111-DTPA anti-p97 scans, 2) heterogeneity of metastases with regard to the localization of these two agents, and 3) improved sensitivity by combining both techniques.

  17. Molecular Classification of Melanoma

    Cancer.gov

    Tissue-based analyses of precursors, melanoma tumors and metastases within existing study populations to further understanding of the heterogeneity of melanoma and determine a predictive pattern of progression for dysplastic nevi.

  18. General Information about Melanoma

    MedlinePlus

    ... Screening Research Melanoma Treatment (PDQ®)–Patient Version General Information About Melanoma Go to Health Professional Version Key ... the PDQ Adult Treatment Editorial Board . Clinical Trial Information A clinical trial is a study to answer ...

  19. Melanoma to the heart

    PubMed Central

    Hall, James A.; Fidone, Erica J.; Mack, Ryan; Metting, Austin L.

    2016-01-01

    Malignant melanoma is the third most common skin cancer yet has the highest mortality rate due to its predilection for metastasis. While the diagnosis of antemortem melanoma with cardiac metastasis is relatively uncommon, diagnosing malignant melanoma itself by first identifying a cardiac metastasis is even more rare. This vignette describes an antemortem diagnosis of melanoma in a 50-year-old woman through identification of metastasis to multiple sites, including the tricuspid valve. PMID:27695188

  20. Melanoma to the heart

    PubMed Central

    Hall, James A.; Fidone, Erica J.; Mack, Ryan; Metting, Austin L.

    2016-01-01

    Malignant melanoma is the third most common skin cancer yet has the highest mortality rate due to its predilection for metastasis. While the diagnosis of antemortem melanoma with cardiac metastasis is relatively uncommon, diagnosing malignant melanoma itself by first identifying a cardiac metastasis is even more rare. This vignette describes an antemortem diagnosis of melanoma in a 50-year-old woman through identification of metastasis to multiple sites, including the tricuspid valve.

  1. Antithyroglobulin antibody

    MedlinePlus

    Thyroglobulin antibody; Thyroiditis - thyroglobulin antibody; Hypothyroidism - thyroglobulin antibody; Thyroiditis - thyroglobulin antibody; Graves disease - thyroglobulin antibody; Underactive thyroid - thyroglobulin antibody

  2. 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

  3. Inhibition of the Myotoxicity Induced by Bothrops jararacussu Venom and Isolated Phospholipases A2 by Specific Camelid Single-Domain Antibody Fragments

    PubMed Central

    Prado, Nidiane D. R.; Pereira, Soraya S.; da Silva, Michele P.; Morais, Michelle S. S.; Kayano, Anderson M.; Moreira-Dill, Leandro S.; Luiz, Marcos B.; Zanchi, Fernando B.; Fuly, André L.; E. F. Huacca, Maribel; Fernandes, Cleberson F.; Calderon, Leonardo A.; Zuliani, Juliana P.; Soares, Andreimar M.; Stabeli, Rodrigo G.; F. C. Fernandes, Carla

    2016-01-01

    Antivenoms, produced using animal hyperimmune plasma, remains the standard therapy for snakebites. Although effective against systemic damages, conventional antivenoms have limited efficacy against local tissue damage. Additionally, the hypersensitivity reactions, often elicited by antivenoms, the high costs for animal maintenance, the difficulty of producing homogeneous lots, and the instability of biological products instigate the search for innovative products for antivenom therapy. In this study, camelid antibody fragments (VHH) with specificity to Bothropstoxin I and II (BthTX-I and BthTX-II), two myotoxic phospholipases from Bothrops jararacussu venom, were selected from an immune VHH phage display library. After biopanning, 28 and 6 clones recognized BthTX-I and BthTX-II by ELISA, respectively. Complementarity determining regions (CDRs) and immunoglobulin frameworks (FRs) of 13 VHH-deduced amino acid sequences were identified, as well as the camelid hallmark amino acid substitutions in FR2. Three VHH clones (KF498607, KF498608, and KC329718) were capable of recognizing BthTX-I by Western blot and showed affinity constants in the nanomolar range against both toxins. VHHs inhibited the BthTX-II phospholipase A2 activity, and when tested for cross-reactivity, presented specificity to the Bothrops genus in ELISA. Furthermore, two clones (KC329718 and KF498607) neutralized the myotoxic effects induced by B. jararacussu venom, BthTX-I, BthTX-II, and by a myotoxin from Bothrops brazili venom (MTX-I) in mice. Molecular docking revealed that VHH CDRs are expected to bind the C-terminal of both toxins, essential for myotoxic activity, and to epitopes in the BthTX-II enzymatic cleft. Identified VHHs could be a biotechnological tool to improve the treatment for snake envenomation, an important and neglected world public health problem. PMID:27028872

  4. In vivo detection of amyloid-β deposits using heavy chain antibody fragments in a transgenic mouse model for Alzheimer's disease.

    PubMed

    Nabuurs, Rob J A; Rutgers, Kim S; Welling, Mick M; Metaxas, Athanasios; de Backer, Maaike E; Rotman, Maarten; Bacskai, Brian J; van Buchem, Mark A; van der Maarel, Silvère M; van der Weerd, Louise

    2012-01-01

    This study investigated the in vivo properties of two heavy chain antibody fragments (V(H)H), ni3A and pa2H, to differentially detect vascular or parenchymal amyloid-β deposits characteristic for Alzheimer's disease and cerebral amyloid angiopathy. Blood clearance and biodistribution including brain uptake were assessed by bolus injection of radiolabeled V(H)H in APP/PS1 mice or wildtype littermates. In addition, in vivo specificity for Aβ was examined in more detail with fluorescently labeled V(H)H by circumventing the blood-brain barrier via direct application or intracarotid co-injection with mannitol. All V(H)H showed rapid renal clearance (10-20 min). Twenty-four hours post-injection (99m)Tc-pa2H resulted in a small yet significant higher cerebral uptake in the APP/PS1 animals. No difference in brain uptake were observed for (99m)Tc-ni3A or DTPA((111)In)-pa2H, which lacked additional peptide tags to investigate further clinical applicability. In vivo specificity for Aβ was confirmed for both fluorescently labeled V(H)H, where pa2H remained readily detectable for 24 hours or more after injection. Furthermore, both V(H)H showed affinity for parenchymal and vascular deposits, this in contrast to human tissue, where ni3A specifically targeted only vascular Aβ. Despite a brain uptake that is as yet too low for in vivo imaging, this study provides evidence that V(H)H detect Aβ deposits in vivo, with high selectivity and favorable in vivo characteristics, making them promising tools for further development as diagnostic agents for the distinctive detection of different Aβ deposits. PMID:22675537

  5. In Vivo Detection of Amyloid-β Deposits Using Heavy Chain Antibody Fragments in a Transgenic Mouse Model for Alzheimer's Disease

    PubMed Central

    Welling, Mick M.; Metaxas, Athanasios; de Backer, Maaike E.; Rotman, Maarten; Bacskai, Brian J.; van Buchem, Mark A.; van der Maarel, Silvère M.; van der Weerd, Louise

    2012-01-01

    This study investigated the in vivo properties of two heavy chain antibody fragments (VHH), ni3A and pa2H, to differentially detect vascular or parenchymal amyloid-β deposits characteristic for Alzheimer's disease and cerebral amyloid angiopathy. Blood clearance and biodistribution including brain uptake were assessed by bolus injection of radiolabeled VHH in APP/PS1 mice or wildtype littermates. In addition, in vivo specificity for Aβ was examined in more detail with fluorescently labeled VHH by circumventing the blood-brain barrier via direct application or intracarotid co-injection with mannitol. All VHH showed rapid renal clearance (10–20 min). Twenty-four hours post-injection 99mTc-pa2H resulted in a small yet significant higher cerebral uptake in the APP/PS1 animals. No difference in brain uptake were observed for 99mTc-ni3A or DTPA(111In)-pa2H, which lacked additional peptide tags to investigate further clinical applicability. In vivo specificity for Aβ was confirmed for both fluorescently labeled VHH, where pa2H remained readily detectable for 24 hours or more after injection. Furthermore, both VHH showed affinity for parenchymal and vascular deposits, this in contrast to human tissue, where ni3A specifically targeted only vascular Aβ. Despite a brain uptake that is as yet too low for in vivo imaging, this study provides evidence that VHH detect Aβ deposits in vivo, with high selectivity and favorable in vivo characteristics, making them promising tools for further development as diagnostic agents for the distinctive detection of different Aβ deposits. PMID:22675537

  6. Inhibition of the Myotoxicity Induced by Bothrops jararacussu Venom and Isolated Phospholipases A2 by Specific Camelid Single-Domain Antibody Fragments.

    PubMed

    Prado, Nidiane D R; Pereira, Soraya S; da Silva, Michele P; Morais, Michelle S S; Kayano, Anderson M; Moreira-Dill, Leandro S; Luiz, Marcos B; Zanchi, Fernando B; Fuly, André L; Huacca, Maribel E F; Fernandes, Cleberson F; Calderon, Leonardo A; Zuliani, Juliana P; Pereira da Silva, Luiz H; Soares, Andreimar M; Stabeli, Rodrigo G; Fernandes, Carla F C

    2016-01-01

    Antivenoms, produced using animal hyperimmune plasma, remains the standard therapy for snakebites. Although effective against systemic damages, conventional antivenoms have limited efficacy against local tissue damage. Additionally, the hypersensitivity reactions, often elicited by antivenoms, the high costs for animal maintenance, the difficulty of producing homogeneous lots, and the instability of biological products instigate the search for innovative products for antivenom therapy. In this study, camelid antibody fragments (VHH) with specificity to Bothropstoxin I and II (BthTX-I and BthTX-II), two myotoxic phospholipases from Bothrops jararacussu venom, were selected from an immune VHH phage display library. After biopanning, 28 and 6 clones recognized BthTX-I and BthTX-II by ELISA, respectively. Complementarity determining regions (CDRs) and immunoglobulin frameworks (FRs) of 13 VHH-deduced amino acid sequences were identified, as well as the camelid hallmark amino acid substitutions in FR2. Three VHH clones (KF498607, KF498608, and KC329718) were capable of recognizing BthTX-I by Western blot and showed affinity constants in the nanomolar range against both toxins. VHHs inhibited the BthTX-II phospholipase A2 activity, and when tested for cross-reactivity, presented specificity to the Bothrops genus in ELISA. Furthermore, two clones (KC329718 and KF498607) neutralized the myotoxic effects induced by B. jararacussu venom, BthTX-I, BthTX-II, and by a myotoxin from Bothrops brazili venom (MTX-I) in mice. Molecular docking revealed that VHH CDRs are expected to bind the C-terminal of both toxins, essential for myotoxic activity, and to epitopes in the BthTX-II enzymatic cleft. Identified VHHs could be a biotechnological tool to improve the treatment for snake envenomation, an important and neglected world public health problem. PMID:27028872

  7. Effects of digoxin-specific antibody Fab fragment (Digibind) on blood pressure and renal water-sodium metabolism in 5/6 reduced renal mass hypertensive rats.

    PubMed

    Kaide, J; Ura, N; Torii, T; Nakagawa, M; Takada, T; Shimamoto, K

    1999-06-01

    The importance of increased endogenous digitalis-like factor (EDLF) in volume-expanded hypertension has been generally agreed. To further clarify the role of EDLF on the development of hypertension and renal water-sodium handling in 5/6 reduced renal mass hypertensive rats (RRM), we studied the effects of acute administration of digoxin-specific antibody Fab fragment (Digibind) in the early phase and the chronic phase of hypertension in RRM. RRM and sham-operated rats were given 1% saline for 1 or 4 weeks. RRM were injected Digibind (60 mg/kg) or vehicle (0.9% saline) intravenously in the first or fourth week under thiobutabarbital anesthesia. All sham-operated rats were administered Digibind under the same condition. Digibind altered neither blood pressure, heart rate, urine volume, nor urinary sodium excretion in sham-operated rats. However, Digibind produced a gradual but significant decline in mean arterial pressure to the level slightly above that in sham-operated rats from 153 +/- 5 to 131 +/- 5 mm Hg in the first week and from 181 +/- 6 to 129 +/- 4 mm Hg in the fourth week without any significant change in heart rate. The decrease in mean arterial pressure at 160 min after Digibind administration in the fourth week (-48 +/- 5 mm Hg) was greater than that in the first week (-22 +/- 4 mm Hg). No differences were observed in urine volume, urinary sodium excretion, or plasma norepinephrine concentration between Digibind and vehicle-treated RRM in either week. These data suggest that EDLF would contribute to both the early and chronic phase in the development of hypertension in RRM.

  8. CTGF is a therapeutic target for metastatic melanoma.

    PubMed

    Finger, E C; Cheng, C-F; Williams, T R; Rankin, E B; Bedogni, B; Tachiki, L; Spong, S; Giaccia, A J; Powell, M B

    2014-02-27

    Metastatic melanoma remains a devastating disease with a 5-year survival rate of less than five percent. Despite recent advances in targeted therapies for melanoma, only a small percentage of melanoma patients experience durable remissions. Therefore, it is critical to identify new therapies for the treatment of advanced melanoma. Here, we define connective tissue growth factor (CTGF) as a therapeutic target for metastatic melanoma. Clinically, CTGF expression correlates with tumor progression and is strongly induced by hypoxia through HIF-1 and HIF-2-dependent mechanisms. Genetic inhibition of CTGF in human melanoma cells is sufficient to significantly reduce orthotopic tumor growth, as well as metastatic tumor growth in the lung of severe combined immunodeficient (SCID) mice. Mechanistically, inhibition of CTGF decreased invasion and migration associated with reduced matrix metalloproteinase-9 expression. Most importantly, the anti-CTGF antibody, FG-3019, had a profound inhibitory effect on the progression of established metastatic melanoma. These results offer the first preclinical validation of anti-CTGF therapy for the treatment of advanced melanoma and underscore the importance of tumor hypoxia in melanoma progression.

  9. Epacadostat and Vaccine Therapy in Treating Patients With Stage III-IV Melanoma

    ClinicalTrials.gov

    2016-10-31

    Mucosal Melanoma; Recurrent Melanoma; Recurrent Uveal Melanoma; Stage IIIA Skin Melanoma; Stage IIIA Uveal Melanoma; Stage IIIB Skin Melanoma; Stage IIIB Uveal Melanoma; Stage IIIC Skin Melanoma; Stage IIIC Uveal Melanoma; Stage IV Skin Melanoma; Stage IV Uveal Melanoma

  10. Vaccine Therapy in Treating Patients With Stage IIC-IV Melanoma

    ClinicalTrials.gov

    2014-05-20

    Ciliary Body and Choroid Melanoma, Medium/Large Size; Ciliary Body and Choroid Melanoma, Small Size; Extraocular Extension Melanoma; Iris Melanoma; Metastatic Intraocular Melanoma; Mucosal Melanoma; Recurrent Intraocular Melanoma; Recurrent Melanoma; Stage IIC Melanoma; Stage IIIA Intraocular Melanoma; Stage IIIA Melanoma; Stage IIIB Intraocular Melanoma; Stage IIIB Melanoma; Stage IIIC Intraocular Melanoma; Stage IIIC Melanoma; Stage IV Intraocular Melanoma; Stage IV Melanoma

  11. Evaluation of the Naturally Acquired Antibody Immune Response to the Pv200L N-terminal Fragment of Plasmodium vivax Merozoite Surface Protein-1 in Four Areas of the Amazon Region of Brazil

    PubMed Central

    Storti-Melo, Luciane M.; Souza-Neiras, Wanessa C.; Cassiano, Gustavo C.; Taveira, Leonardo C.; Cordeiro, Antônio J.; Couto, Vanja S. C. A.; Póvoa, Marinete M.; Cunha, Maristela G.; Echeverry, Diana M.; Rossit, Andréa R. B.; Arévalo-Herrera, Myriam; Herrera, Sócrates; Machado, Ricardo L. D.

    2011-01-01

    Frequency and levels of IgG antibodies to an N-terminal fragment of the Plasmodium vivax MSP-1 (Pv200L) protein, in individuals naturally exposed to malaria in four endemic areas of Brazil, were evaluated by enzyme-linked immunosorbent assay. Plasma samples of 261 P. vivax-infected individuals from communities of Macapá, Novo Repartimento, Porto Velho, and Plácido de Castro in the Amazonian region with different malaria transmission intensities. A high mean number of studied individuals (89.3%) presented with antibodies to the Pv200L that correlated with the number of previous malaria infections; there were significant differences in the frequency of the responders (71.9–98.7) and in the antibody levels (1:200–1:51,200) among the four study areas. Results of this study provide evidence that Pv200L is a naturally immunogenic fragment of the PvMSP-1 and is associated with the degree of exposure to parasites. The fine specificity of antibodies to Pv200L is currently being assessed. PMID:21292879

  12. Melanoma never says die.

    PubMed

    Haass, Nikolas K; Schumacher, Udo

    2014-07-01

    Drug resistance in melanoma is commonly attributed to ineffective apoptotic pathways. Targeting apoptosis regulators is thus considered a promising approach to sensitizing melanoma to therapy. In the previous issue of Experimental Dermatology, Plötz and Eberle discuss the role that apoptosis plays in melanoma progression and drug resistance and the utility of apoptosis-inducing BH3-mimetics as targeted therapy. There are a number of compounds in clinical development and the field seems close to translating recent findings into benefits for patients with melanoma. Thus, this viewpoint is timely and achieves a valuable summary of the current state of apoptosis-inducing therapy of melanoma.

  13. Localization of a tumor cell adhesion domain of laminin by a monoclonal antibody

    SciTech Connect

    Skubitz, A.P.N.; Charonis, A.S.; Tsilibary, E.C.; Furcht, L.T. )

    1987-12-01

    Monoclonal antibodies were prepared to localize the domain(s) of laminin to which tumor cells adhere. Rat Y3-Ag 1.2.3 myeloma cells were fused with spleen cells from a rat immunized with a purified 440-kDa fragment of chymotrypsin-digested laminin. Three monoclonal antibodies (AL-1 to AL-3) that bound to intact laminin in a solid-phase radioimmunoassay were chosen for further analysis. The epitopes recognized by these antibodies were characterized by radioimmunoassays, immunoblotting, radioimmunoprecipitation, and immunoaffinity chromatography. In cell adhesion assays, monoclonal antibody AL-2 inhibited the highly metastatic melanoma cell line, K-1735-M4, to both intact laminin and the 440-kDa fragment of laminin. Electron microscopic examination of laminin-monoclonal antibody interactions showed that monoclonal antibody AL-2 reacted with the long arm of laminin directly below the cross-region. Two monoclonal antibodies that failed to inhibit tumor cell adhesion to laminin reacted with epitopes on the lateral short arms or cross-region of laminin as seen by electron microscopy. These results suggest that a new tumor cell binding domain of laminin may be located close to the cross-region on the long arm of laminin.

  14. AC-1001 H3 CDR peptide induces apoptosis and signs of autophagy in vitro and exhibits antimetastatic activity in a syngeneic melanoma model.

    PubMed

    Rabaça, Aline N; Arruda, Denise C; Figueiredo, Carlos R; Massaoka, Mariana H; Farias, Camyla F; Tada, Dayane B; Maia, Vera C; Silva Junior, Pedro I; Girola, Natalia; Real, Fernando; Mortara, Renato A; Polonelli, Luciano; Travassos, Luiz R

    2016-09-01

    Antibody-derived peptides modulate functions of the immune system and are a source of anti-infective and antitumor substances. Recent studies have shown that they comprise amino acid sequences of immunoglobulin complementarity-determining regions, but also fragments of constant regions. VH CDR3 of murine mAb AC-1001 displays antimetastatic activities using B16F10-Nex2 murine melanoma cells in a syngeneic model. The peptide was cytotoxic in vitro in murine and human melanoma cells inducing reactive oxygen species (ROS) and apoptosis by the intrinsic pathway. Signs of autophagy were also suggested by the increased expression of LC3/LC3II and Beclin 1 and by ultrastructural evidence. AC-1001 H3 bound to both G- and F-actin and inhibited tumor cell migration. These results are important evidence of the antitumor activity of Ig CDR-derived peptides. PMID:27642552

  15. Pembrolizumab for the treatment of melanoma.

    PubMed

    Kumar, Sanjeev Srinivas; McNeil, Catriona Mairi

    2015-01-01

    The immune system plays a vital role in regulating tumor growth, and the oncology community has witnessed an exciting resurgence in clinical research to develop effective immunotherapeutic strategies. The utility of these strategies in advanced melanoma has been at the forefront of these developments. In particular, blockade of programmed cell death protein 1 (PD-1) in advanced melanoma has proven to be a most promising new anticancer strategy. Pembrolizumab is a humanized IgG4 anti-PD-1 antibody that exerts its anti-tumor effect through blocking the interaction of the immune inhibitory molecule PD-1 with its ligands. Its effect has been most convincingly demonstrated in the setting of advanced melanoma, with growing evidence of clinical responses across a broad spectrum of other solid and hematological malignancies.

  16. [Systemic radiotherapy using monoclonal antibodies. Options and problems].

    PubMed

    Sautter-Bihl, M L; Wannenmacher, M; Bihl, H

    1993-06-01

    Radiolabeled monoclonal antibodies (MAbs), by virtue of their tumor specificity, offer the prospect of localized, highly targeted radiation treatment of malignant tumors. To date, a large number of radioimmunotherapy (RIT) studies have been reported in experimental and clinical settings showing the potential of this therapeutic strategy. This includes RIT-trials in hepatoma, cholangiocarcinoma, ovarian carcinoma, brain tumors, melanoma, neuroblastoma and especially Hodgkin's and non-Hodgkin's lymphomas. Despite very promising results in some of these studies, radioimmunotherapy is currently still in a developmental status. Selective accumulation of MAbs at tumor sites-a prerequisite for effective radioimmunotherapy-is a complex process. Many factors such as antigen heterogeneity, distinct antibody features (affinity, subclass, fragment size, etc.), labeling techniques, tumor physiology and competing antigens were identified in the last years using theoretical and experimental tumor models. Strategies to improve these critical parameters are currently under investigation in order to increase the efficacy of radioimmunotherapy.

  17. Immunotherapy for Melanoma: Current Status and Perspectives

    PubMed Central

    Alexandrescu, Doru T.; Ichim, Thomas E.; Riordan, Neil H.; Marincola, Francesco M.; Nardo, Anna Di; Kabigting, Filamer D.; Dasanu, Constantin A.

    2012-01-01

    Summary Immunotherapy is an important modality in the therapy of patients with malignant melanoma. As our knowledge about this disease continues to expand, so does the immunotherapeutic armamentarium. Nevertheless, successful preclinical models do not always translate into clinically meaningful results. The authors give a comprehensive analysis of most recent advances in the immune anti-melanoma therapy, including interleukins, interferons, other cytokines, adoptive immunotherapy, biochemotherapy, as well as the use of different vaccines. We also present the fundamental concepts behind various immune enhancement strategies, passive immunotherapy, as well as the use of immune adjuvants. This review brings into discussion the results of newer and older clinical trials, as well as potential limitations and drawbacks seen with the utilization of various immune therapies in malignant melanoma. Development of novel therapeutic approaches, along with optimization of existing therapies, continues to hold a great promise in the field of melanoma therapy research. Use of anti-CTLA4 and anti-PD1 antibodies, realization of the importance of co-stimulatory signals, which translated into the use of agonist CD40 monoclonal antibodies, as well as activation of innate immunity through enhanced expression of co-stimulatory molecules on the surface of dendritic cells by TLR agonists are only a few items on the list of recent advances in the treatment of melanoma. The need to engineer better immune interactions and to boost positive feedback loops appear crucial for the future of melanoma therapy, which ultimately resides in our understanding of the complexity of immune responses in this disease. PMID:20551839

  18. Preparation, biodistribution and dosimetry of copper-64-labeled anti-colorectal carcinoma monoclonal antibody fragments 1A3-F(ab{prime}){sub 2}

    SciTech Connect

    Anderson, C.J.; Schwarz, S.W.; Connett, J.M. ||

    1995-05-01

    Antibody fragments labeled with a radiometal using bifunctional chelates generally undergo renal clearance followed by trapping of the metabolites, leading to high radiation doses to the kidneys. Copper-64-labeled BAT-2IT-1A3-F(ab{prime}){sub 2} was recently reported to accumulate in colorectal tumors in an animal model, however, kidney uptake was also high. In this study, the preparation of {sup 64}Cu-BAT-2IT-1A3-F(ab{prime}){sub 2} was optimized to reduce the renal uptake. The bifunctional chelate 6-bromoacetamidobenzyl-1,4,8,11-tetraazacyclotetradecane-N,N{prime},N{double_prime},N{prime}{double_prime}-tetraacetic acid (BAT) was conjugated to 1A3-F(ab{prime}){sub 2} using the linking agent 2-iminothiolane (2IT). The conjugation reaction produced 20% of a lower molecular weight impurity found to be TETA-1A3-Fab{prime}. The conjugation procedure was optimized to include FPLC purification of the BAT-2IT-1A3-F(ab{prime}){sub 2} from TETA-1A3-Fab{prime} after conjugation prior to labeling with {sup 64}Cu. The biodistribution of {sup 64}Cu-labeled FPLC-purified and unpurified conjugates was determined in normal Sprague-Dawley rats and tumor-bearing Golden Syrian hamsters. Human absorbed doses were calculated from rat biodistribution data and PET imaging of a baboon. Upon FPLC purification of the BAT-2IT-1A3-F(ab{prime}){sub 2}, the immunoreactivity of {sup 64}Cu-labeled 1A3-F(ab{prime}){sub 2} was significantly improved over that of non-FPLC-purified {sup 64}Cu-BAT-2IT-1A3-F(ab{prime}){sub 2}, and the kidney uptake was decreased in normal rats. The biodistribution in hamsters showed some improvement in both tumor uptake and kidney clearance with FPLC-purified {sup 64}Cu-BAT-2IT-1A3-F(ab{prime}){sub 2}.The improved dosimetry of {sub 64}Cu-labeled FPLC purified BAT-2IT-1A3-F(ab{prime}){sub 2} should more readily allow this agent to be investigated clinically to image colorectal cancer using PET. 33 refs., 7 figs., 3 tabs.

  19. Circulating melanoma cells as a predictive biomarker.

    PubMed

    Karakousis, Giorgos; Yang, Ruifeng; Xu, Xiaowei

    2013-06-01

    The prognosis of patients with metastatic melanoma has improved significantly with targeted therapeutic agents and immunotherapies. Detection of early melanoma recurrence after treatment will be beneficial to switch patients who fail on one therapy to different modalities. Circulating tumor cells (CTCs) are cancer cells released by a tumor into the peripheral blood. These cells hold potential as prognostic, predictive, and pharmacodynamic biomarkers for treatment. In this issue, Khoja et al. report that melanoma CTCs can be detected using Melcam and high molecular weight melanoma-associated antibody. They found that in 101 stage IV melanoma patients, CTC numbers ranged between 0 and 36/7.5 ml blood; 26% of the patients had ≥ 2 CTCs at baseline. The CTC number (≥ 2 CTCs) at baseline was significantly prognostic for median overall survival (OS) in univariate and multivariate analysis. Patients receiving treatment where CTC numbers remained ≥ 2 CTCs during their treatment had shorter median OS than those who maintained <2 CTCs (7 vs. 10 months, hazard ratio 0.34, 95% confidence interval 0.14-0.81, log-rank test P=0.015). The implications of this work are substantial in counseling patients about their prognosis and in helping to assess responses to systemic therapies. PMID:23673501

  20. Specific single chain variable fragment (ScFv) antibodies to angiotensin II AT(2) receptor: evaluation of the angiotensin II receptor expression in normal and tumor-bearing mouse lung.

    PubMed

    Tamura, Masaaki; Yan, Heping; Zegarra-Moro, Ofelia; Edl, Jennifer; Oursler, Stephanie; Chard-Bergstrom, Cindy; Andrews, Gordon; Kanehira, Tsutomu; Takekoshi, Susumu; Mernaugh, Ray

    2008-08-01

    To gain insight into the mechanism by which angiotensin II type 2 receptor (AT(2)) regulates carcinogen-induced lung tumorigenesis, we have newly developed anti-AT(2) single chain variable fragment (ScFv) antibodies using a rodent phage-displayed recombinant antibody library with various peptide fragments of the receptor protein, and investigated the expression of the AT(2) receptor protein. The specificity of the antibodies was verified using AT(2) over-expressing COS-7 cells and AT(2) naturally expressing PC12W cells. In control wild type mouse lung, a stronger immunoreactivity was observed in bronchial epithelial cells. A moderate immunoreactivity was detected in pulmonary vascular walls and vascular endothelial cells. In the lungs possessing tobacco-specific nitrosamine (NNK)-induced tumors, significantly increased AT(2) and AT(1 )immunostaining was observed in adenomatous lesions. These data suggest that the increase in both receptors' expression in the alveolar epithelial cells may be accompanied with the onset of NNK-induced tumorigenesis and hence play important roles in lung tumorigenesis.

  1. Specific Single Chain Variable Fragment (ScFv) Antibodies to Angiotensin II AT2 Receptor: Evaluation of the Angiotensin II Receptor Expression in Normal and Tumor-bearing Mouse Lung

    PubMed Central

    Tamura, Masaaki; Yan, Heping; Zegarra-Moro, Ofelia; Edl, Jennifer; Oursler, Stephanie; Chard-Bergstrom, Cindy; Andrews, Gordon; Kanehira, Tsutomu; Takekoshi, Susumu; Mernaugh, Ray

    2010-01-01

    Summary To gain insight into the mechanism by which angiotensin II type 2 receptor (AT2) regulates carcinogen-induced lung tumorigenesis, we have newly developed anti-AT2 single chain variable fragment (ScFv) antibodies using a rodent phage-displayed recombinant antibody library with various peptide fragments of the receptor protein, and investigated the expression of the AT2 receptor protein. The specificity of the antibodies was verified using AT2 over-expressing COS-7 cells and AT2 naturally expressing PC12W cells. In control wild type mouse lung, a stronger immunoreactivity was observed in bronchial epithelial cells. A moderate immunoreactivity was detected in pulmonary vascular walls and vascular endothelial cells. In the lungs possessing tobacco-specific nitrosamine (NNK)-induced tumors, significantly increased AT2 and AT1 immunostaining was observed in adenomatous lesions. These data suggest that the increase in both receptors' expression in the alveolar epithelial cells may be accompanied with the onset of NNK-induced tumorigenesis and hence play important roles in lung tumorigenesis. PMID:18438736

  2. Development of an immunoassay for determination of 2,4-dichlorophenoxyacetic acid (2,4-D) based upon the recombinant Fab fragment of 2,4-D specific antibody

    NASA Astrophysics Data System (ADS)

    Nguyen, Van C.; Nguyen, Thi D. T.; Dau, Hung A.; Tham, Thu N.; Quyen, Dinh T.; Bachmman, Till; Schmid, Rolf D.

    2001-09-01

    To develop an immunoassay and further an immunosensor for 2,4-D based upon recombinant antibody, the Fab fragments of 2,4-D specific antibody were expressed in E. coli. Western blotting analysis of the periplasmic cell fractions shown that under the non-reducing condition only a single protein band at a molecular mass of 45-kDa, corresponding to the whole Fab fragment was detected. Antigen binding activity for 2,4-D was found only in the extract of cells bearing the 2,4-D plasmid. An immunoassay based on the competitive reaction of 2,4-D and enzyme tracer with 2,4-D Fab fragments immobilized on micro titer plates via rabbit anti-mouse IgC was developed. Using this assay, 2,4-D could be detected at concentration range of 0.5 (mu) g/1 to 10(mu) g/1. The center point of the 2,4-D test was found at a concentration of 5 (mu) g/l. The assay was applied for detection of 2,4-D in spiked orange samples, resulting in recovery rate of 90 percent. The immunoassay could be applied to monitor human exposure to 2,4-D from contamination in fruit samples.

  3. Antibody-gold cluster conjugates

    DOEpatents

    Hainfeld, J.F.

    1988-06-28

    Antibody- or antibody fragment-gold cluster conjugates are shown wherein the conjugate size can be about 5.0 nm. Methods and reagents are disclosed in which antibodies or Fab' fragments thereof are covalently bound to a stable cluster of gold atoms. 2 figs.

  4. Gamma-Secretase/Notch Signalling Pathway Inhibitor RO4929097 in Treating Patients With Stage IV Melanoma

    ClinicalTrials.gov

    2016-05-06

    Acral Lentiginous Malignant Melanoma; Lentigo Maligna Malignant Melanoma; Nodular Malignant Melanoma; Recurrent Melanoma; Solar Radiation-related Skin Melanoma; Stage IV Melanoma; Superficial Spreading Malignant Melanoma

  5. VEGF Trap in Treating Patients With Recurrent Stage III or Stage IV Melanoma That Cannot Be Removed by Surgery

    ClinicalTrials.gov

    2015-02-02

    Ciliary Body and Choroid Melanoma, Medium/Large Size; Extraocular Extension Melanoma; Iris Melanoma; Metastatic Intraocular Melanoma; Recurrent Intraocular Melanoma; Recurrent Melanoma; Stage III Melanoma; Stage IV Melanoma

  6. Ipilimumab-Induced Neutropenia in Melanoma

    PubMed Central

    Ban-Hoefen, Makiko; Burack, Richard; Sievert, Lynn; Sahasrabudhe, Deepak

    2016-01-01

    Ipilimumab is a human monoclonal IgG1 antibody against CTLA-4 that has been shown to prolong the overall survival of advanced melanoma. The most common adverse events associated with ipilimumab are immune-related. Severe hematological toxicity is rare. We report a case of severe neutropenia following ipilimumab therapy that fully resolved after the administration of prednisone, cyclosporine, and anti-thymocyte globulin therapies. PMID:27570779

  7. Ipilimumab-Induced Neutropenia in Melanoma.

    PubMed

    Ban-Hoefen, Makiko; Burack, Richard; Sievert, Lynn; Sahasrabudhe, Deepak

    2016-01-01

    Ipilimumab is a human monoclonal IgG1 antibody against CTLA-4 that has been shown to prolong the overall survival of advanced melanoma. The most common adverse events associated with ipilimumab are immune-related. Severe hematological toxicity is rare. We report a case of severe neutropenia following ipilimumab therapy that fully resolved after the administration of prednisone, cyclosporine, and anti-thymocyte globulin therapies. PMID:27570779

  8. IgG subclass switching and clonal expansion in cutaneous melanoma and normal skin.

    PubMed

    Saul, Louise; Ilieva, Kristina M; Bax, Heather J; Karagiannis, Panagiotis; Correa, Isabel; Rodriguez-Hernandez, Irene; Josephs, Debra H; Tosi, Isabella; Egbuniwe, Isioma U; Lombardi, Sara; Crescioli, Silvia; Hobbs, Carl; Villanova, Federica; Cheung, Anthony; Geh, Jenny L C; Healy, Ciaran; Harries, Mark; Sanz-Moreno, Victoria; Fear, David J; Spicer, James F; Lacy, Katie E; Nestle, Frank O; Karagiannis, Sophia N

    2016-01-01

    B cells participate in immune surveillance in human circulation and tissues, including tumors such as melanoma. By contrast, the role of humoral responses in cutaneous immunity is underappreciated. We report circulating skin-homing CD22+CLA+B cells in healthy volunteers and melanoma patients (n = 73) and CD22+ cells in melanoma and normal skin samples (n = 189). Normal and malignant skin featured mature IgG and CD22 mRNA, alongside mRNA for the transiently-expressed enzyme Activation-induced cytidine Deaminase (AID). Gene expression analyses of publically-available data (n = 234 GEO, n = 384 TCGA) confirmed heightened humoral responses (CD20, CD22, AID) in melanoma. Analyses of 51 melanoma-associated and 29 normal skin-derived IgG sequence repertoires revealed lower IgG1/IgGtotal representation compared with antibodies from circulating B cells. Consistent with AID, comparable somatic hypermutation frequencies and class-switching indicated affinity-matured antibodies in normal and malignant skin. A melanoma-associated antibody subset featured shorter complementarity-determining (CDR3) regions relative to those from circulating B cells. Clonal amplification in melanoma-associated antibodies and homology modeling indicated differential potential antigen recognition profiles between normal skin and melanoma sequences, suggesting distinct antibody repertoires. Evidence for IgG-expressing B cells, class switching and antibody maturation in normal and malignant skin and clonally-expanded antibodies in melanoma, support the involvement of mature B cells in cutaneous immunity. PMID:27411958

  9. IgG subclass switching and clonal expansion in cutaneous melanoma and normal skin

    PubMed Central

    Saul, Louise; Ilieva, Kristina M.; Bax, Heather J.; Karagiannis, Panagiotis; Correa, Isabel; Rodriguez-Hernandez, Irene; Josephs, Debra H.; Tosi, Isabella; Egbuniwe, Isioma U.; Lombardi, Sara; Crescioli, Silvia; Hobbs, Carl; Villanova, Federica; Cheung, Anthony; Geh, Jenny L. C.; Healy, Ciaran; Harries, Mark; Sanz-Moreno, Victoria; Fear, David J.; Spicer, James F.; Lacy, Katie E.; Nestle, Frank O.; Karagiannis, Sophia N.

    2016-01-01

    B cells participate in immune surveillance in human circulation and tissues, including tumors such as melanoma. By contrast, the role of humoral responses in cutaneous immunity is underappreciated. We report circulating skin-homing CD22+CLA+B cells in healthy volunteers and melanoma patients (n = 73) and CD22+ cells in melanoma and normal skin samples (n = 189). Normal and malignant skin featured mature IgG and CD22 mRNA, alongside mRNA for the transiently-expressed enzyme Activation-induced cytidine Deaminase (AID). Gene expression analyses of publically-available data (n = 234 GEO, n = 384 TCGA) confirmed heightened humoral responses (CD20, CD22, AID) in melanoma. Analyses of 51 melanoma-associated and 29 normal skin-derived IgG sequence repertoires revealed lower IgG1/IgGtotal representation compared with antibodies from circulating B cells. Consistent with AID, comparable somatic hypermutation frequencies and class-switching indicated affinity-matured antibodies in normal and malignant skin. A melanoma-associated antibody subset featured shorter complementarity-determining (CDR3) regions relative to those from circulating B cells. Clonal amplification in melanoma-associated antibodies and homology modeling indicated differential potential antigen recognition profiles between normal skin and melanoma sequences, suggesting distinct antibody repertoires. Evidence for IgG-expressing B cells, class switching and antibody maturation in normal and malignant skin and clonally-expanded antibodies in melanoma, support the involvement of mature B cells in cutaneous immunity. PMID:27411958

  10. Melanoma with gastric metastases

    PubMed Central

    Wong, Katherine; Serafi, Sam W.; Bhatia, Abhijit S.; Ibarra, Irene; Allen, Elizabeth A.

    2016-01-01

    An 81-year-old woman with a history of malignant melanoma who presented with dyspnea and fatigue was found to have metastases to the stomach detected on endoscopy. Primary cutaneous malignant melanoma with gastric metastases is a rare occurrence, and it is often not detected until autopsy because of its non-specific manifestations. PMID:27609722

  11. Translational research in melanoma.

    PubMed

    Ray, Madhury; Farma, Jeffrey M; Hsu, Cary

    2013-10-01

    Recent breakthroughs in the fundamental understanding of the cellular and molecular basis of melanoma have culminated in new therapies with unquestionable efficacy. Immunotherapy and targeted therapy strategies have completely transformed the contemporary management of advanced melanoma. The translational research behind these developments is discussed, with an emphasis on immune checkpoint blockade and inhibition of the mitogen-activated protein kinase signaling pathway.

  12. Melanoma with gastric metastases.

    PubMed

    Wong, Katherine; Serafi, Sam W; Bhatia, Abhijit S; Ibarra, Irene; Allen, Elizabeth A

    2016-01-01

    An 81-year-old woman with a history of malignant melanoma who presented with dyspnea and fatigue was found to have metastases to the stomach detected on endoscopy. Primary cutaneous malignant melanoma with gastric metastases is a rare occurrence, and it is often not detected until autopsy because of its non-specific manifestations. PMID:27609722

  13. Positron emission tomographic imaging of tumors using monoclonal antibodies. Progress report, November 1, 1992--October 31, 1993

    SciTech Connect

    Zalutsky, M.R.

    1993-07-29

    The overall goal of this project is to be able to combine the molecular specificity of monoclonal antibodies with the imaging advantages of positron emission tomography. During the past year, were have made progress in a number of areas. This report will focus on our studies evaluating the potential of two different methods for labeling a monoclonal antibody fragment with positron-emitting F-18 both in vitro and in athymic mice bearing subcutaneous D-54 MG human glioma xenografts. The F (a b{prime}){sub 2} fragment of Me1-14, a murine egg{sub 2a} reactive with an epitope of the tumor associated proteoglycan sulfate present in gliomas and melanomas, was used. This antibody is a particular interest because of our ongoing clinical radioimmunotherapy trails using Me1--14 that could ultimately benefit from the determination of quantitative dosimetry using monoclonal antibody PET imaging. Our results demonstrated, for the first time, that MAb fragments could be labeled with F-18 with retention of immunoreactivity and affinity. Further, they show that selective and specific tumor uptake of an F-18 labeled MAb fragment can be achieved in a xenograft model in a time frame compatible with the short half life of this nuclide.

  14. Preparation of the Fv fragment from a short-chain mouse IgG2a anti-dansyl monoclonal antibody and use of selectively deuterated Fv analogues for two-dimensional sup 1 H NMR analyses fo the antigen-antibody interactions

    SciTech Connect

    Takahashi, Hideo; Igarashi, Takako; Shimada, Ichio; Arata, Yoji )

    1991-03-19

    The Fv fragment, a univalent antigen-binding unit with a molecular weight of 25,000, has successfully been prepared in high yield by limited proteolysis with clostripain of a short-chain mouse IgG2a anti-dansyl monoclonal antibody in which the entire C{sub H}1 domain is deleted. The Fv fragment obtained is stable at room temperature and retains its full antigen-binding capability. It has been shown that selective deuterium labeling of the Fv fragment, which is half the size of the Fab fragment, provides {sup 1}H NMR spectral data at a sufficient resolution for a detailed structural analysis of the antigen-combining site. NOESY spectra of an Fv analogue, in which all aromatic protons except for His C2{prime}-H and Tyr C3{prime},5{prime}-H had been deuterated, were measured in the presence of varying amounts of dansyl-L-lysine. On the basis of the NOESY data obtained, it was possible to assign all the ring proton resonances for the dansly group that is bound to the Fv fragment. It was also possible to obtain information about His and Tyr residues of the Fv fragment in the absence and presence of the antigen. On the basis of the NMR data obtained, the authors have shown that at least two Tyr residues along with one of the amide groups are directly involved in antigen binding. The mode of interaction of the dansyl ring with these residues in the Fv fragment has briefly been discussed.

  15. Validation of the VE1 Immunostain for the BRAF V600E Mutation in Melanoma

    PubMed Central

    Pearlstein, Michelle V.; Zedek, Daniel C.; Ollila, David W.; Treece, Amanda; Gulley, Margaret L.; Groben, Pamela A.; Thomas, Nancy E.

    2014-01-01

    BACKGROUND BRAF mutation status, and therefore eligibility for BRAF inhibitors, is currently determined by sequencing methods. We assessed the validity of VE1, a monoclonal antibody against the BRAF V600E mutant protein, in the detection of mutant BRAF V600E melanomas as classified by DNA pyrosequencing. METHODS The cases were 76 metastatic melanoma patients with only one known primary melanoma who had had BRAF codon 600 pyrosequencing of either their primary (n=19), metastatic (n=57) melanoma, or both (n=17). All melanomas (n=93) were immunostained with the BRAF VE1 antibody using a red detection system. The staining intensity of these specimens was scored from 0 – 3+ by a dermatopathologist. Scores of 0 and 1+ were considered as negative staining while scores of 2+ and 3+ were considered positive. RESULTS The VE1 antibody demonstrated a sensitivity of 85% and a specificity of 100% as compared to DNA pyrosequencing results. There was 100% concordance between VE1 immunostaining of primary and metastatic melanomas from the same patient. V600K, V600Q, and V600R BRAF melanomas did not positively stain with VE1. CONCLUSIONS This hospital-based study finds high sensitivity and specificity for the BRAF VE1 immunostain in comparison to pyrosequencing in detection of BRAF V600E in melanomas. PMID:24917033

  16. Vaccine Therapy With or Without Sargramostim in Treating Patients Who Have Undergone Surgery for Melanoma

    ClinicalTrials.gov

    2015-04-14

    Ciliary Body and Choroid Melanoma, Medium/Large Size; Extraocular Extension Melanoma; Iris Melanoma; Stage IIB Melanoma; Stage IIC Melanoma; Stage IIIA Melanoma; Stage IIIB Melanoma; Stage IIIC Melanoma; Stage IV Melanoma

  17. Radioimmunodetection of lymph node invasion in prostatic cancer. The use of iodine 123 (123I)-labeled monoclonal anti-prostatic acid phosphatase (PAP) 227 A F(ab')2 antibody fragments in vivo.

    PubMed

    Leroy, M; Teillac, P; Rain, J D; Saccavini, J C; Le Duc, A; Najean, Y

    1989-07-01

    The therapeutic indications in prostatic cancer depend on the regional and distant extension of the cancer and are difficult to assess before lymphadenectomy. Radioimmunodetection of lymph node involvement with monoclonal anti-prostatic acid phosphatase (PAP) antibodies can be proposed as a noninvasive alternative to lymphadenectomy. Fifteen patients with various stages of histologically proven prostatic cancer were examined by immunolymphoscintigraphy (ILS) before treatment to detect lymph node metastases. These patients had Stage A (n = 7), Stage B (n = 3), Stage C (n = 2), and Stage D (n = 3) tumors. They received between 100 and 400 micrograms of monoclonal antibody 227 A in the form of F(ab')2 fragments labeled with iodine 123 (123I). The antibody was injected directly into the periprostatic area. ILS images were obtained after 1, 3, 6, and 24 hours. Three days later, each patient underwent a lymphadenectomy for histologic examination. The results of the histologic examination and ILS were compared. In ten patients, the examination did not show any images capable of being interpreted as lymphadenopathy and histologic examination confirmed the integrity of the nodes examined. In five cases, scintigraphy suggested the presence of lymph node invasion by prostatic cancer and this was confirmed by histologic examination in three of the five cases. Overall, in terms of lymphadenopathy, this examination had a sensitivity of 100% and a specificity of 83%. Therefore, ILS appears to be capable of detecting lymph node metastases in prostatic cancer.

  18. 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

  19. [Melanoma secondary prevention].

    PubMed

    Suppa, M; Daxhelet, M; del Marmol, V

    2015-09-01

    Melanoma represents a major public health problem. Its incidence is constantly rising and the mortality rate can be frightfully important if the diagnosis is delayed. Melanoma also exerts a significant economical burden on the society. Therefore there is a need of concrete and pragmatic public health strategies in order to enhance melanoma prevention. Primary prevention of melanoma consists in avoiding excessive exposure to ultraviolet rays,'which represent the main risk factor for the disease occurrence. Secondary prevention is a synonym of melanoma early diagnosis and can be obtained by means of two methods : patients' self-examination and medical examination. Both these examinations must be routinely and thoroughly performed, must be based on the ABCDE rule and the ugly duckling sign, and ideally must be aided by the use of total-body photography. Current international guidelines suggest that all cutaneous screenings should be performed using dermoscopy, a non-invasive imaging technique that allows improving considerably the diagnostic performance. More sophisticated imaging techniques, such as confocal microscopy, are also available in specialised centres. The current scientific evidence supports the efficacy of melanoma primary and secondary prevention programs as a tool to decrease melanoma mortality. Many skin cancer prevention campaigns have been organised worldwide. The most famous and successful in Europe is Euromelanoma.

  20. MDM2 SNP309 promoter polymorphism confers risk for hereditary melanoma.

    PubMed

    Thunell, Lena K; Bivik, Cecilia; Wäster, Petra; Fredrikson, Mats; Stjernström, Annika; Synnerstad, Ingrid; Rosdahl, Inger; Enerbäck, Charlotta

    2014-06-01

    The p53 pathway regulates stress response, and variations in p53, MDM2, and MDM4 may predispose an individual to tumor development. The aim of this study was to study the impact of genetic variation on sporadic and hereditary melanoma. We have analyzed a combination of three functionally relevant variants of the p53 pathway in 258 individuals with sporadic malignant melanomas, 50 with hereditary malignant melanomas, and 799 healthy controls. Genotyping was performed by PCR-restriction fragment length polymorphism, pyrosequencing, and allelic discrimination. We found an increased risk for hereditary melanoma in MDM2 GG homozygotes, which was more pronounced among women (P=0.035). In the event of pairwise combinations of the single nucleotide polymorphisms, a risk elevation was shown for MDM2 GG homozygotes/p53 wild-type Arg in hereditary melanoma (P=0.01). Individuals with sporadic melanomas of the superficial spreading type, including melanoma in situ, showed a slightly higher frequency of the MDM2 GG genotype compared with those with nodular melanomas (P=0.04). The dysplastic nevus phenotype, present in the majority of our hereditary melanoma cases and also in some sporadic cases, further enhanced the effect of the MDM2 GG genotype on melanoma risk (P=0.005). In conclusion, the results show an association between MDM2 SNP309 and an increased risk for hereditary melanoma, especially among women. Analysis of sporadic melanoma also shows an association between MDM2 and the superficial spreading melanoma subtype, as well as an association with the presence of dysplastic nevi in sporadic melanoma. PMID:24625390

  1. A generic strategy for subcloning antibody variable regions from the scFv phage display vector pCANTAB 5 E into pASK85 permits the economical production of F(ab) fragments and leads to improved recombinant immunoglobulin stability.

    PubMed

    Kramer, Karl; Fiedler, Markus; Skerra, Arne; Hock, Bertold

    2002-04-01

    Apart from the decisive sensitivity and specificity of immunosensors, the employed antibodies essentially contribute to additional key factors like fabrication costs for sensor chips and sensor stability. A production scheme for recombinant antibody fragments has been optimised with respect to these particular issues of biosensor development. The phagemid vector pCANTAB 5 E is widely used for the selection of antibody fragments from corresponding libraries. However, large-scale production of the selected single-chain F(v) (scFv) fragments is substantially restricted by the high cost for the inducer IPTG and the anti-E-tag antibody. The latter is needed in significant amounts for the purification of the recombinant protein. A generic strategy was established for subcloning scFv variable regions from pCANTAB 5 E into the plasmid pASK85 for the expression of F(ab) fragments. pASK85 bears coding sequences for murine constant domains including a His(6) tag at the carboxyl-terminal end of the constant heavy chain domain. The anti-s-triazine antibody K47H served as a model system in this study. Biosynthesis of the F(ab) fragment in a high cell density fermenter was induced by addition of anhydrotetracycline. The F(ab) fragment was subsequently purified from the periplasmic extract in a single step by immobilized metal affinity chromatography (IMAC). A yield of 100 microg/lxOD(550) purified F(ab) fragment was obtained employing a standard fermentation scheme. The sensitivity and cross-reactivity of the F(ab) was comparable to the parent scFv when assayed by enzyme immunoassay. However, the F(ab) fragment exhibited significantly improved long-term stability.

  2. Intraoral malignant melanoma

    PubMed Central

    Babburi, Suresh; Subramanyam, R. V.; Aparna, V.; Sowjanya, P.

    2013-01-01

    Primary oral mucosal melanoma is a rare aggressive neoplasm and accounts for only 0.2-8% of all reported melanomas. It is a malignant neoplasm of melanocytes that may arise from a benign melanocytic lesion or de novo from melanocytes within normal skin or mucosa. It is considered to be the most deadly and biologically unpredictable of all human neoplasms, having the worst prognosis. In this article, we report a case of oral melanoma in a 52-year-old female patient with a chief complaint of black discolouration of the maxillary gingiva and palate. PMID:24249959

  3. Coordinate role for cell surface chondroitin sulfate proteoglycan and alpha 4 beta 1 integrin in mediating melanoma cell adhesion to fibronectin

    PubMed Central

    1992-01-01

    Cellular recognition and adhesion to the extracellular matrix (ECM) has a complex molecular basis, involving both integrins and cell surface proteoglycans (PG). The current studies have used specific inhibitors of chondroitin sulfate proteoglycan (CSPG) synthesis along with anti- alpha 4 integrin subunit monoclonal antibodies to demonstrate that human melanoma cell adhesion to an A-chain derived, 33-kD carboxyl- terminal heparin binding fragment of human plasma fibronectin (FN) involves both cell surface CSPG and alpha 4 beta 1 integrin. A direct role for cell surface CSPG in mediating melanoma cell adhesion to this FN fragment was demonstrated by the identification of a cationic synthetic peptide, termed FN-C/H-III, within the fragment. FN-C/H-III is located close to the amino terminal end of the fragment, representing residues #1721-1736 of intact FN. FN-C/H-III binds CSPG directly, can inhibit CSPG binding to the fragment, and promotes melanoma cell adhesion by a CSPG-dependent, alpha 4 beta 1 integrin- independent mechanism. A scrambled version of FN-C/H-III does not inhibit CSPG binding or cell adhesion to the fragment or to FN-C/H-III, indicating that the primary sequence of FN-C/H-III is important for its biological properties. Previous studies have identified three other synthetic peptides from within this 33-kD FN fragment that promote cell adhesion by an arginyl-glycyl-aspartic acid (RGD) independent mechanism. Two of these synthetic peptides (FN-C/H-I and FN-C/H-II) bind heparin and promote cell adhesion, implicating cell surface PG in mediating cellular recognition of these two peptides. Additionally, a third synthetic peptide, CS1, is located in close proximity to FN-C/H-I and FN-C/H-II and it promotes cell adhesion by an alpha 4 beta 1 integrin-dependent mechanism. In contrast to FN-C/H-III, cellular recognition of these three peptides involved contributions from both CSPG and alpha 4 integrin subunits. Of particular importance are observations

  4. Novel Human Three-Domain Antibody Fragments Against sTNFα as Well as tmTNFα with High Affinity Generated by the Combination of Ribosome Display and E. coli Expression System.

    PubMed

    Zhao, X-L; Tian, L-F; Zhang, S-J; Li, J-M; Feng, H; Wang, L-M; Wang, S; Wang, J; Wang, T; Chen, W-Q

    2016-04-01

    Human tumour necrosis factor α (hTNFα) has been proved to be a validated therapeutic target in a number of immune-mediated inflammatory diseases (IMIDs). Fully human monoclonal antibodies (mAbs) that can neutralize soluble hTNFα (sTNFα) as well as transmembrane hTNFα (tmTNFα) are more desirable hTNFα antagonists. Here, we report that novel anti-hTNFα human low-molecular-weight MAbs have been selected and identified using both sTNFα and tmTNFα as target antigens by the combination of ribosome display and E. coli expression system for the first time. As a newly born engineering small molecular antibody, three-domain antibody fragment (VH /κ) provides an alternative promising molecular principle to generate biological agents for TNFα-dependent IMIDs. In this study, a panel of novel human VH /κs (F09, F21, F49 and F409) with high affinity (10(-10) -10(-9) mol/l) to neutralize sTNFα as well as tmTNFα was generated by the combination of ribosome display and E. coli expression system. Among the four clones, F21 and F409 could reduce cytotoxicity on L929 cells induced by sTNFα as well as tmTNFα effectively, and both of them had great potential to inhibit hTNFα-mediated NF-κB activation. Soluble F21 and F409 were also able to inhibit the binding of hTNFα to TNFR1 and TNFR2. The new human antibodies described here have desirable capability to neutralize sTNFα as well as tmTNFα effectively with high affinity and reasonable stability; this may provide an alternative approach for patients who are not responding adequately to currently available anti-TNFα agents. PMID:26860639

  5. Structure of the Fab fragment of the anti-murine EGFR antibody 7A7 and exploration of its receptor binding site.

    PubMed

    Talavera, Ariel; Mackenzie, Jenny; Garrido, Greta; Friemann, Rosmarie; López-Requena, Alejandro; Moreno, Ernesto; Krengel, Ute

    2011-07-01

    The EGF receptor is an important target of cancer immunotherapies. The 7A7 monoclonal antibody has been raised against the murine EGFR, but it cross-reacts with the human receptor. The results from experiments using immune-competent mice can therefore, in principle, be extrapolated to the corresponding scenario in humans. In this work we report the crystal structure of the 7A7 Fab at an effective resolution of 1.4Å. The antibody binding site comprises a deep pocket, located at the interface between the light and heavy chains, with major contributions from CDR loops H1, H2, H3 and L1. Binding experiments show that 7A7 recognizes a site on the EGFR extracellular domain that is not accessible in its most stable conformations, but that becomes exposed upon treatment with a tyrosine kinase inhibitor. This suggests a recognition mechanism similar to that proposed for mAb 806. PMID:21592580

  6. Future perspectives in melanoma research. Meeting report from the “Melanoma Bridge. Napoli, December 2nd-4th 2012”

    PubMed Central

    2013-01-01

    Recent insights into the genetic and somatic aberrations have initiated a new era of rapidly evolving targeted and immune-based treatments for melanoma. After decades of unsuccessful attempts to finding a more effective cure in the treatment of melanoma now we have several drugs active in melanoma. The possibility to use these drugs in combination to improve responses to overcome the resistance, to potentiate the action of immune system with the new immunomodulating antibodies, and identification of biomarkers that can predict the response to a particular therapy represent new concepts and approaches in the clinical management of melanoma. The third “Melanoma Research: “A bridge from Naples to the World” meeting, shortened as “Bridge Melanoma Meeting” took place in Naples, December 2 to 4th, 2012. The four topics of discussion at this meeting were: advances in molecular profiling and novel biomarkers, combination therapies, novel concepts toward integrating biomarkers and therapies into contemporary clinical management of patients with melanoma across the entire spectrum of disease stage, and the knowledge gained from the biology of tumor microenvironment across different tumors as a bridge to impact on prognosis and response to therapy in melanoma. This international congress gathered more than 30 international faculty members who in an interactive atmosphere which stimulated discussion and exchange of their experience regarding the most recent advances in research and clinical management of melanoma patients. PMID:23731854

  7. Construction of prokaryotic expression system of 2 148-bp fragment from cagA gene and detection of cagA gene, CagA protein in Helicobacter pylori isolates and its antibody in sera of patients

    PubMed Central

    Yan, Jie; Wang, Yuan; Shao, Shi-He; Mao, Ya-Fei; Li, Hua-Wen; Luo, Yi-Hui

    2004-01-01

    AIM: To construct a prokaryotic expression system of a Helicobacter pylori (H pylori) cagA gene fragment and establish enzyme-linked immunosorbent assays (ELISA) for detecting CagA and its antibody, so as to understand the manner in which the infection of CagA-expressing H pylori (CagA+ H pylori) isolates cause diseases. METHODS: H pylori strains in gastric biopsy specimens from 156 patients with positive results in rapid urease test were isolated. PCR was used to detect the frequency of cagA gene in the 109 H pylori isolates and to amplify a 2 148-bp fragment (cagA1) of cagA gene from a clinical strain Y06. A prokaryotic expression system of cagA1 gene was constructed, and the expression of the target recombinant protein (rCagA1) was examined by SDS-PAGE. Western blotting and immunodiffusion assay were employed to determine the immunoreactivity and antigenicity of rCagA1, respectively. Two ELISAs were established to detect CagA expression in 109 H pylori isolates and the presence of CagA antibody in the corresponding patients’ sera, and the correlations between infection with CagA+ H pylori and gastritis as well as peptic ulcer were analyzed. RESULTS: Of all the clinical specimens obtained, 80.8% (126/156) were found to have H pylori isolates and 97.2% of the isolates (106/109) were positive for cagA gene. In comparison with the reported data, the cloned cagA1 fragment possessed 94.83% and 93.30% homologies with the nucleotide and putative amino acid sequences, respectively. The output of rCagA1 produced by the constructed recombinant prokaryotic expression system was approximately 30% of the total bacterial protein. rCagA1 was able to bind to the commercial antibody against the whole-cells of H pylori and to induce the immunized rabbits to produce antibody with an immunodiffusion titer of 1:4. A proportion as high as 92.6% of the H pylori isolates (101/109) expressed CagA and 88.1% of the patients’ serum samples (96/109) were CagA antibody-positive. The

  8. 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

  9. Melanoma International Foundation

    MedlinePlus

    ... Exam Card Download a Patient Navigation Card Events, Webinars & Videos Events, Webinars & Videos Patient Experience Video Events Host an Event Past Webinars Upcoming Webinars Volunteer Blog Blog Melanoma? The Doctor ...

  10. Melanoma Epidemiology and Prevention.

    PubMed

    Berwick, Marianne; Buller, David B; Cust, Anne; Gallagher, Richard; Lee, Tim K; Meyskens, Frank; Pandey, Shaily; Thomas, Nancy E; Veierød, Marit B; Ward, Sarah

    2016-01-01

    The epidemiology of melanoma is complex, and individual risk depends on sun exposure, host factors, and genetic factors, and in their interactions as well. Sun exposure can be classified as intermittent, chronic, or cumulative (overall) exposure, and each appears to have a different effect on type of melanoma. Other environmental factors, such as chemical exposures-either through occupation, atmosphere, or food-may increase risk for melanoma, and this area warrants further study. Host factors that are well known to be important are the numbers and types of nevi and the skin phenotype. Genetic factors are classified as high-penetrant genes, moderate-risk genes, or low-risk genetic polymorphisms. Subtypes of tumors, such as BRAF-mutated tumors, have different risk factors as well as different therapies. Prevention of melanoma has been attempted using various strategies in specific subpopulations, but to date optimal interventions to reduce incidence have not emerged. PMID:26601858

  11. Anorectal melanoma. An update.

    PubMed

    Reina, Angel; Errasti, José; Espín, Eloy

    2014-10-01

    Anorectal melanoma is an uncommon and aggressive disease. Because the patients often present with non specific complaints, a high clinical suspicion is important to avoid a delayed diagnosis. Patients undergoing radical surgery have no significant survival difference compared to those undergoing wide local excision. Abdominoperineal resection should be reserved for selected patients in whom local excision is not technically possible or cannot obtain a clear margin. The indiscriminate use of groin dissection is not advisable in anorectal melanoma and should be use in selected cases. Systemic chemotherapy is generally a non effective treatment and continues be studied. Radiation therapy can be used as hypofractionated radiation therapy combined with local excision or in a palliative setting. The oncological outcomes in anorectal melanoma are very poor. The aim of the present study is to review clinicopathology features and management of anorectal melanoma.

  12. Melanoma and Hawaii's youth.

    PubMed

    Williams, Laura

    2004-03-01

    Hawaii's sandy beaches, warm crystal waters, and mild climate attract tourists and residents alike to enjoy hours of outdoor activities under the sun. As frequent participants of these sun related activities, Hawaii's youth are exposed to high levels and duration of ultraviolet radiation throughout their early lives. This study aims to define occurrence trends of cutaneous malignant melanoma in Hawaii in correlation to increased childhood ultraviolet exposure. This paper addresses trends in melanoma incidence during 1979-2002 for Hawaii residents < 25 years of age. Data obtained from this review were analyzed by age group and ethnicity. Results show that although the incidence of melanoma is increasing for Hawaii residents over 25 years of age, the rate of melanoma occurrence in Hawaii's youth (< 25 years) is not increasing. PMID:15124743

  13. Booster Vaccination in Preventing Disease Recurrence in Previously Vaccinated Patients With Melanoma That Has Been Removed By Surgery

    ClinicalTrials.gov

    2015-01-23

    Recurrent Melanoma; Stage IA Skin Melanoma; Stage IB Skin Melanoma; Stage IIA Skin Melanoma; Stage IIB Skin Melanoma; Stage IIC Skin Melanoma; Stage IIIA Skin Melanoma; Stage IIIB Skin Melanoma; Stage IIIC Skin Melanoma; Stage IV Skin Melanoma

  14. Intravital Microscopy for Identifying Tumor Vessels in Patients With Stage IA-IV Melanoma That is Being Removed by Surgery

    ClinicalTrials.gov

    2016-01-13

    Recurrent Melanoma; Stage IA Skin Melanoma; Stage IB Skin Melanoma; Stage IIA Skin Melanoma; Stage IIB Skin Melanoma; Stage IIC Skin Melanoma; Stage IIIA Skin Melanoma; Stage IIIB Skin Melanoma; Stage IIIC Skin Melanoma; Stage IV Skin Melanoma

  15. Familial cutaneous melanoma.

    PubMed

    Hansson, Johan

    2010-01-01

    Approximately 5-10 % of all cutaneous melanomas occur in families with hereditary melanoma predisposition. Worldwide, approximately 20-40% of kindreds with familial elanoma harbor germline mutations in the CDKN2A gene, located on chromosome 9p21, which encodes two different proteins, p16INK4 and p14ARF, both involved in regulation of cell cycle progression and induction of senescence. In different populations several recurring CDKN2A founder mutations have been described. The risk of melanoma in CDKN2A mutations carriers varies between populations and is higher in regions with high sun exposure and high incidence of melanoma in the general population. Some CDKN2A mutations have been associated not only with melanoma but also with increased risk of other malignancies--most notably pancreatic carcinoma. A much smaller number of families have germline mutations in the CDK4 gene on chromosome 12q14, encoding a cyclin dependent kinase which normally interacts with p16INK4A. The management of families with hereditary melanoma is discussed. PMID:20687502

  16. Identification of cells initiating human melanomas

    PubMed Central

    Schatton, Tobias; Murphy, George F.; Frank, Natasha Y.; Yamaura, Kazuhiro; Waaga-Gasser, Ana Maria; Gasser, Martin; Zhan, Qian; Jordan, Stefan; Duncan, Lyn M.; Weishaupt, Carsten; Fuhlbrigge, Robert C.; Kupper, Thomas S.; Sayegh, Mohamed H.; Frank, Markus H.

    2012-01-01

    Tumour-initiating cells capable of self-renewal and differentiation, which are responsible for tumour growth, have been identified in human haematological malignancies1,2 and solid cancers3–6. If such minority populations are associated with tumour progression in human patients, specific targeting of tumour-initiating cells could be a strategy to eradicate cancers currently resistant to systemic therapy. Here we identify a subpopulation enriched for human malignant-melanoma-initiating cells (MMIC) defined by expression of the chemoresistance mediator ABCB5 (refs 7, 8) and show that specific targeting of this tumorigenic minority population inhibits tumour growth. ABCB5+ tumour cells detected in human melanoma patients show a primitive molecular phenotype and correlate with clinical melanoma progression. In serial human-to-mouse xenotransplantation experiments, ABCB5+ melanoma cells possess greater tumorigenic capacity than ABCB5− bulk populations and re-establish clinical tumour heterogeneity. In vivo genetic lineage tracking demonstrates a specific capacity of ABCB5+ sub-populations for self-renewal and differentiation, because ABCB5+ cancer cells generate both ABCB5+ and ABCB5− progeny, whereas ABCB5− tumour populations give rise, at lower rates, exclusively to ABCB5− cells. In an initial proof-of-principle analysis, designed to test the hypothesis that MMIC are also required for growth of established tumours, systemic administration of a monoclonal antibody directed at ABCB5, shown to be capable of inducing antibody-dependent cell-mediated cytotoxicity in ABCB5+ MMIC, exerted tumour-inhibitory effects. Identification of tumour-initiating cells with enhanced abundance in more advanced disease but susceptibility to specific targeting through a defining chemoresistance determinant has important implications for cancer therapy. PMID:18202660

  17. Crystals of a ternary complex of human immunodeficiency virus type 1 reverse transcriptase with a monoclonal antibody Fab fragment and double-stranded DNA diffract x-rays to 3.5-A resolution.

    PubMed Central

    Jacobo-Molina, A; Clark, A D; Williams, R L; Nanni, R G; Clark, P; Ferris, A L; Hughes, S H; Arnold, E

    1991-01-01

    Two crystal forms of complexes have been grown that contain human immunodeficiency virus type 1 reverse transcriptase and a monoclonal antibody Fab fragment. One of the crystal forms (form II, space group P3112, a = 168.7 A, c = 220.3 A) diffracts x-rays to 3.5-A resolution and appears suitable for moderate-resolution structure determination. The form II crystals have the unusual property that their maximum resolution of diffraction and resistance to radiation damage are enhanced by either crystallization in the presence of or soaking with double-stranded DNA primer-template mimics. These crystals may permit structural studies of catalytically relevant complexes and eventually enable us to experimentally observe successive steps in the reverse transcription process. Images PMID:1720554

  18. Human Parathyroid Hormone IMMUNOLOGICAL CHARACTERIZATION OF ANTIBODIES AGAINST A GLANDULAR EXTRACT AND THE SYNTHETIC AMINO-TERMINAL FRAGMENTS 1-12 AND 1-34 AND THEIR USE IN THE DETERMINATION OF IMMUNOREACTIVE HORMONE IN HUMAN SERA

    PubMed Central

    Fischer, Jan A.; Binswanger, Ulrich; Dietrich, Felix M.

    1974-01-01

    Antibodies to a urea-trichloroacetic acid extract [hPTH-(TCA)] of human parathyroid tumors and to the synthetic NH2-terminal fragments of human parathyroid hormone hPTH-(1-12) and -(1-34) were developed in goats to characterize immunochemically various PTH preparations and to estimate immunoreactive PTH (iPTH) in human sera. They were quantitated on the basis of their capacity to bind [131I]-hPTH-(1-12), [131I]hPTH-(1-34) or [131I]bovine PTH (bPTH-(1-84)). The quality of the antibodies was assessed by reference to inhibition of their interaction with labeled peptides by synthetic hPTH comprising 34 NH2-terminal amino acid residues or fragments thereof [hPTH-(1-12), -(13-34), -(18-34), -(25-34), -(18-24)] or by the Sephadex G-100-purified full-length peptide hPTH-(1-84) [hPTH-(1-84)G-100]. The synthetic peptides used in this work correspond in their structure to the NH2-terminal amino acid sequence 1-34, as elucidated by Brewer and collaborators (1972. Proc. Natl. Acad. Sci. U. S. A.69: 3583-3588). Inhibition studies were also carried out with bPTH-(1-34) and bPTH-(1-84). Anti-hPTH-(TCA) exhibited specificities directed to determinants in the COOH-terminal and NH2-terminal part of hPTH-(1-84) and exhibited cross-reactivity with bPTH-(1-84). Anti-hPTH-(1-34), on the other hand, showed immunological specificities mainly directed to antigenic determinants located in the COOH-terminal half of hPTH-(1-34). In addition, some reactivity with the NH2-terminal hPTH-(1-12) and with the extractive full-length peptides of human and bovine origin was observed. Antibodies to hPTH-(1-12) cross-reacted with hPTH-(1-34) and -(1-84)G-100. iPTH was radioimmunologically determined in human sera by the following systems: (a) [131I]bPTH-(1-84), anti-hPTH-(TCA) and hPTH-(1-84)G-100 as standard; (b) [131I]hPTH-(1-34), anti-hPTH-(1-34) and hPTH-(1-34) as standard. With system (a), COOH-terminal fragments of hPTH-(1-84) having a molecular weight of approximately 7,000 were detected, and

  19. An electrochemical immunosensing method for detecting melanoma cells.

    PubMed

    Seenivasan, Rajesh; Maddodi, Nityanand; Setaluri, Vijaysaradhi; Gunasekaran, Sundaram

    2015-06-15

    An electrochemical immunosensing method was developed to detect melanoma cells based on the affinity between cell surface melanocortin 1 receptor (MC1R) antigen and anti-MC1R antibody (MC1R-Ab). The MC1R-Abs were immobilized in amino-functionalized silica nanoparticles (n-SiNPs)-polypyrrole (PPy) nanocomposite modified on working electrode surface of screen-printed electrode (SPE). Cyclic voltammetry was employed, with the help of redox mediator ([Fe(CN)6](3-)), to measure the change in anodic oxidation peak current arising due to the specific interaction between MC1R antigens and MC1R-Abs when the target melanoma cells are present in the sample. Various factors affecting the sensor performance, such as the amount of MC1R-Abs loaded, incubation time with the target melanoma cells, the presence of interfering non-melanoma cells, were tested and optimized over different expected melanoma cell loads in the range of 50-7500 cells/2.5 mL. The immunosensor is highly sensitive (20 cells/mL), specific, and reproducible, and the antibody-loaded electrode in ready-to-use stage is stable over two weeks. Thus, in conjunction with a microfluidic lab-on-a-chip device our electrochemical immunosensing approach may be suitable for highly sensitive, selective, and rapid detection of circulating tumor cells (CTCs) in blood samples.

  20. An electrochemical immunosensing method for detecting melanoma cells

    PubMed Central

    Seenivasan, Rajesh; Maddodi, Nityanand; Setaluri, Vijaysaradhi; Gunasekaran, Sundaram

    2015-01-01

    An electrochemical immunosensing method was developed to detect melanoma cells based on the affinity between cell surface melanocortin 1 receptor (MC1R) antigen and anti-MC1R antibody (MC1R-Ab). The MC1R-Abs were immobilized in amino-functionalized silica nanoparticles (n-SiNPs)-polypyrrole (PPy) nanocomposite modified on working electrode surface of screen-printed electrode (SPE). Cyclic voltammetry was employed, with the help of redox mediator ([Fe(CN)6]3−), to measure the change in anodic oxidation peak current arising due to the specific interaction between MC1R antigens and MC1R-Abs when the target melanoma cells are present in the sample. Various factors affecting the sensor performance, such as the amount of MC1R-Abs loaded, incubation time with the target melanoma cells, the presence of interfering non-melanoma cells, were tested and optimized over different expected melanoma cell loads in the range of 50–7500 cells/2.5 mL. The immunosensor is highly sensitive (20 cells/mL), specific, and reproducible, and the antibody-loaded electrode in ready-to-use stage is stable over two weeks. Thus, in conjunction with a microfluidic lab-on-a-chip device our electrochemical immunosensing approach may be suitable for highly sensitive, selective, and rapid detection of circulating tumor cells (CTCs) in blood samples. PMID:25636023

  1. Overexpression and mutations of p53 in metastatic malignant melanomas.

    PubMed

    Hartmann, A; Blaszyk, H; Cunningham, J S; McGovern, R M; Schroeder, J S; Helander, S D; Pittelkow, M R; Sommer, S S; Kovach, J S

    1996-07-29

    Alterations of the p53 tumor suppressor gene are the most frequent genetic abnormalities in human malignancies, but the role of p53 in the etiology of malignant melanomas is unclear. Fifty unselected malignant melanomas were analyzed for p53 overexpression by immunohistochemistry using 3 monoclonal antibodies (MAbs). Fifteen tumors (29.4%) showed positive staining with at least 2 different antibodies. In the first 20 consecutive tumors exons 5-9 and adjacent splice sites of the p53 gene were analyzed by genomic sequencing. There were 4 mutations in 20 metastatic melanomas. Three of 4 mutations were C:G-->T:A transitions. A search of our database of p53 mutations revealed that out of 8 p53 mutations reported by others, 4 are C:G-->T:A transitions at dipyrimidine sites, and one is a tandem CC-->TT mutation. This mutational pattern is comparable with the pattern of p53 mutations in squamous cell and basal cell carcinomas of the skin and is related to exposure to ultraviolet B (UV-B) wavelength radiation. Taken together with a predominance of UV-induced mutations in the CDKN2/ p16 gene demonstrated in melanoma cell lines, our data support a role of sunlight exposure in the etiology of malignant melanoma. The low frequency of p53 mutants in melanomas compared with other types of skin cancers suggests that although mutations in this gene are likely to be involved in the development of some malignant melanomas, they do not play as large a role as in squamous and basal cell carcinomas of the skin. PMID:8707401

  2. What Is Melanoma Skin Cancer?

    MedlinePlus

    ... that can become melanoma. They make a brown pigment called melanin , which gives the skin its tan ... to the sun, melanocytes make more of the pigment, causing the skin to tan or darken. Melanoma ...

  3. Melanoma-associated antigen synthesized in vitro for active specific immunotherapy.

    PubMed

    Hayashibe, K

    1992-11-01

    The immunogenicity of the antigen molecule is a prerequisite for active specific immunotherapy for melanoma. Since most of the melanoma-associated antigens recognized by the murine immune system are known to be not immunogenic in man, a detection and analysis system for melanoma-associated antigens is required to reflect in vivo immune responses in patients with melanoma. One of the promising approaches, an attempt to develop human monoclonal antibodies from B lymphocytes of patients with melanoma, has met with limited success due to the difficulties of producing large amounts of antibodies and using them in immunochemical assays, because most of them belong to the IgM class and have low affinity. Our approach is to utilize the screening of a cDNA expression library constructed from mRNA extracted from cultured melanoma cells with antibodies from patients with melanoma. The cloned cDNA, designated as D-1, had 1029 bp and showed no significant homology with viral and mammalian sequences stored in GENETYX. cDNA D-1 hybridized to a 2.0 kb mRNA species from 3 different cell lines of human melanoma, neuroblastoma, erythroleukemia, B lymphoid, and T lymphoid cells, but not from a renal carcinoma cell line, normal peripheral lymphocytes, or normal fibroblasts. The in vivo expression and distribution of mRNA related to cDNA D-1 has been examined in tissue specimens by in situ hybridization and shown to be rather restricted on melanoma cells. The polypeptide antigen encoded by cDNA D-1 may be a valuable immunogen for implementing active specific immunotherapy in patients with melanoma.

  4. Uveal melanoma: Estimating prognosis

    PubMed Central

    Kaliki, Swathi; Shields, Carol L; Shields, Jerry A

    2015-01-01

    Uveal melanoma is the most common primary malignant tumor of the eye in adults, predominantly found in Caucasians. Local tumor control of uveal melanoma is excellent, yet this malignancy is associated with relatively high mortality secondary to metastasis. Various clinical, histopathological, cytogenetic features and gene expression features help in estimating the prognosis of uveal melanoma. The clinical features associated with poor prognosis in patients with uveal melanoma include older age at presentation, male gender, larger tumor basal diameter and thickness, ciliary body location, diffuse tumor configuration, association with ocular/oculodermal melanocytosis, extraocular tumor extension, and advanced tumor staging by American Joint Committee on Cancer classification. Histopathological features suggestive of poor prognosis include epithelioid cell type, high mitotic activity, higher values of mean diameter of ten largest nucleoli, higher microvascular density, extravascular matrix patterns, tumor-infiltrating lymphocytes, tumor-infiltrating macrophages, higher expression of insulin-like growth factor-1 receptor, and higher expression of human leukocyte antigen Class I and II. Monosomy 3, 1p loss, 6q loss, and 8q and those classified as Class II by gene expression are predictive of poor prognosis of uveal melanoma. In this review, we discuss the prognostic factors of uveal melanoma. A database search was performed on PubMed, using the terms “uvea,” “iris,” “ciliary body,” “choroid,” “melanoma,” “uveal melanoma” and “prognosis,” “metastasis,” “genetic testing,” “gene expression profiling.” Relevant English language articles were extracted, reviewed, and referenced appropriately. PMID:25827538

  5. Capture and On-chip analysis of Melanoma Cells Using Tunable Surface Shear forces

    PubMed Central

    Tsao, Simon Chang-Hao; Vaidyanathan, Ramanathan; Dey, Shuvashis; Carrascosa, Laura G.; Christophi, Christopher; Cebon, Jonathan; Shiddiky, Muhammad J. A.; Behren, Andreas; Trau, Matt

    2016-01-01

    With new systemic therapies becoming available for metastatic melanoma such as BRAF and PD-1 inhibitors, there is an increasing demand for methods to assist with treatment selection and response monitoring. Quantification and characterisation of circulating melanoma cells (CMCs) has been regarded as an excellent non-invasive candidate but a sensitive and efficient tool to do these is lacking. Herein we demonstrate a microfluidic approach for melanoma cell capture and subsequent on-chip evaluation of BRAF mutation status. Our approach utilizes a recently discovered alternating current electrohydrodynamic (AC-EHD)-induced surface shear forces, referred to as nanoshearing. A key feature of nanoshearing is the ability to agitate fluid to encourage contact with surface-bound antibody for the cell capture whilst removing nonspecific cells from the surface. By adjusting the AC-EHD force to match the binding affinity of antibodies against the melanoma-associated chondroitin sulphate proteoglycan (MCSP), a commonly expressed melanoma antigen, this platform achieved an average recovery of 84.7% from biological samples. Subsequent staining with anti-BRAFV600E specific antibody enabled on-chip evaluation of BRAFV600E mutation status in melanoma cells. We believe that the ability of nanoshearing-based capture to enumerate melanoma cells and subsequent on-chip characterisation has the potential as a rapid screening tool while making treatment decisions. PMID:26815318

  6. 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

  7. Thrombus imaging with indium-111 and iodine-131-labeled fibrin-specific monoclonal antibody and its F(ab')2 and Fab fragments

    SciTech Connect

    Rosebrough, S.F.; Grossman, Z.D.; McAfee, J.G.; Kudryk, B.J.; Subramanian, G.; Ritter-Hrncirik, C.A.; Witanowski, L.S.; Tillapaugh-Fay, G.; Urrutia, E.; Zapf-Longo, C.

    1988-07-01

    We have previously reported successful imaging of fresh (2-4 hr old) and aged (1-5 days old) canine thrombi with /sup 131/I-labeled intact monoclonal antibody (MAb) specific for fibrin. We now report thrombus imaging with /sup 131/I-labeled F(ab')2 and Fab and /sup 111/In-labeled intact MAb, F(ab')2, and Fab. Indium-111-labeled F(ab')2 proved to be the best imaging agent due to less nonspecific binding in the liver than whole IgG. Image quality was improved by the higher administered dose permissible with /sup 111/In and its better physical characteristics for imaging, compared to /sup 131/I. Immunofluorescence of fresh human histologic sections showed intact MAb and F(ab')2 binding to thrombi, pulmonary emboli, and atherosclerotic plaques, strengthening the feasibility of clinical thrombus imaging.

  8. Inhibition of human immunodeficiency virus type 1 integrase by the Fab fragment of a specific monoclonal antibody suggests that different multimerization states are required for different enzymatic functions.

    PubMed

    Barsov, E V; Huber, W E; Marcotrigiano, J; Clark, P K; Clark, A D; Arnold, E; Hughes, S H

    1996-07-01

    We have characterized a murine monoclonal antibody (MAb 35), which was raised against human immunodeficiency virus type 1 (HIV-1) integration protein (IN), and the corresponding Fab 35. Although MAb 35 does not inhibit HIV-1 IN, Fab 35 does. MAb 35 (and Fab 35) binds to an epitope in the C-terminal region of HIV-1 IN. Fab 35 inhibits 3'-end processing, strand transfer, and disintegration; however, DNA binding is not affected. The available data suggest that Fab 35 inhibits enzymatic activities of IN by interfering with the ability of IN to form multimers that are enzymatically active. This implies that the C-terminal region of HIV-1 IN participates in interactions that are essential for the multimerization of IN. Titration of the various IN-mediated enzymatic activities suggests that different degrees of multimerization are required for different activities of HIV-1 IN.

  9. Culturing Uveal Melanoma Cells.

    PubMed

    Angi, Martina; Versluis, Mieke; Kalirai, Helen

    2015-04-01

    A major challenge in cancer research is the use of appropriate models with which to study a specific biological question. Cell lines have long been used to study cellular processes and the effects of individual molecules because they are easy to use, grow rapidly, produce reproducible results and have a strong track record in research. In uveal melanoma in particular, the absence of animal models that faithfully replicate the behavior of the human disease has propagated the generation and use of numerous cell lines by individual research groups. This in itself, however, can be viewed as a problem due to the lack of standardization when characterizing these entities to determine how closely they reflect the genetic and phenotypic characteristics of this disease. The alternative is to use in vitro primary cultures of cells obtained directly from uveal melanoma patient samples, but this too has its difficulties. Primary cell cultures are difficult to use, hard to obtain and can show considerable heterogeneity. In this article, we review the following: (1) the uveal melanoma cell lines that are currently available, discussing the importance of establishing a bank of those that represent the molecular heterogeneity of uveal melanoma; (2) the methods used to isolate and perform short-term cultures of primary uveal melanoma cells, and (3) the establishment of 3D tissue culture models that bridge the gap between 2D in vitro systems and in vivo models with which to dissect cancer biology and perform therapeutic screens. PMID:27171555

  10. Methods of Melanoma Detection.

    PubMed

    Leachman, Sancy A; Cassidy, Pamela B; Chen, Suephy C; Curiel, Clara; Geller, Alan; Gareau, Daniel; Pellacani, Giovanni; Grichnik, James M; Malvehy, Josep; North, Jeffrey; Jacques, Steven L; Petrie, Tracy; Puig, Susana; Swetter, Susan M; Tofte, Susan; Weinstock, Martin A

    2016-01-01

    Detection and removal of melanoma, before it has metastasized, dramatically improves prognosis and survival. The purpose of this chapter is to (1) summarize current methods of melanoma detection and (2) review state-of-the-art detection methods and technologies that have the potential to reduce melanoma mortality. Current strategies for the detection of melanoma range from population-based educational campaigns and screening to the use of algorithm-driven imaging technologies and performance of assays that identify markers of transformation. This chapter will begin by describing state-of-the-art methods for educating and increasing awareness of at-risk individuals and for performing comprehensive screening examinations. Standard and advanced photographic methods designed to improve reliability and reproducibility of the clinical examination will also be reviewed. Devices that magnify and/or enhance malignant features of individual melanocytic lesions (and algorithms that are available to interpret the results obtained from these devices) will be compared and contrasted. In vivo confocal microscopy and other cellular-level in vivo technologies will be compared to traditional tissue biopsy, and the role of a noninvasive "optical biopsy" in the clinical setting will be discussed. Finally, cellular and molecular methods that have been applied to the diagnosis of melanoma, such as comparative genomic hybridization (CGH), fluorescent in situ hybridization (FISH), and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), will be discussed. PMID:26601859

  11. Selection of antibodies from synthetic antibody libraries.

    PubMed

    Harel Inbar, Noa; Benhar, Itai

    2012-10-15

    More than 2 dozen years had passed since the field of antibody engineering was established, with the first reports of bacterial [1-3] and mammalian cells [4] expression of recombinant antibody fragments, and in that time a lot of effort was dedicated to the development of efficient technological means, intended to assist in the creation of therapeutic monoclonal antibodies (mAbs). Research focus was given to two intertwined technological aspects: the selection platform and the recombinant antibody repertoires. In accordance with these areas of interest, it is the goal of this chapter to describe the various selection tools and antibody libraries existing, with emphasis on the later, and their applications. This chapter gives a far from exhaustive, subjective "historic account" of the field, describing the selection platforms, the different formats of antibody repertoires and the applications of both for selecting recombinant antibodies. Several excellent books provide detailed protocols for constructing antibody libraries and selecting antibodies from those libraries [5-13]. Such books may guide a newcomer to the field in the fine details of antibody engineering. We would like to offer advice to the novice: although seemingly simple, effective library construction and antibody isolation provide best benefits in the hands of professionals. It is an art as much as it is science.

  12. Lymphoscintigraphy in malignant melanoma

    SciTech Connect

    Berman, C.G.; Norman, J.; Cruse, C.W.; Reintgen, D.S.; Clark, R.A. )

    1992-01-01

    The development and rationale for the use of lymphoscintigraphy in the preoperative evaluation of patients with malignant melanoma being considered for elective lymph node dissection is reviewed. This overview is updated by an analysis of 135 patients with early stage malignant melanoma involving the head, neck, shoulders, and trunk at Moffitt Cancer Center and Research Institute at the University of South Florida (Tampa, FL). High discordancy rates (overall, 41%) were seen between drainage patterns predicted from historical anatomical guidelines and those revealed by the lymphoscintigraphic examination. The high discordancy rate was most pronounced in the head (64%) and the neck (73%). Surgical management was changed in 33% of the patients, overall. A preoperative lymphoscintigram is recommended for all patients with melanoma with head, neck, and truncal lesions evaluated for elective lymph node dissection as the lymphatic drainage patterns are often unpredictable and variable.

  13. Vitamins and Melanoma

    PubMed Central

    Russo, Irene; Caroppo, Francesca; Alaibac, Mauro

    2015-01-01

    A tremendous amount of information was published over the past decades in relation to the role of vitamins in various neoplastic diseases. In particular, several studies showed an inverse relationship between selected vitamins intake and cancer risk. In this review we will focus on the role played by vitamins in melanoma with particular regard to vitamin A, D, K, E and C. Given that vitamin supplementation is easy, convenient, and readily accepted by patients, in the future the use of vitamins in chemoprevention and therapy of melanoma could be encouraged if supported by pre-clinical and clinical evidence. PMID:26213971

  14. The Role of Intralesional Therapies in Melanoma.

    PubMed

    Agarwala, Sanjiv S

    2016-05-01

    The US Food and Drug Administration has been rapidly approving new checkpoint inhibitors and targeted therapies for melanoma and other tumors. Recently, it approved the first intralesional therapy, talimogene laherparepvec (T-VEC), for the treatment of metastatic melanoma lesions in the skin and lymph nodes. Several other intralesional therapies (PV-10, interleukin-12 electroporation, coxsackievirus A21 [CVA21]) are entering later-stage testing. Locally injected agents have clearly shown their ability to produce local responses that can be durable. The possibility that they also stimulate a regional and even systemic immune response is exciting, as this potential effect may have utility in combination regimens; such regimens are an area of active research. Favorable responses with minimal toxicities in monotherapy trials have led to the first melanoma studies of T-VEC in combination with the cytotoxic T-lymphocyte-associated antigen 4 inhibitor ipilimumab and, separately, with the programmed death 1-blocking antibody pembrolizumab. Studies of PV-10 with pembrolizumab and of CVA21 with pembrolizumab are also being initiated. Preliminary analyses of the results of the first combination trials, which show higher response rates than with either agent alone, offer some optimism that these locoregional therapies will find application--as treatment for patients who cannot tolerate systemic immunotherapies, to alleviate locoregional morbidity, and perhaps even to "prime" the immune system.

  15. Cutaneous melanoma: new advances in treatment*

    PubMed Central

    Foletto, Michele Ceolin; Haas, Sandra Elisa

    2014-01-01

    Cutaneous melanoma is a challenge to treat. Over the last 30 years, no drug or combination of drugs demonstrated significant impact to improve patient survival. From 1995 to 2000, the use of cytokines such as interferon and interleukin become treatment options. In 2011, new drugs were approved by the U.S. Food and Drug Administration, including peginterferon alfa-2b for patients with stage III disease, vemurafenib for patients with metastatic melanoma with the BRAF V600E mutation, and ipilimumab, a monoclonal antibody directed to the CTLA-4 T lymphocyte receptor, to combat metastatic melanoma in patients who do not have the BRAF V600E mutation. Both ipilimumab and vemurafenib showed results in terms of overall survival. Other trials with inhibitors of other genes, such as the KIT gene and MEK, are underway in the search for new discoveries. The discovery of new treatments for advanced or metastatic disease aims to relieve symptoms and improve patient quality of life. PMID:24770508

  16. Early intervention in the 3xTg-AD mice with an amyloid β-antibody fragment ameliorates first hallmarks of Alzheimer disease.

    PubMed

    Giménez-Llort, Lydia; Rivera-Hernández, Geovanny; Marin-Argany, Marta; Sánchez-Quesada, José L; Villegas, Sandra

    2013-01-01

    The single-chain variable fragment, scFv-h3D6, has been shown to prevent in vitro toxicity induced by the amyloid β (Aβ) peptide in neuroblastoma cell cultures by withdrawing Aβ oligomers from the amyloid pathway. Present study examined the in vivo effects of scFv-h3D6 in the triple-transgenic 3xTg-AD mouse model of Alzheimer disease. Prior to the treatment, five-month-old female animals, corresponding to early stages of the disease, showed the first behavioral and psychological symptoms of dementia -like behaviors. Cognitive deficits included long- and short-term learning and memory deficits and high swimming navigation speed. After a single intraperitoneal dose of scFv-h3D6, the swimming speed was reversed to normal levels and the learning and memory deficits were ameliorated. Brain tissues of these animals revealed a global decrease of Aβ oligomers in the cortex and olfactory bulb after treatment, but this was not seen in the hippocampus and cerebellum. In the untreated 3xTg-AD animals, we observed an increase of both apoJ and apoE concentrations in the cortex, as well as an increase of apoE in the hippocampus. Treatment significantly recovered the non-pathological levels of these apolipoproteins. Our results suggest that the benefit of scFv-h3D6 occurs at both behavioral and molecular levels.

  17. Immune evasion by murine melanoma mediated through CC chemokine receptor-10.

    PubMed

    Murakami, Takashi; Cardones, Adela R; Finkelstein, Steven E; Restifo, Nicholas P; Klaunberg, Brenda A; Nestle, Frank O; Castillo, S Sianna; Dennis, Phillip A; Hwang, Sam T

    2003-11-01

    Human melanoma cells frequently express CC chemokine receptor (CCR)10, a receptor whose ligand (CCL27) is constitutively produced by keratinocytes. Compared with B16 murine melanoma, cells rendered more immunogenic via overexpression of luciferase, B16 cells that overexpressed both luciferase and CCR10 resisted host immune responses and readily formed tumors. In vitro, exposure of tumor cells to CCL27 led to rapid activation of Akt, resistance to cell death induced by melanoma antigen-specific cytotoxic T cells, and phosphatidylinositol-3-kinase (PI3K)-dependent protection from apoptosis induced by Fas cross-linking. In vivo, cutaneous injection of neutralizing antibodies to endogenous CCL27 blocked growth of CCR10-expressing melanoma cells. We propose that CCR10 engagement by locally produced CCL27 allows melanoma cells to escape host immune antitumor killing mechanisms (possibly through activation of PI3K/Akt), thereby providing a means for tumor progression. PMID:14581607

  18. Small Molecules Antagonise the MIA-Fibronectin Interaction in Malignant Melanoma

    PubMed Central

    Yip, King Tuo; Zhong, Xue Yin; Seibel, Nadia; Pütz, Stefanie; Autzen, Jasmin; Gasper, Raphael; Hofmann, Eckhard; Scherkenbeck, Jürgen; Stoll, Raphael

    2016-01-01

    Melanoma inhibitory activity (MIA), an extracellular protein highly expressed by malignant melanoma cells, plays an important functional role in melanoma development, progression, and metastasis. After its secretion, MIA directly interacts with extracellular matrix proteins, such as fibronectin (FN). By this mechanism, MIA actively facilitates focal cell detachment from surrounding structures and strongly promotes tumour cell invasion and migration. Hence, the molecular understanding of MIA’s function provides a promising target for the development of new strategies in malignant melanoma therapy. Here, we describe for the first time the discovery of small molecules that are able to disrupt the MIA-FN complex by selectively binding to a new druggable pocket, which we could identify on MIA by structural analysis and fragment-based screening. Our findings may inspire novel drug discovery efforts aiming at a therapeutically effective treatment of melanoma by targeting MIA. PMID:27151361

  19. Basic and clinical aspects of malignant melanoma

    SciTech Connect

    Nathanson, L. )

    1987-01-01

    This book contains the following 10 chapters: The role of oncogenes in the pathogenesis of malignant melanoma; Laminin and fibronectin modulate the metastatic activity of melanoma cells; Structure, function and biosynthesis of ganglioside antigens associated with human tumors derived from the neuroectoderm; Epidemiology of ocular melanoma; Malignant melanoma: Prognostic factors; Endocrine influences on the natural history of human malignant melanoma; Psychosocial factors associated with prognostic indicators, progression, psychophysiology, and tumor-host response in cutaneous malignant melanoma; Central nervous system metastases in malignant melanoma; Interferon trials in the management of malignant melanoma and other neoplasms: an overview; and The treatment of malignant melanoma by fast neutrons.

  20. Advances in Antibody Design.

    PubMed

    Tiller, Kathryn E; Tessier, Peter M

    2015-01-01

    The use of monoclonal antibodies as therapeutics requires optimizing several of their key attributes. These include binding affinity and specificity, folding stability, solubility, pharmacokinetics, effector functions, and compatibility with the attachment of additional antibody domains (bispecific antibodies) and cytotoxic drugs (antibody-drug conjugates). Addressing these and other challenges requires the use of systematic design methods that complement powerful immunization and in vitro screening methods. We review advances in designing the binding loops, scaffolds, domain interfaces, constant regions, post-translational and chemical modifications, and bispecific architectures of antibodies and fragments thereof to improve their bioactivity. We also highlight unmet challenges in antibody design that must be overcome to generate potent antibody therapeutics. PMID:26274600

  1. 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

  2. Recombinant Monovalent Llama-Derived Antibody Fragments (VHH) to Rotavirus VP6 Protect Neonatal Gnotobiotic Piglets against Human Rotavirus-Induced Diarrhea

    PubMed Central

    Vlasova, Anastasia N.; Chattha, Kuldeep S.; Gómez-Sebastián, Silvia; Nuñez, Carmen; Alvarado, Carmen; Lasa, Rodrigo; Escribano, José M.; Garaicoechea, Lorena L.; Fernandez, Fernando; Bok, Karin; Wigdorovitz, Andrés; Saif, Linda J.; Parreño, Viviana

    2013-01-01

    Group A Rotavirus (RVA) is the leading cause of severe diarrhea in children. The aims of the present study were to determine the neutralizing activity of VP6-specific llama-derived single domain nanoantibodies (VHH nanoAbs) against different RVA strains in vitro and to evaluate the ability of G6P[1] VP6-specific llama-derived single domain nanoantibodies (VHH) to protect against human rotavirus in gnotobiotic (Gn) piglets experimentally inoculated with virulent Wa G1P[8] rotavirus. Supplementation of the daily milk diet with 3B2 VHH clone produced using a baculovirus vector expression system (final ELISA antibody -Ab- titer of 4096; virus neutralization -VN- titer of 256) for 9 days conferred full protection against rotavirus associated diarrhea and significantly reduced virus shedding. The administration of comparable levels of porcine IgG Abs only protected 4 out of 6 of the animals from human RVA diarrhea but significantly reduced virus shedding. In contrast, G6P[1]-VP6 rotavirus-specific IgY Abs purified from eggs of hyperimmunized hens failed to protect piglets against human RVA-induced diarrhea or virus shedding when administering similar quantities of Abs. The oral administration of VHH nanoAb neither interfered with the host's isotype profiles of the Ab secreting cell responses to rotavirus, nor induced detectable host Ab responses to the treatment in serum or intestinal contents. This study shows that the oral administration of rotavirus VP6-VHH nanoAb is a broadly reactive and effective treatment against rotavirus-induced diarrhea in neonatal pigs. Our findings highlight the potential value of a broad neutralizing VP6-specific VHH nanoAb as a treatment that can complement or be used as an alternative to the current strain-specific RVA vaccines. Nanobodies could also be scaled-up to develop pediatric medication or functional food like infant milk formulas that might help treat RVA diarrhea. PMID:23658521

  3. 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

  4. 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.

  5. 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.

  6. Management of a patient with advanced BRAF-mutant melanoma.

    PubMed

    Ashworth, Michelle T; Daud, Adil

    2014-03-01

    A 49-year-old man initially diagnosed in 1995 with cutaneous melanoma presented to the authors' institution in 2009 with metastatic, BRAF V600E-mutant melanoma. His treatment course to date has included surgery, adjuvant radiotherapy, and interferon, metastasectomies, granulocyte-macrophage colony-stimulating factors, a clinical trial with the BRAF inhibitor vemurafenib (PLX-4032), clinical trial with combination BRAF plus MEK inhibition with vemurafenib plus GDC-0973, and combination targeted and immune therapy with vemurafenib plus the anti-CTLA4 antibody ipilimumab. This case report illustrates the long-term management of a patient with metastatic melanoma using targeted and immune therapy, evolution in treatment guidelines, next directions in research, and the critical role of clinical trials in advancement of patient care.

  7. Melanoma immunotherapy: historical precedents, recent successes and future prospects.

    PubMed

    Raaijmakers, Marieke I G; Rozati, Sima; Goldinger, Simone M; Widmer, Daniel S; Dummer, Reinhard; Levesque, Mitchell P

    2013-02-01

    The idea of cancer immunotherapy has been around for more than a century; however, the first immunotherapeutic ipilimumab, an anti-CTLA-4 antibody, has only recently been approved by the US FDA for melanoma. With an increasing understanding of the immune response, it is expected that more therapies will follow. This review aims to provide a general overview of immunotherapy in melanoma. We first explain the development of cancer immunotherapy more than a century ago and the general opinions about it over time. This is followed by a general overview of the immune reaction in order to give insight into the possible targets for therapy. Finally, we will discuss the current therapies for melanoma, their shortcomings and why it is important to develop patient stratification criteria. We conclude with an overview of recent discoveries and possible future therapies.

  8. Uveal Melanoma Expression of Indoleamine 2, 3 Deoxygenase: Establishment of an Immune Privileged Environment by Tryptophan Depletion

    PubMed Central

    Chen, Peter W.; Mellon, Jessamee K.; Mayhew, Elizabeth; Wang, Shixuan; He, Yu Guang; Hogan, Nick; Niederkorn, Jerry Y.

    2009-01-01

    The enzyme indoleamine 2, 3-dioxygenase (IDO) catalyzes degradation of tryptophan, an essential amino acid required for lymphocyte activation and proliferation. Many tumors express IDO which implied that it acts as a mechanism to evade T cell-mediated immune attack, and also to establish an immunosuppressive tumor microenvironment. The purpose of this study was to determine whether primary and metastatic uveal melanoma expressed the IDO gene and whether uveal melanoma cells could deplete tryptophan. In situ expression of IDO in primary uveal melanoma from tumor bearing eyes and metastatic uveal melanoma liver tissues was determined by immunohistostaining with IDO-specific antibody. Reverse transcription PCR was used to assess IDO gene transcription by primary and metastatic uveal melanoma cell lines. IDO protein expression was determined by Western blot of uveal melanoma cell protein lysate. IDO catalytic activity was assessed by measuring the presence of kynurenine, a product generated by tryptophan degradation, in uveal melanoma culture supernatants. Primary uveal melanoma from tumor-bearing eyes and metastatic uveal melanoma from the liver did not express IDO in situ. IDO was not constitutively expressed in either primary or metastatic uveal melanoma cell lines. However, stimulation of primary and metastatic uveal melanoma cell cultures with interferon-gamma (IFN-γ) universally upregulated both IDO gene and protein expression. Culture supernatants from IFN-γ treated primary and metastatic uveal melanoma cell cultures contained elevated levels of kynurenine. Addition of the IDO inhibitor 1-methyl DL-tryptophan significantly diminished kynurenine levels in IFN-γ treated uveal melanoma cell cultures. The results from this study suggest that IFN-γ inducible IDO upregulation by primary and metastatic uveal melanoma may generate a local immune privileged microenvironment to promote escape from T cell-mediated immune surveillance. PMID:17870068

  9. Expression of the human endogenous retrovirus-K transmembrane envelope, Rec and Np9 proteins in melanomas and melanoma cell lines.

    PubMed

    Büscher, Kristina; Hahn, Silvia; Hofmann, Maja; Trefzer, Uwe; Ozel, Muhsin; Sterry, Wolfram; Löwer, Johannes; Löwer, Roswitha; Kurth, Reinhard; Denner, Joachim

    2006-06-01

    The human endogenous retrovirus-K encodes two potential tumor proteins, Rec and Np9. Rec is related to the Rev protein of HIV-1 and has been shown to be associated with tumor development in nude mice. Having shown the expression of human endogenous retrovirus-K in human melanomas and melanoma cell lines, tools were developed to allow the expression of the transmembrane envelope, Rec and Np9 mRNA and proteins to be studied in more detail. The expression of spliced env, rec and np9 was investigated by reverse transcriptase-polymerase chain reaction using a set of primers developed to discriminate between full-length and spliced mRNA. Env-specific, Rec-specific and Np9-specific antisera were produced, characterized and used to study protein expression in melanomas and melanoma cell lines by immunohistochemistry, immunofluorescence and Western blot analyses. Existence of human endogenous retrovirus-K Rec and Np9-specific antibodies in the sera of melanoma patients were analyzed by Western blot of immunofluorescence studies. The expression of both spliced env and rec mRNA was detected in 39% of the melanomas and in 40% of the melanoma cell lines and np9 mRNA was detected in 29 and 21%, respectively. In normal neonatal melanocytes, spliced rec mRNA was detected in the absence of spliced env mRNA. Using antisera specific for Rec and Np9, Rec protein was found in 14% of the melanomas but Np9 in none. In addition, cell surface expression of the putatively immunosuppressive transmembrane envelope protein and release of virus particles were shown. Antibodies specific for neither Rec nor Np9 were detected. The transmembrane envelope protein, Rec and Np9 proteins are expressed in melanoma cells with a pattern similar to that seen in teratocarcinoma cell lines. Additional experiments are needed to determine their involvement, if any, in cell proliferation and tumor progression.

  10. 21 CFR 866.5540 - Immunoglobulin G (Fd fragment specific) immunological test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... fragment) of the heavy chain (a subunit) of the immunoglobulin antibody molecule in serum. Measurement of immunoglobulin G Fd fragments aids in the diagnosis of plasma antibody-forming cell abnormalities....

  11. Raman spectroscopy detects melanoma and the tissue surrounding melanoma using tissue-engineered melanoma models

    PubMed Central

    Yorucu, Ceyla; Lau, Katherine; Mittar, Shweta; Green, Nicola H.; Raza, Ahtasham; Rehman, Ihtesham Ur; MacNeil, Sheila

    2016-01-01

    ABSTRACT Invasion of melanoma cells from the primary tumor involves interaction with adjacent tissues and extracellular matrix. The extent of this interaction is not fully understood. In this study Raman spectroscopy was applied to cryo-sections of established 3D models of melanoma in human skin. Principal component analysis was used to investigate differences between the tumor and normal tissue and between the peri-tumor area and the normal skin. Two human melanoma cells lines A375SM and C8161 were investigated and compared in 3D melanoma models. Changes were found in protein conformations and tryptophan configurations across the entire melanoma samples, in tyrosine orientation and in more fluid lipid packing only in tumor dense areas, and in increased glycogen content in the peri-tumor areas of melanoma. Raman spectroscopy revealed changes around the perimeter of a melanoma tumor as well as detecting differences between the tumor and the normal tissue. PMID:27158185

  12. Melanoma of the Skin

    MedlinePlus

    ... at a Glance Show More At a Glance Estimated New Cases in 2016 76,380 % of All New Cancer Cases 4.5% Estimated Deaths in 2016 10,130 % of All Cancer ... of This Cancer : In 2013, there were an estimated 1,034,460 people living with melanoma of ...

  13. Spice Blocks Melanoma Growth

    ERIC Educational Resources Information Center

    Science Teacher, 2005

    2005-01-01

    Curcumin, the pungent yellow spice found in both turmeric and curry powders, blocks a key biological pathway needed for development of melanoma and other cancers, according to a study that appears in the journal Cancer. Researchers from The University of Texas M. D. Anderson Cancer Center demonstrate how curcumin stops laboratory strains of…

  14. Melanoma Risk Prediction Models

    Cancer.gov

    Developing statistical models that estima