Human-human hybridoma producing monoclonal antibodies against colorectal cancer-associated antigens

Lymphocytes from lymph nodes draining the tumor region in patients with colorectal cancer were fused with two different human B-lymphoblastoid cell lines, LICR-LON-HMy-2 (HMy-2) and WI-L2-729-HF2 (729-HF2), to generate hybridomas synthesizing antibodies reacting with tumor-associated antigens. In this way 220 hybridomas were obtained which produce antibody reacting with colon cancer cells. All established clones produced IgM. Four human monoclonal antibodies have been further analyzed. The cell lines producing these antibodies are all hybrids based on DNA analysis. Three of the antibodies (G4146, B9165 and D4213) showed binding to differentiation antigens by immunocytochemical analysis on different cancer cell lines and normal human leucocytes and by immunohistochemical analysis on sections of frozen malignant and normal tissues, while the fourth (F11348) showed a reaction with all cells and tissues tested. Western blots of tumor extracts showed binding of G4146 to two components from colon cancer cells with Mr of 59 K and 61 K, while B9165 bound to a 43 K component and F11348 to several components with Mr from 30 to 200K. D4213 showed no binding in this analysis. The results obtained demonstrate the successful application of hybridoma technology to produce human monoclonals with reactivity to differentiation antigens.     

Generation of monoclonal antibodies to human lymphocyte cell surface antigens using insect cells expressing recombinant proteins.

We have expressed human CD40 and human B7 in insect cells using the baculovirus expression system and have used these insect cells to immunize mice for the generation of monoclonal antibodies. We demonstrate here that specific monoclonal antibodies to human CD40 and human B7 were obtained using this approach. One significant advantage of this method is that immunizing mice with insect cells did not evoke an immune response to human cells and, therefore, EBV-transformed human B cells could be used to screen for specific antibody production by the hybridoma clones.     

Characterization of polymyositis infiltrates using monoclonal antibodies to human leucocyte antigens.

Frozen serial sections of muscle from 15 patients with polymyositis and three normal controls were studied by indirect immunofluorescence with a panel of mouse monoclonal antibodies to various human leucocyte components. The results showed good correlation with conventional histology. In addition, large numbers of T lymphocytes were identified in those cases with a marked inflammatory infiltrate. Many of the T cells probably bear HLA-DR antigen as the anti-HLA-DR antibody stained as many cells as the anti-leucocyte antiserum. This strongly suggests that the T cells present are 'activated'. In two patients HLA-DR-positive material was identified apparently diffusing from the infiltrates into muscle fibres suggesting its release as a soluble factor. In one case, structures with the appearance of giant cells were seen. The method promises to provide new information on the nature of infiltrating leucocytes which may provide more accurate diagnostic and prognostic information than conventional histology alone.     

Generation and characterization of human monoclonal scFv antibodies against Helicobacter pylori antigens

Infection with Helicobacter pylori is chronic despite a vigorous cellular and humoral immune response and causes severe pathology in some patients. In this study, phage display was used as a new approach in order to investigate the role of the host's humoral immune response in the pathogenesis of H. pylori gastritis. Human monoclonal single-chain Fv (scFv) antibody fragments against H. pylori cell lysate and the H. pylori urease were isolated from an immune phage display library, constructed from peripheral blood lymphocytes of an H. pylori-infected patient. After affinity selection, 23% of the clones tested showed binding activity against a lysate of the H. pylori Sydney strain in enzyme-linked immunosorbent assay (ELISA) and 9% bound the H. pylori urease. Further characterization by PCR-fingerprint analysis and sequencing revealed that two closely related H. pylori binders and one antiurease scFv could be isolated. The selected scFvs were highly specific as analyzed by ELISA and immunoblots using various bacterial lysates and recombinant proteins. Analysis of the humoral immune response following H. pylori infection using human monoclonal antibodies might contribute to a better understanding of the pathogenesis of the disease. Moreover, using immune phage display libraries, it might be possible for relevant epitopes of H. pylori antigens to be determined, which might be of use for vaccine development.     

Immunodetection of cell-bound antigens using both mouse and human monoclonal antibodies

A micro enzyme-linked immunoassay (EIA) has been developed for the rapid and sensitive detection of either human or mouse monoclonal antibodies reactive with cell bound antigens. Whole intact cells are immobilized onto 96-well flat bottom microtiter plates by drying in an oven at 37 degrees C overnight prior to the start of the assay. This method of attachment was suitable for all cell types tested, regardless of origin, size and chromosomal content. The dried cells were then rehydrated, incubated with the appropriate test hybridoma supernatant, followed with subsequent analysis by EIA. The plates can be stored at 4 degrees C up to 1 month for future EIA analysis. This assay offers high sensitivity, requires only small amounts of target cells and test hybridoma supernatant, and can be completed within 3 h. This EIA is well suited for the rapid screening of large numbers of hybridoma supernatants and can also be adapted to include cells of any species, providing the appropriate antibody reagents are available.     

Reactivity of monoclonal antibodies against human leucocyte antigens with lymphocytes of non-human primate origin

The phylogenetic distribution of antigens present on human lymphocytes was investigated by incubating human or simian cells with murine anti-human monoclonal antibodies and then determining the level of reactivity with a radiolabelled anti-murine IgG reagent. The monoclonal antibodies used were specific for a T-cell antigen, lymphoid and lymphoid:myeloid antigens, Ia antigens, and beta 2 microglobulin. The cells examined included B- and T-lymphoblastoid cell lines and fresh peripheral blood lymphocytes separated by sheep erythrocyte rosetting into T-cell and non T-cell fractions. Results of these studies showed that the antibodies gave complete cross-reactivity with gorilla and chimpanzee cells while B-cell lines of orangutan origin had lost lymphoid and beta 2 microglobulin markers. Gibbon cells and cells of Old World and New World monkeys reacted strongly only with monoclonal antibodies against Ia antigenic determinants. These Ia antigens were found on the non T-cell fraction of fresh peripheral lymphocytes, on B-cell lines and on some virus induced T-cell tumour lines. Immunoprecipitation analysis using the anti-Ia antibodies showed a degree of molecular diversity on owl monkey and marmoset cells compared to the Ia antigens associated with human cells.     

Generation and preliminary characterization of three mouse monoclonal antibodies reacting with human B lymphocytes

The paper describes generation and characterization of three monoclonal antibodies (BR3, BR16, BR31) specific for human B lymphocytes. BR3 and BR31 stain highly purified population of B lymphocytes but fail to react with granulocytes, monocytes and E+ lymphocytes. BR16 in addition to B cells also stains 10% peripheral T cells and 70% granulocytes. BR3 staining pattern closely correlates that obtained with antihuman Ig both in peripheral blood and in lymphoid tissues. BR31 recognizes a subpopulation of B lymphocytes (about 50%).     

Cytofluorometric analysis of major histocompatibility antigens on human monocytes using monoclonal antibodies

The expression of HLA-A, B, C, and DR antigens was investigated on monocyte preparations by flow cytometry using various monoclonal antibodies. Essentially all human monocytes, either freshly isolated or after culture for several days, were stained for HLA-A, B, C, and DR antigens. When monocytes were incubated with Con-A-stimulated lymphocyte supernatants, an increase in HLA-A, B, C, and DR staining was observed. No increase was noted when two other monoclonal antibodies against non-HLA-related monocyte antigens (63D3 and 61D3) were studied under the same culture conditions. These results indicate that soluble factor(s) present in Con-A-stimulated lymphocyte supernatants modulate the expression of the major histocompatibility antigens on the surface of human monocytes.     

Studies of murine malaria antigens using monoclonal antibodies

A panel of ten monoclonal antibodies made againstPlasmodium chabaudi andPlasmodium yoelii infected mouse erythrocytes were used for characterization of antigens present in murine malaria. Screening of the antibodies in ELISA with different fractions of infected erythrocytes revealed both species-specific and fraction-specific monoclonal antibodies (MAbs), but also MAbs cross-reacting between the species. Two MAbs bound normal erythrocyte components. Subcellular localization of the target antigens was studied by immunofluorescence and their molecular identity by immunoblotting after SDS-PAGE. Of the MAbs toP. yoelii, one reacted with a cytoplasmic granule component of 137 k and two others reacted with vacuole-associated antigens of 26 k and 25/70/73 k, respectively. The latter antibodies cross-reacted withP. chabaudi antigens. Of the MAbs toP. chabaudi, all were species specific, one reacting with parasite surface antigens of 79 and 250 k and two with a vacuole-associated antigen of 70 k.     

Serological and biochemical analyses of monoclonal antibodies to human melanoma-associated antigens

Monoclonal antibodies submitted and exchanged by participating laboratories were analyzed serologically and biochemically in accordance with the goals and aims of the Workshop on Monoclonal Antibodies to Melanoma. Serological evaluation of the workshop panel revealed that when a variety of tumor and normal cell lines were tested by enzyme-linked immunoassay, a majority of antibodies reacted preferentially with melanoma cells. Indirect immunoprecipitation analysis of those antibodies that bound staphylococcal protein A provided molecular weight estimates for some antigens and additional information that suggested possible commonalities among antigens recognized by antibodies 9.2.27, 225.28S and 763.24TS. Competitive binding analyses demonstrated that antibodies 225.288 and 763.24TS bound separate and distinct epitopes from that recognized by antibody 9.2.27 on the core glycoprotein of melanoma-associated chondroitin sulfate proteoglycans. Immunoperoxidase staining of fresh frozen melanoma tissue with antibody 9.2.27 revealed a limited tissue distribution and preferential association with the cell surface of human melanoma cells.     

Analysis of the NIH workshop monoclonal antibodies to human melanoma antigens

Reagents exchanged at the 2nd workshop on monoclonal antibodies (MoAb) to human melanoma antigens were analyzed using both serological and immunochemical assays. The analysis by laboratories participating in the workshop of our MoAb 225.28S, 345.134S, 376.96S, 465.12S, and 763.24TS reaffirms our own analysis of these reagents in that (1) they all react with the majority of melanoma cell lines tested and (2) the reactivity of MoAb 225.28S and 763.24TS is much more restricted than that of MoAb 345.134S 376.96S, and 465.12S. Our serological analysis revealed that the majority of workshop reagents reacted with cultured melanoma cells. Immunochemical analysis of these monoclonal antibodies allowed for their division into three groups according to the molecular weights of the antigens recognized in immunoprecipitation experiments, greater than 100 kd, 80-100 kd, and DR antigens. Further analysis of the first two groups of monoclonal antibodies by immunodepletion and antibody binding inhibition assays revealed that MoAb 9.2.27, 225.28S, and 763.24TS recognize distinct determinants with a heterogeneous distribution on subpopulations of a high molecular weight melanoma associated antigen. MoAb 376.96S and 705.F6 recognize either the same or spatially close determinant(s).     

Generation of monoclonal antibodies against surface antigens of Moraxella bovis.

Six hybridoma lines producing monoclonal antibodies (MAbs) against Moraxella bovis were established from fusions between the SP2/0 myeloma cells and BALB/c mice splenocytes. Three antibodies were of the IgG1 isotype, two were IgG2a, and one was IgG2b. The specificity of the antibodies was determined by indirect enzyme-linked immunosorbent assay (ELISA) using whole cells of M. bovis and of other Gram-negative bacteria, and lipopolysaccharide (LPS) from M. bovis JUR2 and E. coli as antigens. Ascitic fluid produced by the six hybridoma lines inhibited hemagglutination by M. bovis GF9. One MAb (35F) reacted specifically with purified M. bovis LPS in the ELISA test. The MAb panel detected heterogeneity among the isolates recovered from different geographical regions.     

Two distinct antigen systems in human B lymphocytes: identification of cell surface and intracellular antigens using monoclonal antibodies.

Two distinct antigen systems (L26 and L27) specifically expressed in human B lymphocytes were identified using TB2-2B3 (2B3) and T3-5B3 (5B3) monoclonal antibodies, respectively. Whereas L26 antigen defined by 2B3 were rarely expressed on the surface of B cells but abundant in the cytoplasm, 127 antigens detected by 5B3 was clearly expressed on the cell surface. These two antigens appeared to be restricted in their expression to B cells, as they were found in most B cells but not other cell types including thymocytes, T cells, monocytes and granulocytes. Functional studies demonstrated that L27 was more easily lost from B cells after activation with pokeweed mitogen than was L26. Likewise, plasma cell myeloma, as well as normal plasma cells, was devoid of both L26 and L27, whereas immunoblastic sarcoma of B cell type expressed L26 but not L27. These two antigens co-existed in the same B cell lines including Epstein-Barr virus transformed B cell lines, B cell type acute lymphatic leukaemia (B-ALL) cell line, Burkitt's lymphoma cell lines and myeloma cell lines, but pre-B and common ALL cell lines were entirely negative for both L26 and L27. Immunoprecipitation studies showed that L26 consisted of at least two polypeptide chains with molecular weights of 30K and 33K daltons, which were clearly distinct from HLA-DR antigens. The antigen L27 is presently under study.     

Demonstration of cross-reactivity between bacterial antigens and class I human leukocyte antigens by using monoclonal antibodies to Shigella flexneri

Bacterial envelope proteins which share immunodeterminants with the human leukocyte antigen (HLA) class I histocompatibility antigen HLA-B27 may invoke spondyloarthritic disease through the process of molecular mimicry in patients expressing this phenotype. Monoclonal antibodies generated by the immunization of BALB/c mice with envelope proteins of Shigella flexneri type 2a were tested for reactivity against cultured lymphoblastoid cell lines of defined HLA phenotype. As measured by flow microfluorometry, four immunoglobulin M monoclonal antibodies reacted preferentially with HLA-B27-positive lymphocytes (HOM-2, MM) as compared with a B27-loss mutant line (1065) or cells lacking major histocompatibility complex class I antigen (Daudi, K562). Monoclonal antibodies also reacted with mouse EL-4 cells transfected with and expressing the HLA-B7 gene. Western immunoblot analysis of isolated enterobacterial envelopes demonstrated that the reactive epitope was present on bacterial proteins with an apparent relative molecular mass of 36 and 19 kilodaltons. The structural basis for the cross-reactivity of bacterial antigen and HLA-B27 appeared to reside in the portion of the HLA molecule that is responsible for allotypic specificity (amino acids 63 through 83), since monoclonal antibodies were positive by enzyme-linked immunosorbent assay with synthetic polypeptides corresponding to this segment.     

抗人肿瘤相关核仁抗原单克隆抗体的建立

本研究以A549细胞系和人肺腺癌组织细胞核仁为抗原,建立了7株McAbs,并用ELISA技术对其反应性进行了初步分析。结果表明:各株McAb均能与人癌细胞核仁起反应,但各自的抗原却不尽相同。MA1、MA2、MA3和MA6株的抗原可能是HMNA类物质;MA4、MAS和ML1株的抗原可能是属于核仁的正常成份,但优势表达于癌细胞中。本组抗体的建立,对于研究肿瘤细胞核仁的分子组成和生物学功能,并进而利用HMNA为临床肿瘤病理服务可能有重要意义。     

Activation of human monocytes and granulocytes by monoclonal antibodies to glycosylphosphatidelinositol-anchored antigens

The present study investigated possible receptor-like characteristics of glycosyl-phosphatidylinositol (GPI)-linked antigens on human monocytes and granulocytes by measuring cytoplasmic calcium fluxes and the oxidative burst in cells following cross-linking of GPI-linked antigens. Cross-linking of cell-bound anti-CD14, -CDw52 and -CD55 induced cytoplasmic calcium fluxes and oxidative bursts in unprimed human monocytes similar to those observed following FcyR cross-linking. In granulocytes primed with 200 nM N-formyl-Met-Leu-Phe (FMLP), cross-linking of cell-bound anti-CD16, -CD24, -CD59 and -CD67 led to calcium fluxes and activation of the oxidative burst. The oxidative bursts mediated by GPI-linked antigens were stronger than those induced by 200 nM FMLP, even though FMLP induced a larger increase in cytoplasmic calcium concentration. The responses were likely to be independent of FcyR interactions as F(ab′)2 fragments of IgG or IgM antibodies were used in the experiments. Activating effects of monoclonal antibody to GPI-linked antigens were not observed in cells from patients with paroxysmal nocturnal hemoglobinuria, which are deficient in GPI-linked antigens. In addition, treatment with GPI-specific phospholipase C led to inhibition of cell activation through GPI-linked antigens but not through transmembrane receptors. Cross-linking of a number of non-GPI-linked antigens (CD11a, CD18, CD31, CD35, CD43, and CD45) neither induced calcium fluxes, nor activated the oxidative burst. The results indicate that most, if not all, GPI-linked surface glycoproteins on myeloid cells are capable of mediating cell activation and suggest that the GPI anchor is a structure facilitating signal transduction.     

Immunization of normal human splenocytes in vitro to produce human monoclonal antibodies to cellular antigens

We have obtained human monoclonal antibodies to polymorphic cell surface determinants by immunizing normal human splenecytomy in vitro to allogeneic cells. Splenocytes from young patients undergoing splenectomy secondary to traumatic injury are separated into T and B lymphocyte populations. The T lymphocytes are irradiated with 1500 rad to selectively inactivate T suppressors. Responder T and B cells are then recombined at a 1:1 ratio. Maximal IgM and IgG production is obtained when pokeweed mitogen and irradiated stimulator cells are added to the cultures. Stimulator cell specific antibody levels peak at day 7 of in vitro immunization, and fall thereafter. Fusion of the immunized splenocytes to a human fusion partner as early as day 4 results in hybridomas secreting antibodies to cellular antigens. Transformation by EBV, expansion and fusion of the immunized cell line also yield hybridomas secreting stimulator cell specific antibody.