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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

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

    2015-01-01

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

  2. 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

  3. 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.

  4. 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.

  5. 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)

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

    PubMed Central

    Holliger, P; Prospero, T; Winter, G

    1993-01-01

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

  7. 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.

  8. 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.

  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. Production of antibody fragments in Escherichia coli.

    PubMed

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

    2012-01-01

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

  11. 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

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

    PubMed Central

    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; Hellström, I

    1986-01-01

    Fab fragments of monoclonal antibodies (MoAb) to melanoma, radiolabeled with 131I, 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 131I 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 on biopsied lesions 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 131I (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, 4/8 (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 and was possibly related to antigen shedding. 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. Melanoma metastases can be identified

  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. 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

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

    SciTech Connect

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

    1984-10-23

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

  16. Two new trifunctional antibodies for the therapy of human malignant melanoma.

    PubMed

    Ruf, Peter; Jäger, Michael; Ellwart, Joachim; Wosch, Susanne; Kusterer, Elisabeth; Lindhofer, Horst

    2004-02-20

    Trifunctional antibodies are able to redirect T cells and Fcgamma receptor(+) accessory immune cells to tumor targets. The simultaneous activation of these different classes of effector cells results in efficient killing of the tumor cells by different mechanisms such as phagocytosis and perforin-mediated cytotoxicity. Here, we introduce 2 new trifunctional antibodies specific for human melanoma. These trifunctional antibodies recognize with one binding arm CD3 on human T cells. The other binding arm is directed against melanoma-associated proteoglycans or melanoma-associated gangliosides (GD2 as well as GD3). They mediate specific lysis of various melanoma cell lines in correlation with the level of antigen expression in short-term cytotoxicity experiments. A combination of the 2 trifunctional antibodies was equally or even more efficient. Moreover, they induced a strong Th1 cytokine pattern with high amounts of IFN-gamma and low or no IL-4. Accordingly, CD4(+) and especially CD8(+) T cells expanded, whereas B cells, NK cells and monocytes decreased. The cytokine response was up to 16-fold higher when tumor cells were present. IFN-gamma reached cytotoxic concentrations for SK-MEL-23 melanoma cells. The induction of a T-cell-activatory and melanoma cell-inhibitory cytokine milieu together with the redirection of T-cell- and accessory cell-mediated cytotoxicity are interesting features of these trifunctional antibodies. They may be a new option for the therapy of human malignant melanoma. PMID:14696099

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

    PubMed

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

    2016-07-01

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

  18. 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

  19. 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.

  20. Melanoma

    MedlinePlus

    Melanoma is the most serious type of skin cancer. Often the first sign of melanoma is a change in the size, shape, color, or feel of a mole. Most melanomas have a black or black-blue area. Melanoma ...

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

    PubMed Central

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

    2003-01-01

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

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

    PubMed

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

    2009-02-01

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

  3. 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

  4. Production of recombinant antibody fragments in Bacillus megaterium

    PubMed Central

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

    2007-01-01

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

  5. 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

  6. 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.

  7. Therapeutic Monoclonal Antibodies and Fragments: Bevacizumab.

    PubMed

    Klein, Ainat; Loewenstein, Anat

    2016-01-01

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

  8. Progress in programming antibody fragments to crystallize.

    PubMed

    Edmundson, A B; Borrebaeck, C A

    1998-01-01

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

  9. Elastin fragments induce IL-1beta upregulation via NF-kappaB pathway in melanoma cells.

    PubMed

    Debret, Romain; Le Naour, Richard R; Sallenave, Jean-Michel; Deshorgue, Aurelie; Hornebeck, William G; Guenounou, Moncef; Bernard, Philippe; Antonicelli, Frank D

    2006-08-01

    In a previous work, we reported the influence of elastin fragments (EFs) on matrix metalloproteinases-2 and -14 expression and activation in melanoma cells in vitro. We hypothesized that EFs might also modulate expression of other mediators involved during melanoma progression. Therefore we investigated the contribution of EFs on IL-1beta expression, a cytokine playing a key role in melanoma cells activation. Our results evidenced that high tumorigenic melanoma cells (M3Da cells) treated with EFs led to IL-1beta mRNA and protein upregulation. The effects of EFs on M3Da cells were found to be mediated by receptor (spliced galactosidase) occupancy, as being suppressed by lactose and reproduced by cell stimulation with the VGVAPG peptide. Binding of EFs to their receptor induced a rapid activation of extracellular signal-regulated kinase 1/2; and p38 mitogen-activated protein kinase pathways. However, these pathways were not associated with IL-1beta mRNA upregulation by EFs. Concomitantly, we demonstrated that EFs stimulation induced NF-kappaB nuclear translocation and DNA binding on IL-1beta promoter region whereas inhibition of NF-kappaB with the specific chemical inhibitor SN-50 or by overexpression of IkappaB, the endogenous inhibitor of NF-kappaB pathway, totally abolished EFs-mediated IL-1beta mRNA overexpression. These results demonstrate that EFs induce NF-kappaB activation, leading to IL-1beta upregulation in invasive melanoma cells. PMID:16675961

  10. 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

  11. 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

  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. Thermodynamic stability and flexibility characteristics of antibody fragment complexes

    PubMed Central

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

    2015-01-01

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

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

    PubMed

    Eisenbeis, Andrea M; Grau, Stefan J

    2012-01-01

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

  15. Monoclonal antibodies and Fc fragments for treating solid tumors

    PubMed Central

    Eisenbeis, Andrea M; Grau, Stefan J

    2012-01-01

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

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

    PubMed

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

    2016-08-01

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

  17. 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

  18. Guiding the Killer and Bringing in Accomplices: Bispecific Antibody Treatment for Malignant Melanoma.

    PubMed

    Szegezdi, Eva; Leverkus, Martin

    2016-02-01

    Discovery of oncogene and immune checkpoint targeting has transformed melanoma therapy in the last 5 years. However, treatment of primary or secondary drug-resistant melanoma remains a challenge. Agents designed to activate the cell death machinery directly, for example by activating the death receptors expressed by melanoma cells, could break drug resistance, and they may achieve long-lasting therapeutic success. He et al. report their studies of an MCSPxDR5 bispecific, tetravalent antibody that can simultaneously target death receptor 5 (DR5, TRAIL-R2) and melanoma-associated chondroitin sulfate proteoglycan (MCSP). This antibody can exert strong and selective DR5-dependent cytotoxic activity against MCSP-expressing melanoma cells. Crosslinking of the antibody with Fcγ-receptors increased the cytotoxic potential further, without compromising its selectivity. This approach offers a novel immunotherapeutic tool via coupling of three cooperating processes: delivering the death receptor agonist to the malignant cell population, potent activation of DR5-mediated cell death signaling, and recruitment of Fcγ-receptor-carrying immune cells that can mount an immune response against the tumor cells. PMID:26802233

  19. 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

  20. The Melanosomal Protein PMEL17 as a Target for Antibody Drug Conjugate Therapy in Melanoma

    PubMed Central

    Chen, Youjun; Chalouni, Cecile; Tan, Christine; Clark, Robyn; Venook, Rayna; Ohri, Rachana; Raab, Helga; Firestein, Ron; Mallet, William; Polakis, Paul

    2012-01-01

    Melanocytes uniquely express specialized genes required for pigment formation, some of which are maintained following their transformation to melanoma. Here we exploit this property to selectively target melanoma with an antibody drug conjugate (ADC) specific to PMEL17, the product of the SILV pigment-forming gene. We describe new PMEL17 antibodies that detect the endogenous protein. These antibodies help define the secretory fate of PMEL17 and demonstrate its utility as an ADC target. Although newly synthesized PMEL17 is ultimately routed to the melanosome, we find substantial amounts accessible to our antibodies at the cell surface that undergo internalization and routing to a LAMP1-enriched, lysosome-related organelle. Accordingly, an ADC reactive with PMEL17 exhibits target-dependent tumor cell killing in vitro and in vivo. PMID:22613716

  1. Monoclonal Antibodies Inhibit the Adhesion of Mouse B 16 Melanoma Cells in vitro and Block Lung Metastasis in vivo

    NASA Astrophysics Data System (ADS)

    Vollmers, H. Peter; Birchmeier, Walter

    1983-06-01

    Seven monoclonal antibodies against mouse B 16 melanoma cells (produced in syngeneic C57BL/6 mice) were selected that blocked the adhesion of melanoma cells to tissue culture dishes. These antibodies were found to be directed against antigens on the surface of mouse B 16 melanoma cells but not on normal mouse cells such as 3T3 fibroblasts. Similarly, the antigens could not be detected in normal mouse tissues (e.g., lung, kidney, liver) but were found in lungs colonized by B 16 melanoma cells. Significantly, three of these antibodies virtually abolished lung colonization of highly invasive B 16 sublines injected into the animals' bloodstream. They exerted their effect both when preabsorbed by the melanoma cell in vitro and when delivered to the animals prior to the tumor cells. It is suggested that monoclonal antibodies might be a promising tool for preventing metastasis.

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

    PubMed

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

    2016-07-01

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

  3. Melanoma

    MedlinePlus

    ... to other parts of the body very quickly. Melanoma treatment can cause side effects, including pain, nausea, and ... Livingstone; 2013:chap 69. National Cancer Institute: PDQ Melanoma Treatment. Bethesda, MD: National Cancer Institute. Last modified March ...

  4. Monoclonal antibody against a lactose epitope of glycosphingolipids binds to melanoma tumour cells.

    PubMed

    Ding, K; Ekberg, T; Zeuthen, J; Teneberg, S; Karlsson, K A; Rosén, A

    1993-10-01

    Mice were immunized with a neoglycoprotein consisting of a chemically modified carbohydrate moiety (reductively aminated 3'-sialyllactose) linked to human serum albumin. By this procedure an antibody response to the normally non-immunogenic carbohydrate structure was obtained. Hybridomas were established, and monoclonal antibodies were selected in ELISA based on their binding to the saccharide hapten, or to a lactosylceramide-mimicking neoglycolipid, lactose-bis-sulfone. One of the selected antibodies, 2H4, was of particular interest, since it also bound to glycolipids present on melanoma cells. FACS analysis of a panel of 14 melanoma cell lines showed that the 2H4 antibody bound to the majority of these. In frozen, non-fixed sections or paraffin sections of biopsies the monoclonal antibody 2H4 stained melanoma cells, but not tumour infiltrating lymphocytes or normal skin. Detailed immunochemical analysis of 2H4, using thin layer chromatography revealed that it recognized an internal lactose epitope in several glycosphingolipids. PMID:7507746

  5. Specific biodetection of B16 mouse melanoma in vivo by syngeneic monoclonal antibody

    SciTech Connect

    Yamasaki, T.; Wakabayashi, S.; Inoue, O.; Ando, K.; Kusakabe, K.; Kawasaki, Y.; Okamoto, S.; Taniguchi, M.

    1987-09-01

    The specific detection of tumors in vivo using a radiolabeled syngeneic monoclonal antibody made by fusion of P3U1 (BALB/c myeloma cells) and C57BL/6 spleen cells primed with syngeneic B16 melanoma cells was investigated by color imaging, autoradiography, and biodistribution. The radiolabeled antimelanoma antibody specifically accumulated only in the tumor lesions, whereas no radioactivity was observed in normal tissues or organs. The distribution patterns of the radioactive antibody in the tumor lesions depended on the sizes of the tumor. Almost the entire region of the small metastatic tumor in lymph nodes was labeled, whereas the radioactive antibody was irregularly localized mainly in the center of the medium-sized tumor. However, only the peripheral region of the large primary tumor was labeled. The highest uptake of radioactivity (tumor:blood ratio) was observed in the small lymph node metastatic tumor lesions rather than in the large primary tumor. Furthermore, high resolution color imaging of B16 melanoma was also obtained by using /sup 125/I-labeled monoclonal antibody. Tumor location was specifically visible without subtraction or enhancement methods 3-5 days after injection of the radiolabeled antibody.

  6. 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

  7. 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. PMID:26313415

  8. 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.

  9. An Uncoordinated-5 Homolog B Receptor Monoclonal Antibody Regulates A375 Melanoma Cell Migration

    PubMed Central

    Zuo, Yuanling; He, Jun; Zhao, Yiming; He, Yang

    2014-01-01

    Uncoordinated-5 homolog B receptor (UNC5B) was first found to mediate neural chemorepulsive effects by binding to its ligand netrin-1 in the nervous system. Newer evidence indicated that UNC5B also has functions outside the nervous system. In this study, we report on the generation of a monoclonal antibody specific to the outer-membrane immunoglobulin-like domains of UNC5B using the hybridoma technique. Western blot, immunofluorescence, and flow cytometry analyses showed that the antibody specifically bound to UNC5B protein. Interestingly, the antibody blocked the Netrin-1-induced inhibitory effect on the mobility of melanoma A375 cells by wound healing assay and transwell migration assay, whereas it had no effects on cell proliferation measured by CCK-8 assay. Thus, the functional antibody may provide a useful tool for the study of UNC5B expression profiles and functions outside the nervous system. PMID:25171009

  10. 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

  11. Dacarbazine and the Agonistic TRAIL Receptor-2 Antibody Lexatumumab Induce Synergistic Anticancer Effects in Melanoma

    PubMed Central

    Engesæter, Birgit; Engebraaten, Olav; Flørenes, Vivi Ann; Mælandsmo, Gunhild Mari

    2012-01-01

    Mapatumumab and lexatumumab (targeting death receptor 4 (DR4) and 5 (DR5), respectively) are agonistic TRAIL receptor antibodies that induce apoptosis in a wide range of cancer cells. The potency of mapatumumab and lexatumumab was assessed in mono therapy protocols, and the ability to sensitize for dacarbazine (DTIC) treatment was explored in ten different melanoma cell lines. Our data indicated that melanoma cell lines tend to be resistant to mapatumumab, most likely due to low expression of DR4, while a dose dependent response to lexatumumab was observed. Combining DTIC and lexatumumab induced an additive or synergistic effect on cell death in the various melanoma cell lines. The synergistic effect observed in the FEMX-1 cell line was related to enhanced cleavage of Bid in parallel with elevated expression of the pro-apoptotic proteins Bim, Bax and Bak. Furthermore, the anti-apoptotic proteins Bcl-XL, cIAP-1, XIAP and livin were down regulated. Cleavage of Bid and down regulation of cIAP-2 and livin were observed in vivo. Altogether, these data suggest a change in the balance between pro- and anti-apoptotic proteins favoring induction of apoptosis. In the more therapy resistant cell line, HHMS, no changes in the pro- and anti-apoptotic proteins were observed. FEMX-1 xenografts treated with DTIC and lexatumumab showed reduced growth and increased level of apoptosis compared to the control groups, providing arguments for further evaluation of this combination in melanoma patients. PMID:23029050

  12. 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.

  13. 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

  14. 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.

  15. 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

  16. 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

  17. 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 ...

  18. 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.

  19. Comparison of two radionuclide doses in melanoma imaging with anti P-97 monoclonal antibody

    SciTech Connect

    Murray, J.L.; Lamki, L.; Haynie, T.P.; Glenn, H.J.; Hersh, E.M.

    1985-05-01

    38 patients were included in phase I study of localization of melanoma with Indium-111 labeled P96.5 monoclonal antibody reactive to melanoma antigen P97. 21 patients were studied using 2.5 mCi of In-111 and 17 patients using 5 mCi of In-111. Results of the two doses of In-111 were compared with results of scans. In 9 pats. receiving doses of 2.5 mCi In-111/20 mg stable antibody, out of 54 evaluable sites of disease, 19 sites were seen on scans performed 48 to 72 h after injection (Sensitivity 35%). In 12 pats. studied with 5.0 mCi of In-111/20 mg stable antibody there were 43 known sites of disease of which 30 were identified giving a sensitivity of 70%. As there was a high percentage of lesions greater than 1 cm in diameter in the group receiving 5 mCi and since tumor size has been previously shown to be related to sensitivity of imaging, the data was reanalyzed after excluding lesions less than 1 cm. When only lesions > 2 cm in diameter were considered there were 17 out of 21 (81%) imaged with the 2.5 mCi tracer dose and 27 out of 29 (93%) with 5.0 mCi dose. These differences are significant by chi square test with P < 0.05. Absorbed radiation dose from a 5 mCi dose of In-111 antibody has been calculated at 1.36 rads for the total body and 6.96 rads for the liver (critical organ). Although these doses could be lowered by using lower tracer dose, the results reported here suggest that the sensitivity would be significantly impaired at the smaller dose of 2.5 mCi. We conclude that the current maximum dose of 5.0 mCi should be used in evaluating In-111 labeled anti-P97 antibody imaging of melanoma.

  20. 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

  1. 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

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

    PubMed Central

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

    2015-01-01

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

  3. 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

  4. [Melanoma].

    PubMed

    Uhara, Hisashi

    2016-04-01

    Since 2011, several effective drugs for patients with metastatic melanoma, including BRAF inhibitors, MEK inhibitors, and immune checkpoint inhibitors, have been approved. The combination of BRAF and MEK inhibitors achieve response rates of 70% and a median progression-free survival of >11 months in patients. The combination of ipilimumab and nivolumab has shown response rates of up to 60-70% and a median progression-free survival of 11-14 months, despite increased toxicities. Moreover, many clinical trials for new combination therapies are still ongoing. PMID:27220785

  5. Effects of a novel Nodal-targeting monoclonal antibody in melanoma

    PubMed Central

    Margaryan, Naira V.; Focà, Annalia; Sanguigno, Luca; Bodenstine, Thomas M.; Chandler, Grace S.; Reed, David W.; Gilgur, Alina; Seftor, Elisabeth A.; Seftor, Richard E.B.; Khalkhali-Ellis, Zhila; Leonardi, Antonio; Ruvo, Menotti; Hendrix, Mary J.C.

    2015-01-01

    Nodal is highly expressed in various human malignancies, thus supporting the rationale for exploring Nodal as a therapeutic target. Here, we describe the effects of a novel monoclonal antibody (mAb), 3D1, raised against human Nodal. In vitro treatment of C8161 human melanoma cells with 3D1 mAb shows reductions in anchorage-independent growth and vasculogenic network formation. 3D1 treated cells also show decreases of Nodal and downstream signaling molecules, P-Smad2 and P-ERK and of P-H3 and CyclinB1, with an increase in p27. Similar effects were previously reported in human breast cancer cells where Nodal expression was generally down-regulated; following 3D1 mAb treatment, both Nodal and P-H3 levels are reduced. Noteworthy is the reduced growth of human melanoma xenografts in Nude mice treated with 3D1 mAb, where immunostaining of representative tumor sections show diminished P-Smad2 expression. Similar effects both in vitro and in vivo were observed in 3D1 treated A375SM melanoma cells harboring the active BRAF(V600E) mutation compared to treatments with IgG control or a BRAF inhibitor, dabrafenib. Finally, we describe a 3D1-based ELISA for the detection of Nodal in serum samples from cancer patients. These data suggest the potential of 3D1 mAb for selecting and targeting Nodal expressing cancers. PMID:26460952

  6. 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.

  7. 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.

  8. Enhancement of monoclonal antibody binding to melanoma with single dose radiation or hyperthermia

    SciTech Connect

    Stickney, D.R.; Gridley, D.S.; Kirk, G.A.; Slater, J.M.

    1987-01-01

    We undertook this study to determine whether radiation (10 Gray, single dose) or water bath hyperthermia (41 degrees C, 45 min) could enhance binding of /sup 111/In-labeled anti-p97a monoclonal antibody (MAb) to human melanoma tumors transplanted subcutaneously into nude mice. Sixty animals were given injections of 1-2 X 10(7) Brown C5513 melanoma cells. At 1-2 weeks postinjection, two-thirds of the mice were treated (one-third served as controls). Within 3 hours after treatment, each animal was given iv 2 muCi /sup 111/In-anti-p97a MAb. At 24 and 48 hours thereafter, whole-body scans were done with the use of a MaxiCamera 300 A/M unit, and the ratio of activity at the tumor and liver was determined. Some animals were kept for 7 days posttreatment, whereas others were taken after the 48-hour scan for determination of biodistribution of the radiolabeled complex. Enhancement of MAb binding was demonstrated by either modality, although enhancement was more consistent with radiation. The therapeutic efficacy of MAb may be enhanced with increased binding of radioactive MAb complexes through single dose radiation or hyperthermia.

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

    PubMed

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

    2016-01-01

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

  10. IgG4 subclass antibodies impair antitumor immunity in melanoma

    PubMed Central

    Karagiannis, Panagiotis; Gilbert, Amy E.; Josephs, Debra H.; Ali, Niwa; Dodev, Tihomir; Saul, Louise; Correa, Isabel; Roberts, Luke; Beddowes, Emma; Koers, Alexander; Hobbs, Carl; Ferreira, Silvia; Geh, Jenny L.C.; Healy, Ciaran; Harries, Mark; Acland, Katharine M.; Blower, Philip J.; Mitchell, Tracey; Fear, David J.; Spicer, James F.; Lacy, Katie E.; Nestle, Frank O.; Karagiannis, Sophia N.

    2013-01-01

    Host-induced antibodies and their contributions to cancer inflammation are largely unexplored. IgG4 subclass antibodies are present in IL-10–driven Th2 immune responses in some inflammatory conditions. Since Th2-biased inflammation is a hallmark of tumor microenvironments, we investigated the presence and functional implications of IgG4 in malignant melanoma. Consistent with Th2 inflammation, CD22+ B cells and IgG4+-infiltrating cells accumulated in tumors, and IL-10, IL-4, and tumor-reactive IgG4 were expressed in situ. When compared with B cells from patient lymph nodes and blood, tumor-associated B cells were polarized to produce IgG4. Secreted B cells increased VEGF and IgG4, and tumor cells enhanced IL-10 secretion in cocultures. Unlike IgG1, an engineered tumor antigen-specific IgG4 was ineffective in triggering effector cell–mediated tumor killing in vitro. Antigen-specific and nonspecific IgG4 inhibited IgG1-mediated tumoricidal functions. IgG4 blockade was mediated through reduction of FcγRI activation. Additionally, IgG4 significantly impaired the potency of tumoricidal IgG1 in a human melanoma xenograft mouse model. Furthermore, serum IgG4 was inversely correlated with patient survival. These findings suggest that IgG4 promoted by tumor-induced Th2-biased inflammation may restrict effector cell functions against tumors, providing a previously unexplored aspect of tumor-induced immune escape and a basis for biomarker development and patient-specific therapeutic approaches. PMID:23454746

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

    PubMed Central

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

    2014-01-01

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

  12. 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

  13. 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.

  14. Immune-mediated adverse events of anticytotoxic T lymphocyte-associated antigen 4 antibody therapy in metastatic melanoma.

    PubMed

    Quirk, Shannon K; Shure, Anna K; Agrawal, Devendra K

    2015-11-01

    Ipilimumab, an antibody that blocks cytotoxic T lymphocyte-associated antigen 4 (CTLA-4; CD152), was approved by the Food and Drug Administration in 2011 for the treatment of unresectable stage III or IV malignant melanoma. Although the addition of this particular immunotherapy has broadened treatment options, immune-related adverse events (irAEs) are associated with ipilimumab therapy, including dermatologic effects, colitis and diarrhea, endocrine effects, hepatotoxicity, ocular effects, renal effects, neurologic effects, and others. In this article, a critical evaluation of the underlying mechanisms of irAEs associated with anti-CTLA-4 therapy is presented. Additionally, potentially beneficial effects of combinational therapies to alleviate ipilimumab-induced irAEs in malignant melanoma are discussed. Future research is warranted to elucidate the efficacy of such combination therapies and specific biomarkers that would help to predict a clinical response to ipilimumab in patients with malignant melanoma. PMID:26118951

  15. 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

  16. 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.

  17. 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

  18. 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

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

    PubMed

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

    2015-01-01

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

  20. 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.

  1. 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

  2. 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...

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

    SciTech Connect

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

    1989-02-01

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

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

    PubMed

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

    2012-01-01

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

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

    PubMed

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

    2011-01-01

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

  7. 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.

  8. 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

  9. 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

  10. 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

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

    PubMed

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

    1975-01-01

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

  12. 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

  13. 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

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

    PubMed Central

    Pisarchick, M L; Thompson, N L

    1990-01-01

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

  16. 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

  17. 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

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

    PubMed

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

    2012-03-01

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

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

    PubMed

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

    2014-01-01

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

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

    PubMed

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

    2014-01-01

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

  1. 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

  2. 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

  3. 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

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

    PubMed

    Vaks, Lilach; Benhar, Itai

    2014-01-01

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

  5. 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

  6. Phase I/II Study of the Antibody-Drug Conjugate Glembatumumab Vedotin in Patients With Advanced Melanoma

    PubMed Central

    Ott, Patrick A.; Hamid, Omid; Pavlick, Anna C.; Kluger, Harriet; Kim, Kevin B.; Boasberg, Peter D.; Simantov, Ronit; Crowley, Elizabeth; Green, Jennifer A.; Hawthorne, Thomas; Davis, Thomas A.; Sznol, Mario; Hwu, Patrick

    2014-01-01

    Purpose The antibody-drug conjugate glembatumumab vedotin links a fully human immunoglobulin G2 monoclonal antibody against the melanoma-related glycoprotein NMB (gpNMB) to the potent cytotoxin monomethyl auristatin E. This study evaluated the safety and activity of glembatumumab vedotin in patients with advanced melanoma. Patients and Methods Patients received glembatumumab vedotin every 3 weeks (schedule 1) in a dose escalation and phase II expansion at the maximum-tolerated dose (MTD). Dosing during 2 of 3 weeks (schedule 2) and weekly (schedule 3) was also assessed. The primary end points were safety and pharmacokinetics. The secondary end points included antitumor activity, gpNMB expression, and immunogenicity. Results One hundred seventeen patients were treated using schedule 1 (n = 79), schedule 2 (n = 15), or schedule 3 (n = 23). The MTDs were 1.88, 1.5, and 1.0 mg/kg for schedules 1, 2, and 3, respectively. Grade 3/4 treatment-related toxicities that occurred in two or more patients included rash, neutropenia, fatigue, neuropathy, arthralgia, myalgia, and diarrhea. Three treatment-related deaths (resulting from pneumococcal sepsis, toxic epidermal necrolysis, and renal failure) occurred at doses exceeding the MTDs. In the schedule 1 phase II expansion cohort (n = 34), five patients (15%) had a partial response and eight patients (24%) had stable disease for ≥ 6 months. The objective response rate (ORR) was 2 of 6 (33%) for the schedule 2 MTD and 3 of 12 (25%) for the schedule 3 MTD. Rash was correlated with a greater ORR and improved progression-free survival. Conclusion Glembatumumab vedotin is active in advanced melanoma. The schedule 1 MTD (1.88 mg/kg once every 3 weeks) was associated with a promising ORR and was generally well tolerated. More frequent dosing was potentially associated with a greater ORR but increased toxicity. PMID:25267741

  7. 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

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

    PubMed

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

    2015-03-01

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

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

    PubMed

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

    2016-09-01

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

  10. 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

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

    PubMed

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

    2016-05-10

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

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

    PubMed

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

    1993-04-01

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

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

    PubMed Central

    2015-01-01

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

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

    PubMed

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

    2010-04-01

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

  15. 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

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

    PubMed

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

    2004-09-01

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

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

    PubMed Central

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

    2009-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  19. 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

  20. 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

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

    PubMed

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

    1994-11-01

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

  2. 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

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

    PubMed

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

    2004-09-01

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

  4. 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

  5. Local distribution and concentration of intravenously injected sup 131 I-9. 2. 27 monoclonal antibody in human malignant melanoma

    SciTech Connect

    Del Vecchio, S.; Reynolds, J.C.; Carrasquillo, J.A.; Blasberg, R.G.; Neumann, R.D.; Lotze, M.T.; Bryant, G.J.; Farkas, R.J.; Larson, S.M. )

    1989-05-15

    Regional measurements of {sup 131}I-9.2.27 distribution in human melanoma tumors were obtained using quantitative autoradiography. Tumors were removed from patients 72-96 h after they had received an i.v. injection of 9.15 mCi (100 mg) of {sup 131}I-9.2.27. The autoradiographic images showed that the radioactivity reaching the tumor was heterogeneously distributed. Areas of relative high and low uptake were selected in each tumor. Regions of high activity contained from 51 to 1371 nCi/g, while areas with low uptake had radioactivity ranging from 12 to 487 nCi/g. The reliability of the autoradiographic measurements was demonstrated by the strong positive correlation with direct tissue sample counting (r = 0.994 P less than 0.001). Since comparative immunocytochemistry showed a homogeneous and diffuse staining of target antigen on viable tumor cells, variability of monoclonal antibody uptake within individual tumors was not primarily due to heterogeneity of antigen expression in these cases. However, antigen levels accounted for some of the variation from tumor to tumor. When immunoperoxidase staining was repeated on adjacent sections without the addition of 9.2.27, it confirmed the nonuniform distribution of monoclonal antibody found at autoradiography. Thus, quantitative autoradiography gives information about the distribution and the local concentration of radioactive antibody in tumors allowing calculation of the radiation dose delivered to small regions within tumors.

  6. 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

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

    PubMed Central

    2011-01-01

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

  8. 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

  9. 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).

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    1990-01-01

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

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

    PubMed

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

    2015-12-01

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

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

    PubMed

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

    2016-04-13

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

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

    PubMed

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

    2015-05-01

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

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

    PubMed

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

    2014-02-01

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

  16. 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

  17. 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

  18. 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

  19. 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

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2009-02-01

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

  3. 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

  4. 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

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

    PubMed

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

    2012-11-01

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

  6. 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.

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

    PubMed Central

    2013-01-01

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

  8. 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.

  9. 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...

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

    PubMed

    Crivianu-Gaita, Victor; Thompson, Michael

    2016-11-15

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

  11. 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. PMID:25415286

  12. Proteins of the cystic fibrosis respiratory tract. Fragmented immunoglobulin G opsonic antibody causing defective opsonophagocytosis.

    PubMed Central

    Fick, R B; Naegel, G P; Squier, S U; Wood, R E; Gee, J B; Reynolds, H Y

    1984-01-01

    In the disease cystic fibrosis (CF), pulmonary infection with Pseudomonas aeruginosa is a common clinical complication that determines most morbidity and almost all excess mortality. We postulated that in this disease a defect in Pseudomonas-reactive IgG antibodies may contribute to chronic Pseudomonas infections. Bronchoalveolar lavages were performed upon 13 patients with CF, 7 patients with chronic bronchitis characterized by recurrent Pseudomonas infections, and 4 normal volunteers. The levels of various proteins important to host defenses and proteases were determined; enzyme inhibition studies were performed. CF respiratory immunoglobulin levels were significantly elevated when compared with both normals and patients with chronic bronchitis (P less than 0.05). Albumin and transferrin levels were decreased in the CF lung fluids. CF elastolytic activity was strikingly elevated (means = 6.02 micrograms/mg total protein) and the inhibitory profile suggested such activity resembled a serine-proteinase. Alpha-1-antitrypsin antigenic levels were not altered in CF respiratory fluids. There was a tendency for the lavage IgG to fall as elastase levels rose (r = -0.29). IgG opsonins for two Pseudomonas immunotypes were isolated with affinity chromatography for functional and immunochemical studies. Bacterial phagocytic rates in the presence of these Pseudomonas-reactive IgG opsonins derived from CF lavage fluid were depressed (0.3% uptake/unit time) when compared with similarly titered positive controls (uptake = 1.3%/unit time, P less than 0.001). Additionally, normal pulmonary macrophage intracellular killing of Pseudomonas was severely altered in the presence of opsonins derived from CF respiratory fluids. At some time points, less than 30% of the bacteria were killed. CF IgG opsonins contain a cleavage fragment (100,000 D, 5S sedimentation coefficient) with antigenic determinants similar to the Fab portion of IgG. The presence of such a fragment was inversely

  13. 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

  14. Purification of antibody and antibody-fragment from E. coli homogenate using 6,9-diamino-2-ethoxyacridine lactate as precipitation agent.

    PubMed

    Persson, Josefine; Lester, Philip

    2004-08-01

    To obtain a more efficient purification process for antibody fragments from an Escherichia coli homogenate, the precipitant, Ethodin (6,9-diamino-2-ethoxyacridine lactate) was introduced to the homogenate. By adding the precipitant a drastic reduction of host cell protein was obtained. The majority of the proteins were recovered in a precipitate with the cell debris, while the antibody or antibody-fragment was recovered in the clarified supernatant. In addition, DNA was also efficiently precipitated when using Ethodin as a precipitation agent. The improved purity of the clarified extract obtained by using the precipitant allows for the use of smaller chromatography columns and may reduce the number of chromatographic steps required in the recovery process. The effect of Ethodin concentration, pH, temperature, and conductivity were investigated. The investigation was performed on two different antibody-fragments, e.g., F(ab')(2) molecules and a full-length antibody produced in E. coli. The two F(ab')(2) proteins were F(ab')(2)A and F(ab')(2)B, which have a similar molecular mass (100 kDa) but different isoelectric points (pIs), i.e., 8.9 and 7.5, respectively. The full-length antibody, Ab (the full IgG form of F(ab')(2)B) has a pI of 7.8 and molecular mass of 150 kDa. The investigation showed that the highest purification factors were obtained at neutral pH, low conductivity, and Ethodin concentrations of 0.6%. PMID:15281116

  15. Characterization of Poliovirus Neutralization Escape Mutants of Single-Domain Antibody Fragments (VHHs)

    PubMed Central

    Schotte, Lise; Thys, Bert; Strauss, Mike; Filman, David J.; Rombaut, Bart

    2015-01-01

    To complete the eradication of poliovirus and to protect unvaccinated people subsequently, the development of one or more antiviral drugs will be necessary. A set of five single-domain antibody fragments (variable parts of the heavy chain of a heavy-chain antibody [VHHs]) with an in vitro neutralizing activity against poliovirus type 1 was developed previously (B. Thys, L. Schotte, S. Muyldermans, U. Wernery, G. Hassanzadeh-Ghassabeh, and B. Rombaut, Antiviral Res 87:257–264, 2010, http://dx.doi.org/10.1016/j.antiviral.2010.05.012), and their mechanisms of action have been studied (L. Schotte, M. Strauss, B. Thys, H. Halewyck, D. J. Filman, M. Bostina, J. M. Hogle, and B. Rombaut, J Virol 88:4403–4413, 2014, http://dx.doi.org/10.1128/JVI.03402-13). In this study, neutralization escape mutants were selected for each VHH. Sequencing of the P1 region of the genome showed that amino acid substitutions are found in the four viral proteins of the capsid and that they are located both in proximity to the binding sites of the VHHs and in regions further away from the canyon and hidden beneath the surface. Characterization of the mutants demonstrated that they have single-cycle replication kinetics that are similar to those of their parental strain and that they are all drug (VHH) independent. Their resistant phenotypes are stable, as they do not regain full susceptibility to the VHH after passage over HeLa cells in the absence of VHH. They are all at least as stable as the parental strain against heat inactivation at 44°C, and three of them are even significantly (P < 0.05) more resistant to heat inactivation. The resistant variants all still can be neutralized by at least two other VHHs and retain full susceptibility to pirodavir and 35-1F4. PMID:26014941

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

    PubMed Central

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

    2011-01-01

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

  17. Comparison of gallium-67 versus indium-111 monoclonal antibody (96. 5, ZME-018) in detection of human melanoma in athymic mice

    SciTech Connect

    Chan, S.M.; Hoffer, P.B.; Maric, N.; Zoghbi, S.S.; Kirkwood, J.M.; Ernstoff, M.S.; Duray, P.H.; Gerich, B.

    1987-09-01

    We compared the biodistribution of two radiolabeled, whole, tumor selective monoclonal antibodies (( /sup 111/In)96.5, (/sup 111/In)ZME-018) to /sup 67/Ga in nude mice bearing a human melanoma known to express p97 antigen. Localization of gallium was determined 48 hr following i.v. injection. Localization of the radiolabeled antibodies was determined at 3 days and 7 days following i.v. injection. All agents showed more or less similar absolute tumor uptake which varied between 22% and 36% of the injected dose per gram of tumor. Only the tumor uptake of (/sup 111/In)96.5 antibody at 7 days was significantly lower than the /sup 67/Ga uptake at 48 hr. However, uptake in normal tissues was generally higher for both antibodies at 3 and 7 days than for /sup 67/Ga uptake at 48 hr. Therefore, the tumor-to-blood ratio for /sup 67/Ga was tenfold higher than that for either antibody, the tumor-to-muscle ratio was twofold higher. Bone was the only organ in which the tumor-to-organ ratio was consistently higher with radiolabeled antibody than with /sup 67/Ga. The tumor-to-liver and tumor-to-intestine ratios were comparable. Localization of the two tumor selective antibodies was greater than a nonspecific control antibody (( /sup 111/In)CEA) and change in specific activity from 0.17 mCi/mg to 3.3 mCi/mg did not influence localization. From these animal data it may be anticipated that tumor imaging with (/sup 111/In)96.5 or (/sup 111/In)ZME-018 will not be superior to imaging with 67Ga for detection of melanoma.

  18. 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

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

    PubMed Central

    2012-01-01

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

  20. 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

  1. 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.

  2. 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

  3. 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.

  4. A new type of pseudothrombocytopenia: EDTA-mediated agglutination of platelets bearing Fab fragments of a chimaeric antibody.

    PubMed

    Christopoulos, C G; Machin, S J

    1994-07-01

    In vitro agglutination of platelets leading to low automated platelet counts was observed in EDTA-anticoagulated blood from human volunteers receiving infusions of Fab fragments of a chimaeric monoclonal antibody to platelet glycoprotein IIb-IIIa. This pseudothrombocytopenia depended on the presence of chimaeric Fab on the platelet surface and was not seen when sodium citrate was used as anticoagulent. Preliminary evidence suggests that this phenomenon might be mediated by immunoglobulin G reactive with the human component of the chimaeric Fab. It is important to exclude pseudothrombocytopenia when low automated platelet counts are reported in association with the administration of chimaeric anti-platelet antibodies. PMID:7993813

  5. 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

  6. Injectable formulations for an intravitreal sustained-release application of a novel single-chain VEGF antibody fragment.

    PubMed

    Asmus, Lutz R; Grimshaw, John P A; Richle, Philipp; Eicher, Barbara; Urech, David M; Gurny, Robert; Möller, Michael

    2015-09-01

    Sustained-release formulations of a single-chain anti-VEGF-A antibody fragment were investigated in vitro toward their potential use for intravitreal applications. The hydrophobic polyester hexylsubstituted poly(lactic acid) (hexPLA) was selected as the sustained-release excipient for its biodegradability and semi-solid aggregate state, allowing an easy and mild formulation procedure. The lyophilized antibody fragment ESBA903 was micronized and incorporated into the liquid polymer matrix by cryo-milling, forming homogeneous and injectable suspensions. The protein showed excellent compatibility with the hexPLA polymer and storage stability at 4°C for 10 weeks. Additionally, hexPLA shielded the incorporated active substance from the surrounding medium, resulting in a better stability of ESBA903 inside the polymer than after its release in the buffer solution. Formulations of ESBA903 with hexPLA having drug loadings between 1.25% and 5.0% and polymer molecular weights of 1500 g/mol, 2500 g/mol, 3500 g/mol and 5000 g/mol were investigated regarding their in vitro release. All formulations except with the highest molecular weight formed spherical depots in aqueous buffer solutions and released the antibody fragment for at least 6-14 weeks. The polymer viscosity derived from the molecular weight strongly influenced the release rate, while the drug loading had minor influence, allowing customization of the release profile and the daily drug release. Size exclusion chromatography and SDS-PAGE revealed that the antibody fragment structure was kept intact during incorporation and release from the liquid matrix. Furthermore, the released protein monomer maintained its high affinity to human VEGF-A, as measured by surface plasmon resonance analysis. Formulations of ESBA903 in hexPLA meet the basic needs to be used for intravitreal sustained-release applications in age-related macular degeneration treatment. PMID:25779352

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

    PubMed

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

    2014-12-12

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

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

    PubMed Central

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

    2009-01-01

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

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

    PubMed

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

    2016-04-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-10-01

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

  12. 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

  13. 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

  14. Anti-MET immunoPET for non-small cell lung cancer using novel fully human antibody fragments

    PubMed Central

    Li, Keyu; Tavaré, Richard; Zettlitz, Kirstin A.; Mumenthaler, Shannon M.; Mallick, Parag; Zhou, Yu; Marks, James D.; Wu, Anna M.

    2014-01-01

    MET, the receptor of hepatocyte growth factor, plays important roles in tumorigenesis and drug resistance in numerous cancers including non-small cell lung cancer. As increasing numbers of MET inhibitors are being developed for clinical applications, antibody fragment based immuno-positron emission tomography (immunoPET) has the potential to rapidly quantify in vivo MET expression levels for drug response evaluation and patient stratification for these targeted therapies. Here, fully human single-chain variable fragments (scFvs) isolated from a phage display library were re-formatted into bivalent cys-diabodies (scFv-cys dimers) with affinities to MET ranging from 0.7 nM to 5.1 nM. The candidate with the highest affinity, H2, was radiolabeled with 89Zr for immunoPET studies targeting non-small cell lung cancer xenografts: low MET expressing Hcc827 and the gefitinib-resistant Hcc827-GR6 with 4-fold MET over-expression. ImmunoPET at as early as 4 hours post injection produced high contrast images, and ex vivo biodistribution analysis at 20 hours post injection showed about 2-fold difference in tracer uptake levels between the parental and resistant tumors (p < 0.01). Further immunoPET studies using a larger fragment, the H2 minibody (scFv-CH3 dimer) produced similar results at later time points. Two of the antibody clones (H2 and H5) showed in vitro growth inhibitory effects on MET-dependent gefitinib-resistant cell lines, while no effects were observed on resistant lines lacking MET activation. In conclusion, these fully human antibody fragments inhibit MET-dependent cancer cells and enable rapid immunoPET imaging to assess MET expression levels, showing potential for both therapeutic and diagnostic applications. PMID:25143449

  15. 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

  16. 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

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

    PubMed

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

    2015-05-01

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

  18. 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.

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

    PubMed Central

    Hu, Guodong; Chen, L. Y.

    2010-01-01

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

  20. Gluten-specific antibodies of celiac disease gut plasma cells recognize long proteolytic fragments that typically harbor T-cell epitopes.

    PubMed

    Dørum, Siri; Steinsbø, Øyvind; Bergseng, Elin; Arntzen, Magnus Ø; de Souza, Gustavo A; Sollid, Ludvig M

    2016-01-01

    This study aimed to identify proteolytic fragments of gluten proteins recognized by recombinant IgG1 monoclonal antibodies generated from single IgA plasma cells of celiac disease lesions. Peptides bound by monoclonal antibodies in complex gut-enzyme digests of gluten treated with the deamidating enzyme transglutaminase 2, were identified by mass spectrometry after antibody pull-down with protein G beads. The antibody bound peptides were long deamidated peptide fragments that contained the substrate recognition sequence of transglutaminase 2. Characteristically, the fragments contained epitopes with the sequence QPEQPFP and variants thereof in multiple copies, and they typically also harbored many different gluten T-cell epitopes. In the pull-down setting where antibodies were immobilized on a solid phase, peptide fragments with multivalent display of epitopes were targeted. This scenario resembles the situation of the B-cell receptor on the surface of B cells. Conceivably, B cells of celiac disease patients select gluten epitopes that are repeated multiple times in long peptide fragments generated by gut digestive enzymes. As the fragments also contain many different T-cell epitopes, this will lead to generation of strong antibody responses by effective presentation of several distinct T-cell epitopes and establishment of T-cell help to B cells. PMID:27146306

  1. 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

  2. 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

  3. 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

  4. Advancing the global proteome survey platform by using an oriented single chain antibody fragment immobilization approach.

    PubMed

    Säll, Anna; Persson, Helena; Ohlin, Mats; Borrebaeck, Carl A K; Wingren, Christer

    2016-09-25

    Increasing the understanding of a proteome and how its protein composition is affected by for example different diseases, such as cancer, has the potential to improve strategies for early diagnosis and therapeutics. The Global Proteome Survey or GPS is a method that combines mass spectrometry and affinity enrichment with the use of antibodies. The technology enables profiling of complex proteomes in a species independent manner. The sensitivity of GPS, and other methods relying on affinity enrichment, is largely affected by the activity of the exploited affinity reagent. We here present an improvement of the GPS platform by utilizing an antibody immobilization approach which ensures a controlled immobilization process of the antibody to the magnetic bead support. More specifically, we make use of an antibody format that enables site-directed biotinylation and use this in combination with streptavidin coated magnetic beads. The performance of the expanded GPS platform was evaluated by profiling yeast proteome samples. We demonstrate that the oriented antibody immobilization strategy increases the ability of the GPS platform and results in larger fraction of functional antibodies. Additionally, we show that this new antibody format enabled in-solution capture, i.e. immobilization of the antibodies after sample incubation. A workflow has been established that permit the use of an oriented immobilization strategy for the GPS platform. PMID:26703809

  5. 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...

  6. Doxorubicin conjugated with a monoclonal antibody directed to a human melanoma-associated proteoglycan suppresses the growth of established tumor xenografts in nude mice.

    PubMed Central

    Yang, H M; Reisfeld, R A

    1988-01-01

    Doxorubicin (DXR) was covalently conjugated to a monoclonal antibody (mAb), 9.2.27 (IgG2a), which recognizes a chondroitin sulfate proteoglycan expressed preferentially on the surface of human melanoma cells. Immunoconjugates with a molar ratio of DXR to mAb ranging from 2:1 to 10:1 were obtained by coupling the drug via an acid-sensitive linker, cis-aconitic anhydride. The immunoreactivity of mAb 9.2.27 was well retained after conjugation. DXR-mAb 9.2.27 conjugates were found to be 2 orders of magnitude more potent in killing tumor cells in vitro (IC50 = 0.1 microM) than free drug targeted to drug receptor(s). Most significantly, DXR-mAb 9.2.27 immunoconjugates specifically suppressed the growth of established tumors in vivo and prolonged the life-span of tumor-bearing nude mice. This suppression of melanoma growth achieved by the immunoconjugate was both tumor and antibody specific. A biodistribution study indicated that DXR-mAb 9.2.27 conjugates delivered at least 4 times more DXR (3.7% total injected dose per g of tumor) as compared to free DXR alone (0.8% total injected dose per g of tumor) in tumor-bearing nude mice 48 hr postinjection. The tumor-suppressive effects of DXR-mAb 9.2.27 conjugates are even more remarkable since free DXR did not suppress tumor growth in vivo and also because this drug per se is known to be quite ineffective for the treatment of human melanoma. Images PMID:3422487

  7. Galactosylated streptavidin for improved clearance of biotinylated intact and F(ab')2 fragments of an anti-tumour antibody.

    PubMed Central

    Marshall, D.; Pedley, R. B.; Melton, R. G.; Boden, J. A.; Boden, R.; Begent, R. H.

    1995-01-01

    Persistence of high levels of radiolabelled antibody in the circulation is a major limitation of radioimmunotherapy. Biotinylation of the radiolabelled anti-tumour antibody followed by administration of streptavidin is known to give much improved tumour to blood ratios as the radioantibody is complexed and subsequently cleared via the reticuloendothelial system, although prolonged splenic uptake is a problem. We have investigated the effect on the clearance pattern and tumour localisation of a 125I-labelled biotinylated anti-CEA antibody (A5B7) after administration of a galactosylated form of streptavidin (gal-streptavidin) in nude mice bearing a human colon carcinoma xenograft. Fifteen minutes to 1 h after gal-streptavidin administration the complexes were cleared via the liver alone (as opposed to liver and spleen after native streptavidin). Twenty-four hours after administration of gal-streptavidin, the tumour to blood ratio for biotinylated A5B7 IgG increased from 2.9 to 13.2 and for biotinylated F(ab')2 fragments an increase from 4.9 to 33.2 was achieved. The reduction in tumour accumulation of F(ab')2 24 h after injection of the clearing agent was less than that seen with intact antibody. Injection of asialofetuin inhibited clearance, confirming that removal of the gal-streptavidin-biotinylated antibody complexes from the blood was via the asialoglycoprotein receptor on liver hepatocytes. Therefore, galactosylation of the streptavidin clearing agent allows rapid removal of radiolabelled biotinylated antibodies via the liver asialoglycoprotein receptor, as opposed to the reticuloendothelial system. Images Figure 5 Figure 6 Figure 7 PMID:7529526

  8. 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

  9. 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

  10. Recombinant anti-CD20 antibody fragments for microPET imaging of B-cell lymphoma

    PubMed Central

    Olafsen, Tove; Betting, David; Kenanova, Vania E.; Salazar, Felix B.; Clarke, Pat; Said, Jonathan; Raubitschek, Andrew A.; Timmerman, John M.; Wu, Anna M.

    2010-01-01

    The CD20 cell surface antigen is expressed at high levels by over 90% of B cell non-Hodgkin lymphomas (NHL), and is the target of the anti-CD20 monoclonal antibody rituximab. To provide more sensitive, tumor-specific positron emission tomography (PET) imaging of NHL, we sought to develop PET imaging agents targeting CD20. Methods Two recombinant anti-CD20 rituximab fragments, a minibody (scFv-CH3 dimer, 80 kDa) and a modified scFv-Fc fragment (105 kDa), designed to clear rapidly, were generated. Both fragments were radiolabeled with 124I, and the minibody was additionally radiometal labeled with 64Cu following conjugation to 1,4,7,10-tetraazacyclododecane-N,N’,N’’,N’’’-tetraacetic acid (DOTA). The radioiodinated fragments and the radiometal labeled minibody were evaluated in mice as microPET imaging agents for in vivo imaging of human CD20-expressing lymphomas. Results Rapid and specific localization to CD20-positive tumors was observed with the radioiodinated fragments. However, their tumor uptakes and blood activities differed, resulting in different levels of contrast in the images. The best candidate was the minibody, with superior uptake (2-fold higher than the scFv-Fc) in CD20-positive tumor and low uptake in CD20-negative tumor. Positive tumor to negative tumor ratios were 7.0(±3.1) and 3.9(±0.7) for the minibody and scFv-Fc, respectively at 21 hours. About a 5-fold lower ratio was achieved with the 64Cu-DOTA-minibody at 19 hours due to higher residual background activity in CD20 negative tumor. Conclusion Radioiodinated minibody and scFv-Fc fragment produced excellent, high-contrast images in vivo. These new immunoPET agents may prove useful for the imaging CD20 positive lymphomas in preclinical models and in humans with NHL. PMID:19690034

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

    PubMed

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

    2013-08-30

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

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

    PubMed

    Toleikis, Lars; Frenzel, André

    2012-01-01

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

  13. 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

  14. Absence of SV40 antibodies or DNA fragments in prediagnostic mesothelioma serum samples.

    PubMed

    Kjaerheim, Kristina; Røe, Oluf Dimitri; Waterboer, Tim; Sehr, Peter; Rizk, Raeda; Dai, Hong Yan; Sandeck, Helmut; Larsson, Erik; Andersen, Aage; Boffetta, Paolo; Pawlita, Michael

    2007-06-01

    The rhesus monkey virus Simian Virus 40 (SV40) is a member of the polyomavirus family. It was introduced inadvertently to human populations through contaminated polio vaccine during the years 1956-1963, can induce experimental tumors in animals and transform human cells in culture. SV40 DNA has been identified in mesothelioma and other human tumors in some but not all studies. We tested prediagnostic sera from 49 mesothelioma cases and 147 matched controls for antibodies against the viral capsid protein VP1 and the large T antigen of SV40 and of the closely related human polyomaviruses BK and JC, and for SV40 DNA. Cases and controls were identified among donors to the Janus Serum Bank, which was linked to the Cancer Registry of Norway. Antibodies were analyzed by recently developed multiplex serology based on recombinantly expressed fusions of glutathione-S transferase with viral proteins as antigens combined with fluorescent bead technology. BKV and JCV specific antibodies cross- reactive with SV40 were preabsorbed with the respective VP1 proteins. Sera showing SV40 reactivity after preabsorption with BKV and JCV VP1 were further analyzed in SV40 neutralization assays. SV40 DNA was analyzed by SV40 specific polymerase chain reactions. The odds ratio for being a case when tested positive for SV40 VP1 in the antibody capture assay was 1.5 (95% CI 0.6-3.7) and 2.0 (95% CI 0.6-7.0) when only strongly reactive sera where counted as positive. Although some sera could neutralize SV40, preabsorption with BKV and JCV VP1 showed for all such sera that this neutralizing activity was due to cross-reacting antibodies and did not represent truly SV40-specific antibodies. No viral DNA was found in the sera. No significant association between SV40 antibody response in prediagnostic sera and risk of mesothelioma was seen. PMID:17315193

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

    PubMed

    Martínez Cristancho, Carlos Andrés; Seidel-Morgenstern, Andreas

    2016-02-19

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

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

    PubMed

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

    2015-12-15

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

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

    PubMed

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

    2011-05-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

  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. PMID:26963935

  2. Optimizing the expression of a monoclonal antibody fragment under the transcriptional control of the Escherichia coli lac promoter.

    PubMed

    Donovan, R S; Robinson, C W; Glick, B R

    2000-06-01

    The expression of a monoclonal antibody Fab fragment in Escherichia coli strain RB791/pComb3, induced with either lactose or isopropyl-beta-D-thiogalactoside (IPTG), was compared to determine if lactose might provide an inexpensive alternative to induction with IPTG. Induction of Fab expression imposed a metabolic load on the recombinant cells, resulting in lower final cell yields compared to the non-induced controls. An IPTG concentration of 0.05 mM was sufficient to achieve maximal expression of soluble Fab protein when inducing in the early-, mid-, or late-log phases of batch cultures grown using either glucose or glycerol as a carbon source. The largest overall yield of Fab fragments when using 0.05 mM IPTG was achieved by increasing the final yield of cells through glycerol feeding following induction in late-log phase. Lactose was as effective as IPTG for inducing Fab expression in E. coli RB791/pComb3. The greatest overall level of Fab expression was found when cells grown on glycerol were induced with 2 g/L lactose in late-log phase. Since the cost of 0.05 mM of IPTG is significantly greater than the cost of 2 g/L lactose, lactose provides an inexpensive alternative to IPTG for inducing the expression of Fab fragments, and possibly other recombinant proteins, from the E. coli lac promoter. PMID:10913975

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

    PubMed

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

    2015-06-01

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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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.

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

    PubMed Central

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

    2010-01-01

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

  10. Crystallization and preliminary X-ray structure determination of jack bean urease with a bound antibody fragment.

    PubMed

    Sheridan, Louisa; Wilmot, Carrie M; Cromie, Karen D; van der Logt, Paul; Phillips, Simon E V

    2002-02-01

    Urease allows organisms to use exogenous and internally generated urea as a nitrogen source, by catalyzing the hydrolysis of urea to form ammonia and carbon dioxide. Urease may also participate in the systemic nitrogen-transport pathways and possibly acts as a toxic defence protein. Jack bean urease (JBU) was the first nickel-metalloenzyme identified and was crystallized as early as 1926. Despite this, the structure has not yet been determined. An antibody fragment, Fv, that has a high affinity for JBU has been used to aid crystallization. The complex, which retains full enzyme activity, forms very small crystals that diffract weakly to 3.3 A. The crystals belong to the rhombohedral space group R32, with unit-cell parameters a = b = 228.6, c = 130.9 A. The structure of the urease molecule has been solved by molecular replacement using the structure of homogenous enzyme from Klebsiella aerogenes as a search model. PMID:11807281

  11. Melanoma Cancer Stem Cells: Markers and Functions

    PubMed Central

    Parmiani, Giorgio

    2016-01-01

    The discovery of cancer stem cells (CSCs) in human solid tumors has allowed a better understanding of the biology and neoplastic transformation of normal melanocytes, and the possible mechanisms by which melanoma cells acquire tumorigenicity. In this review I summarize the literature findings on the potential biomarkers of melanoma CSCs, their presence in the melanoma cell populations, the interaction with the immune system (with both T and NK cells) and the role of melanoma CSCs in the clinics. Given the extraordinary progress in the therapy of melanoma caused by immune checkpoint antibodies blockade, I discuss how these antibodies can work by the activation of melanoma infiltrating T cells specifically recognizing neo-antigens expressed even by melanoma CSCs. This is the mechanism that can induce a regression of the metastatic melanomas. PMID:26978405

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

    PubMed

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

    2015-10-01

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

  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. Extreme scale-down of expanded bed adsorption: Purification of an antibody fragment directly from recombinant E. coli culture.

    PubMed

    Willoughby, Nik; Martin, Phillip; Titchener-Hooker, Nigel

    2004-09-01

    Scale-down is a methodology that combines the use of very small volumes of process fluid in dedicated devices to predict accurately the behaviour of process-scale biotechnological unit operations and for the production of comparable material for use in further devices which, taken together, facilitate the mimic of a complete full-scale process. This article provides the rationale behind the development of a small-scale mimic and demonstrates the use of a highly scaled-down expanded bed to predict hydrodynamic, kinetic, and adsorptive performance using less than 5-mL sample volumes. Data acquired on a specially developed 1.9 mm ID column was compared with that obtained in a standard 25 mm ID column. A homogenised E. coli system expressing an antibody fragment (F(ab)) adsorbed onto an rProtein A matrix was used to characterise the full adsorptive performance. Breakthrough curve studies using BSA in buffer were used to characterise binding kinetics. Performance at the two scales was comparable both in terms of expansion, axial dispersion, binding isotherms, and elution behaviour of the antibody fragment. The eluted F(ab) material was further purified by ion exchange chromatography to demonstrate the similarity between the profile of the product material obtained at both scales. The high level of scale-down (approximately 200-fold) provides for rapid process evaluation early in development, where material is at a premium and where a fast appreciation of the likely merits of one process strategy will lead to greater confidence in process selection and more robust flowsheets. PMID:15352062

  15. 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

  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. PMID:26700095

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

    PubMed Central

    2011-01-01

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

  18. Novel human 3-domain disulfide-stabilized antibody fragment against glycoprotein of rabies virus.

    PubMed

    Cai, Kun; Wang, Hui; Bao, Shizhong; Shi, Jing; Hou, Xiaojun; Gao, Xiang; Liu, Hao; Yin, Jun

    2008-04-01

    Mutated disulfide bond sites VH (Cys44) and VL (Cys100) were constructed in variable domains (Fvs) of the human anti-glycoprotein antigen of the rabies virus (anti-GPRV), and the light chain variable (VL) and heavy chain variable (VH) fragments were linked using the heavy chain constant region 1 (CH1) of the human immunoglobulin (Ig) to successfully construct a 3-domain disulfide-stabilized fragment of variables (3d-dsFv). 3d-dsFv was mainly expressed as an inclusion body. After refolding by the conventional dilution method, 3d-dsFv was purified using a nickel-nitrilotriacetic acid (Ni-NTA) column. Enyzme-linked immunosorbent assay (ELISA) was used to determine the binding activity of 3d-dsFv to GPRV. Flow cytometry studies and rapid fluorescent focus inhibition test were used to evaluate the function of 3d-dsFv. The results showed that the stability of 3d-dsFv was improved notably in some aspects such as thermal kinetics, ability to withstand urea denaturation, etc. 3d-dsFv could bind specially to infective cells and the GPRV. The titration of 3d-dsFv to RV-CVS is 83.3 IU/mg, and it can easily reach 2.5IU/mL, which is the value suggested by the WHO as effective for neutralization titration of the rabies virus. PMID:18424153

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

    PubMed

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

    2009-01-01

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

  20. Neutralization Analysis of a Chicken Single-Chain Variable Fragment Derived from an Immune Antibody Library Against Infectious Bronchitis Virus.

    PubMed

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

    2015-09-01

    Avian infectious bronchitis virus (IBV), which is prevalent in many countries causing severe economic loss to the poultry industry, causes infectious bronchitis (IB) in birds. Recombinant single-chain variable fragments (scFvs) have been proven to effectively inhibit many viruses, both in vitro and in vivo, and they could be a potential diagnostic and therapeutic reagent to control IB. In this study, six anti-IBV chicken scFvs, ZL.10, ZL.64, ZL.78, ZL.80, ZL.138, and ZL.256, were obtained by screening random clones from an immune antibody library. An analysis of nucleotide sequences revealed that they represented distinctive genetic sequences and greatly varied in complementarity-determining region three of the heavy chain. Neutralization tests showed that ZL.10, which bound the S1 protein in western blots, inhibited the formation of syncytia in Vero cells 48 h post IBV infection and decreased the transcriptional level of nucleoprotein mRNA to 17.2%, while the other five scFvs, including ZL.78 and ZL.256, that bound the N protein did not. In conclusion, the results suggested that specific and neutralizing chicken scFvs against IBV, which can be safe and economical antibody reagents, can be produced in vitro through prokaryotic expression. PMID:26090700

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

    PubMed

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

    2016-06-01

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

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

    PubMed

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

    2015-11-01

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

  3. 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

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

    PubMed

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

    2014-10-15

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

  5. 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

  6. Crystal Structure of Snake Venom Acetylcholinesterase in Complex with Inhibitory Antibody Fragment Fab410 Bound at the Peripheral Site

    PubMed Central

    Bourne, Yves; Renault, Ludovic; Marchot, Pascale

    2015-01-01

    The acetylcholinesterase found in the venom of Bungarus fasciatus (BfAChE) is produced as a soluble, non-amphiphilic monomer with a canonical catalytic domain but a distinct C terminus compared with the other vertebrate enzymes. Moreover, the peripheral anionic site of BfAChE, a surface site located at the active site gorge entrance, bears two substitutions altering sensitivity to cationic inhibitors. Antibody Elec410, generated against Electrophorus electricus acetylcholinesterase (EeAChE), inhibits EeAChE and BfAChE by binding to their peripheral sites. However, both complexes retain significant residual catalytic activity, suggesting incomplete gorge occlusion by bound antibody and/or high frequency back door opening. To explore a novel acetylcholinesterase species, ascertain the molecular bases of inhibition by Elec410, and document the determinants and mechanisms for back door opening, we solved a 2.7-Å resolution crystal structure of natural BfAChE in complex with antibody fragment Fab410. Crystalline BfAChE forms the canonical dimer found in all acetylcholinesterase structures. Equally represented open and closed states of a back door channel, associated with alternate positions of a tyrosine phenol ring at the active site base, coexist in each subunit. At the BfAChE molecular surface, Fab410 is seated on the long Ω-loop between two N-glycan chains and partially occludes the gorge entrance, a position that fully reflects the available mutagenesis and biochemical data. Experimentally based flexible molecular docking supports a similar Fab410 binding mode onto the EeAChE antigen. These data document the molecular and dynamic peculiarities of BfAChE with high frequency back door opening, and the mode of action of Elec410 as one of the largest peptidic inhibitors targeting the acetylcholinesterase peripheral site. PMID:25411244

  7. Biokinetics of a radioiodinated antibreast carcinoma monoclonal antibody and fragment in humans

    SciTech Connect

    Zalutsky, M.R.; Noska, M.; Kaplan, W.D.; Hayes, D.; Colcher, D.; Schlom, J.; Kufe, D.

    1984-01-01

    Monoclonal antibody B6.2 is promising for the detection of breast cancers; it binds to >80% of human breast carcinoma (hbc) lines, its antigen is not seen in serum, and it localizes selectively in hbc xenografts in nude mice. The authors have studied the biokinetics of I-131 activity following injection of I-131-IgG and F(ab')/sub 2/ each in 4 patients (pts). The antibody was labeled using iodogen and tested for specific binding to hbc membrane extracts prior to injection. Pts received 0.6-1.1 mCi of I-131 and 50-100 ..mu..g of protein. Blood clearance of I-131 activity was biphasic with half times of 2 and 15.4 hrs for IgG; 1 and 30 hrs for F(ab')/sub 2/. Dehalogenation was noted: by 72 hrs post-injection, 22% (IgG) and 21% (F(ab')/sub 2/) of the injected dose of I-131 was found in the urine. In 2 pts receiving I-131 IgG, stomach uptake was 7-10% at 24 hrs. Protein associated activity in the blood was >90% for the first 8 hrs and gradually decreased to 79% (IgG) and 58% (F(ab')/sub 2/) at 48 hrs. High liver uptake, reported with other antibody systems, was not observed; <20% of the activity was seen in the liver at all time points for both proteins. In 1/4 pts receiving IgG and 4/4 receiving F(ab')/sub 2/, bone marrow uptake was clearly noted. In these pts, >20% of the activity present in blood was cell associated. This is not inconsistent with the observation that B6.2 binds to granulocytes in vitro. Increased binding to cells in the blood for F(ab')/sub 2/ probably accounts for the anomolously longer blood clearance half times observed for F(ab')/sub 2/ vs IgG and low liver accumulation most likely reflects the absence of hepatic or circulating B6.2 antigen.

  8. 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 ...

  9. 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.

  10. Immunotherapy of Melanoma.

    PubMed

    Snyder, Alexandra; Zamarin, Dmitriy; Wolchok, Jedd D

    2015-01-01

    The history of immunotherapy is rooted in the treatment of melanoma and therapy with immune checkpoint-blocking agents is now a cornerstone for the treatment of metastatic melanoma. The first effective immunotherapies approved by the US Food and Drug Administration in melanoma included interleukin-2 for metastatic disease and interferon alpha in the adjuvant setting. These were followed by a group of new therapies, including checkpoint-blocking antibodies targeting cytotoxic T lymphocyte-associated protein 4 and programmed cell death protein 1. Therapies intended to 'reeducate' T cells, such as tumor-infiltrating lymphocyte therapy, oncolytic viruses and tumor vaccines, have yielded promising results and are under development. Finally, the integration of the above therapies as well as development of new coinhibitory and costimulatory agents, though in early stages, appear very promising and likely represent the next phase in drug development for the treatment of metastatic melanoma. PMID:26376963

  11. Cytokine Activation by Antibody Fragments Targeted to Cytokine-Receptor Signaling Complexes.

    PubMed

    Kuruganti, Srilalitha; Miersch, Shane; Deshpande, Ashlesha; Speir, Jeffrey A; Harris, Bethany D; Schriewer, Jill M; Buller, R Mark L; Sidhu, Sachdev S; Walter, Mark R

    2016-01-01

    Exogenous cytokine therapy can induce systemic toxicity, which might be prevented by activating endogenously produced cytokines in local cell niches. Here we developed antibody-based activators of cytokine signaling (AcCS), which recognize cytokines only when they are bound to their cell surface receptors. AcCS were developed for type I interferons (IFNs), which induce cellular activities by binding to cell surface receptors IFNAR1 and IFNAR2. As a potential alternative to exogenous IFN therapy, AcCS were shown to potentiate the biological activities of natural IFNs by ∼100-fold. Biochemical and structural characterization demonstrates that the AcCS stabilize the IFN-IFNAR2 binary complex by recognizing an IFN-induced conformational change in IFNAR2. Using IFN mutants that disrupt IFNAR1 binding, AcCS were able to enhance IFN antiviral potency without activating antiproliferative responses. This suggests AcCS can be used to manipulate cytokine signaling for basic science and possibly for therapeutic applications. PMID:26546677

  12. 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

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

    PubMed

    Yi, Li; Cheng, Shipeng

    2015-08-01

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

  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. Enhanced glutathione PEGylated liposomal brain delivery of an anti-amyloid single domain antibody fragment in a mouse model for Alzheimer's disease.

    PubMed

    Rotman, Maarten; Welling, Mick M; Bunschoten, Anton; de Backer, Maaike E; Rip, Jaap; Nabuurs, Rob J A; Gaillard, Pieter J; van Buchem, Mark A; van der Maarel, Silvère M; van der Weerd, Louise

    2015-04-10

    Treatment of neurodegenerative disorders such as Alzheimer's disease is hampered by the blood-brain barrier (BBB). This tight cerebral vascular endothelium regulates selective diffusion and active transport of endogenous molecules and xenobiotics into and out of the brain parenchyma. In this study, glutathione targeted PEGylated (GSH-PEG) liposomes were designed to deliver amyloid-targeting antibody fragments across the BBB into the brain. Two different formulations of GSH-PEG liposomes based on 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and egg-yolk phosphatidylcholine (EYPC) were produced. Both formulations encapsulate 15kDa amyloid beta binding llama single domain antibody fragments (VHH-pa2H). To follow the biodistribution of VHH-pa2H rather than the liposome, the antibody fragment was labeled with the radioisotope indium-111. To prolong the shelf life of the construct beyond the limit of radioactive decay, an active-loading method was developed to efficiently radiolabel the antibody fragments after encapsulation into the liposomes, with radiolabeling efficiencies of up to 68% after purification. The radiolabeled liposomes were administered via a single intravenous bolus injection to APPswe/PS1dE9 double transgenic mice, a mouse model of Alzheimer's disease, and their wildtype littermates. Both GSH-PEG DMPC and GSH-PEG EYPC liposomes significantly increased the standard uptake values (SUV) of VHH-pa2H in the blood of the animals compared to free VHH-pa2H. Encapsulation in GSH-PEG EYPC liposomes resulted in the highest increase in SUV in the brains of transgenic animals. Overall, these data provide evidence that GSH-PEG liposomes may be suitable for specific delivery of single domain antibody fragments over the BBB into the brain. PMID:25668771

  16. Melanoma Treatments: Advances and Mechanisms.

    PubMed

    Marzuka, Alexander; Huang, Laura; Theodosakis, Nicholas; Bosenberg, Marcus

    2015-11-01

    Advances in the understanding of the molecular pathogenesis of melanoma and in cancer immunology have led to the rational design and recent clinical implementation of a variety of novel therapies for metastatic melanoma. BRAF and MEK inhibitors that target the MAPK pathway have high rates of clinical response in BRAF-mutant melanoma. Therapies that modulate the immune response to melanoma, including monoclonal antibodies that interfere with pathways that inhibit T-cell function, have been shown to be effective in melanoma. Lessons learned from these encouraging results are driving the development of new treatments for melanoma and other cancers. This review will focus on the science and clinical findings related to targeted therapies that inhibit BRAF or MEK as well as the immunotherapies that block the CTLA-4 or PD-1 pathways. Other experimental and combinatorial therapeutic approaches will also be discussed. PMID:25899612

  17. PET Imaging of CD105/Endoglin Expression with a 61/64Cu-Labeled Fab Antibody Fragment

    PubMed Central

    Zhang, Yin; Hong, Hao; Orbay, Hakan; Valdovinos, Hector F.; Nayak, Tapas R.; Theuer, Charles P.; Barnhart, Todd E.; Cai, Weibo

    2013-01-01

    Purpose The goal of this study was to generate and characterize the Fab fragment of TRC105, a monoclonal antibody that binds with high affinity to human and murine CD105 (i.e. endoglin), and investigate its potential for positron emission tomography (PET) imaging of tumor angiogenesis in a small animal model after 61/64Cu-labeling. Methods TRC105-Fab was generated by enzymatic papain digestion. The integrity and CD105 binding affinity of TRC105-Fab was evaluated before NOTA (i.e., 1,4,7-triazacyclononane-1,4,7-triacetic acid) conjugation and 61/64Cu-labeling. Serial PET imaging and biodistribution studies were carried out in the syngeneic 4T1 murine breast cancer model to quantify tumor targeting efficacy and normal organ distribution of 61/64Cu-NOTA-TRC105-Fab. Blocking studies with unlabeled TRC105 were performed to confirm CD105 specificity of the tracer in vivo. Immunofluorescence staining was also conducted to correlate tracer uptake in the tumor and normal tissues with CD105 expression. Results TRC105-Fab was produced with high purity through papain digestion of TRC105, as confirmed by SDS-PAGE, HPLC analysis, and mass spectrometry. 61/64Cu-labeling of NOTA-TRC105-Fab was achieved with ~50% yield (specific activity: ~44 GBq/µmol). PET imaging revealed rapid uptake of 64Cu-NOTA-TRC105-Fab in the 4T1 tumor (3.6 ± 0.4, 4.2 ± 0.5, 4.9 ± 0.3, 4.4 ± 0.7, and 4.6 ± 0.8 %ID/g at 0.5, 2, 5, 16, and 24 h post-injection respectively; n = 4). Since tumor uptake peaked soon after tracer injection, 61Cu-labeled TRC105-Fab was also able to provide tumor contrast at 3 and 8 h post-injection. CD105 specificity of the tracer was confirmed with blocking studies and histological examination. Conclusion Herein we report PET imaging of CD105 expression with 61/64Cu-NOTA-TRC105-Fab, which exhibited prominent and target specific uptake in the 4T1 tumor. The use of a Fab fragment led to much faster tumor uptake (which peaked at a few hours after tracer injection) compared to

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

    PubMed Central

    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. PMID:26426340

  19. 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

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

    PubMed

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

    2015-11-01

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

  1. 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.

  2. 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

  3. 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

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

    PubMed

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

    2014-11-01

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

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

    PubMed Central

    2011-01-01

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

  6. 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

  7. 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

  8. Melanoma (image)

    MedlinePlus

    ... tumor that involves the skin cells that produce pigment (melanin). The risk of melanoma increases with age, but frequently effects young, otherwise healthy people. Melanoma is an aggressive type of cancer that can spread very rapidly.

  9. [One amino acid mutation in an anti-CD20 antibody fragment that affects the yield bacterial secretion and the affinity].

    PubMed

    Liu, Yin-Xing; Xiong, Dong-Sheng; Fan, Dong-Mei; Shao, Xiao-Feng; Xu, Yuan-Fu; Zhu, Zhen-Ping; Yang, Chun-Zheng

    2003-05-01

    Monoclonal antibodies (mAb) directed against CD20, either unmodified or in radiolabeled forms, have been successfully exploited in clinic as effective therapeutic agents in the management of non-Hodgkin's B-cell lymphoma. The antibody fragment is a potential agent in image and therapy of tumor. To further improve the soluble expression of anti-CD20 antibody Fab' fragment, PCR was used to mutate the anti-CD20 VL and VH genes and its biological activity was identified. The expression vector of chimeric antibody Fab' was constructed and expressed in E. coli. The data of mutant clone DNA sequence showed that the amino acid of light chain gene of the parent anti-CD20 antibody (H47) was successful mutated as Ser (GAG)-Asn (CAG). The soluble expression of mutated anti-CD20 Fab' (CD20-7) was 3.8 mg/g dry cell weight, while the parent (CD20-2) was 1.3 mg/g dry cell weight. The affinity constant Ka of CD20-7 was 2.2 x 10(9) L/mol. The primary results of competitive assays by FACS showed that CD20-7 could partially block the sites through which parent antibody (HI47) bind to Raji cells. There was difference in the Raji cells (CD20+)-binding activity between the mutant CD20-7 and parent CD20-2. The site mutation of anti-CD20 Fab' gene make it possible that the anti-CD20 antibody fragment was succeeded to obtain higher expression. In this thesis, we succeeded in completing mutation and expression of anti-CD20 Fab' genes, distinguishing its biological activity, and obtaining its highly expression. These period results will lay a foundation for development of other kind of anti-CD20 engineering antibody (for instance: Fab' Diabody and miniantibody), and make it possible for anti-CD20 antibody to be applied to tumor therapy in civil in the future. PMID:15969005

  10. Bone marrow dosimetry in rats using direct tissue counting after injection of radio-iodinated intact monoclonal antibodies or F(ab')2 fragments

    SciTech Connect

    Buchegger, F.; Chalandon, Y.; Pelegrin, A.; Hardman, N.; Mach, J.P. )

    1991-07-01

    Normal rats were injected intravenously with 131I- and 125I-labeled intact murine and chimeric mouse-human monoclonal antibodies directed against carcinoembryonic antigen or with the corresponding F(ab')2 fragments. At different times after injection, individual animals were killed and radioactivity of blood and major organs, including bones and bone marrow, was determined. Ratios comparing radioactivity concentration in different tissues with that of bone marrow were calculated and found to remain stable during several effective half-lives of the antibodies. Mean bone marrow radioactivity was 35% (range, 29%-40%) of that of blood and 126% (range, 108%-147%) of that of liver after injection of intact Mabs or F(ab')2 fragments. In nude rats bearing human colon carcinoma xenografts producing carcinoembryonic antigen, relative bone marrow radioactivity was slightly lower than that in normal rats.

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

    PubMed Central

    Mendler, Claudia T; Friedrich, Lars; Laitinen, Iina; Schlapschy, Martin; Schwaiger, Markus; Wester, Hans-Jürgen; Skerra, Arne

    2015-01-01

    Although antigen-binding fragments (Fabs) of antibodies constitute established tracers for in vivo radiodiagnostics, their functionality is hampered by a very short circulation half-life. PASylation, the genetic fusion with a long, conformationally disordered amino acid chain comprising Pro, Ala and Ser, provides a convenient way to expand protein size and, consequently, retard renal filtration. Humanized αHER2 and αCD20 Fabs were systematically fused with 100 to 600 PAS residues and produced in E. coli. Cytofluorimetric titration analysis on tumor cell lines confirmed that antigen-binding activities of the parental antibodies were retained. The radio-iodinated PASylated Fabs were studied by positron emission tomography (PET) imaging and biodistribution analysis in mouse tumor xenograft models. While the unmodified αHER2 and αCD20 Fabs showed weak tumor uptake (0.8% and 0.2% ID/g, respectively; 24 h p.i.) tumor-associated radioactivity was boosted with increasing PAS length (up to 9 and 26-fold, respectively), approaching an optimum for Fab-PAS400. Remarkably, 6- and 5-fold higher tumor-to-blood ratios compared with the unmodified Fabs were measured in the biodistribution analysis (48 h p.i.) for αHER2 Fab-PAS100 and Fab-PAS200, respectively. These findings were confirmed by PET studies, showing high imaging contrast in line with tumor-to-blood ratios of 12.2 and 5.7 (24 h p.i.) for αHER2 Fab-PAS100 and Fab-PAS200. Even stronger tumor signals were obtained with the corresponding αCD20 Fabs, both in PET imaging and biodistribution analysis, with an uptake of 2.8% ID/g for Fab-PAS100 vs. 0.24% ID/g for the unmodified Fab. Hence, by engineering Fabs via PASylation, plasma half-life can be tailored to significantly improve tracer uptake and tumor contrast, thus optimally matching reagent/target interactions. PMID:25484039

  12. PET Imaging of Abdominal Aortic Aneurysm with 64Cu-Labeled Anti-CD105 Antibody Fab Fragment

    PubMed Central

    Shi, Sixiang; Orbay, Hakan; Yang, Yunan; Graves, Stephen A.; Nayak, Tapas R.; Hong, Hao; Hernandez, Reinier; Luo, Haiming; Goel, Shreya; Theuer, Charles P.; Nickles, Robert J.; Cai, Weibo

    2015-01-01

    The critical challenge in abdominal aortic aneurysm (AAA) research is the accurate diagnosis and assessment of AAA progression. Angiogenesis is a pathological hallmark of AAA, and CD105 is highly expressed on newly formed vessels. Our goal was to use 64Cu-labeled anti-CD105 antibody Fab fragment for noninvasive assessment of angiogenesis in the aortic wall in a murine model of AAA. Methods Fab fragment of TRC105, a mAb that specifically binds to CD105, was generated by enzymatic papain digestion and conjugated to NOTA for 64Cu-labeling. Binding affinity/specificity of NOTA-TRC105-Fab was evaluated by flow cytometry and various ex vivo studies. BALB/c mice were anesthetized and treated with calcium phosphate to induce AAA, which underwent weekly PET scans using 64Cu-NOTA-TRC105-Fab. Biodistribution and autoradiography studies were also performed to confirm the accuracy of PET results. Results NOTA-TRC105-Fab exhibited high purity and specifically bound to CD105 in vitro. Uptake of 64Cu-NOTA-TRC105-Fab increased from a control level of 3.4 ± 0.1 to 9.5 ± 0.4 %ID/g at 6 h p.i. on Day 5, and decreased to 7.2 ± 1.4 %ID/g on Day 12 which correlated well with biodistribution and autoradiography studies (i.e. much higher tracer uptake in AAA than normal aorta). Of note, enhanced AAA contrast was achieved, due to the minimal background in the abdominal area of mice. Degradation of elastic fibers and highly expressed CD105 were observed in ex vivo studies. Conclusion 64Cu-NOTA-TRC105-Fab cleared rapidly through kidneys, which enabled noninvasive PET imaging of the aorta with enhanced contrast and showed increased angiogenesis (CD105 expression) during AAA. 64Cu-NOTA-TRC105-Fab PET may potentially be used for future diagnosis and prognosis of AAA. PMID:25883125

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

    PubMed

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

    2013-12-01

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

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

    PubMed

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

    2016-06-15

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

  15. 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

  16. 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

  17. Using the local immune response from the natural buffalo host to generate an antibody fragment library that binds the early larval stages of Schistosoma japonicum.

    PubMed

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

    2015-09-01

    Antibodies isolated from the local draining inguinal lymph node of field exposed-water buffaloes following challenge with Schistosoma japonicum cercariae showed high reactivity towards S. japonicum antigen preparations and bound specifically to formaldehyde-fixed S. japonicum schistosomules. Using this specific local immune response we produced a series of single-chain antibody Fv domain libraries from the same lymph nodes. Removal of phage that cross reacted with epitopes on adult parasites yielded a single-chain antibody Fv domain-phage library that specifically bound to whole formaldehyde-fixed and live S. japonicum schistosomules. DNA sequencing indicated clear enrichment of the single-chain antibody Fv domain library for buffalo B-cell complementarity determining regions post-selection for schistosomule binding. This study also revealed that long heavy chain complementarity determining regions appear to be an important factor when selecting for antibody binding fragments against schistosomule proteins. The selected single-chain antibody Fv domain-phage were used to probe a schistosome-specific protein microarray, which resulted in the recognition of many proteins expressed across all schistosome life-cycle stages. Following absorption to adult worms, the single-chain antibody Fv domain-phage library showed significantly reduced binding to most proteins, whilst two proteins (NCBI GenBank accession numbers AY915878 and AY815196) showed increased binding. We have thus developed a unique set of host derived single-chain antibody Fv domains comprising buffalo B-cell variable regions that specifically bind to early S. japonicum life-stages. PMID:26116907

  18. Conserved stem fragment from H3 influenza hemagglutinin elicits cross-clade neutralizing antibodies through stalk-targeted blocking of conformational change during membrane fusion.

    PubMed

    Gong, Xin; Yin, He; Shi, Yuhua; Guan, Shanshan; He, Xiaoqiu; Yang, Lan; Yu, Yongjiao; Kuai, Ziyu; Jiang, Chunlai; Kong, Wei; Wang, Song; Shan, Yaming

    2016-04-01

    Currently available influenza vaccines typically fail to elicit/boost broadly neutralizing antibodies due to the mutability of virus sequences and conformational changes during protective immunity, thereby limiting their efficacy. This problem needs to be addressed by further understanding the mechanisms of neutralization and finding the desired neutralizing site during membrane fusion. This study specifically focused on viruses of the H3N2 subtype, which have persisted as a principal source of influenza-related morbidity and mortality in humans since the 1968 influenza pandemic. Through sequence alignment and epitope prediction, a series of highly conserved stem fragments (spanning 47 years) were found and coupled to the Keyhole Limpet Hemocyanin (KLH) protein. By application of a combinatorial display library and crystal structure modeling, a stem fragment immunogen, located at the turning point of the HA neck undergoing conformational change during membrane fusion with both B- and T-cell epitopes, was identified. After synthesis of the optimal stem fragment using a multiple antigen peptide (MAP) system, strong humoral immune responses and cross-clade neutralizing activities against strains from the H3 subtype of group 2 influenza viruses after animal immunizations were observed. By detection of nuclear protein immunofluorescence with acid bypass treatment, antisera raised against MAP4 immunogens of the stem fragment showed the potential to inhibit the conformational change of HA in stem-targeted virus neutralization. The identification of this conserved stem fragment provides great potential for exploitation of this site of vulnerability in therapeutic and vaccine design. PMID:26875772

  19. Engineering, purification and applications of His-tagged recombinant antibody fragments with specificity for the major birch pollen allergen, bet v1.

    PubMed

    Flicker, S; Laffer, S; Steinberger, P; Alhani, B; Zhu, Y; Laukkanen, M L; Keinänen, K; Kraft, D; Valenta, R

    2000-01-01

    Type I allergy, an immunodisorder affecting almost 20% of the population worldwide, is based on the production of IgE antibodies against per se harmless allergens. We report the expression of hexahistidine-tagged antibody fragments (Fabs) with specificity for Bet v1, the major birch pollen allergen, in Escherichia coli. The cDNA coding for the heavy chain fragment of a mouse monoclonal anti-Bet v1 antibody, Bip 1, was engineered by PCR to contain a hexahistidine-encoding 3' end. The modified Bip1 heavy chain cDNA was co-expressed in E. coli XL-1 Blue with the Bip 1 light chain cDNA using the combinatorial plasmid pComb3H. His-tagged recombinant (r) Bip 1 Fabs were isolated by nickel affinity chromatography and rBip 1 Fabs without His-tag were purified via affinity to rBet v1. rBip 1 Fabs with and without His-tag bound specifically to rBet v1 and, like Bet v1 -specific human serum IgE and rabbit-anti rBet v1 antibodies, cross-reacted with Bet v1-related allergens in other plant-species (alder, oak, hazelnut). We demonstrate the usefulness of His-tagged rBip 1 Fabs (1) for the identification of pollen samples containing Bet v 1 by particle blotting, (2) forthe detection of Bet v1-specific IgE antibodies in human serum samples by sandwich ELISA and (3) for the quantification of Bet v1 in solution. Based on these examples we suggest to use rBip 1 Fabs for the detection of Bet v1 and Bet v1-related allergens in natural allergen sources for allergy prevention, as well as for the standardization of natural allergen extracts produced for diagnosis and immunotherapy of birch pollen allergy. PMID:10722049

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

    PubMed

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

    2015-10-01

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

  1. 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

  2. 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

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

    PubMed

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

    2016-01-22

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

  4. A mycobacterial lipoarabinomannan specific monoclonal antibody and its F(ab′)2 fragment prolong survival of mice infected with Mycobacterium tuberculosis

    PubMed Central

    HAMASUR, B; HAILE, M; PAWLOWSKI, A; SCHRÖDER, U; KÄLLENIUS, G; SVENSON, S B

    2004-01-01

    Lipoarabinomannan (LAM) is a major structural carbohydrate antigen of the outer surface of Mycobacterium tuberculosis. High antibody titres against LAM are often seen in active tuberculosis (TB). The role of such LAM-specific antibodies in the immune response against TB is unknown. Here we have investigated a monoclonal antibody (MoAb) SMITB14 of IgG1 subclass and its corresponding F(ab′)2 fragment directed against LAM from M. tuberculosis strain H37Rv. MoAb SMITB14 was shown by immunofluorescence to bind to whole cells of the clinical isolate M. tuberculosis strain Harlingen as well as to M. tuberculosis H37Rv. The binding of MoAb SMITB14 to LAM was inhibited by arabinomannan (AM) and oligosaccharides (5·2 kDa) derived from LAM, showing that the MoAb binds specifically to the AM carbohydrate portion of LAM. In passive protection experiments BALB/c mice were infected intravenously with M. tuberculosis Harlingen. MoAb SMITB14 was added intravenously either prior to, or together with, the bacteria. The antibody proved to be protective against the M. tuberculosis infection in terms of a dose-dependent reduction in bacterial load in spleens and lungs, reduced weight loss and, most importantly, increased long-term survival. PMID:15373902

  5. 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.

  6. 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

  7. Antibody Fragments Directed against Different Portions of the Human Neural Cell Adhesion Molecule L1 Act as Inhibitors or Activators of L1 Function

    PubMed Central

    Wang, Yan; Loers, Gabriele; Pan, Hong-Chao; Gouveia, Ricardo; Zhao, Wei-Jiang; Shen, Yan-Qin; Kleene, Ralf; Costa, Julia; Schachner, Melitta

    2012-01-01

    The neural cell adhesion molecule L1 plays important roles in neuronal migration and survival, neuritogenesis and synaptogenesis. L1 has also been found in tumors of different origins, with levels of L1 expression correlating positively with the metastatic potential of tumors. To select antibodies targeting the varied functions of L1, we screened the Tomlinson library of recombinant human antibody fragments to identify antibodies binding to recombinant human L1 protein comprising the entire extracellular domain of human L1. We obtained four L1 binding single-chain variable fragment antibodies (scFvs), named I4, I6, I13, and I27 and showed by enzyme-linked immunosorbent assay (ELISA) that scFvs I4 and I6 have high affinity to the immunoglobulin-like (Ig) domains 1–4 of L1, while scFvs I13 and I27 bind strongly to the fibronectin type III homologous (Fn) domains 1–3 of L1. Application of scFvs I4 and I6 to human SK-N-SH neuroblastoma cells reduced proliferation and transmigration of these cells. Treatment of SK-N-SH cells with scFvs I13 and I27 enhanced cell proliferation and migration, neurite outgrowth, and protected against the toxic effects of H2O2 by increasing the ratio of Bcl-2/Bax. In addition, scFvs I4 and I6 inhibited and scFvs I13 and I27 promoted phosphorylation of src and Erk. Our findings indicate that scFvs reacting with the immunoglobulin-like domains 1–4 inhibit L1 functions, whereas scFvs interacting with the fibronectin type III domains 1–3 trigger L1 functions of cultured neuroblastoma cells. PMID:23272240

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

    PubMed

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

    2014-06-01

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

  9. Characterization of a Lipopolysaccharide-Targeted Monoclonal Antibody and Its Variable Fragments as Candidates for Prophylaxis against the Obligate Intracellular Bacterial Pathogen Coxiella burnetii

    PubMed Central

    Peng, Ying; Schoenlaub, Laura; Elliott, Alexandra; Mitchell, William J.

    2014-01-01

    Our previous study demonstrated that treatment of Coxiella burnetii with the phase I lipopolysaccharide (PI-LPS)-targeted monoclonal antibody (MAb) 1E4 significantly inhibited C. burnetii infection in mice, suggesting that 1E4 is a protective MAb. To determine whether passive transfer of antibodies (Abs) can provide protection against C. burnetii natural infection, we examined if passive transfer of 1E4 would protect SCID mice against C. burnetii aerosol infection. The results indicated that 1E4 conferred significant protection against aerosolized C. burnetii, suggesting that 1E4 may be useful for preventing C. burnetii natural infection. To further understand the mechanisms of 1E4-mediated protection and to test the possibility of using humanized 1E4 to prevent C. burnetii infection, we examined whether the Fab fragment of 1E4 (Fab1E4), a recombinant murine single-chain variable fragment (muscFv1E4), and a humanized single-chain variable fragment (huscFv1E4) retained the ability of 1E4 to inhibit C. burnetii infection. The results indicated that Fab1E4, muscFv1E4, and huscFv1E4 were able to inhibit C. burnetii infection in mice but that their ability to inhibit C. burnetii infection was lower than that of 1E4. In addition, treatment of C. burnetii with Fab1E4, muscFv1E4, or huscFv1E4 can block C. burnetii infection of macrophages. Interestingly, treatment of C. burnetii with huscFv1E4 can significantly reduce C. burnetii infectivity in human macrophages. This report provides the first evidence to demonstrate that the humanized variable fragments of an LPS-specific MAb can neutralize C. burnetii infection and appears to be a promising step toward the potential use of a humanized MAb as emergency prophylaxis against C. burnetii exposure. PMID:25114119

  10. 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.

  11. Characterization of a human antibody fragment Fab and its calcium phosphate nanoparticles that inhibit rabies virus infection with vaccine.

    PubMed

    Liu, Xinjian; Lin, Hong; Tang, Qi; Li, Chen; Yang, Songtao; Wang, Zhongcan; Wang, Changjun; He, Qing; Cao, Brian; Feng, Zhenqing; Guan, Xiaohong; Zhu, Jin

    2011-01-01

    Recombinant antibody phage display technology has been used to mimic many aspects of the processes that govern the generation and selection of high-affinity natural human antibodies in the human immune system, especially for infectious disease prophylaxis. An anti-rabies virus immunized phage-display Fab library was constructed from peripheral blood lymphocytes from vaccinated volunteers. The immunized antibody library, with a diversity of 6.7×10(8), was used to select and produce antibodies that bound to rabies virus glycoprotein. After five rounds of immobilized fixed rabies virion panning, four unique DNA sequences were found in the higher binding clones, and only one, Fab094, showed neutralization activity. Fab094 components were analyzed by ELISA, immunoprecipitation and immunofluorescent staining. ELISA and immunofluorescence showed that Fab094 bound specifically to rabies virions. Immunoprecipitation and mass spectrometry showed that Fab094 reacted with rabies virus glycoprotein. To improve the penetration power of Fab094 antibodies, we developed Fab094 calcium phosphate nanoparticles (Fab094-CPNPs) and tested their efficacy. The rapid fluorescent focus inhibition test indicated that the neutralizing antibody titers of Fab094 and Fab094-CPNPs were reached at 200.17 IU/Kg and 246.12 IU/Kg, respectively. These findings were confirmed in vivo in a Kunming mouse challenge model. Our results demonstrate that human Fab094 and Fab094-CPNPs are efficacious candidate drugs to replace rabies immunoglobulin in post-exposure prophylaxis (PEP). PMID:21573024

  12. 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

  13. 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

  14. 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

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

    PubMed

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

    2015-12-01

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

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

    PubMed

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

    2011-06-30

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

  17. 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.

  18. 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.

  19. Functional domains on von Willebrand factor. Recognition of discrete tryptic fragments by monoclonal antibodies that inhibit interaction of von Willebrand factor with platelets and with collagen.

    PubMed Central

    Sixma, J J; Sakariassen, K S; Stel, H V; Houdijk, W P; In der Maur, D W; Hamer, R J; de Groot, P G; van Mourik, J A

    1984-01-01

    We have identified two functional domains on the von Willebrand factor (VWF) moiety of the Factor VIII-von Willebrand factor complex (FVIII-VWF), one interacting with blood platelets, and one interacting with vessel wall collagens, by means of two monoclonal antibodies directed against the VWF molecule, CLB-RAg 35 and CLB-RAg 201. The monoclonal antibody CLB-RAg 35 inhibited virtually all platelet adherence to artery subendothelium and to purified vessel wall collagen type III, at relatively high wall shear rates. CLB-RAg 35 also inhibited the ristocetin-induced platelet aggregation and the binding of FVIII-VWF to the platelet in the presence of ristocetin but did not affect the binding of FVIII-VWF to collagen. The monoclonal antibody CLB-RAg 201 inhibited the binding of FVIII-VWF to purified vessel wall collagen type I and III and all platelet adherence to collagen type III and the platelet adherence to subendothelium that was mediated by FVIII-VWF in plasma. The two functional domains on FVIII-VWF that were recognized by CLB-RAg 35 and CLB-RAg 201 were identified by means of immunoprecipitation studies of trypsin-digested FVIII-VWF. The domains resided on different polypeptide fragments, with a Mr of 48,000 for the collagen binding domain and a Mr of 116,000 for the platelet binding domain. The 116,000-mol wt fragment consisted of subunits of 52,000/56,000 mol wt and 14,000 mol wt after reduction. The 52,000/56,000-mol wt subunits possessed the epitope for CLB-RAg 35. Images PMID:6332119

  20. 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

  1. 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

  2. Translational fusion and redirection to thylakoid lumen as strategies to improve the accumulation of a camelid antibody fragment in transplastomic tobacco.

    PubMed

    Lentz, Ezequiel M; Garaicoechea, Lorena; Alfano, E Federico; Parreño, Viviana; Wigdorovitz, Andrés; Bravo-Almonacid, Fernando F

    2012-08-01

    Fragments from camelid single-chain antibodies known as VHHs or nanobodies represent a valuable tool in diagnostics, investigation and passive immunity therapy. Here, we explored different strategies to improve the accumulation of a neutralizing VHH antibody against rotavirus in tobacco transplastomic plants. First, we attempted to express the VHH in the chloroplast stroma and then two alternative strategies were carried out to improve the expression levels: expression as a translational fusion to the β-glucuronidase enzyme (GUS-E-VHH), and redirection of the VHH into the thylakoid lumen (pep-VHH). Every attempt to produce transplastomic plants expressing the VHH in the stroma was futile. The transgene turned out to be unstable and the presence of the VHH protein was almost undetectable. Although pep-VHH plants also presented some of the aforementioned problems, higher accumulation of the nanobody was observed (2-3% of the total soluble proteins). The use of β-glucuronidase as a partner protein turned out to be a successful strategy and expression levels reached 3% of the total soluble proteins. The functionality of the VHHs produced by pep-VHH and GUS-E-VHH plants was studied and compared with that of the antibody produced in Escherichia coli. This work contributes to optimizing the expression of VHH in transplastomic plants. Recombinant proteins could be obtained either by accumulation in the thylakoid lumen or as a fusion protein with β-glucuronidase, and both strategies allow for further optimization. PMID:22526499

  3. 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

  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. Differential analyses of major allergen proteins in wild-type rice and rice producing a fragment of anti-rotavirus antibody.

    PubMed

    Yuki, Yoshikazu; Kurokawa, Shiho; Kozuka-Hata, Hiroko; Tokuhara, Daisuke; Mejima, Mio; Kuroda, Masaharu; Oyama, Masaaki; Nishimaki-Mogami, Tomoko; Teshima, Reiko; Kiyono, Hiroshi

    2016-04-01

    To develop oral antibody therapy against rotavirus infection, we previously produced a recombinant fragment of llama heavy-chain antibody to rotavirus (ARP1) in rice seeds (MucoRice-ARP1). We intend to use a purification-free rice powder for clinical application but needed to check whether MucoRice-ARP1 had increased levels of known allergen proteins. For this purpose, we used two-dimensional fluorescence difference gel electrophoresis to compare the allergen protein levels in MucoRice-ARP1 and wild-type rice. We detected no notable differences, except in the levels of α-amylase/trypsin inhibitor-like family proteins. Because by this approach we could not completely separate ARP1 from the proteins of this family, we confirmed the absence of changes in the levels of these allergens by using shotgun mass spectrometry as well as immunoblot. By using immunoelectron microscopy, we also showed that RAG2, a member of the α-amylase/trypsin inhibitor-like protein family, was relocated from protein bodies II to the plasma membrane or cell wall in MucoRice-ARP1 seed. The relocation did not affect the level of RAG2. We demonstrated that most of the known rice allergens were not considerably upregulated by the genetic modification in MucoRice-ARP1. Our data suggest that MucoRice-ARP1 is a potentially safe oral antibody for clinical application. PMID:26851506

  6. 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

  7. Pharmacokinetics of the monoclonal antibody B72. 3 and its fragments labeled with either /sup 125/I or /sup 111/In

    SciTech Connect

    Brown, B.A.; Comeau, R.D.; Jones, P.L.; Liberatore, F.A.; Neacy, W.P.; Sands, H.; Gallagher, B.M.

    1987-02-15

    A comparison of the pharmacokinetics of intact B72.3 (a murine monoclonal antibody specific for human breast and colon carcinoma) with F(ab')2 and Fab fragments labeled with /sup 111/In and /sup 125/I was done in athymic mice bearing target (LS174T) and non-target (HCT-15) tumors. IgG B72.3 labeled with either isotype imaged LS174T. Biodistributions of both labels were similar in all organs except liver. F(ab')2 also imaged the LS174T tumor, while Fab bearing either isotype did not. The blood clearance was Fab greater than F(ab')2 greater than immunoglobulin G B72.3 for both isotopes. /sup 111/In-labeled fragments yielded large accumulations in the kidneys which persisted for 2 days. The different patterns of biodistribution for the various forms of B72.3 labeled with the two isotopes suggest that the most desirable combination of fragment and isotope will depend on the intended use.

  8. 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.

  9. Management of advanced melanoma

    SciTech Connect

    Nathanson, L. )

    1986-01-01

    This book presents papers on the subject of management of advanced melanoma. The topics covered are: non-investigational cytotoxic agents; high-dosage chemotherapy in antologous bone marrow transplantation; Radiotherapy of melanomas; hyperthermia; ureal melanoma; surgical treatment of recurrent a metastatic melanoma; role of interferons in management of melanoma and molecular genetics of melanoma.

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

    PubMed Central

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

    2010-01-01

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

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

    PubMed

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

    2013-12-01

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

  12. 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

  13. 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

  14. 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.

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

    PubMed

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

    2015-05-01

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

  17. 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.

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

    PubMed

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

    2016-10-01

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

  19. 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

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

    PubMed

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

    2014-10-01

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

  1. An Exopolysaccharide-Deficient Mutant of Lactobacillus rhamnosus GG Efficiently Displays a Protective Llama Antibody Fragment against Rotavirus on Its Surface

    PubMed Central

    Álvarez, Beatriz; Krogh-Andersen, Kasper; Tellgren-Roth, Christian; Martínez, Noelia; Günaydın, Gökçe; Lin, Yin; Martín, M. Cruz; Álvarez, Miguel A.; Hammarström, Lennart

    2015-01-01

    Rotavirus is the leading cause of infantile diarrhea in developing countries, where it causes a high number of deaths among infants. Two vaccines are available, being highly effective in developed countries although markedly less efficient in developing countries. As a complementary treatment to the vaccines, a Lactobacillus strain producing an anti-rotavirus antibody fragment in the gastrointestinal tract could potentially be used. In order to develop such an alternative therapy, the effectiveness of Lactobacillus rhamnosus GG to produce and display a VHH antibody fragment (referred to as anti-rotavirus protein 1 [ARP1]) on the surface was investigated. L. rhamnosus GG is one of the best-characterized probiotic bacteria and has intrinsic antirotavirus activity. Among four L. rhamnosus GG strains [GG (CMC), GG (ATCC 53103), GG (NCC 3003), and GG (UT)] originating from different sources, only GG (UT) was able to display ARP1 on the bacterial surface. The genomic analysis of strain GG (UT) showed that the genes welE and welF of the EPS cluster are inactivated, which causes a defect in exopolysaccharide (EPS) production, allowing efficient display of ARP1 on its surface. Finally, GG (UT) seemed to confer a level of protection against rotavirus-induced diarrhea similar to that of wild-type GG (NCC 3003) in a mouse pup model, indicating that the EPS may not be involved in the intrinsic antirotavirus activity. Most important, GG (EM233), a derivative of GG (UT) producing ARP1, was significantly more protective than the control strain L. casei BL23. PMID:26092449

  2. An Exopolysaccharide-Deficient Mutant of Lactobacillus rhamnosus GG Efficiently Displays a Protective Llama Antibody Fragment against Rotavirus on Its Surface.

    PubMed

    Álvarez, Beatriz; Krogh-Andersen, Kasper; Tellgren-Roth, Christian; Martínez, Noelia; Günaydın, Gökçe; Lin, Yin; Martín, M Cruz; Álvarez, Miguel A; Hammarström, Lennart; Marcotte, Harold

    2015-09-01

    Rotavirus is the leading cause of infantile diarrhea in developing countries, where it causes a high number of deaths among infants. Two vaccines are available, being highly effective in developed countries although markedly less efficient in developing countries. As a complementary treatment to the vaccines, a Lactobacillus strain producing an anti-rotavirus antibody fragment in the gastrointestinal tract could potentially be used. In order to develop such an alternative therapy, the effectiveness of Lactobacillus rhamnosus GG to produce and display a VHH antibody fragment (referred to as anti-rotavirus protein 1 [ARP1]) on the surface was investigated. L. rhamnosus GG is one of the best-characterized probiotic bacteria and has intrinsic antirotavirus activity. Among four L. rhamnosus GG strains [GG (CMC), GG (ATCC 53103), GG (NCC 3003), and GG (UT)] originating from different sources, only GG (UT) was able to display ARP1 on the bacterial surface. The genomic analysis of strain GG (UT) showed that the genes welE and welF of the EPS cluster are inactivated, which causes a defect in exopolysaccharide (EPS) production, allowing efficient display of ARP1 on its surface. Finally, GG (UT) seemed to confer a level of protection against rotavirus-induced diarrhea similar to that of wild-type GG (NCC 3003) in a mouse pup model, indicating that the EPS may not be involved in the intrinsic antirotavirus activity. Most important, GG (EM233), a derivative of GG (UT) producing ARP1, was significantly more protective than the control strain L. casei BL23. PMID:26092449

  3. Positron Emission Tomography Imaging of Tumor Angiogenesis with a 61/64Cu-Labeled F(ab')2 Antibody Fragment

    PubMed Central

    Hong, Hao; Zhang, Yin; Orbay, Hakan; Valdovinos, Hector F.; Nayak, Tapas R.; Bean, Jero; Theuer, Charles P.; Barnhart, Todd E.; Cai, Weibo

    2013-01-01

    The objective of this study was to characterize the in vitro and in vivo properties of the F(ab')2 fragment of TRC105, a human/murine chimeric IgG1 monoclonal antibody that binds with high avidity to human and murine CD105 (i.e. endoglin), and investigate its potential for positron emission tomography (PET) imaging of tumor angiogenesis after 61/64Cu-labeling. TRC105-F(ab')2 of high purity was produced by pepsin digestion of TRC105, which was confirmed by SDS-PAGE, HPLC analysis, and mass spectrometry. 61/64Cu-labeling of NOTA-TRC105-F(ab')2 (NOTA denotes 1,4,7-triazacyclononane-1,4,7-triacetic acid) was achieved with yields of > 75% (specific activity: ~115 GBq/μmol). PET imaging revealed rapid tumor uptake of 64Cu-NOTA TRC105-F(ab')2 in the 4T1 murine breast cancer model (5.8 ± 0.8, 7.6 ± 0.6, 5.6 ± 0.4, 5.0 ± 0.6, and 3.8 ± 0.7 %ID/g at 0.5, 3, 16, 24, and 48 h post-injection respectively; n = 4). Since tumor uptake peaked at 3 h post-injection, 61Cu-NOTA-TRC105-F(ab')2 also gave good tumor contrast at 3 and 8 h post-injection. CD105 specificity of the tracers was confirmed by blocking studies and histopathology. In conclusion, the use of a F(ab')2 fragment led to more rapid tumor uptake (which peaked at 3 h post-injection) than radiolabeled intact antibody (which often peaked after 24 h post-injection), which may allow for same day immunoPET imaging in future clinical studies. PMID:23316869

  4. Positron emission tomography imaging of tumor angiogenesis with a (61/64)Cu-labeled F(ab')(2) antibody fragment.

    PubMed

    Hong, Hao; Zhang, Yin; Orbay, Hakan; Valdovinos, Hector F; Nayak, Tapas R; Bean, Jero; Theuer, Charles P; Barnhart, Todd E; Cai, Weibo

    2013-02-01

    The objective of this study was to characterize the in vitro and in vivo properties of the F(ab')(2) fragment of TRC105, a human/murine chimeric IgG1 monoclonal antibody that binds with high avidity to human and murine CD105 (i.e., endoglin), and investigate its potential for positron emission tomography (PET) imaging of tumor angiogenesis after (61/64)Cu-labeling. TRC105-F(ab')(2) of high purity was produced by pepsin digestion of TRC105, which was confirmed by SDS-PAGE, HPLC analysis, and mass spectrometry. (61/64)Cu-labeling of NOTA-TRC105-F(ab')(2) (NOTA denotes 1,4,7-triazacyclononane-1,4,7-triacetic acid) was achieved with yields of >75% (specific activity: ∼115 GBq/μmol). PET imaging revealed rapid tumor uptake of (64)Cu-NOTA-TRC105-F(ab')(2) in the 4T1 murine breast cancer model (5.8 ± 0.8, 7.6 ± 0.6, 5.6 ± 0.4, 5.0 ± 0.6, and 3.8 ± 0.7% ID/g at 0.5, 3, 16, 24, and 48 h postinjection respectively; n = 4). Since tumor uptake peaked at 3 h postinjection, (61)Cu-NOTA-TRC105-F(ab')(2) also gave good tumor contrast at 3 and 8 h postinjection. CD105 specificity of the tracers was confirmed by blocking studies and histopathology. In conclusion, the use of a F(ab')(2) fragment led to more rapid tumor uptake (which peaked at 3 h postinjection) than radiolabeled intact antibody (which often peaked after 24 h postinjection), which may allow for same day immunoPET imaging in future clinical studies. PMID:23316869

  5. Epitope Mapping of Antigenic MUC1 Peptides to Breast Cancer Antibody Fragment B27.29: A Heteronuclear NMR Study

    SciTech Connect

    Grinstead, Jeffrey S.; Schuman, Jason T.; Campbell, Ann P.

    2003-11-13

    MUC1 mucin is a breast cancer-associated transmembrane glycoprotein, of which the extracellular domain is formed by the repeating 20-amino acid sequence GVTSAPDTRPAPGSTAPPAH. In neoplastic breast tissue, the highly immunogenic sequence PDTRPAP (in bold above) is exposed. Antibodies raised directly against MUC1-expressing tumors offer unique access to this neoplastic state, as they represent immunologically relevant ''reverse templates'' of the tumor-associated mucin. In a previous study [Grinstead, J. S., et al. (2002) Biochemistry 41, 9946-9961], 1H NMR methods were used to correlate the effects of cryptic glycosylation outside of the PDTRPAP core epitope sequence on the recognition and binding of Mab B27.29, a monoclonal antibody raised against breast tumor cells. In the study presented here, isotope-edited NMR methods, including 15N and 13C relaxation measurements, were used to probe the recognition and binding of the PDTRPAP epitope sequence to Fab B27.29. Two peptides were studied: a one-repeat MUC1 16mer peptide of the sequence GVTSAPDTRPAPGSTA and a two-repeat MUC1 40mer peptide of the sequence (VTSAPDTRPAPGSTAPPAHG)2. 15N and 13C NMR relaxation parameters were measured for both peptides free in solution and bound to Fab B27.29. The 13CR T1 values best represent changes in the local correlation time of the peptide epitope upon binding antibody, and demonstrate that the PDTRPAP sequence is immobilized in the antibody-combining site. This result is also reflected in the appearance of the 15N- and 13C-edited HSQC spectra, where line broadening of the same peptide epitope resonances is observed. The PDTRPAP peptide epitope expands upon the peptide epitope identified previously in our group as PDTRP by homonuclear NMR experiments [Grinstead, J. S., et al. (2002) Biochemistry 41, 9946-9961], and illustrates the usefulness of the heteronuclear NMR experiments. The implications of these results are discussed within the context of MUC1 breast cancer vaccine design.

  6. 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.

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

    PubMed

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

    2016-05-15

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

  8. 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

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

    PubMed

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

    2014-01-01

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

  10. 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

  11. 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

  12. 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

  13. 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.

  14. Malignant Melanoma

    PubMed Central

    Perera, Eshini; Gnaneswaran, Neiraja; Jennens, Ross; Sinclair, Rodney

    2013-01-01

    Melanomas are a major cause of premature death from cancer. The gradual decrease in rates of morbidity and mortality has occurred as a result of public health campaigns and improved rates of early diagnosis. Survival of melanoma has increased to over 90%. Management of melanoma involves a number of components: excision, tumor staging, re-excision with negative margins, adjuvant therapies (chemo, radiation or surgery), treatment of stage IV disease, follow-up examination for metastasis, lifestyle modification and counseling. Sentinel lymph node status is an important prognostic factor for survival in patients with a melanoma >1 mm. However, sentinel lymph node biopsies have received partial support due to the limited data regarding the survival advantage of complete lymph node dissection when a micrometastasis is detected in the lymph nodes. Functional mutations in the mitogen-activated pathways are commonly detected in melanomas and these influence the growth control. Therapies that target these pathways are rapidly emerging, and are being shown to increase survival rates in patients. Access to these newer agents can be gained by participation in clinical trials after referral to a multidisciplinary team for staging and re-excision of the scar. PMID:27429256

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

    PubMed

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

    2011-05-01

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

  16. 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.

  17. 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

  18. 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.

  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. 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

  1. 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

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

    PubMed

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

    2016-07-01

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

  3. Evaluating Biomarkers in Melanoma

    PubMed Central

    Karagiannis, Panagiotis; Fittall, Matthew; Karagiannis, Sophia N.

    2015-01-01

    The incidence of cutaneous melanoma has more than doubled over the last decades making it one of the fastest rising cancers worldwide. Improved awareness and early detection of malignant moles now permit earlier diagnosis aiming to decrease the likelihood of recurrence. However, it is difficult to identify those patients initially diagnosed with localized melanoma who subsequently develop metastatic disease. For this group, prognosis remains poor and clinical outcomes are variable and challenging to predict. Considerable efforts have focused on the search for novel prognostic tools, with numerous markers evaluated in the circulation and in tumor lesions. The most reliable predictors of patient outcome are the clinical and histological features of the primary tumor such as Breslow thickness, ulceration status, and mitotic rate. Elevated serum levels of the enzyme lactate dehydrogenase, likely to indicate active metastatic disease, are also routinely used to monitor patients. The emergence of novel immune and checkpoint antibody treatments for melanoma and increasing appreciation of key roles of the immune system in promoting or halting cancer progression have focused attention to immunological biomarkers. Validation of the most promising of these may have clinical applications in assisting prognosis, assessing endpoints in therapy, and monitoring responses during treatment. PMID:25667918

  4. Adjuvant Therapy: Melanoma

    PubMed Central

    Davar, Diwakar; Tarhini, Ahmad; Kirkwood, John M.

    2011-01-01

    With an incidence that is increasing at 2–5% per year, cutaneous melanoma is an international scourge that disproportionately targets young individuals. Despite much research, the treatment of advanced disease is still quite challenging. Immunotherapy with high-dose interferon-α2b or interleukin-2 benefits a select group of patients in the adjuvant and metastatic settings, respectively, with significant attendant toxicity. Advances in the biology of malignant melanoma and the role of immunomodulatory therapy have produced advances that have stunned the field. In this paper, we review the data for the use of interferon-α2b in various dosing ranges, vaccine therapy, and the role of radiotherapy in the adjuvant setting for malignant melanoma. Recent trials in the metastatic setting using anticytoxic T-lymphocyte antigen-4 (anti-CTLA-4) monoclonal antibody therapy and BRAF inhibitor therapy have demonstrated clear benefit with prolongation of survival. Trials investigating combinations of these novel agents with existing immunomodulators are at present underway. PMID:22220281

  5. Novel Camelid Antibody Fragments Targeting Recombinant Nucleoprotein of Araucaria hantavirus: A Prototype for an Early Diagnosis of Hantavirus Pulmonary Syndrome

    PubMed Central

    Pereira, Soraya S.; Moreira-Dill, Leandro S.; Morais, Michelle S. S.; Prado, Nidiane D. R.; Barros, Marcos L.; Koishi, Andrea C.; Mazarrotto, Giovanny A. C. A.; Gonçalves, Giselle M.; Zuliani, Juliana P.; Calderon, Leonardo A.; Soares, Andreimar M.; Pereira da Silva, Luiz H.; Duarte dos Santos, Claudia N.; Fernandes, Carla F. C.; Stabeli, Rodrigo G.

    2014-01-01

    In addition to conventional antibodies, camelids produce immunoglobulins G composed exclusively of heavy chains in which the antigen binding site is formed only by single domains called VHH. Their particular characteristics make VHHs interesting tools for drug-delivery, passive immunotherapy and high-throughput diagnosis. Hantaviruses are rodent-borne viruses of the Bunyaviridae family. Two clinical forms of the infection are known. Hemorrhagic Fever with Renal Syndrome (HFRS) is present in the Old World, while Hantavirus Pulmonary Syndrome (HPS) is found on the American continent. There is no specific treatment for HPS and its diagnosis is carried out by molecular or serological techniques, using mainly monoclonal antibodies or hantavirus nucleoprotein (N) to detect IgM and IgG in patient serum. This study proposes the use of camelid VHHs to develop alternative methods for diagnosing and confirming HPS. Phage display technology was employed to obtain VHHs. After immunizing one Lama glama against the recombinant N protein (prNΔ85) of a Brazilian hantavirus strain, VHH regions were isolated to construct an immune library. VHHs were displayed fused to the M13KO7 phage coat protein III and the selection steps were performed on immobilized prNΔ85. After selection, eighty clones recognized specifically the N protein. These were sequenced, grouped based mainly on the CDRs, and five clones were analyzed by western blot (WB), surface plasmon resonance (SPR) device, and ELISA. Besides the ability to recognize prNΔ85 by WB, all selected clones showed affinity constants in the nanomolar range. Additionaly, the clone KC329705 is able to detect prNΔ85 in solution, as well as the native viral antigen. Findings support the hypothesis that selected VHHs could be a powerful tool in the development of rapid and accurate HPS diagnostic assays, which are essential to provide supportive care to patients and reduce the high mortality rate associated with hantavirus infections. PMID

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2011-01-01

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

  9. A Camelid-derived Antibody Fragment Targeting the Active Site of a Serine Protease Balances between Inhibitor and Substrate Behavior.

    PubMed

    Kromann-Hansen, Tobias; Oldenburg, Emil; Yung, Kristen Wing Yu; Ghassabeh, Gholamreza H; Muyldermans, Serge; Declerck, Paul J; Huang, Mingdong; Andreasen, Peter A; Ngo, Jacky Chi Ki

    2016-07-15

    A peptide segment that binds the active site of a serine protease in a substrate-like manner may behave like an inhibitor or a substrate. However, there is sparse information on which factors determine the behavior a particular peptide segment will exhibit. Here, we describe the first x-ray crystal structure of a nanobody in complex with a serine protease. The nanobody displays a new type of interaction between an antibody and a serine protease as it inserts its complementary determining region-H3 loop into the active site of the protease in a substrate-like manner. The unique binding mechanism causes the nanobody to behave as a strong inhibitor as well as a poor substrate. Intriguingly, its substrate behavior is incomplete, as 30-40% of the nanobody remained intact and inhibitory after prolonged incubation with the protease. Biochemical analysis reveals that an intra-loop interaction network within the complementary determining region-H3 of the nanobody balances its inhibitor versus substrate behavior. Collectively, our results unveil molecular factors, which may be a general mechanism to determine the substrate versus inhibitor behavior of other protease inhibitors. PMID:27226628

  10. 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

  11. Identification of framework residues in a secreted recombinant antibody fragment that control production level and localization in Escherichia coli.

    PubMed

    Forsberg, G; Forsgren, M; Jaki, M; Norin, M; Sterky, C; Enhörning, A; Larsson, K; Ericsson, M; Björk, P

    1997-05-01

    The monoclonal antibody 5T4, directed against a human tumor-associated antigen, was expressed as a secreted Fab superantigen fusion protein in Escherichia coli. The product is a putative agent for immunotherapy of non-small cell lung cancer. During fermentation, most of the fusion protein leaked out from the periplasm to the growth medium at a level of approximately 40 mg/liter. This level was notably low compared with similar products containing identical CH1, CL, and superantigen moieties, and the Fv framework was therefore engineered. Using hybrid molecules, the light chain was found to limit high expression levels. Substituting five residues in VL increased the level almost 15 times, exceeding 500 mg/liter in the growth medium. Here, the substitutions Phe-10 --> Ser, Thr-45 --> Lys, Thr-77 --> Ser, and Leu-78 --> Val were most powerful. In addition, replacing four VH residues diminished cell lysis during fermentation. Thereby the product was preferentially located in the periplasm instead of the growth medium, and the total yield was more than 700 mg/liter. All engineered products retained a high affinity for the tumor-associated antigen. It is suggested that at least some of the identified framework residues generally have to be replaced to obtain high level production of recombinant Fab products in E. coli. PMID:9139690

  12. [Vulvar melanoma].

    PubMed

    Chokoeva, A; Tchernev, G; Wollina, U

    2015-01-01

    Malignant melanoma of the vulva is a rare disease with aggressive behavior and poor prognosis. It consist < 5% of all cases of melanoma in females, as the ratio of its manifestation, compared with the cutaneous melanoma is 1:71. Higher risk of developing melanoma of the vulva is established in white women, as the peak of the incidence is between 60 and 70 years of age. Clinically, MM of the vulva manifests as asymptomatic pigmented, rarely a pigmented lesion, as the usual clinical form is superficial spreading MM and much less common nodular MM, which is associated with a poorer prognosis in. general. The diagnosis is confirmed by histological examination. Conduction of PCR and DNA analysis for detection of BRAF mutations, NRAS mutations and KIT amplification is also appropriate. Advanced age, black race, tumor size, tumor thickness, ulceration, presence of satellite lesions, involvement of adjacent organs (vagina, urethra), and the presence of regional or distant metastases are identified as the most important prognostic markers. Radical wide excision followed by bilateral lymphadenectomy id considered as the optimal therapeutic approach. PMID:25909143

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

    PubMed

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

    2016-03-01

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

  16. 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

  17. Camelid single-domain antibody fragments: Uses and prospects to investigate protein misfolding and aggregation, and to treat diseases associated with these phenomena.

    PubMed

    Pain, Coralie; Dumont, Janice; Dumoulin, Mireille

    2015-04-01

    The deposition of misfolded peptides and proteins in the form of amyloid fibrils is the hallmark of nearly fifty medical disorders, including Alzheimer's disease, Parkinson's disease, prion diseases and type II diabetes. These disorders, referred to as amyloidoses, generally become apparent late in life. Their psycho-sociological and economic incidence in western societies will be therefore considerable in the coming decades due to the ageing of the population. Neither preventing nor curative treatments are available yet. These disorders constitute therefore a medical challenge of great importance. Thus, an extensive research is being carried out to understand, at the molecular level, (i) how amyloidogenic proteins misfold and convert from their soluble form into amyloid fibrils, and (ii) how these aggregates or some of their oligomeric precursor species are toxic. The formation of amyloid fibrils proceeds through a complex nucleation/polymerisation mechanism with the formation of various species, including small oligomers. In this review, we focus on how VHHs or nanobodies, the antigen-binding domains of camelid heavy-chain antibodies, are being increasingly used to characterise each of the species formed on the pathway of fibril formation in terms of structure, stability, kinetics of formation and toxicity. We first introduce the characteristic features of nanobodies compared to those of conventional antibody fragments. Thereafter, we discuss how nanobodies, due to their unique properties, are used as probes to dissect the molecular mechanisms of misfolding and aggregation of six proteins associated with diseases, i.e. human lysozyme, β2-microglobulin, α-synuclein, prion, polyadenylate binding protein nuclear 1 and amyloid β-peptide. A brief general presentation of each disease and the associated peptide/protein is also provided. In addition, we discuss how nanobodies could be used as early diagnostic tools and as novel strategies to treat diseases associated

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

    PubMed

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

    2012-03-01

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

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

    PubMed

    Martineau, P; Betton, J M

    1999-10-01

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

  20. 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

  1. 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. PMID:23475508

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

    PubMed

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

    2012-11-01

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

  3. Malicious masquerade: myxoid melanoma.

    PubMed

    Hitchcock, M G; White, W L

    1998-08-01

    The morphological spectrum of malignant melanoma is broad. Unusual stromal changes can distract pathologists from the correct diagnosis. Fibroblastic, chondroid, osteoid, and myxoid stroma have been documented in melanomas. Myxoid melanoma is problematic--introducing carcinomas and soft tissue sarcomas into the differential diagnosis. This review examines the clinicopathologic aspects of myxoid malignant melanoma, with emphasis on its differential diagnosis. PMID:9711669

  4. Aquaporin 1 protein expression is associated with BRAF V600 mutation and adverse prognosis in cutaneous melanoma.

    PubMed

    Imrédi, Eleonóra; Tóth, Béla; Doma, Viktória; Barbai, Tamás; Rásó, Erzsébet; Kenessey, István; Tímár, József

    2016-06-01

    Despite experimental findings suggesting the prognostic significance of Aquaporin 1 (AQP1) in human melanoma, no published clinical data are available. We studied the expression of AQP1 protein in cutaneous melanoma, correlated our findings with standard histological and genetic markers, and long-term clinical follow-up. Our study evaluated the AQP1 protein expression in 78 melanoma patients, representing two predefined risk cohorts using the immune labeling technique with commercially available anti-AQP1 antibodies on routinely formalin-fixed and paraffin-embedded tumor tissue samples. BRAF V600E mutation analyses were carried out successfully in 70 patients using PCR and restriction fragment length polymorphism analyses, followed by confirmatory analysis with the Sanger sequencing technique. AQP1-expressing melanoma cells were found in 52 cases (66.7%, median H-score=124.24). Significantly higher AQP1 H-scores (P=0.047) were found in the 'high-risk' patients. No correlations were found with the established histological markers, such as mitotic index (P=0.42), Clark level (P=0.95), and Breslow thickness (P=0.51). BRAF V600 mutation analyses were successful in 89%, and showed a two times higher mutation frequency in the 'high-risk' group. The BRAF V600 mutations were significantly associated with AQP1 expression (P=0.014). Long-term follow-up indicated a reduced progression-free survival (P=0.036) and overall survival (P=0.017) for the AQP1-positive cutaneous melanoma patients. AQP1 expression is likely to be associated with an adverse prognosis in cutaneous melanoma. PMID:26848795

  5. 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. PMID:25081390

  6. 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. PMID:21034988

  7. 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

  8. 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

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

    PubMed

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

    2013-01-01

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

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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

  16. Novel melanoma therapies and their side effects.

    PubMed

    González, Noelani; Ratner, Désirée

    2016-06-01

    In the last few years, melanoma treatment has been revolutionized by the development of immune checkpoint-blocking antibodies or immune checkpoint inhibitors including ipilimumab, vemurafenib, dabrafenib, trametinib, nivolumab, and pembrolizumab. Although they have shown promising results, they also have caused multiple adverse events (AEs), particularly immune-related AEs (irAEs). Specialists should be familiar with these AEs. PMID:27416087

  17. 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.

  18. 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

  19. Melanoma Cell-Intrinsic PD-1 Receptor Functions Promote Tumor Growth.

    PubMed

    Kleffel, Sonja; Posch, Christian; Barthel, Steven R; Mueller, Hansgeorg; Schlapbach, Christoph; Guenova, Emmanuella; Elco, Christopher P; Lee, Nayoung; Juneja, Vikram R; Zhan, Qian; Lian, Christine G; Thomi, Rahel; Hoetzenecker, Wolfram; Cozzio, Antonio; Dummer, Reinhard; Mihm, Martin C; Flaherty, Keith T; Frank, Markus H; Murphy, George F; Sharpe, Arlene H; Kupper, Thomas S; Schatton, Tobias

    2015-09-10

    Therapeutic antibodies targeting programmed cell death 1 (PD-1) activate tumor-specific immunity and have shown remarkable efficacy in the treatment of melanoma. Yet, little is known about tumor cell-intrinsic PD-1 pathway effects. Here, we show that murine and human melanomas contain PD-1-expressing cancer subpopulations and demonstrate that melanoma cell-intrinsic PD-1 promotes tumorigenesis, even in mice lacking adaptive immunity. PD-1 inhibition on melanoma cells by RNAi, blocking antibodies, or mutagenesis of melanoma-PD-1 signaling motifs suppresses tumor growth in immunocompetent, immunocompromised, and PD-1-deficient tumor graft recipient mice. Conversely, melanoma-specific PD-1 overexpression enhances tumorigenicity, as does engagement of melanoma-PD-1 by its ligand, PD-L1, whereas melanoma-PD-L1 inhibition or knockout of host-PD-L1 attenuate growth of PD-1-positive melanomas. Mechanistically, the melanoma-PD-1 receptor modulates downstream effectors of mTOR signaling. Our results identify melanoma cell-intrinsic functions of the PD-1:PD-L1 axis in tumor growth and suggest that blocking melanoma-PD-1 might contribute to the striking clinical efficacy of anti-PD-1 therapy. PMID:26359984

  20. Immune checkpoint inhibitors: therapeutic advances in melanoma

    PubMed Central

    Márquez-Rodas, Ivan; Cerezuela, Pablo; Soria, Ainara; Berrocal, Alfonso; Riso, Aldo; Martín-Algarra, Salvador

    2015-01-01

    In recent years, new strategies for treating melanoma have been introduced, improving the outlook for this challenging disease. One of the most important advances has been the development of immunotherapy. The better understanding of the role of the immunological system in tumor control has paved the way for strategies to enhance the immune response against cancer cells. Monoclonal antibodies (mAbs) against the immune checkpoints cytotoxic T-lymphocyte antigen-4 (CTLA-4) and programmed cell death protein 1 (PD-1) and its ligand (PD-L1) have demonstrated high activity in melanoma and other tumors. Ipilimumab, an anti CTLA-4 antibody, was the first drug of this class that was approved. Although the response rate with ipilimumab is low (less than 20% of patients have objective responses), 20% of patients have long survival, with similar results in the first and second line settings. Nivolumab and pembrolizumab, both anti PD-1 inhibitors, have been approved for the treatment of melanoma, with response rates of 40% and a demonstrated survival advantage in phase III trials. This has marked a new era in the treatment of metastatic melanoma and much research is now ongoing with other drugs targeting checkpoint inhibitors. In addition, the agonist of activating molecules on T cells and their combinations are being investigated. Herein we review the clinical development of checkpoint inhibitors and their approval for treatment of metastatic melanoma. PMID:26605313

  1. 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.

  2. Nivolumab in the treatment of malignant melanoma: review of the literature

    PubMed Central

    Mashima, Emi; Inoue, Akiha; Sakuragi, Yumiko; Yamaguchi, Takashi; Sasaki, Natsuko; Hara, Yoko; Omoto, Daisuke; Ohmori, Shun; Haruyama, Sanehito; Sawada, Yu; Yoshioka, Manabu; Nishio, Daisuke; Nakamura, Motonobu

    2015-01-01

    Nivolumab was developed as a monoclonal antibody against programmed death receptor-1, an immune checkpoint inhibitor which negatively regulates T-cell proliferation and activation. Intravenous administration of nivolumab was approved for the treatment of unresectable malignant melanoma in 2014 in Japan. When advanced melanoma patients were treated with nivolumab, median overall survival became longer. Overall survival rate was significantly better in nivolumab-treated melanoma patients than dacarbazine-treated melanoma patients. Nivolumab had an acceptable long-term tolerability profile, with 22% of patients experiencing grade 3 or 4 adverse events related to the drug. Therefore, nivolumab can become an alternative therapy for advanced malignant melanoma. PMID:26273207

  3. 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.

  4. Malignant melanoma (image)

    MedlinePlus

    ... tumor that involves the skin cells that produce pigment (melanin). The risk of melanoma increases with age, but frequently affects young, otherwise healthy people. Melanoma is the number one cause of cancer death in women aged 25 to 30.

  5. 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 ...

  6. Melanoma - neck (image)

    MedlinePlus

    This melanoma on the neck is variously colored with a very darkly pigmented area found centrally. It has irregular ... be larger than 0.5 cm. Prognosis in melanoma is best defined by its depth on resection.

  7. Melanoma International Foundation

    MedlinePlus

    ... Gershenwald, MD May 09, 2015 Our Awards Melanoma International Foundation Our Mission: To develop personalized strategies with ... the state of Pennsylvania, certificate #29498 © 2013 Melanoma International Foundation. All Rights Reserved. Privacy Policy | Terms of ...

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

    ClinicalTrials.gov

    2016-05-12

    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

  9. Tumor immunotherapy in melanoma: strategies for overcoming mechanisms of resistance and escape.

    PubMed

    Zigler, Maya; Villares, Gabriel J; Lev, Dina C; Melnikova, Vladislava O; Bar-Eli, Menashe

    2008-01-01

    The incidence of melanoma has been steadily increasing over the last 3 decades. Currently, there are several approved treatments for metastatic melanoma, including chemotherapy and biologic therapy as both single treatments and in combination, but none is associated with a significant increase in survival. The chemotherapeutic agent dacarbazine is the standard treatment for metastatic melanoma, with a response rate of 15-20%, although most responses are not sustained. One of the main problems with melanoma treatment is chemotherapeutic resistance. The mechanisms of resistance of melanoma cells to chemotherapy have yet to be elucidated. Following treatment with dacarbazine, melanoma cells activate the extracellular signal-regulated kinase pathway, which results in over-expression and secretion of interleukin (IL)-8 and vascular endothelial growth factor. Melanoma cells utilize this mechanism to escape from the cytotoxic effect of the drug. We have previously reported on the development of fully human neutralizing antibodies against IL-8 (anti-IL-8-monoclonal-antibody [ABX-IL8]). In preclinical studies, ABX-IL8 inhibited tumor growth, angiogenesis, and metastasis of human melanoma in vivo. We propose that combination treatment with dacarbazine and IL-8 will potentiate the cytotoxic effect of the drug. Furthermore, formation of metastasis is a multistep process that includes melanoma cell adhesion to endothelial cells. Melanoma cell adhesion molecule (MUC18) mediates these processes in melanoma and is therefore a good target for eliminating metastasis. We have developed a fully human antibody against MUC18 that has shown promising results in preclinical studies. Since resistance is one of the major obstacles in the treatment of melanoma, we propose that utilization of antibodies against IL-8 or MUC18 alone, or as part of a 'cocktail' in combination with dacarbazine, may be a new treatment modality for metastatic melanoma that overcomes resistance of the disease to

  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. Elastin-derived peptides enhance melanoma growth in vivo by upregulating the activation of Mcol-A (MMP-1) collagenase

    PubMed Central

    Devy, J; Duca, L; Cantarelli, B; Joseph-Pietras, D; Scandolera, A; Rusciani, A; Parent, L; Thevenard, J; Pasco, S Brassart; Tarpin, M; Martiny, L; Debelle, L

    2010-01-01

    Background: Elastin peptides possess several biological activities and in vitro data suggest they could be involved in the early phase of melanoma growth. Methods: Using diverse in vitro and in vivo techniques (cell proliferation, invasion and migration assays, zymography, western blots, collagen degradation assay, reverse transcription PCR, melanoma allographs and immunohistochemistry), we analysed the effect of elastin-derived peptides (EDPs) on B16F1 melanoma growth and invasion, as well as on the proteolytic systems involved. Results: We found that EDPs dramatically promote in vivo tumour development of B16F1 melanoma, as well as their in vitro migration and invasion. The inhibition of serine proteases and matrix metalloproteinases (MMPs) activities, by aprotinin and galardin, respectively, demonstrated that these enzymes were involved in these processes. However, we found that EDPs did not increase urokinase-type plasminogen activator, tissue-type plasminogen activator or MMP-2 expression and/or activation, neither in vitro nor in vivo. Nevertheless, we observed a strong increase of pro-MMP-9 secretion in EDPs-treated tumours and, more importantly, an increase in the expression and activation of the murine counterpart of MMP-1, named murine collagenase-A (Mcol-A). Moreover, we show that plasminogen system inhibition decreases collagen degradation by this enzyme. Finally, the use of a specific blocking antibody against Mcol-A abolished EDP-induced B16F1 invasion in vitro, showing that this MMP was directly involved in this process. Conclusion: Our data show that in vivo, EDPs are involved in melanoma growth and invasion and reinforced the concept of elastin fragmentation as a predictive factor. PMID:20959825

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

    PubMed Central

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

    2016-01-01

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

  13. Immune based therapy for melanoma

    PubMed Central

    Ancuceanu, Robert; Neagu, Monica

    2016-01-01

    A few years ago therapeutic options in advanced melanoma were very limited and the prognosis was somber. Although recent progresses are far from providing a cure for advanced melanoma, yet these have kindled new hopes and searching for a cure does not seem unreasonable. Seven new medicines have been authorized in various regions of the world in the recent past in the therapy of advanced melanoma, over half of them acting by mechanisms involving the immune system of the host. The anti-CTLA-4 (cytotoxic T lymphocyte associated protein-4) ipilimumab has been followed by anti-PD1 (programmed death1) inhibitors, more effective and safer. Very recently, the first oncolytic immunotherapy, talimogene laherparepvec (T-VEC) has been authorized for placing on the market and a variety of combinations of the new therapies are currently being evaluated or considered. Besides, a plethora of other molecules and approaches, especially monoclonal antibodies, are in the preliminary phases of clinical investigation and are likely to bring new benefits for the treatment of this potentially fatal form of cancer. PMID:27121512

  14. Malignant melanoma maxilla

    PubMed Central

    Devi, Seema; Sinha, Richi; Singh, Rakesh Kumar

    2015-01-01

    A malignant melanoma is a highly lethal melanocytic neoplasm. A neoplasm usually affects the skin. Malignant melanomas in the head and neck region are rare, accounting for less than 1% of all melanomas. Malignant melanoma of the nose and paranasal sinuses is an aggressive disease typically presenting at an advanced stage, with a 5-year survival rate ranging 20-30%. Melanomas are tumors arising from melanocytes, which are neuroectodermally derived cells located in the basal layers of the skin. This is a case report of a 35-year-old male, who presented with very aggressive disease and developed liver metastasis. PMID:26668467

  15. 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

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

    PubMed

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

    2012-11-01

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

  17. 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

  18. 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

  19. 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

  20. 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

  1. Cutaneous Melanoma in Women

    PubMed Central

    Roh, Mi Ryung; Eliades, Philip; Gupta, Sameer; Tsao, Hensin

    2015-01-01

    The incidence of cutaneous melanoma (CM) continues to increase in the Caucasian population in the United States. In 2014, women only accounted for 42% of the 76,100 new melanoma cases and only 33% of the 9,710 deaths associated with CM in the US.1 These trends are consistently observed in populations around the world. Indeed, gender disparity in melanoma outcome is so consistently observed that gender has been suggested as an important prognostic factor in melanoma, despite not being formerly incorporated in staging algorithms.2 The source of this gender disparity in melanoma remains unclear but likely represents both biological and behavioral etiologies. Herein, we review the current knowledge of how melanoma differs between men and women. PMID:25844396

  2. 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

  3. 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

  4. Llama Antibody Fragments Recognizing Various Epitopes of the CD4bs Neutralize a Broad Range of HIV-1 Subtypes A, B and C

    PubMed Central

    Aasa-Chapman, Marlèn; Gorlani, Andrea; Forsman Quigley, Anna; Hulsik, David Lutje; Chen, Lei; Weiss, Robin; de Haard, Hans; Verrips, Theo

    2012-01-01

    Many of the neutralising antibodies, isolated to date, display limited activities against the globally most prevalent HIV-1 subtypes A and C. Therefore, those subtypes are considered to be an important target for antibody-based therapy. Variable domains of llama heavy chain antibodies (VHH) have some superior properties compared with classical antibodies. Therefore we describe the application of trimeric forms of envelope proteins (Env), derived from HIV-1 of subtype A and B/C, for a prolonged immunization of two llamas. A panel of VHH, which interfere with CD4 binding to HIV-1 Env were selected with use of panning. The results of binding and competition assays to various Env, including a variant with a stabilized CD4-binding state (gp120Ds2), cross-competition experiments, maturation analysis and neutralisation assays, enabled us to classify the selected VHH into three groups. The VHH of group I were efficient mainly against viruses of subtype A, C and B′/C. The VHH of group II resemble the broadly neutralising antibody (bnmAb) b12, neutralizing mainly subtype B and C viruses, however some had a broader neutralisation profile. A representative of the third group, 2E7, had an even higher neutralization breadth, neutralizing 21 out of the 26 tested strains belonging to the A, A/G, B, B/C and C subtypes. To evaluate the contribution of certain amino acids to the potency of the VHH a small set of the mutants were constructed. Surprisingly this yielded one mutant with slightly improved neutralisation potency against 92UG37.A9 (subtype A) and 96ZM651.02 (subtype C). These findings and the well-known stability of VHH indicate the potential application of these VHH as anti-HIV-1 microbicides. PMID:22438910

  5. Checkpoint Blockade for the Treatment of Advanced Melanoma.

    PubMed

    Callahan, Margaret K; Flaherty, Catherine R; Postow, Michael A

    2016-01-01

    Immunotherapy with immune checkpoint inhibition has been improving the outcomes of patients with many different types of malignancies. Immune checkpoint inhibition has been most extensively studied in patients with advanced melanoma and there are three FDA approved antibodies already widely used in clinical practice (ipilimumab, nivolumab, and pembrolizumab). In this chapter, we review the mechanistic basis behind the development of immune checkpoint blocking antibodies. We then discuss specifics regarding each agent, unique clinical considerations in treating patients with this approach, and future directions, including combination strategies. This chapter is focused on melanoma, but the principles related to this immunotherapy approach are applicable to patients with many types of malignancies. PMID:26601865

  6. Melanoma in Immunosuppressed Patients

    PubMed Central

    Kubica, Agnieszka W.; Brewer, Jerry D.

    2012-01-01

    The immunogenic characteristics of malignant melanoma are intriguing. To date, multiple studies exist regarding the immunogenicity of melanoma. In this article, we summarize data in the literature on the role of immunosuppression in melanoma and discuss several immunocompromised patient populations in detail. A comprehensive PubMed search was conducted with no date limitation. The following search terms were used: melanoma in combination with immunosuppression, immunocompromised, genetics, antigen processing, UV radiation, organ transplantation, organ transplant recipients, lymphoproliferative disease, lymphoma, CLL, NHL, radiation, and HIV/AIDS. Although no formal criteria were used for inclusion of studies, most pertinent studies on the topic were reviewed, with the exception of smaller case reports and case series. The included studies were generally large (≥1000 patients in organ transplant recipient studies; ≥500 patients in lymphoma studies), with a focus on institutional experiences, or population-based national or international epidemiologic studies. Melanoma-induced immunosuppression, the role of UV radiation in melanoma development, and the epidemiology, clinical course, and prognosis of melanoma in immunocompromised patients are highlighted. Organ transplant recipients, patients with lymphoproliferative disorders, patients with iatrogenic immunosuppression, and patients with human immunodeficiency virus infection/AIDS are also highlighted. Recommendations are proposed for the care and monitoring of immunosuppressed patients with melanoma. With better understanding of the molecular microenvironment and clinical course of melanoma in immunosuppressed patients, novel therapies could be developed and outcomes potentially affected in these patients. PMID:23036673

  7. 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

  8. A single-dose cytomegalovirus-based vaccine encoding tetanus toxin fragment C induces sustained levels of protective tetanus toxin antibodies in mice.

    PubMed

    Tierney, Rob; Nakai, Toru; Parkins, Christopher J; Caposio, Patrizia; Fairweather, Neil F; Sesardic, Dorothea; Jarvis, Michael A

    2012-04-26

    The current commercially available vaccine used to prevent tetanus disease following infection with the anaerobic bacterium Clostridium tetani is safe and effective. However, tetanus remains a major source of mortality in developing countries. In 2008, neonatal tetanus was estimated to have caused >59,000 deaths, accounting for 1% of worldwide infant mortality, primarily in poorer nations. The cost of multiple vaccine doses administered by injection necessary to achieve protective levels of anti-tetanus toxoid antibodies is the primary reason for low vaccine coverage. Herein, we show that a novel vaccine strategy using a cytomegalovirus (CMV)-based vaccine platform induces protective levels of anti-tetanus antibodies that are durable (lasting >13 months) in mice following only a single dose. This study demonstrates the ability of a 'single-dose' CMV-based vaccine strategy to induce durable protection, and supports the potential for a tetanus vaccine based on CMV to impact the incidence of tetanus in developing countries. PMID:22414558

  9. 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

  10. 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

  11. 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

  12. Uveal melanoma: estimating prognosis.

    PubMed

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

    2015-02-01

    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

  13. Cutaneous melanoma: uncommon presentations.

    PubMed

    Beyeler, Mirjam; Dummer, Reinhard

    2005-01-01

    Melanoma is the most important nonepithelial skin cancer. The diagnosis is usually made by clinical examination including dermatoscopy and histology. There are, however, variants of melanoma that miss the characteristic signs of pigmented lesions which are easily detectable when using the ABCD rule. PMID:16325067

  14. Genetic epidemiology of melanoma.

    PubMed

    Ribero, Simone; Glass, Dan; Bataille, Veronique

    2016-08-01

    The field of melanoma genetics is moving at great pace with new platforms to investigate single nucleotide polymorphism, genome sequencing, gene expression, and methylation. Melanoma incidence is still rising mainly because of screening campaigns, which has increased the number of reported melanomas. However, mortality due to melanoma is not decreasing. Many cutaneous phenotypic risk factors have been linked to melanoma, but the association with UV radiation is very complex. The level of vitamin D affects both the risk of melanoma and prognosis, but more studies are needed. The genetics of melanoma involves genes involved in pigmentation and naevi, as well as genes involved in the cell cycle and senescence, which have been identified via genome-wide association studies over the last 10 years. One area of research highly relevant to melanoma is telomere biology with further links to reduced senescence. At the somatic level, new gene pathways are being explored with many new therapeutic targets, and boosting immune responses against the tumour appears to offer the best long-term outcome. PMID:27436815

  15. Isolation of melanoma cell subpopulations using negative selection

    PubMed Central

    Slipicevic, Ana; Somasundaram, Rajasekharan; Sproesser, Katrin; Herlyn, Meenhard

    2014-01-01

    Melanomas are phenotypically and functiwonally heterogeneous tumors comprising of distinct subpopulations that drive disease progression and are responsible for resistance to therapy. Identification and characterization of such subpopulations are highly important to develop novel targeted therapies. However, this can be a challenging task as there is a lack of clearly defined markers to distinguish the melanoma subpopulations from a general tumor cell population. Also, there is a lack of optimal isolation methods and functional assays that can fully recapitulate their phenotype. Here we describe a method for isolating tumor cells from fresh human tumor tissue specimens using an antibody coupled magnetic bead sorting technique that is well established in our laboratory. Thus, melanoma cells are enriched by negative cell sorting and elimination of non-tumor cell population such as erythrocytes, leukocytes, and endothelial cells. Enriched unmodified tumor cells can be further used for phenotypic and functional characterization of melanoma subpopulations. PMID:24258995

  16. 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

  17. Malignant melanoma S3-guideline "diagnosis, therapy and follow-up of melanoma".

    PubMed

    Pflugfelder, Annette; Kochs, Corinna; Blum, Andreas; Capellaro, Marcus; Czeschik, Christina; Dettenborn, Therese; Dill, Dorothee; Dippel, Edgar; Eigentler, Thomas; Feyer, Petra; Follmann, Markus; Frerich, Bernhard; Ganten, Maria-Katharina; Gärtner, Jan; Gutzmer, Ralf; Hassel, Jessica; Hauschild, Axel; Hohenberger, Peter; Hübner, Jutta; Kaatz, Martin; Kleeberg, Ulrich R; Kölbl, Oliver; Kortmann, Rolf-Dieter; Krause-Bergmann, Albrecht; Kurschat, Peter; Leiter, Ulrike; Link, Hartmut; Loquai, Carmen; Löser, Christoph; Mackensen, Andreas; Meier, Friedegund; Mohr, Peter; Möhrle, Matthias; Nashan, Dorothee; Reske, Sven; Rose, Christian; Sander, Christian; Satzger, Imke; Schiller, Meinhard; Schlemmer, Heinz-Peter; Strittmatter, Gerhard; Sunderkötter, Cord; Swoboda, Lothar; Trefzer, Uwe; Voltz, Raymond; Vordermark, Dirk; Weichenthal, Michael; Werner, Andreas; Wesselmann, Simone; Weyergraf, Ansgar J; Wick, Wolfgang; Garbe, Claus; Schadendorf, Dirk

    2013-08-01

    This first German evidence-based guideline for cutaneous melanoma was developed under the auspices of the German Dermatological Society (DDG) and the Dermatologic Cooperative Oncology Group (DeCOG) and funded by the German Guideline Program in Oncology. The recommendations are based on a systematic literature search, and on the consensus of 32 medical societies, working groups and patient representatives. This guideline contains recommendations concerning diagnosis, therapy and follow-up of melanoma. The diagnosis of primary melanoma based on clinical features and dermoscopic criteria. It is confirmed by histopathologic examination after complete excision with a small margin. For the staging of melanoma, the AJCC classification of 2009 is used. The definitive excision margins are 0.5 cm for in situ melanomas, 1 cm for melanomas with up to 2 mm tumor thickness and 2 cm for thicker melanomas, they are reached in a secondary excision. From 1 mm tumor thickness, sentinel lymph node biopsy is recommended. For stages II and III, adjuvant therapy with interferon-alpha should be considered after careful analysis of the benefits and possible risks. In the stage of locoregional metastasis surgical treatment with complete lymphadenectomy is the treatment of choice. In the presence of distant metastasis mutational screening should be performed for BRAF mutation, and eventually for CKIT and NRAS mutations. In the presence of mutations in case of inoperable metastases targeted therapies should be applied. Furthermore, in addition to standard chemotherapies, new immunotherapies such as the CTLA-4 antibody ipilimumab are available. Regular follow-up examinations are recommended for a period of 10 years, with an intensified schedule for the first three years. PMID:24028775

  18. Main roads to melanoma

    PubMed Central

    Palmieri, Giuseppe; Capone, Mariaelena; Ascierto, Maria Libera; Gentilcore, Giusy; Stroncek, David F; Casula, Milena; Sini, Maria Cristina; Palla, Marco; Mozzillo, Nicola; Ascierto, Paolo A

    2009-01-01

    The characterization of the molecular mechanisms involved in development and progression of melanoma could be helpful to identify the molecular profiles underlying aggressiveness, clinical behavior, and response to therapy as well as to better classify the subsets of melanoma patients with different prognosis and/or clinical outcome. Actually, some aspects regarding the main molecular changes responsible for the onset as well as the progression of melanoma toward a more aggressive phenotype have been described. Genes and molecules which control either cell proliferation, apoptosis, or cell senescence have been implicated. Here we provided an overview of the main molecular changes underlying the pathogenesis of melanoma. All evidence clearly indicates the existence of a complex molecular machinery that provides checks and balances in normal melanocytes. Progression from normal melanocytes to malignant metastatic cells in melanoma patients is the result of a combination of down- or up-regulation of various effectors acting on different molecular pathways. PMID:19828018

  19. Main roads to melanoma.

    PubMed

    Palmieri, Giuseppe; Capone, Mariaelena; Ascierto, Maria Libera; Gentilcore, Giusy; Stroncek, David F; Casula, Milena; Sini, Maria Cristina; Palla, Marco; Mozzillo, Nicola; Ascierto, Paolo A

    2009-01-01

    The characterization of the molecular mechanisms involved in development and progression of melanoma could be helpful to identify the molecular profiles underlying aggressiveness, clinical behavior, and response to therapy as well as to better classify the subsets of melanoma patients with different prognosis and/or clinical outcome. Actually, some aspects regarding the main molecular changes responsible for the onset as well as the progression of melanoma toward a more aggressive phenotype have been described. Genes and molecules which control either cell proliferation, apoptosis, or cell senescence have been implicated. Here we provided an overview of the main molecular changes underlying the pathogenesis of melanoma. All evidence clearly indicates the existence of a complex molecular machinery that provides checks and balances in normal melanocytes. Progression from normal melanocytes to malignant metastatic cells in melanoma patients is the result of a combination of down- or up-regulation of various effectors acting on different molecular pathways. PMID:19828018

  20. Synchronous anorectal melanoma

    PubMed Central

    Balicevic, Drinko; Tomic, Karla; Bekavac-Beslin, Miroslav; Kovacevic, Igor; Mijic, August; Belicza, Mladen; Kruslin, Bozo

    2006-01-01

    Anorectal melanoma is a very rare tumor with poor prognosis. Rectal bleeding is the most frequent symptom and surgical treatment ranges from local excision to radical abdominoperineal resection. We report a case of a 75-years-old male patient who presented with a history of recurrent rectal bleeding, and whose histopathological diagnosis was melanoma. Macroscopically, we found two distinct tumors in anorectal region, 0.5 cm and 1.5 cm from dentate line. The first one was pedunculated, on a thin stalk, measuring 1 cm in greatest diameter, and the second one was sessile and nodular measuring up to 2.8 cm in largest diameter. Microscopic examination and immunohistochemical analysis of both tumors confirmed the diagnosis of melanoma. This case represents multiple synchronous primary melanoma of the anorectal region, with a possibility that one of the lesions is primary melanoma and the second one is a satellite lesion. PMID:16733870

  1. Cutaneous manifestations associated with melanoma.

    PubMed

    Vyas, Ritva; Selph, Jacqueline; Gerstenblith, Meg R

    2016-06-01

    Melanoma is a malignancy most commonly arising from the skin; therefore, primary melanoma characteristics are usually the first cutaneous manifestations of melanoma. Cutaneous metastases, which can occur locally or diffusely, are important to detect in a timely manner as treatments for advanced melanoma that impact survival are now available. Melanoma can be associated with local or diffuse pigmentation changes, including depigmentation associated with the leukodermas and hyperpigmentation associated with diffuse melanosis cutis. The leukodermas occur frequently, illustrate the immunogenic nature of melanoma, and may impact prognosis. Paraneoplastic syndromes in association with melanoma are rare, though can occur. PMID:27178692

  2. [Melanoma brain metastases : Treatment options].

    PubMed

    Rauschenberg, R; Tabatabai, G; Troost, E G C; Garzarolli, M; Beissert, S; Meier, F

    2016-07-01

    The majority of patients with metastatic melanoma will develop brain metastases, which are the most common cause of death. Until recently, local therapies (e. g., neurosurgery, radiotherapy) were the only options for brain metastases; however, effective systemic treatment options are now available. Upon suspicion of brain metastases, diagnostic staging with brain MRI and a neurological investigation are indicated. Prognostic factors such as number of cerebral metastases and symptoms, serum lactate dehydrogenase and S‑100 levels, extracerebral metastases, and ECOG status are considered during therapeutic planning. Treatment planning and therapeutic interventions should be based on an interdisciplinary and multimodal approach. Established treatments for singular brain metastases are neurosurgical resection and stereotactic radiotherapy, which can prolong survival. In patients with asymptomatic BRAF V600E-mutant brain metastases, the BRAF inhibitors dabrafenib, vemurafenib, and immunotherapy with ipilimumab are used. In the case of multiple symptomatic brain metastases, palliative whole-brain radiotherapy is used for treatment, although it has failed to show an overall survival benefit. Increased intracranial pressure and epileptic seizures are addressed with corticosteroids and anticonvulsants. Current clinical studies for melanoma patients with brain metastases are investigating new treatment options such as PD-1 antibodies, combined ipilimumab and nivolumab, combined BRAF inhibitors and MEK inhibitors, and stereotactic radiation in combination with immunotherapy or targeted therapy. PMID:27206449

  3. 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. PMID:22244834

  4. Highly immunoreactive antibodies against the rHup-F2 fragment (aa 63-161) of the iron-regulated HupB protein of Mycobacterium tuberculosis and its potential for the serodiagnosis of extrapulmonary and recurrent tuberculosis.

    PubMed

    Sritharan, N; Choudhury, M; Sivakolundu, S; Chaurasia, R; Chouhan, N; Rao, P P; Sritharan, M

    2015-01-01

    HupB is an iron-regulated protein in Mycobacterium tuberculosis that functions as a positive regulator of mycobactin biosynthesis. It is essential for the growth and survival of the pathogen inside macrophages. Previously, using the full-length rHupB of M. tuberculosis, we demonstrated high levels of anti-HupB antibodies in the serum of pulmonary tuberculosis (TB) and, interestingly, extrapulmonary TB patients with negligible levels in household contacts and healthy controls. Here, we used three antigenic fragments of HupB, namely the recombinant HupB-F1 (aa 1-71), HupB-F2 (aa 63-161) and HupB-F3 (aa 164-214), as antigens in enzyme-linked immunosorbent assay (ELISA) to screen serum from TB patients. HupB-F2 showed enhanced immunoreactivity with serum from patients with pulmonary TB (three groups consisting of new cases, defaulters and recurrent cases) and extrapulmonary TB, with negligible levels in normal healthy controls. The negative correlation of the anti-(HupB-F2) antibodies with serum iron was maximal, with a Pearson's correlation coefficient value of -0.415. The study, in addition to strengthening the diagnostic potential of HupB, reflected the superior performance of HupB-F2 as an antigen in screening pulmonary and extrapulmonary TB. PMID:25037869

  5. 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

  6. Enhanced detection of circulating melanoma cells using gold nanoparticles as photoacoustic contrasting agents

    NASA Astrophysics Data System (ADS)

    McCormack, Devin R.; Bhattacharyya, Kiran; Kannan, Raghuraman; Katti, Kattesh; Viator, John A.

    2010-02-01

    Nanotechnology and the various properties of gold nanoparticles (AuNPs) are quickly changing the field of cancer detection and treatment. Photoacoustic detection methods show an increase in sensitivity using gold nanoparticle antibody conjugation, which selectively targets melanoma cancer cells. Instead of targeting melanoma tumors, we tag single cells, analogous to circulating metastatic melanoma cells. Using an in vitro, stationary cell system and planar samples, we demonstrate an average of 24% improved optical detectability of melanoma cells tagged with AuNPs over unprocessed melanoma cells. Tagged cells showed a raised plateau of absorbance from 470nm to 550nm. Untagged cells showed a general decline in absorption as wavelength increased. The results of our study have the potential to not only better develop photoacoustic detection of melanoma, but also extend the viability and use of photoacoustics into detection of otherwise unpigmented cancers.

  7. 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

  8. Monoclonal antibody specific for a pigmentation associated antigen

    SciTech Connect

    Thomson, T.M.; Mattes, M.J.; Old, L.J.; Lloyd, K.O

    1989-01-17

    Monoclonal antibody TA99, which specifically binds to a pigmentation associated antigen present on melanoma cells is described. Additionally, the hybridoma cell line deposited with the ATCC under Accession Number HB 8704 from which the antibody is derived, as well as methods for using the antibody are described.

  9. Optimization of an antibreast carcinoma monoclonal antibody as a tumor imaging agent

    SciTech Connect

    Zalutsky, M.R.; Colcher, D.; Kaplan, W.D.; Schlom, J.; Kufe, D.

    1984-01-01

    The authors have previously reported that monoclonal antibody B6.2 and its fragments labeled with I-125 selectively localizes in human breast tumor (bt) xeongrafts in nude mice. Herein the authors compare I-125 B6.2 and its fragments with regard to (a) in vitro binding to bt extracts and (b) blood clearance. Antibody B6.2 and fragments were labeled using iodogen and then incubated with cell extracts of a human bt metastasis to the liver (Met.173), MCF-7 bt line, and normal human liver. Scatchard analysis of the data revealed that I-125-B6.2 and its fragments bound to both breast tumors with affinity constants of the order of 10/sup 9/M/sup -1/; no specific binding to normal liver was observed. The affinity constant for both divalent fragments was higher than that observed for the monovalent Fab' fragment. Serial sampling of blood from tumor bearing mice indicated that the blood clearance of F(ab')/sub 2/ was more rapid than IgG and that Fab' cleared considerably faster still. A comparison of the biodistribution at 0.1 and 5 ..mu..g protein per mouse suggests that with 1 gm tumors, lower doses do not necessarily result in better tumor-to-tissue ratios. When the blood clearance of I-125-B6.2 was compared to that of a non-specific IgG (MOPC), a much faster clearance of I-125 activity, significantly greater than that resultant from uptake in the tumor, was observed. Accelerated blood clearance may be due to selective catabolism of the specific antibody. When I-125 labeled B6.2 was injected into mice bearing breast and melanoma tumors, the thyroid uptake of I-125 activity was 2-3 times greater in the bt mice. The authors conclude that catabolism may be an important factor in determining the optimal radiolabel for immunoscintigraphy.

  10. 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

  11. Crystallization and preliminary X-ray diffraction analysis of the interleukin-3 alpha receptor bound to the Fab fragment of antibody CSL362

    PubMed Central

    Broughton, Sophie E.; Hercus, Timothy R.; Nero, Tracy L.; Dhagat, Urmi; Owczarek, Catherine M.; Hardy, Matthew P.; Fabri, Louis J.; Scotney, Pierre D.; Nash, Andrew D.; Wilson, Nicholas J.; Lopez, Angel F.; Parker, Michael W.

    2014-01-01

    Interleukin-3 (IL-3) is a member of the beta common family of cytokines that regulate multiple functions of myeloid cells. The IL-3 receptor-specific alpha subunit (IL3Rα) is overexpressed on stem cells/progenitor cells of patients with acute myeloid leukaemia, where elevated receptor expression correlates clinically with a reduced patient survival rate. The monoclonal antibody (MAb) CSL362 is a humanized MAb derived from the murine MAb 7G3, originally identified for its ability to specifically recognize the human IL-3 receptor and for blocking the signalling of IL-3 in myeloid and endothelial cells. In order to elucidate the molecular mechanism of CSL362 antagonism, a preliminary structure of human IL3Rα in complex with the MAb CSL362 has been determined. PMID:24598927

  12. Crystallization and preliminary X-ray diffraction analysis of the interleukin-3 alpha receptor bound to the Fab fragment of antibody CSL362.

    PubMed

    Broughton, Sophie E; Hercus, Timothy R; Nero, Tracy L; Dhagat, Urmi; Owczarek, Catherine M; Hardy, Matthew P; Fabri, Louis J; Scotney, Pierre D; Nash, Andrew D; Wilson, Nicholas J; Lopez, Angel F; Parker, Michael W

    2014-03-01

    Interleukin-3 (IL-3) is a member of the beta common family of cytokines that regulate multiple functions of myeloid cells. The IL-3 receptor-specific alpha subunit (IL3Rα) is overexpressed on stem cells/progenitor cells of patients with acute myeloid leukaemia, where elevated receptor expression correlates clinically with a reduced patient survival rate. The monoclonal antibody (MAb) CSL362 is a humanized MAb derived from the murine MAb 7G3, originally identified for its ability to specifically recognize the human IL-3 receptor and for blocking the signalling of IL-3 in myeloid and endothelial cells. In order to elucidate the molecular mechanism of CSL362 antagonism, a preliminary structure of human IL3Rα in complex with the MAb CSL362 has been determined. PMID:24598927

  13. Simultaneous blocking of IL-6 and IL-8 is sufficient to fully inhibit CAF-induced human melanoma cell invasiveness.

    PubMed

    Jobe, Njainday Pulo; Rösel, Daniel; Dvořánková, Barbora; Kodet, Ondřej; Lacina, Lukáš; Mateu, Rosana; Smetana, Karel; Brábek, Jan

    2016-08-01

    Tumour microenvironment plays a critical role in cell invasion and metastasis. To investigate the role of cancer-associated fibroblasts (CAFs) in melanoma cell invasiveness, we used 3D spheroid invasion assay. The effect of conditioned media from normal fibroblasts and CAFs cultivated alone or co-cultivated with melanoma cells on BLM or A2058 melanoma spheroid invasion was analysed. We found that conditioned media from CAFs and CAFs co-cultured with melanoma cells, especially, promote invasion and migration, without significant effect on melanoma cell proliferation. We further analysed the expression of pro-invasive cytokines IL-8 and IL-6 in media and found that melanoma cells are dominant producers of IL-8 and fibroblasts are dominant producers of IL-6 in 2D monocultures, while co-cultivation of CAFs with melanoma cells induces production/secretion of IL-6 and IL-8 into the media. The analyses of IL-6 levels in 3D cultures and human melanoma samples, however, revealed that at least in some cases IL-6 is also produced directly by melanoma cells. Analysis of the role of IL-6 and IL-8 in CAF-induced melanoma invasion, using neutralising antibodies, revealed that simultaneous blocking of IL-6 and IL-8 is sufficient to fully inhibit CAF-induced human melanoma cell invasiveness. In summary, these experiments indicate the important role of CAFs and IL-8 and IL-6 cytokines in melanoma cell invasiveness. PMID:27102177

  14. Epidemiology of malignant melanoma.

    PubMed

    Liu, T; Soong, S J

    1996-12-01

    Descriptive epidemiology of melanoma indicates increases in both incidence and mortality over the past two to three decades. A moderation in both rates began to emerge in several regions after the 1980s, especially in younger age groups. Recent improvement in survival rates is more likely due to earlier diagnosis than to real improvement in treatment. This suggests the potential effectiveness of secondary prevention. Continued health education efforts to improve awareness about signs and symptoms of melanoma should lead to earlier diagnosis and may increase incidence for a certain period of time. However, reduction in mortality will eventually be achieved owing to thinner melanoma at time of diagnosis. Etiologic studies indicate that the most important environmental risk factor for melanoma is extensive exposure to the sun. Primary prevention efforts should target public education about the risk of sun exposure and the benefit of wearing hats and adequate clothing. Specific prevention and control programs should be implemented among high-risk groups, such as those with light complexions and those sensitive to sunburn. In view of the long latency of melanoma, as much as 10 years, past exposure to the risk factors continues to cause melanoma, and any benefits of preventive efforts do not appear for some time. Although a dramatic decline is not expected in melanoma rates immediately, continuous preventive efforts ultimately should lead to a reduction in incidence and mortality. PMID:8977547

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

    PubMed

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

    2014-02-01

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

  16. Melanoma and genetics.

    PubMed

    Nelson, Andrew A; Tsao, Hensin

    2009-01-01

    As the incidence of malignant melanoma continues to increase and with the completion of the sequencing of the human genome, there have been increasing efforts to identify the "melanoma gene(s)." Although some patients and families have significantly increased risks due to genetic predisposition, most melanoma cases are sporadic and likely result from low to moderate risk genetic factors. This review focuses on the genes that cover the greatest risk of developing melanoma. It is important to remember that many--if not most--cases of melanoma are the result of undiscovered variants. The strongest genetic risk for the development of melanoma results from heritable alterations in cyclin-dependent kinase inhibitor 2A (CDKN2A) gene, which encodes two separate but related proteins, p16/INK4a and p14/ARF. These proteins help regulate cell division and apoptosis, both of which are necessary to maintain cellular homeostasis. Other important genes include CDK4/6 and retinoblastoma (RB1), which encode downstream proteins in the same pathway as p16/INK4a and p14/ARF. Finally, we discuss the relative importance of the melanocortin 1 receptor (MC1R) gene as a moderate risk factor for melanoma. Although great advances have been made in understanding the molecular basis and genetic predisposition of melanoma, many questions still remain to be answered. Someday soon, it will be possible to predict a patient's risk of melanoma by DNA analysis; however, it is important to reconcile our tremendous technologic capabilities with documented clinical utility. PMID:19095153

  17. 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

  18. 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

  19. Early intervention in the 3xTg-AD mice with an amyloid β-antibody fragment ameliorates first hallmarks of Alzheimer disease

    PubMed Central

    Giménez-Llort, Lydia; Rivera-Hernández, Geovanny; Marín-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. PMID:23884018

  20. Sunburn and malignant melanoma.

    PubMed Central

    Green, A.; Siskind, V.; Bain, C.; Alexander, J.

    1985-01-01

    We investigated the relationship between cutaneous malignant melanoma and multiple sunburns in the Queensland population. Interview data were gathered from 236 case-control pairs concerning their lifetime experience of severe sunburns, their occupational and recreational sun exposure, and their skin type. Excluding the lentigo maligna melanoma subtype, an association between multiple sunburns and melanoma was evident. After controlling for other major risk factors there was a significant dose-response relationship (P less than 0.05): the estimated relative risk associated with 2-5 sunburns in life was 1.5, and with 6 or more was 2.4. This observation extends the hitherto circumstantial evidence of a causal relationship between exposure to solar ultraviolet radiation and melanoma, and suggests that precautionary measures could prevent the development of this disease in a proportion of cases in fair-skinned populations. PMID:3970815

  1. 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

  2. Drugs Approved for Melanoma

    MedlinePlus

    ... are not listed here. Drugs Approved for Melanoma Aldesleukin Cobimetinib Cotellic (Cobimetinib) Dabrafenib Dacarbazine DTIC-Dome (Dacarbazine) IL-2 (Aldesleukin) Imlygic (Talimogene Laherparepvec) Interleukin-2 (Aldesleukin) Intron A ( ...

  3. 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

  4. Melanoma of the eye

    MedlinePlus

    ... modified July 9, 2015. www.cancer.gov/types/eye/hp/intraocular-melanoma-treatment-pdq . Accessed October 7, 2015. Read ... by: Yi-Bin Chen, MD, Leukemia/Bone Marrow Transplant Program, Massachusetts General Hospital, Boston, MA. Also reviewed ...

  5. 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

  6. 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

  7. Thrombospondin 1 promotes an aggressive phenotype through epithelial-to-mesenchymal transition in human melanoma

    PubMed Central

    Jayachandran, Aparna; Anaka, Matthew; Prithviraj, Prashanth; Hudson, Christopher; McKeown, Sonja J; Lo, Pu-Han; Vella, Laura J; Goding, Colin R; Cebon, Jonathan; Behren, Andreas

    2014-01-01

    Epithelial-to-mesenchymal transition (EMT), in which epithelial cells loose their polarity and become motile mesenchymal cells, is a determinant of melanoma metastasis. We compared gene expression signatures of mesenchymal-like melanoma cells with those of epithelial-like melanoma cells, and identified Thrombospondin 1 (THBS1) as highly up-regulated in the mesenchymal phenotype. This study investigated whether THBS1, a major physiological activator of transforming growth factor (TGF)-beta, is involved in melanoma EMT-like process. We sought to examine expression patterns in distinct melanoma phenotypes including invasive, de-differentiated, label-retaining and drug resistant populations that are putatively associated with an EMT-like process. Here we show that THBS1 expression and secretion was elevated in melanoma cells exhibiting invasive, drug resistant, label retaining and mesenchymal phenotypes and correlated with reduced expression of genes involved in pigmentation. Elevated THBS1 levels were detected in Vemurafenib resistant melanoma cells and inhibition of THBS1 led to significantly reduced chemoresistance in melanoma cells. Notably, siRNA-mediated silencing of THBS1 and neutralizing antibody to THBS1 reduced invasion in mesenchymal-like melanoma cells, while ectopic THBS1 expression in epithelial-like melanoma cells enhanced invasion. Furthermore, the loss of THBS1 inhibited in vivo motility of melanoma cells within the embryonic chicken neural tube. In addition, we found aberrant THBS1 protein expression in metastatic melanoma tumor biopsies. These results implicate a role for THBS1 in EMT, and hence THBS1 may serve as a novel target for strategies aimed at the treatment of melanoma invasion and drug resistance. PMID:25051363

  8. Genetics of melanoma predisposition.

    PubMed

    Lin, J; Hocker, T L; Singh, M; Tsao, H

    2008-08-01

    Over the past 10 years, our understanding of melanoma at the molecular level has blossomed with the advent of genomic technologies. The enormous enthusiasm for the Human Genome Project is slowly being replaced by an even greater excitement for the unravelling of disease genomes, including melanoma. In this review, we will consider some of the clinical implications of these genetic findings for both diagnostics and therapeutics. PMID:18547303

  9. Genetics of melanoma

    PubMed Central

    Wangari-Talbot, Janet; Chen, Suzie

    2013-01-01

    Genomic variation is a trend observed in various human diseases including cancer. Genetic studies have set out to understand how and why these variations result in cancer, why some populations are pre-disposed to the disease, and also how genetics affect drug responses. The melanoma incidence has been increasing at an alarming rate worldwide. The burden posed by melanoma has made it a necessity to understand the fundamental signaling pathways involved in this deadly disease. Signaling cascades such as mitogen-activated protein kinase and PI3K/AKT have been shown to be crucial in the regulation of processes that are commonly dysregulated during cancer development such as aberrant proliferation, loss of cell cycle control, impaired apoptosis, and altered drug metabolism. Understanding how these and other oncogenic pathways are regulated has been integral in our challenge to develop potent anti-melanoma drugs. With advances in technology and especially in next generation sequencing, we have been able to explore melanoma genomes and exomes leading to the identification of previously unknown genes with functions in melanomagenesis such as GRIN2A and PREX2. The therapeutic potential of these novel candidate genes is actively being pursued with some presenting as druggable targets while others serve as indicators of therapeutic responses. In addition, the analysis of the mutational signatures of melanoma tumors continues to cement the causative role of UV exposure in melanoma pathogenesis. It has become distinctly clear that melanomas from sun-exposed skin areas have distinct mutational signatures including C to T transitions indicative of UV-induced damage. It is thus necessary to continue spreading awareness on how to decrease the risk factors of developing the disease while at the same time working for a cure. Given the large amount of information gained from these sequencing studies, it is likely that in the future, treatment of melanoma will follow a highly

  10. 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

  11. High frequencies of circulating melanoma-reactive CD8+ T cells in patients with advanced melanoma.

    PubMed

    Letsch, A; Keilholz, U; Schadendorf, D; Nagorsen, D; Schmittel, A; Thiel, E; Scheibenbogen, C

    2000-09-01

    To determine whether circulating tumor-reactive T cells are present in melanoma patients, unstimulated T cells from peripheral blood were tested for recognition of HLA-A2- or HLA-A1-matched melanoma cell lines using the ELISPOT assay. Eleven out of 19 patients with metastatic melanoma had a T-cell response with up to 0.81%, 0.78%, 0. 53%, 0.12%, 0.10%, 0.09%, 0.07%, 0.06%, 0.06%, 0.04%, and 0.04% of peripheral blood mononuclear cells (PBMC) secreting IFNgamma upon exposure to various HLA-A2- or HLA-A1-matched melanoma cell lines. These T-cell responses were mediated by CD8+ T cells and could specifically be blocked by an anti-HLA-A2 antibody in HLA-A2-positive patients. Separation experiments performed in one melanoma patient showed tumor-reactive T cells in both the CD8+ effector T cell (CD45RA+/IFNgamma+) as well as the CD8+ memory T-cell compartment (CD45RO+/IFNgamma+). In 3 out of 5 patients, in whom autologous cell lines were available, similar frequencies of T cells in response to HLA-A1- or HLA-A2-matched allogeneic and autologous tumor cells were observed, while 2 patients had a T-cell response restricted to either the autologous or the allogeneic cell lines. These results give evidence for the presence of tumor-reactive CD8+ T cells in more than half of melanoma patients tested. Although some of these patients have clinical evidence for an immunological-mediated tumor control, several patients have growing tumors suggesting presence of escape mechanisms. PMID:10925359

  12. Targeted therapy in melanoma.

    PubMed

    Kudchadkar, Ragini R; Smalley, Keiran S M; Glass, L Frank; Trimble, James S; Sondak, Vernon K

    2013-01-01

    Since the discovery of activating mutations in the BRAF oncogene in melanoma, there has been remarkable progress in the development of targeted therapies for unresectable and metastatic melanoma. We review the latest developments in our understanding of the role of BRAF/MEK/ERK pathway signaling in melanoma, and the development of inhibitors of this pathway. We also explore alternative mutations seen in melanoma, such as NRAS, KIT, GNAQ, and GNA11, and the drug development that is ongoing based on this biology. Strategies for the management of the vexing clinical problem of BRAF inhibitor resistance, primarily via combination therapy, are outlined. With the recent approval of the BRAF inhibitor vemurafenib for stage IV metastatic melanoma, use of this agent is expanding in the United States. Thus, management of the skin toxicities of this agent, such as squamous cell carcinomas, "acneiform" eruptions, hand-foot syndrome, and panniculitis, will be a growing problem facing dermatologists today. We discuss the toxicities of targeted agents in use for melanoma, in particular the dermatologic effects and the management of these skin toxicities. PMID:23438383

  13. 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

  14. 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

  15. 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

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

    PubMed

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

    2010-08-31

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

  17. 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

  18. Altered Morphology and Immunohistochemical Characteristics in Metastatic Malignant Melanoma After Therapy With Vemurafenib.

    PubMed

    Powell, Matthew R; Sheehan, Daniel J; Kleven, Daniel T

    2016-09-01

    Metastatic melanoma is traditionally diagnosed using classic morphologic features in addition to immunohistochemical studies. The authors report a case of metastatic malignant melanoma where both morphology and immunohistochemistry were altered after treatment. This 51-year-old patient presented with metastatic melanoma to the brain and axilla. Initially, both metastases showed classic morphology and diffuse staining with the pan-melanoma antibody cocktail. This cocktail is a combination of 3 antibodies commonly used to diagnose melanocytic neoplasms: Melan-A (MART-1), tyrosinase, and HMB-45. In combination, the cocktail is highly sensitive for detecting melanocytic neoplasms and is commonly used to diagnose metastatic melanoma. Her tumor was positive for the BRAF 1799T>A (V600E) mutation, and she was treated with BRAF inhibitor therapy (vemurafenib). However, the axillary tumor recurred after treatment with vemurafenib. The recurrent tumor showed a markedly different morphology and complete loss of staining with the pan-melanoma antibody cocktail. This loss of staining accompanied by the change in morphology was an observation not previously documented after therapy with vemurafenib. This case demonstrates a potential pitfall in the diagnosis of metastatic or recurrent malignant melanoma. PMID:27541173

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

    PubMed Central

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

    2015-01-01

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

  20. Monoclonal antibody OKB7, which identifies the 14OKd complement receptor type 2 (CR/sub 2/), also identifies a 72Kd secreted fragment of CR/sub 2/ that contains the C3d-binding site

    SciTech Connect

    Myones, B.L.; Ross, G.D.

    1986-03-05

    CR/sub 2/ is a 140-145Kd glycoprotein expressed on B lymphocytes which binds both C3d and Epstein-Barr virus (EBV). OKB7, an IgG/sub 2a/ monoclonal antibody to CR/sub 2/, blocks C3d and EBV binding, while HB-5, another monoclonal IgG/sub 2a/ anti-CR/sub 2/, does not. A 72Kd C3d-binding glycoprotein (gp72), isolated from Raji cell media, was previously thought to be CR/sub 2/ because a polyclonal rabbit anti-gp72 inhibited EC3d rosettes. ELISA assay demonstrated that OKB7, but not HB-5, bound to purified gp72 fixed to microtiter wells. Insoluble and soluble gp72 blocked Raji cell uptake of /sup 125/I-labeled OKB7, but not labeled anti-B2 or HB-5. Rabbit anti-gp72 immunoprecipitated bands at 140Kd and 72Kd from /sup 125/I-labelled and solubilized B cell membranes. Culture media from Raji cells grown in the presence /sup 3/H-labeled amino acids was sequentially immunoprecipitated by irrelevant antibody, OKB7, and HB-5. A single 72Kd radiolabeled band was demonstrated only with OKB7, and this was identical to that produced by the immunoprecipitation of /sup 125/I-labeled gp72 with rabbit anti-gp72. Thus, OKB7, which identifies the 140Kd CR/sub 2/ molecule, also identifies a 72Kd shed fragment of CR/sub 2/ isolated from Raji cell media, which contains the C3d-binding site.

  1. Targeting the replisome with transduced monoclonal antibodies triggers lethal DNA replication stress in cancer cells.

    PubMed

    Desplancq, Dominique; Freund, Guillaume; Conic, Sascha; Sibler, Annie-Paule; Didier, Pascal; Stoessel, Audrey; Oulad-Abdelghani, Mustapha; Vigneron, Marc; Wagner, Jérôme; Mély, Yves; Chatton, Bruno; Tora, Laszlo; Weiss, Etienne

    2016-03-15

    Although chemical inhibition of the DNA damage response (DDR) in cancer cells triggers cell death, it is not clear if the fork blockade achieved with inhibitors that neutralise proteins of the replisome is sufficient on its own to overcome the DDR. Monoclonal antibodies to PCNA, which block the DNA elongation process in vitro, have been developed. When these antibodies were transduced into cancer cells, they are able to inhibit the incorporation of nucleoside analogues. When co-delivered with anti-PCNA siRNA, the cells were flattened and the size of their nuclei increased by up to 3-fold, prior to cell death. Analysis of these nuclei by super-resolution microscopy revealed the presence of large numbers of phosphorylated histone H2AX foci. A senescence-like phenotype of the transduced cells was also observed upon delivery of the corresponding Fab molecules or following PCNA gene disruption or when the Fab fragment of an antibody that neutralises DNA polymerase alpha was used. Primary melanoma cells and leukaemia cells that are resistant to chemical inhibitors were similarly affected by these antibody treatments. These results demonstrate that transduced antibodies can trigger a lethal DNA replication stress, which kills cancer cells by abolishing the biological activity of several constituents of the replisome. PMID:26968636

  2. A Mathematical Model of Immune-System-Melanoma Competition

    PubMed Central

    Pennisi, Marzio

    2012-01-01

    We present a mathematical model developed to reproduce the immune response entitled with the combined administration of activated OT1 cytotoxic T lymphocytes (CTLs) and Anti-CD137 monoclonal antibodies. The treatment is directed against melanoma in B16 OVA mouse models exposed to a specific immunotherapy strategy. We model two compartments: the injection point compartment where the treatment is administered and the skin compartment where melanoma tumor cells proliferate. To model the migration of OT1 CTLs and antibodies from the injection to the skin compartment, we use delay differential equations (DDEs). The outcomes of the mathematical model are in good agreement with the in vivo results. Moreover, sensitivity analysis of the mathematical model underlines the key role of OT1 CTLs and suggests that a possible reduction of the number of injected antibodies should not affect substantially the treatment efficacy. PMID:22701144

  3. Melanoma Development and Progression Are Associated with Rad6 Upregulation and β-Catenin Relocation to the Cell Membrane

    PubMed Central

    Mehregan, Darius R.; Abrams, Judith; Haynes, Brittany; Shekhar, Malathy P. V.

    2014-01-01

    We have previously demonstrated that Rad6 and β-catenin enhance each other's expression through a positive feedback loop to promote breast cancer development/progression. While β-catenin has been implicated in melanoma pathogenesis, Rad6 function has not been investigated. Here, we examined the relationship between Rad6 and β-catenin in melanoma development and progression. Eighty-eight cutaneous tumors, 30 nevi, 29 primary melanoma, and 29 metastatic melanomas, were immunostained with anti-β-catenin and anti-Rad6 antibodies. Strong expression of Rad6 was observed in only 27% of nevi as compared to 100% of primary and 96% of metastatic melanomas. β-Catenin was strongly expressed in 97% of primary and 93% of metastatic melanomas, and unlike Rad6, in 93% of nevi. None of the tumors expressed nuclear β-catenin. β-Catenin was exclusively localized on the cell membrane of 55% of primary, 62% of metastatic melanomas, and only 10% of nevi. Cytoplasmic β-catenin was detected in 90% of nevi, 17% of primary, and 8% of metastatic melanoma, whereas 28% of primary and 30% of metastatic melanomas exhibited β-catenin at both locations. These data suggest that melanoma development and progression are associated with Rad6 upregulation and membranous redistribution of β-catenin and that β-catenin and Rad6 play independent roles in melanoma development. PMID:24891954

  4. [Histological spectrum of malignant melanoma].

    PubMed

    Brenn, T

    2015-02-01

    The diagnosis of melanocytic tumors is one of the most problematic areas in dermatology and diagnostic pathology. Melanoma is a malignant melanocytic tumor and the risk for metastasis and associated mortality is mainly dependent on tumor thickness and depth of invasion. Early recognition and correct diagnosis is therefore important for successful and effective treatment. The correct diagnosis of melanoma is, however, challenging due to the wide morphological spectrum. Historically, the disease was subdivided into superficial spreading, nodular, lentigo maligna and acral lentiginous melanoma but many more subtypes have subsequently been added. Some of these melanoma variants also show differences relating to the genetic background, clinical presentation, prognosis and treatment and may be associated with a specific differential diagnosis. In this article four of these melanoma variants, desmoplastic melanoma, nevoid melanoma, malignant blue nevus and pigment synthesizing melanoma will be discussed in more detail. PMID:25589353

  5. 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 ...

  6. 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

  7. Recombinant DNA technology for melanoma immunotherapy: anti-Id DNA vaccines targeting high molecular weight melanoma-associated antigen.

    PubMed

    Barucca, A; Capitani, M; Cesca, M; Tomassoni, D; Kazmi, U; Concetti, F; Vincenzetti, L; Concetti, A; Venanzi, F M

    2014-11-01

    Anti-idiotypic MK2-23 monoclonal antibody (anti-Id MK2-23 mAb), which mimics the high molecular weight melanoma-associated antigen (HMW-MAA), has been used to implement active immunotherapy against melanoma. However, due to safety and standardization issues, this approach never entered extensive clinical trials. In the present study, we investigated the usage of DNA vaccines as an alternative to MK2-23 mAb immunization. MK2-23 DNA plasmids coding for single chain (scFv) MK2-23 antibody were constructed via the insertion of variable heavy (V H) and light (V L) chains of MK2-23 into the pVAC-1mcs plasmids. Two alternative MK2-23 plasmids format V H/V L, and V L/V H were assembled. We demonstrate that both polypeptides expressed by scFv plasmids in vitro retained the ability to mimic HMW-MAA antigen, and to elicit specific anti-HMW-MAA humoral and cellular immunoresponses in immunized mice. Notably, MK2-23 scFv DNA vaccines impaired the onset and growth of transplantable B16 melanoma cells not engineered to express HMW-MAA. This pilot study suggests that optimized MK2-23 scFv DNA vaccines could potentially provide a safer and cost-effective alternative to anti-Id antibody immunization, for melanoma immunotherapy. PMID:25027754

  8. Immunity to the melanoma inhibitor of apoptosis protein (ML-IAP; livin) in patients with malignant melanoma.

    PubMed

    Zhou, Jun; Yuen, Noah K; Zhan, Qian; Velazquez, Elsa F; Murphy, George F; Giobbie-Hurder, Anita; Hodi, F Stephen

    2012-05-01

    Therapeutic targeting of melanoma antigens frequently focuses on the melanocyte differentiation or cancer-testis families. Antigen-loss variants can often result, as these antigens are not critical for tumor cell survival. Exploration of functionally relevant targets has been limited. The melanoma inhibitor of apoptosis protein (ML-IAP; livin) is overexpressed in melanoma, contributing to disease progression and treatment resistance. Improved understanding of the significance of ML-IAP immune responses in patients has possible therapeutic applications. We found ML-IAP frequently expressed in melanoma metastases by immunohistochemistry. To assess spontaneous immunity to ML-IAP, an overlapping peptide library representing full-length protein was utilized to screen cellular responses in stage I-IV patients and healthy controls by ELISPOT. A broad array of CD4(+) and CD8(+) cellular responses against ML-IAP was observed with novel class I and class II epitopes identified. Specific HLA-A*0201 epitopes were analyzed further for frequency of reactivity. The generation of specific CD4(+) and cytotoxic T cells revealed potent functional capability including cytokine responsiveness to melanoma cell lines and tumor cell killing. In addition, recombinant ML-IAP protein used in an ELISA demonstrated high titer antibody responses in a subset of patients. Several melanoma patients who received CTLA-4 blockade with ipilimumab developed augmented humoral immune responses to ML-IAP as a function of treatment which was associated with beneficial clinical outcomes. High frequency immune responses in melanoma patients, associations with favorable treatment outcomes, and its essential role in melanoma pathogenesis support the development of ML-IAP as a disease marker and therapeutic target. PMID:22033581

  9. Boronated monoclonal antibody conjugates for neutron capture therapy

    SciTech Connect

    Borg, D.C.; Elmore, J.J. Jr.; Ferrone, S.

    1986-01-01

    This paper describes the effectiveness of /sup 10/B-labeled monoclonal antibodies against Colo-38 human melanoma in vitro. The authors obtained high boron to antibody ratios while maintaining antibody activity by using dextran intermediate carriers to link /sup 10/B to the antibody. They developed a double cell quasi-competitive binding bioassay to minimize the effects of nonspecific binding of boronated complexes to cells. 1 fig., 2 tabs.

  10. Activation of CCR9/CCL25 in Cutaneous Melanoma Mediates Preferential Metastasis to the Small Intestine

    PubMed Central

    Amersi, Farin F.; Terando, Alicia M.; Goto, Yasufumi; Scolyer, Richard A.; Thompson, John F.; Tran, Andy N.; Faries, Mark B.; Morton, Donald L.; Hoon, Dave S.B.

    2009-01-01

    Purpose Specific chemokines and their respective receptors have been implicated in distant tumor cell metastasis. Cutaneous melanoma has a distinct pattern of metastasis, preferentially targeting the submucosa of the small intestine. However, the underlying pathogenic mechanism remains unknown. Migration of CCR9(+) lymphocytes to the small intestine is known to occur in response to the chemoattractant effects of CCL25 (thymus-expressed chemokine). The integrin heterodimers αβ are also known to be important mediators of cellular adhesion. We hypothesize that the mechanism of small intestinal metastasis by melanoma is via the CCR9-CCL25 axis and specific integrins. Experimental Design Quantitative reverse transcription-PCR, flow cytometry, and immunohistochemistry were used to assess melanoma tumors for CCR9 and CCL25. Integrin expression was assessed using flow cytometry. CCR9 expression by quantitative reverse transcription-PCR was assessed in primary (n = 23) and metastatic (n = 198) melanomas, and melanoma lines derived from small intestinal metastases (n = 23). Results We showed CCR9 expression in 88 of 102 paraffin-embedded metastatic melanomas from the small intestine, 8 of 8 melanoma lines derived from metastases in the small intestine, and 0 of 96 metastatic melanomas from other sites. In vitro migration and invasion studies done on CCR9(+) melanoma lines showed migration in response to CCL25 that was inhibited by anti-CCR9 antibody or by short interfering RNA CCR9. Flow cytometric analysis confirmed CCR9 expression by melanomas to the small intestine and showed concomitant α4β1 integrin expression. Conclusions Our findings show that functionally active CCR9 on melanoma cells facilitates metastasis to the small intestine. The CCR9-CCL25 axis may explain the high incidence of melanoma metastasis to this specific location. PMID:18245522

  11. Genotyping of cutaneous melanoma

    PubMed Central

    Glitza, Isabella C.; Davies, Michael A.

    2014-01-01

    Until recently, treatment options for patients with metastatic melanoma were very limited. This landscape has evolved dramatically since the discovery of activating mutations in the BRAF gene in ~45% of cutaneous melanomas. Vemurafenib, dabrafenib, and trametinib have all received regulatory approval for the treatment of metastatic melanoma patients with a BRAFV600 mutation. Based on the necessity to document the presence of a BRAFV600 mutation to prescribe these agents, molecular testing is now the standard of care in this disease. However, the options and rationale for testing are evolving rapidly due to an improved understanding of the molecular drivers and heterogeneity of melanoma. Such testing may identify rational combinatorial approaches to prevent or overcome resistance for the approved BRAF inhibitors. In addition, new clinical strategies have been identified for a number of other molecular changes that are detected in this disease, including somatic changes in NRAS, PTEN, CDKN2A, and c-KIT, among others. This review summarizes the current understanding of the genetic landscape of mutations in melanoma, their associations with clinicopathological features, and their implications for clinical testing and treatment. PMID:25632386

  12. 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

  13. 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

  14. 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

  15. Effects of BRAF mutations and BRAF inhibition on immune responses to melanoma

    PubMed Central

    Ilieva, Kristina M.; Correa, Isabel; Josephs, Debra H.; Karagiannis, Panagiotis; Egbuniwe, Isioma U.; Cafferkey, Michiala J.; Spicer, James F.; Harries, Mark; Nestle, Frank O.; Lacy, Katie E.; Karagiannis, Sophia N.

    2014-01-01

    Malignant melanoma is associated with poor clinical prognosis; however, novel molecular and immune therapies are now improving patient outcomes. Almost 50% of melanomas harbor targetable activating mutations of BRAF which promote RAS-RAF-MEK-ERK pathway activation and melanoma proliferation. Recent evidence also indicates that melanomas bearing mutant BRAF may also have altered immune responses, suggesting additional avenues for treatment of this patient group. The small molecule inhibitors selective for mutant BRAF induce significant but short-lived clinical responses in a proportion of patients, but also lead to immune stimulatory bystander events, which then subside with the emergence of resistance to inhibition. Simultaneous BRAF and MEK inhibition, and especially combination of BRAF inhibitors with new immunotherapies such as checkpoint blockade antibodies, may further enhance immune activation, or counteract immunosuppressive signals. Pre-clinical evaluation and ongoing clinical trials should provide novel insights into the role of immunity in the therapy of BRAF-mutant melanoma. PMID:25385327

  16. 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

  17. 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. PMID:27188674

  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. Small Molecules Antagonise the MIA-Fibronectin Interaction in Malignant Melanoma.

    PubMed

    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

  20. Melanoma in Black Patients

    PubMed Central

    Smith, Robert J.

    1982-01-01

    Melanoma in black patients is uncommon but not rare. This paper reports six cases seen in one general surgeon's practice in Arkansas during a 14-year period. A review of the current literature regarding melanoma in blacks is given. Characteristically, melanoma in blacks is found on the soles of the feet, palms of the hands, or mucous membranes. The tumor has a deadly potential unless it is treated at an early stage; four of the patients reported have died of metastatic disease. A public health program is needed to make physicians and the public aware of the incidence and location of this tumor. ImagesFigure 1Figure 2Figure 3Figure 4 PMID:7120473

  1. [External ear melanoma].

    PubMed

    Amando García, L; Suárez Nieto, C; Madrigal Rubiales, B; García García, J

    2003-02-01

    Cutaneous melanomas are the tumours that have increased more their incidence in the last fifty years. Melanomas arising from the external auditory canal are extraordinariously unfrequent. These tumours show an aggressive and silent behaviour, and due to this the diagnosis is frequently made in an advanced stage. A male with a malignant melanoma arising from his left external auditory canal was attended in our department, suspecting an epidermoid carcinoma. The clinical findings and the extension of the lesion required a lateral temporal bone resection, parotidectomy and neck dissection to achieve a total resection. We present a review of the literature about this entity and an analysis of the incidence, significance of the lymph node metastases and value of the elective neck dissection. PMID:12802982

  2. 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.

  3. 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

  4. Melanoma of the Foot.

    PubMed

    Bristow, Ivan; Bower, Chris

    2016-07-01

    Melanoma is a rare form of skin cancer that is responsible for most skin cancer deaths globally. Tumors arising on the foot continue to be a particular challenge. Patients present later and lesions are frequently misdiagnosed, leading to more advanced disease with an overall poorer prognosis then melanoma elsewhere. In order to improve early recognition, this article reviews the clinical features of the disease along with published algorithms. Emerging assessment techniques such as dermoscopy are also discussed as tools to improve clinical decision making. Contemporary drug therapies in the treatment of advanced disease are also discussed. PMID:27215160

  5. Pathology of melanoma.

    PubMed

    Elder, David E

    2015-04-01

    The pathology of melanoma is discussed in relation to surgical diagnosis and management. Pitfalls that may result in problems of clinicopathologic communication are emphasized. A compelling vision for the future is that all tumors will be characterized by their driving oncogenes, suppressor genes, and other genomic factors. The success of targeted therapy directed against individual oncogenes, despite its limitations, suggests that a repertoire of targeted agents will be developed that can be used against a variety of different tumors, depending on the results of genetic testing. Nevertheless, histopathologic diagnosis and surgical therapy will remain mainstays of management for melanoma. PMID:25769708

  6. Angiogenesis and Melanoma

    PubMed Central

    Ribatti, Domenico; Annese, Tiziana; Longo, Vito

    2010-01-01

    Angiogenesis occurs in pathological conditions, such as tumors, where a specific critical point in tumor progression is the transition from the avascular to the vascular phase. Tumor angiogenesis depends mainly on the release by neoplastic cells of growth factors specific for endothelial cells, which are able to stimulate the growth of the host’s blood vessels. This article summarizes the literature concerning the relationship between angiogenesis and human melanoma progression. The recent applications of antiangiogenic agents which interfere with melanoma progression are also described. PMID:24281035

  7. 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.

  8. Magma Fragmentation

    NASA Astrophysics Data System (ADS)

    Gonnermann, Helge M.

    2015-05-01

    Magma fragmentation is the breakup of a continuous volume of molten rock into discrete pieces, called pyroclasts. Because magma contains bubbles of compressible magmatic volatiles, decompression of low-viscosity magma leads to rapid expansion. The magma is torn into fragments, as it is stretched into hydrodynamically unstable sheets and filaments. If the magma is highly viscous, resistance to bubble growth will instead lead to excess gas pressure and the magma will deform viscoelastically by fracturing like a glassy solid, resulting in the formation of a violently expanding gas-pyroclast mixture. In either case, fragmentation represents the conversion of potential energy into the surface energy of the newly created fragments and the kinetic energy of the expanding gas-pyroclast mixture. If magma comes into contact with external water, the conversion of thermal energy will vaporize water and quench magma at the melt-water interface, thus creating dynamic stresses that cause fragmentation and the release of kinetic energy. Lastly, shear deformation of highly viscous magma may cause brittle fractures and release seismic energy.

  9. Targeting NEU Protein in Melanoma Cells with Non-Thermal Atmospheric Pressure Plasma and Gold Nanoparticles.

    PubMed

    Choi, Byul Bora; Kim, Myung Soo; Kim, Uk Kyu; Hong, Jin Woo; Lee, Hae June; Kim, Gyoo Cheon

    2015-05-01

    Non-thermal atmospheric pressure plasma effectively kills cancer cells, but it cannot selectively kill cancer cells. The authors targeted NEU (human epidermal growth factor receptor 2) protein, which is frequently over-expressed in the cell membrane of melanoma cells, using anti-NEU antibody-labeled gold nanoparticles. The labeled nanoparticles preferentially targeted melanoma cells rather than normal keratinocytes. After the addition of labeled gold nanoparticles to melanoma and normal keratinocyte cells, both cells were exposed to non-thermal atmospheric pressure plasma. The death rate of melanoma cells was significantly higher than that of normal keratinocyte cells; many vacuoles, indicative of cell death, were observed in melanoma cells treated with anti-NEU antibody labeled gold nanoparticles and plasma. This selective cancer cell death was attributed to the selective destruction of NEU protein and a downstream effector of NEU. Our study findings show that treatment with a combination of non-thermal atmospheric pressure plasma and anti-NEU antibody-labeled gold nanoparticles effectively and selectively kills melanoma cells. PMID:26349401

  10. Targeting antibodies to the cytoplasm

    PubMed Central

    Marschall, Andrea L J; Frenzel, André; Schirrmann, Thomas; Schüngel, Manuela

    2011-01-01

    A growing number of research consortia are now focused on generating antibodies and recombinant antibody fragments that target the human proteome. A particularly valuable application for these binding molecules would be their use inside a living cell, e.g., for imaging or functional intervention. Animal-derived antibodies must be brought into the cell through the membrane, whereas the availability of the antibody genes from phage display systems allows intracellular expression. Here, the various technologies to target intracellular proteins with antibodies are reviewed. PMID:21099369

  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. Radiotherapy of malignant melanoma

    SciTech Connect

    Cooper, J.S.

    1985-04-01

    The role of radiotherapy in the treatment of malignant melanoma is limited, and surgery generally forms the mainstay of medical practice. However, there are some circumstances in which radiotherapy should be considered the treatment of choice. Symptomatic metastatic lesions in bone or brain can effectively be palliated in a substantial proportion of instances. At the current stage of our knowledge, conventionally fractionated treatment of such lesions forms the standard against which other treatments should be measured. In contrast, metastatic lesions to skin or lymph nodes that do not overlie critical normal structures probably are better treated by high-dose-per-fraction techniques. Radiotherapy may play a definitive role in the treatment of lentigo maligna. The precise optimal energy of the beam to be used remains to be defined. Slightly more penetrating radiation appears to be required for lentigo maligna melanomas. Here, too, the optimal energy remains to be defined. The treatment of nonlentigenous melanomas primarily by radiotherapy is unproved in my opinion. Certainly, the data from the Princess Margaret Hospital is exciting, but I believe it must be corroborated by a well-designed trial before it can be accepted without question. Future directions in treatment of malignant melanoma are likely to include further trials of unconventional fractionation and the use of radiosensitizing agents in conjunction with radiotherapy. The time for dermatologists and radiation therapists to cooperate in such studies is at hand.

  14. Melanoma Risk Prediction Models

    Cancer.gov

    Developing statistical models that estimate the probability of developing melanoma cancer over a defined period of time will help clinicians identify individuals at higher risk of specific cancers, allowing for earlier or more frequent screening and counseling of behavioral changes to decrease risk.

  15. 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…

  16. Immunotherapy of melanoma: present options and future promises.

    PubMed

    Rotte, Anand; Bhandaru, Madhuri; Zhou, Youwen; McElwee, Kevin J

    2015-03-01

    Metastatic melanoma is notorious for its immune evasion and resistance to conventional chemotherapy. The recent success of ipilimumab, a human monoclonal antibody against cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), in increasing the median survival time and stabilizing the disease progression renewed, hopes in treatment for melanoma. Currently, ipilimumab and high-dose interleukin-2 (IL-2; Aldesleukin) are approved as monotherapies for the treatment of patients with unresectable advanced melanoma, and pegylated interferon-α2b (p-IFN-α2b) is approved as an adjuvant for the treatment of patients with surgically resected high-risk melanoma. The present review describes the currently approved immune-modulators and the promising immune-based interventions that are currently in clinical trials. We present the four commonly used strategies to boost immune responses against the tumors; monoclonal antibodies, cytokines, cancer vaccines, and adoptive T cell transfer. The corresponding lists of ongoing clinical trials include details of the trial phase, target patients, intervention details, status of the study, and expected date of completion. Further, our review discusses the challenges faced by immunotherapy and the various strategies adopted to overcome them. PMID:25589384

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

    PubMed

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

    2016-02-01

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

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

    PubMed

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

    2011-03-01

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

  19. Topical skin treatment with Fab fragments of an allergen-specific IgG1 monoclonal antibody suppresses allergen-induced atopic dermatitis-like skin lesions in mice.

    PubMed

    Sae-Wong, Chutha; Mizutani, Nobuaki; Kangsanant, Sureeporn; Yoshino, Shin

    2016-05-15

    Fab fragments (Fabs), which lack effector functions due to the absence of the Fc portion, maintain the ability to bind to specific allergens. In the present study, we examined whether Fabs of an allergen-specific IgG1 monoclonal antibody (mAb) were able to regulate allergen-induced atopic dermatitis-like skin lesions in mice.