Human antibodies and hybridomas最新文献

{"title":"A humanized antibody with specificity for hepatitis B surface antigen.","authors":"C. Ryu, E. Padlan, B. R. Jin, O. Yoo, H. Hong","doi":"10.3233/HAB-1996-7304","DOIUrl":"https://doi.org/10.3233/HAB-1996-7304","url":null,"abstract":"A murine monoclonal antibody H67 was characterized for the binding specificity, which showed that H67 recognizes a disulfide-bond-dependent conformational epitope of common a antigenic determinant on the hepatitis B surface antigen. The result suggested that this antibody may have the potential of replacing hepatitis B immune globulin in the prevention of hepatitis B virus (HBV) infection. Therefore, we have constructed the humanized antibody HuS10 by grafting the complementarity determining regions and some framework amino acid residues of H67 onto the most homologous human antibody variable regions, 21/28 for heavy chain variable region and B1 and J kappa 2 for light chain variable region, followed by combining with human constant regions C gamma 1 and C kappa. The affinity of the HuS10 was the same as that of the H67, 8 x 10(8) x 10(8)M-1, and the HuS10 neutralized the in vitro infection of adult human hepatocyte primary culture by adr or ayw subtype of HBV. The neutralization assay showed that the HuS10 had approximately 2,000-times higher specific activity than commercially available polyclonal HBIG. These results suggest that the humanized antibody will be useful in the prevention or treatment of HBV infection.","PeriodicalId":77166,"journal":{"name":"Human antibodies and hybridomas","volume":"7 3 1","pages":"113-22"},"PeriodicalIF":0.0,"publicationDate":"1996-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3233/HAB-1996-7304","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"69906693","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Humanization of an anti-human IL-6 mouse monoclonal antibody glycosylated in its heavy chain variable region.","authors":"K. Sato, T. Ohtomo, Y. Hirata, H. Saito, T. Matsuura, T. Akimoto, K. Akamatsu, Y. Koishihara, Y. Ohsugi, M. Tsuchiya","doi":"10.3233/HAB-1996-7405","DOIUrl":"https://doi.org/10.3233/HAB-1996-7405","url":null,"abstract":"Interleukin-6 (IL-6) inhibitors are good potential therapeutic agents in human patients, and anti-IL-6 antibodies are among the best candidates. Here, we have successfully humanized mouse monoclonal antibody SK2, which specifically binds to IL-6 and strongly inhibits IL-6 functions. Since this antibody possesses N-linked carbohydrates on Asn-30 of VH region, which seems to be very close to an antigen-binding site, influence of these carbohydrates on antigen-binding was investigated. A biosensor study showed that the mouse SK2 Fab and its deglycosylated fragments had almost equal Kd (Kon/Koff), 26.8 nM (1.05 x 10(6)/2.81 x 10(-2)) and 24.7 nM (1.28 x 10(6)/3.15 x 10(-2)), respectively. Furthermore, a mutant chimeric SK2 antibody, in which the N-glycosylation site was removed from the VH region, showed a Kd of 11 nM, almost similar to that of the original chimeric SK2 antibody, determined by Scatchard analysis with 125I-IL-6. These data indicate the carbohydrates of mouse SK2 VH region do not significantly influence antigen-binding activity. In the next step, two versions of each humanized SK2 VL and VH regions were carefully designed based on the amino acid sequences of human REI and DAW, respectively. Only one alteration, Tyr to Phe, was made at position 71 in the two light chains, according to the canonical residue for LI. A N-glycosylation site was introduced on the two heavy chains, by changing Ser to Asn at position 30. All four combinations of humanized light and heavy chains could bind to IL-6 as well as the chimeric SK2 antibody. The light chain first version, however, could not efficiently inhibit IL-6 binding to its receptor, indicating the importance of the LI loop conformation for the inhibitory activity of SK2 antibody. In contrast, both versions of the heavy chains were comparable, in yielding good humanized SK2 antibodies, suggesting that the glycosylation of the SK2 VH region has no influence in recreating a functional antigen-binding site in this humanization.","PeriodicalId":77166,"journal":{"name":"Human antibodies and hybridomas","volume":"7 4 1","pages":"175-83"},"PeriodicalIF":0.0,"publicationDate":"1996-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3233/HAB-1996-7405","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"69906869","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Reactivity to tyrosinase: expression in cancer (melanoma) and autoimmunity (vitiligo).","authors":"O Merimsky,&nbsp;Y Shoenfeld,&nbsp;E Baharav,&nbsp;R Zigelman,&nbsp;P Fishman","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>Anti-tyrosinase antibodies are found in the sera of patients with diffuse vitiligo, metastatic melanoma and in sera of patients with melanoma and hypopigmentation (MAH). The autoantigen is tyrosinase itself, the enzyme that participates in pigment (melanin) formation by both melanocytes and melanoma cells. The production of autoantibodies in both diseases is associated with the development of white patches on the patients' skin. The presence of these autoantibodies in patients with melanoma may suggest a better prognosis. Cross-antigenicity between melanoma cells and normal melanocytes is most probably the key mechanism leading to the appearance of MAH. Anti-tyrosinase antibodies are absorbed by melanocytes and by melanoma cells in all the 3 situations (melanoma, vitiligo, MAH). However, since the production of antibodies in vitiligo exceeds that in melanoma or MAH, the antibodies are detected in significantly higher levels only in vitiligo. It is suggested here that anti-tyrosinase antibodies may be responsible, or at least participate in destruction of normal melanocytes during the immune response to melanoma antigens. This mechanism may be responsible for the phenomenon of MAH in patients with melanoma, and for the formation of the autoimmune vitiligo. Anti-tyrosinase antibodies may serve for two clinical applications. One is a marker for monitoring and follow up of patients with melanoma treated by immune therapy. The second is active (or passive) immunotherapy. We have recently shown that C57BL/6J mice immunized with tyrosinase generated a high titer of antityrosinase antibodies, and following the inoculation of melanoma cells developed lower number of lung metastases, compared to the unvaccinated control group.</p>","PeriodicalId":77166,"journal":{"name":"Human antibodies and hybridomas","volume":"7 4","pages":"151-6"},"PeriodicalIF":0.0,"publicationDate":"1996-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"20088757","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Immunogenetics of human IgE.","authors":"R E Snow,&nbsp;C J Chapman,&nbsp;S T Holgate,&nbsp;F K Stevenson","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>Immunoglobulin E plays a central role in mediating the pathology of allergic disease. Conversely, it is involved in the normal protective immune responses against parasite infection. Both these biological processes depend on interaction between the variable regions (VH and VL) of IgE antibodies and target antigen. It is now feasible to investigate the molecular nature of VH regions used to encode IgE at the genetic level. Using this technology to analyze IgE in patients with asthma has revealed features which may have relevance for allergic disease. First, preferential choice of VH genes, with dominance of the small VH5 family, particularly the VH32 gene, has been found. This may implicate a B cell superantigen (superallergen) selectively driving the use of these genes. Second, VH5 genes in IgE are somatically mutated with clear hot spots of mutational activity. Mutational hotspots, which are a feature of the VH5 gene, are supplemented in IgE by ongoing mutations which may be involved in affinity maturation. Third, a single B cell can switch to either IgE or IgG4, with both variants coexisting in blood. These findings may provide clues to the mechanism by which IgE is generated, and suggest options for therapeutic intervention.</p>","PeriodicalId":77166,"journal":{"name":"Human antibodies and hybridomas","volume":"7 4","pages":"157-66"},"PeriodicalIF":0.0,"publicationDate":"1996-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"20088758","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A comparison of the performance in vitro of precision cut tissue slices and suspensions of human spleen with special reference to immunoglobulin and cytokine production.","authors":"K. James, G. Skibinski, P. Hoffman","doi":"10.3233/HAB-1996-7401","DOIUrl":"https://doi.org/10.3233/HAB-1996-7401","url":null,"abstract":"During the past decade our knowledge of the cellular and molecular events associated with key immunological responses has been greatly advanced by the use of isolated subpopulations of immunocompetent cells, cloned cell lines and recombinant derived cytokines. Valuable as these studies have been they do not truly reflect the complex integrative events which take place in both primary and secondary lymphoid tissue both in vivo and in vitro. In order to address this problem we have developed a tissue culture procedure which is a modification of that previously used by others to study T cell maturation in the thymus. This involves culturing precision cut slices of human lymphoid tissue in a sponge culture system. Using this technique we have observed marked differences in both immunoglobulin and cytokine secretion between slices and suspensions of human spleen. In brief, cultured slices (mitogen stimulated or otherwise) consistently secrete higher levels of immunoglobulin, IL-1 beta, IL-6, IL-8 and IL-11 and exhibit much lower proliferation than suspensions of the same tissue. Mitogen stimulated suspensions on the other hand secrete higher levels of IL-2, IL-4, IL-10 and TNF alpha than do slices. These differences are also observed at the intracellular cytokine level. Additional studies reveal that the immunoglobulin and cytokine secretion observed is largely due to the de novo synthesis of these molecules and not as a result of spontaneous secretion of preformed products. Furthermore immunoglobulin secretion in both slices and suspensions can be inhibited by the addition of specific antibodies to IL-1 beta, IL-6 and TNF alpha while IL-6 production can be differentially modulated by a variety of substances. Preliminary studies indicate that close interaction between B cells and stromal cells within explants accounts for some of the observed differences. This review article describes the basic technique, summarises the results we have obtained in this system and outlines the possible basis of the observed differences.","PeriodicalId":77166,"journal":{"name":"Human antibodies and hybridomas","volume":"7 4 1","pages":"138-50"},"PeriodicalIF":0.0,"publicationDate":"1996-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3233/HAB-1996-7401","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"69906783","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Anti-gp210 antibodies in sera of patients with primary biliary cirrhosis. Identification of a 64 kD fragment of gp210 as a major epitope.","authors":"J Wesierska-Gadek,&nbsp;H Hohenauer,&nbsp;E Hitchman,&nbsp;E Penner","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>Patients with primary biliary cirrhosis (PBC) frequently produce autoantibodies against gp210, an integral glycoprotein of the nuclear pores. this protein consists of three main domains: a large glycosylated lumenal domain, a single hydrophobic transmembrane segment and a short cytoplasmic tail. It has been previously shown that autoantibodies from PBC patients exclusively react with the cytoplasmic tail when recombinant rat gp210 expressed in Escherichia coli was used as antigen. Using human gp210 isolated from HeLa cells we found the lumenal domain as the major target. The aim of this study was to further characterize the dominant autoepitopes of gp210. Sera from 88 patients with autoimmune liver disease and 20 controls were used. Gp210 protein was digested with papain or endoglycosidase H and then subjected to immunoblotting. Autoantibodies against gp210 were detected in 12 of 43 (28%) PBC patients, but in none of the autoimmune hepatitis and control sera. Four of 12 (33%) anti-gp210 positive sera reacted with a fragment consisting of the cytoplasmic tail and 8 (66%) sera targeted an epitope located within the large lumenal domain. Furthermore, our data show that antigenic determinant is restricted to the 64 kD glycosylated amino-terminal fragment and that carbohydrate residues are an essential part of this novel epitope. We suggest that antigens possessing both epitopes namely; the glycosylated lumenal domain and the cytoplasmic tail should be used for screening tests in order to detect all sera with anti-gp210 specificity.</p>","PeriodicalId":77166,"journal":{"name":"Human antibodies and hybridomas","volume":"7 4","pages":"167-74"},"PeriodicalIF":0.0,"publicationDate":"1996-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"20088759","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Unsusceptibility of recombinant human Fc fragments of immunoglobulin E to thrombin.","authors":"T Kamiya","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>Human immunoglobulin E (IgE) contains a potential recognition sequence for thrombin protease cleavage at N-terminal end of C epsilon 3 domain responsible for binding to alpha chain of high affinity Fc receptor for IgE (Fc epsilon RI alpha), but it remains unknown for the enzyme susceptibility. The human Fc fragments of IgE (IgE-Fc), consisting of C epsilon 2' (Ala329)-C epsilon 3-C epsilon 4 chains (Fc') and of C epsilon 2' (Thr315-Ala329)-C epsilon 3-C epsilon 4 chains (F(c')2), were expressed in the mammalian COS and Chinese hamster ovary (CHO) cells by placing the IgE-Fc cDNA under the control of the cytomegalovirus (CMV) promoter. Under nonreducing condition F(c')2 was not cleaved by thrombin protease as well as native IgE. Neither treatment of F(c')2 with final 0.1% 2-mercaptoethanol at boiling point for 5 min, with a sulfhydryl-reactive biotin derivative, by which monomers in COS-derived F(c')2 preparations were biotinylated at Cys328, nor with neuraminidase, affected the accessibility to the enzyme. These results suggested that F(c')2, either dimeric or monomeric, had compact conformations.</p>","PeriodicalId":77166,"journal":{"name":"Human antibodies and hybridomas","volume":"7 1","pages":"42-7"},"PeriodicalIF":0.0,"publicationDate":"1996-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19851349","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Membranous nephropathy in primary antiphospholipid syndrome: description of a case and induction of renal injury in SCID mice.","authors":"Y. Levy, L. Ziporen, B. Gilburd, J. George, S. Polak‐Charcon, H. Amital, J. Clèdes, P. Youinou, Y. Shoenfeld","doi":"10.3233/HAB-1996-7301","DOIUrl":"https://doi.org/10.3233/HAB-1996-7301","url":null,"abstract":"Primary antiphospholipid syndrome (PAPS) is a recently recognized clinical entity encompassing the combination of thromboembolic phenomena, thrombocytopenia and recurrent abortions in the presence of antiphospholipid antibodies. We present a patient with PAPS accompanied by renal involvement, manifested as membranous nephropathy, as proven by a renal biopsy. To investigate further the possible association between PAPS and the renal lesions we attempted to induce similar renal manifestations by transferring peripheral blood lymphocytes (PBL) from this patient to severe combined immunodeficiency (SCID) mice. The mice transfused with PBL from the affected patient exemplified antiphospholipid antibodies (aPL) following which a renal lesion consistent with the human membranous nephropathy lesion was precipitated. This study substantiates the role of aPL as possible inducers of renal damage.","PeriodicalId":77166,"journal":{"name":"Human antibodies and hybridomas","volume":"7 3 1","pages":"91-6"},"PeriodicalIF":0.0,"publicationDate":"1996-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3233/HAB-1996-7301","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"69906217","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A comparison of the performance in vitro of precision cut tissue slices and suspensions of human spleen with special reference to immunoglobulin and cytokine production.","authors":"K James,&nbsp;G Skibinski,&nbsp;P Hoffman","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>During the past decade our knowledge of the cellular and molecular events associated with key immunological responses has been greatly advanced by the use of isolated subpopulations of immunocompetent cells, cloned cell lines and recombinant derived cytokines. Valuable as these studies have been they do not truly reflect the complex integrative events which take place in both primary and secondary lymphoid tissue both in vivo and in vitro. In order to address this problem we have developed a tissue culture procedure which is a modification of that previously used by others to study T cell maturation in the thymus. This involves culturing precision cut slices of human lymphoid tissue in a sponge culture system. Using this technique we have observed marked differences in both immunoglobulin and cytokine secretion between slices and suspensions of human spleen. In brief, cultured slices (mitogen stimulated or otherwise) consistently secrete higher levels of immunoglobulin, IL-1 beta, IL-6, IL-8 and IL-11 and exhibit much lower proliferation than suspensions of the same tissue. Mitogen stimulated suspensions on the other hand secrete higher levels of IL-2, IL-4, IL-10 and TNF alpha than do slices. These differences are also observed at the intracellular cytokine level. Additional studies reveal that the immunoglobulin and cytokine secretion observed is largely due to the de novo synthesis of these molecules and not as a result of spontaneous secretion of preformed products. Furthermore immunoglobulin secretion in both slices and suspensions can be inhibited by the addition of specific antibodies to IL-1 beta, IL-6 and TNF alpha while IL-6 production can be differentially modulated by a variety of substances. Preliminary studies indicate that close interaction between B cells and stromal cells within explants accounts for some of the observed differences. This review article describes the basic technique, summarises the results we have obtained in this system and outlines the possible basis of the observed differences.</p>","PeriodicalId":77166,"journal":{"name":"Human antibodies and hybridomas","volume":"7 4","pages":"138-50"},"PeriodicalIF":0.0,"publicationDate":"1996-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"20088756","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Targeting gamma interferon to tumor cells by a genetically engineered fusion protein secreted from myeloma cells.","authors":"J Xiang,&nbsp;Y Qi,&nbsp;D Cook,&nbsp;T Moyana","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>The construction, synthesis and expression of a genetically engineered bifunctional antibody/cytokine fusion protein is described. To target IFN-tau to tumor cells, recombinant antibody techniques were used to construct a RM4/IFN-tau fusion protein containing the chimeric anti-tumor F(ab')2 (RM4) and the IFN-tau moiety. The recombinant cDNA of IFN-tau was linked to 3 prime end of the chimeric heavy-chain gene fragment (M4) containing the VH, the CH1 and the hinge region to form the fused heavy-chain gene fragment M4-IFN-tau. Transfection of the M4-IFN-tau gene fragment into a myeloma derived cell line VKCK which produced the chimeric light-chain of the same antibody, allowed the transfectant secreting the bifunctional fusion protein RM4/IFN-tau. The RM4/IFN-tau was purified by the affinity chromatography. Our data showed that the RM4/IFN-tau retained the TAG72 antigen-binding reactivity as well as the IFN-tau activity as measured in ELISA, FACS analysis of cell-surface TAG72 expression, immunohistochemical study, and up-regulation of cell-surface expression of CEA, HLA class I and class II antigens. Therefore, the bifunctional fusion protein RM4/IFN-tau may prove to be useful in targeting biological effects of the IFN-tau to tumor cells and in this way to stimulate the immune destruction of tumor cells.</p>","PeriodicalId":77166,"journal":{"name":"Human antibodies and hybridomas","volume":"7 1","pages":"2-10"},"PeriodicalIF":0.0,"publicationDate":"1996-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19851344","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}