Characterization of a human-human hybridoma antibody, C-OU1, directed against a colon tumor-associated antigen.

The human hybridoma cell line, B9165, was obtained after fusion of lymphocytes from lymph nodes draining the tumor region in a patient with adenocarcinoma of the colon with the human B-lymphoblastoid cell line WI-L2-729-HF2 (729-HF2). B9165 secretes the human monoclonal antibody, C-OU1 (IgM, kappa). Immunocytochemical and immunohistochemical analysis showed that the antibody bound to a differentiation antigen. Electron microscopy of colonic adenocarcinoma cells, intact tumor and colonic epithelium by the immunogold technique demonstrated that the C-OU1 antibody reacted with a molecule associated with areas of disruption of the intermediate filaments in the cytoplasm of the tumor cells. No reaction was seen with intermediate filaments in normal colonic epithelium. The molecular weight of the antigen was shown to be 43 Kda by SDS-PAGE and Western blotting of tumor extracts, and isoelectric focusing of sonicated extracts demonstrated reaction with molecular species of pI 5.4-6.2. These findings suggest that the C-OU1 antigen is a modified cytokeratin 18. The B9165 cell line has proved to be quite stable, and the antibody is of potential clinical value. Its usefulness for localizing tumors in patients is being investigated.     

Development and characterization of ouabain-resistant human fusion partners.

The ouabain-resistant mutant cell lines, HOA-1 and HOA-20 were developed from WI-L2-729-HF2 by cloning with increasing concentration of ouabain. Both parent and mutant cell lines were resistant to base analogues, 6-thioguanine (6-TG) and 8-azaguanine (8-AG) to the level of 20 micrograms/ml in the culture medium. The parent cell line WI-L2-729-HF2 was highly sensitive to ouabain, whereas HOA-1 and HOA-20 were resistant to ouabain to the level of 1 microM and 20 microM, respectively. However, all the cell lines were sensitive to HAT-selective medium which is essential for hybrid selection after fusion. All three lymphoblastoid cell lines were positive for Epstein-Barr virus nuclear antigen (EBNA), secreted TNF-beta (lymphotoxin) without any external stimulation, secreted trace amounts of IgG(kappa), which was also present in their cytoplasm and had IgM(kappa) as surface bound immunoglobulin. They also expressed the CD20, CD71 (transferrin receptor) as surface antigens. In addition to these antigens, HOA-20 also expressed CD38 antigen. The karyotype analysis of these cell lines revealed modal chromosomal numbers ranging from 40 to 47. The HLA-A, -B and -C antigens expressed by WI-L2-729-HF2 and its mutants HOA-1 and HOA-20 were identical. Both the HOA-1 and HOA-20 mutants were found suitable for the generation of hybrids after fusion with EBV-transformed human B-lymphocytes.     

Functional human T cell-B cell hybridomas established from fusion of normal T cells and an EBV-transformed B cell line

Human T cell-B cell hybridomas containing 92 chromosomes were derived by fusing normal stimulated CD-depleted T cells with the EBV-transformed human B cell line 729-HF. These hybridomas coexpressed the T cell surface markers CD3, CD8, and CD2 and the B cell surface antigens CD19, CD20, CD23, DR, and DQ. They weakly and variably expressed surface IgM and TCR. Genomic analysis revealed the presence of rearranged Ig genes as well as beta-T cell receptor genes that could be ascribed to the B and T cell parent, respectively. Analysis of TCR rearrangement suggests that the T-B hybridomas are, in fact, subclones of a single dominant clone. Although the hybrids expressed CD8 and not CD4, following preincubation with PWM, some the of clones induced IgG synthesis from normal B cells while the parent B cell line failed to demonstrate this activity. Stimulation of the hybridomas with PMA down-regulated the T cell lineage-specific antigen display (CD3, CD8, and TCR) and increased IgM production from the hybrids without changing B cell surface antigen display. These hybridomas may be useful to dissect the steps involved in the ultimate commitment of a cell to the B or T cell lineages and will be made available to interested investigators.     

抗胃癌细胞系MKN—74单克隆抗体的制备及其初步应用

以低分化胃腺癌细胞株MKN—74直接免疫BALB/c小鼠,制得3株抗胃癌表面抗原单克隆抗体,经鉴定及初步临床应用证明,它们所针对的可能是一类新的主要存在于胃瘤细胞表面的肿瘤相关抗原。     

Human hybridoma generation by hypo-osmolar electrofusion: characterization of human monoclonal antibodies to Schistosoma mansoni parasite antigens.

Human monoclonal antibodies which bind Schistosoma mansoni worm and egg antigens were identified and characterized from hybridomas generated using the hypo-osmolar electrofusion technique of somatic cell fusion. Splenocytes from S. mansoni infected individuals were mitogen-activated in vitro and subsequently fused by electrofusion. The greatest number of HAT resistant hybridomas per helical fusion chamber was obtained with unfrozen splenocytes cultured for 4-6 days after introduction of mitogen. Hybridomas secreting IgG antibodies recognizing parasite antigens were identified by ELISA. Twenty-one cloned cell lines secreting IgG antibody were maintained for at least 6 months. Characterization of antigen reactivity by Western blot analysis of nien cloned cell lines revealed antibodies which bound stage specific parasitic antigens. The data show that the technique of hypo-osmolar electrofusion produces stable, antibody producing hybridomas. The human monoclonal antibodies screened represent candidate molecules useful in the investigations of the human pathogen S. mansoni.     

A radioactive antibody binding-inhibition assay,for the detection of cell-membrane related antigens in body fluids

Antisera raised in rabbits against glutaraldehyde-fixed human breast cancer cells contain antibodies to human cell membrane components, as determined by immunofluorescence. Adsorption of such antisera onto polymerized human serum, followed by acid elution, yields purified antibodies reacting with human cell surface antigens, indicating that membrane related antigens are present in the serum. The purified antibodies were radioiodinated and shown to bind to an immunoadsorbent prepared by entrapping in a polyacrylamide gel pleural exudate of breast cancer patients. The specificity of the binding was confirmed by inhibition experiments. Data are presented demonstrating that at least some of the antibodies reacting in this radioimmunoassay are directed against antigens related to cell surface components.     

Monoclonal Antibodies to HLA–DRw Determinants

Two monoclonal antibodies recognizing HLA-DRw (human la) antigens were produced. The DA2 antibody binds a monomorphic determinant, common to all specificities and Genox3.53 antibody binds to a cross-reacting site on the HLA-DRw1,2 and 6 specificities. Both antibodies are IgG1 and show complement dependent cytotoxicity only in the presence of rabbit anti-mouse IgG serum. Specificity of both antibodies for the HLA-DRw molecule was shown by inhibition of antibody binding by preincubation of antibody with detergent solubilized Ia from JY (HLA-DRw4,6) cells and by preincubation of target cells with F(ab')2 fragments of a rabbit anti-la serum. DA2 antibody reacted with all cells of human B cell origin tested and with peripheral blood lymphocytes of several primate species tested. Genox3.53 antibody bound only to human cells expressing HLA-DRw1,2 or 6 antigens, giving a negative reaction with all primates tested. Genox3.53 antibody detected a split in the HLA-DRw6 specificity, showing reduced binding to the Daudi cell (HLA-DRw6) in comparison with binding to several other cell lines typed as HLA-DRw6, under saturating conditions. This low reactivity with Daudi was confirmed by absorption experiments. The ratio of DA2 binding to Genox3.53 binding to homozygous and heterozygous cell lines under saturation conditions was compared. Results suggested that, on some cell lines, DA2 might be reacting with a second population of human Ia antigens in addition to the HLA-DRw antigens. When a mixture of saturating concentrations of DA2 and Genox3.53 antibodies was tested for binding to cells under saturating conditions, the number of counts bound suggested the antibodies could bind simultaneously. Direct binding experiments showed that when each antibody was iodinated, its binding was not inhibited by preincubation with the other antibody, confirming that the DA2 and Genox3.53 determinants are distinct on the Ia molecule.     

Immunopurification of Radiolabelled Antigens of Mycobacterium leprae and Mycobacterium bovis (Bacillus Calmette-Guerin) with Monoclonal Antibodies

Radiolabelled sonicate of Mycobacterium leprae when examined by SDS-PAGE and two-dimensional gel electrophoresis (2-DE) contained fewer antigens than the comparable sonicate from M. Bovis (bacillus Calmette-Guerin) (BCG). A solid-phase immunopurification assay with anti-M. leprae monoclonal antibodies (MoAb) was used to characterize four of these antigens. Three of the MoAb were M. leprae-specific and with them antigens with apparent molecular weights (Mr) of 12,000 (12K), 18K, and 35K were isolated. On 2-DE, the heavily labelled 12K antigen was heterogeneous with a range in pI of 4.8-5.2. The 35K antigen, which was identified by a conformational determinant, and the 18K antigen were also acidic proteins with pI of 5.4 and 5.1. The fourth antigen was purified from both M. leprae and BCG sonicates and had an Mr of 70K and a pI of 5.1. MoAb reacting with the cell wall protein of M. leprae resulted in separation of multiple bands ranging in Mr from 12K to 65K, rather than the dominant 65K protein seen in immunoblots. A similar pattern was obtained with MoAb that reacted with two cell wall polysaccharide antigens, and these antibodies may have co-precipitated the radiolabelled cell wall proteins. Immunoprecipitates of the M. leprae sonicate with human lepromatous leprosy sera, when analysed by 2-DE, were also found to contain the dominant 12K band and the 35K band. Furthermore, half the radiolabelled BCG antigens were precipitated by the same sera.     

Murine monoclonal antibodies specific for virulent Treponema pallidum (Nichols).

Murine anti-Treponema pallidum (Nichols) lymphocyte hybridoma cell lines secreting monoclonal antibodies against a variety of treponemal antigens have been generated. Hybridomas isolated were of three major types: those that were directed specifically against T. pallidum antigens, those that were directed against treponemal group antigens (as evidenced by their cross-reactivity with T. phagedenis biotype Reiter antigens), and those that cross-reacted with both treponemal as well as rabbit host testicular tissue antigens. The majority (31 of 39 clones) of these anti-T. pallidum hybridomas, which produced monoclonal antibodies of mouse isotypes immunoglobulin G1 (IgG1), IgG2a, IgG2b, IgG3 or IgM, were directed specifically against T. pallidum and not other treponemal or rabbit antigens tested by radioimmunoassay. Four of these T. pallidum-specific hybridomas secreted monoclonal antibodies with greater binding affinity for "aged" rather than freshly isolated intact T. pallidum cells, suggesting a possible specificity for "unmasked" surface antigens of T. pallidum. Six anti-T. pallidum hybridomas produced complement-fixing monoclonal antibodies (IgG2a, IgG2b, or IgM) that were capable of immobilizing virulent treponemes in the T. pallidum immobilization (TPI) test; these may represent biologically active monoclonal antibodies against treponemal surface antigens. Three other hybridomas secreted monoclonal antibodies which bound to both T. pallidum and T. phagedenis biotype Reiter antigens, thus demonstrating a possible specificity for treponemal group antigens. Five hybridoma cell lines were also isolated which produced IgM monoclonal antibodies that cross-reacted with all treponemal and rabbit host testicular tissue antigens employed in the radioimmunoassays. This report describes the construction and characteristics of these hybridoma cell lines. The potential applications of the anti-T. pallidum monoclonal antibodies are discussed.     

Monoclonal autoantibodies from insulin-dependent diabetic patients: autoantibodies against beta-cell surface or cytoplasmic antigens.

To investigate the target antigen(s) recognized during the autoimmune process in insulin-dependent diabetes mellitus (IDDM), we produced human monoclonal antibodies by Epstein-Barr virus transformation of peripheral blood lymphocytes from a large number (n = 50) of newly diagnosed IDDM patients. Screening by indirect immunofluorescence assay, using the RINm5F rat insulinoma cell line and eight other human or rat tumour cell lines, was performed to identify monoclonal antibodies that reacted with either membrane or cytoplasmic antigens. Eighteen IgM monoclonal antibodies reacting with cytoplasmic antigens of RIN cells were obtained; 14 of them also showed a staining of the cytoplasm of various non-beta-cell lines, while four displayed a binding restricted to beta-cells among the panel tested. However, among three monoclonal antibodies reacting with the membrane of RIN cells, one (HMD-1) produced an IgG antibody with a binding restricted to the membrane of beta-cells (RIN, HIT, and normal rat islet cells). The membrane antigens of HMD-1 were identified in Western blotting as proteins with molecular weights of 64 and 70 kD. This antibody had no apparent cytotoxic effect on RIN cells. These data suggest that, apart from 'natural autoantibodies,' it is feasible to obtain human monoclonal antibodies from IDDM patients that bind specifically to the beta-cell cytoplasm or to the beta-cell membrane.     

Binding sites for Lewis antigens are expressed by human colon cancer cells and negatively affect their migration

In colon cancer, endothelial cell selectins can promote tumor cell attachment via interactions with sialylated Lewis antigens present at the surface of tumor cells, thereby facilitating tumor cell arrest and transmigration into the extravascular space. However, it is not known whether Lewis antigens interact with colon tumor cells and modify their migration. Our aim was to detect the presence of binding sites on human tumor cells for Lewis(a/x) antigens and their sialylated derivatives in vitro and in vivo and to analyze their influence on migration of colon cancer cells. The immunocytochemical and histochemical levels of expression of the four Lewis antigens were quantitatively determined in four human colon cancer cell lines and in in vivo nude mice xenografts. The levels of expression of specific binding sites for these sugar epitopes were determined by synthetic neoglycoconjugates. The influence of binding of these carbohydrate ligands on cancer cell migration was quantitatively evaluated by computer-assisted phase-contrast videomicroscopy performed on Matrigel culture supports either left uncoated or coated with neoglycoconjugate presenting synthetic Lewis(a), sialyl Lewis(a), Lewis(x), or sialyl Lewis(x) antigens. The influence of the calcium concentration in the culture medium on the Lewis antigen-mediated effects was checked. Human colon cancer cells expressed significant amounts of specific binding sites detected by the synthetic probes in addition to the oligosaccharide epitopes. The expression levels differed considerably between the four cell lines and between in vitro and in vivo specimens. Cell migration analysis revealed that the four Lewis antigens markedly decreased the levels of migration of the HCT-15 and LoVo cancer cells. This effect depends on the calcium concentration in the culture medium. Binding sites for Lewis epitopes are present on colon cancer cells. The functional relevance of these sites is indicated by the negative influence on cell migration of a matrix containing the oligosaccharides as ligand parts.     

Monoclonal antibodies defining markers with apparent selectivity for particular haemopoietic cell types may also detect antigens on cells of neural crest origin

Monoclonal antibodies described as reacting with particular subsets of haemopoietic cells were screened against a variety of neuronal cell lines to further investigate their true specificity. While some reagents, e.g., monoclonal anti-cALL (J5), were found only reactive with haemopoietic cells, other monoclonals, e.g., BA1, BA2, NA134, OKT6, and OKT9, also bound to neuronal cells. Further investigation into the cross-reactivity of these antibodies to a variety of neural crest derived cell lines indicated that, with the exception of OKT9, differential binding patterns to different lines were obtained. This suggests that haemopoietic cell subsets and neural crest derived tissues can share similar differentiation antigens. For monoclonals OKT9 and BA2 this observation was confirmed biochemically, showing that it is not just antigenic determinants but similar molecular weight cell surface antigens that are shared between subsets of the two major cell types. A similar analysis using monoclonal antibodies raised against human neuronal cells or cell lines again indicates that some antigens appear unique to neural tissue while others are shared by haemopoietic cell subsets.     

Human monoclonal IgM antibodies from foetal B-cell hybridomas directed against a surface antigen on human tumour cells.

In order to assess the existence of B lymphocytes capable of producing anti-tumour antibodies in non-tumour-bearing individuals, human lymphocytes derived from foetuses and adults were fused with the heteromyeloma cell line CB-F7. By indirect immunofluorescence, 29 out of 4,472 IgM-producing hybridomas (from 8 foetuses and 8 adults) were shown to produce antibodies which bind to colon carcinoma lines Colo205 and SW620, Raji lymphoma cells and small cell carcinoma of the lung. In vitro growth of tumour cells recognized by these antibodies was inhibited. The antibodies also mediated complement-dependent cytotoxicity. All antibodies tested recognized a cell surface molecule of 55 kDa. Southern blot hybridization analysis of hybridoma DNA with a human JH probe showed that the hybridomas were derived from clonally unrelated B cells. These results demonstrate that human foetal and adult B cells from non-tumour-bearing individuals are able to produce IgM antibodies recognizing defined cell surface molecules expressed on some tumour cells.     

Generation and characterization of recombinant ScFv antibodies detecting Eimeria acervulina surface antigens.

In our previous attempt to generate monoclonal antibodies (MAbs) against coccidia parasites that more accurately reflect the natural avian humoral immune response, we produced two chicken B-cell hybridomas, 5D11 and 2-1. While both cell lines secreted antibodies reactive with sporozoites of Eimeria acervulina, they were produced in yields too low to conduct meaningful in vivo studies. To circumvent this problem, we produced four single chain variable fragment (scFv) antibodies from the V(H) and V(L) genes of hybridomas 5D11 and 2-1. The concentration of these recombinant antibodies expressed in E. coli and purified to homogeneity was 5-6 mg/L. Three of the antibodies exhibited antigen binding specificity to Eimeria surface antigens equivalent to that of the native MAbs. Nucleotide sequence analysis of the V(L) genes from hybridomas 5D11 and 2-1 and genomic DNA revealed vestiges of gene conversion with V(lambda) pseudogenes. These recombinant scFv antibodies will prove useful for further characterization of natural Eimeria surface antigens as potential vaccine candidates.     

Identification of Cytosolic Antigens from GW-39 Adenocarcinoma Cells by Crossed Immunoelectrophoresis and Immunofluorescence

Rabbits were immunized with a cytosolic fraction prepared from human adenocarcinoma cells of the colon (GW-39). The antibodies obtained were analyzed using crossed Immunoelectrophoresis and were found to precipitate 23 distinct antigens in the cytosolic fraction from colon tumor cells. After pre-absorption with human serum and plasma as well as acetone powders prepared from 2 normal human tissues and 4 normal hamster tissues, 1 major immunodominant cytosol antigen (CA-3) and two less intense immunoprecipitin peaks (CA-1 and CA-5) remained detectable by the crossed immunoelectrophoretic method. The preabsorptions with normal tissues were sufficiently complete to remove anti-carcinoembryonic antigen antibodies and showed that antigens CA-1, CA-3 and CA-5 are immunologically distinct from carcinoembryonic antigen.

Indirect immunofluorescence localization studies with pre-absorbed anti-cytosol antibodies and FITC-conjugated second antibody showed that these antigens were expressed in the cytoplasm and at the cell surface in several human colon tumor cell lines, their subclones and in primary colon tumor specimens.     

Identification of cytosolic antigens from GW-39 adenocarcinoma cells by crossed immunoelectrophoresis and immunofluorescence

Rabbits were immunized with a cytosolic fraction prepared from human adenocarcinoma cells of the colon (GW-39). The antibodies obtained were analyzed using crossed immunoelectrophoresis and were found to precipitate 23 distinct antigens in the cytosolic fraction from colon tumor cells. After preabsorption with human serum and plasma as well as acetone powders prepared from 2 normal human tissues and 4 normal hamster tissues, 1 major immunodominant cytosol antigen (CA-3) and two less intense immunoprecipitin peaks (CA-1 and CA-5) remained detectable by the crossed immunoelectrophoretic method. The preabsorptions with normal tissues were sufficiently complete to remove anti-carcinoembryonic antigen antibodies and showed that antigens CA-1, CA-3 and CA-5 are immunologically distinct from carcinoembryonic antigen. Indirect immunofluorescence localization studies with preabsorbed anti-cytosol antibodies and FITC-conjugated second antibody showed that these antigens were expressed in the cytoplasm and at the cell surface in several human colon tumor cell lines, their subclones and in primary colon tumor specimens.     

Selection of anti-cancer antibodies from combinatorial libraries by whole-cell panning and stringent subtraction with human blood cells

Traditional strategies for the identification of cell-surface cancer targets often fall short of their objective. For example, whole-cell panning of antibody libraries to isolate a diverse panel of antibodies directed against targets on cancer cells often identifies all immunogenic and/or abundant cell-surface antigens, not simply tumor-specific or tumor-associated antigens. Here we describe the use of stringent negative selection in combination with positive panning to increase tumor specificity and clinical relevance of selected antibodies. Sera from cancer cell-immunized mice showed strong binding to immunizing cancer cell lines but also cross-reacted strongly with human blood cells. Antisera blood cell binding was considerably decreased after stringent subtraction with human red blood cells (RBCs) and white blood cells (WBCs), yet cancer cell specificity was retained. In order to select for a higher percentage of clinically relevant antibodies for potential therapeutic use, stringent negative selection by RBC subtraction was employed in whole-cell panning of a disease-specific phage displayed antibody library on the prostate cancer cell line, PC-3. Isolated antibodies were found to bind to target antigens implicated in tumorigenicity and cancer cell migration and/or invasion, and included CD26, CDCP1, and the integrin complexes 2/β1, 3/β1, 5/β1, and 6/β4. Compared with traditional cell panning, this method considerably increased the selectivity of antibodies to tumor-associated antigens.     

The significance of HLA studies in human-human hybridomas

The technique of human-human (H + H) hybridoma is employed to generate human monoclonal antibodies. However, in contrast to mouse hybridomas, the human counterpart is very difficult to establish. It is still unclear what the precise reasons are for the failure to establish the human-human hybridoma technique as a routine. Analyses of HLA antigens of seven lymphocyte donors and 22 human cell lines generated by the H + H hybridoma technique have demonstrated the importance of the compatibility between human lymphocytes and the lymphoblastoid cell lines. Furthermore, there is a preferential expression of several HLA on human hybridomas (e.g. B51; B15; B18; B35). Screening several unrelated human lymphoblastoid cell lines available demonstrated preferred HLA antigens (A2, A3; A30/31; B5; B18; B15; B35), despite the fact that these lines were derived from subjects of different ethnic origins. It seems that HLA typing of donor lymphocytes and lymphoblastoid cell lines may help to increase the yield of H + H hybridomas.     

Production and ELISA application of bispecific monoclonal antibodies against fluorescein isothiocyanate (FITC) and horseradish peroxidase (HRP)

Hybrid hybridomas producing bispecific monoclonal antibodies reacting with both horseradish peroxidase (HRP) and fluorescein isothiocyanate (FITC) were obtained by fusing two hybridoma lines and selecting the fused cells using a fluorescence activated cell sorter (FACS). FITC was used to label different monoclonal antibodies and the bispecific antibodies acted as a linking agent between FITC-labelled antibody and the marker enzyme HRP. This system was used in enzyme immunoassays for the detection of different antigens. The results suggest a wide application of bispecific anti-FITC/anti-HRP antibodies as a detection system in EIA.     

Murine Bispecific Antibody 1A10 Directed to Human Transferrin Receptor and a 42-kDa Tumor-Associated Glycoprotein

Previously, we observed that bispecific antibodies (“antigen forks”) that bound to certain pairs of different tumor surface antigens could inhibit cell growth. The chemically linked heteroconjugate of MAb 454A12 (murine IgG1 recognizing human transferrin receptor) and 317G5 (murine IgG1 recognizing a 42-kDa tumor-associated glycoprotein) was particularly inhibitory toward human colorectal cancer cell lines, and the iron-chelating agent deferoxamine was found to augment inhibition of tumor cell growth by this antigen fork. Further experiments revealed that an antigen fork constructed by linking Fab′ fragments instead of whole antibodies retained activity, which led us to construct a fork-secreting hybrid hybridoma. Hybridoma 454A12 was fused with hybridoma 34F2 (murine IgG1 with the same specificity as 317G5). Hybrid hybridomas whose supernatants blocked binding of both 454A12 and 34F2 probes were further tested for the ability to block growth of SW948 human colorectal cancer cells in an MTT growth assay, and were chosen for subcloning. Ascites produced by clone 1A10 was purified by affinity and cation exchange chromatography. Purified 1A10 bispecific antibody showed growth inhibitory activity comparable to that of a chemically linked heteroconjugate of its parental antibodies 34F2 and 454A12. Adding deferoxamine greatly enhanced the inhibitory activity of 1A10 and effectively prevented regrowth of tumor cellsin vitro.By heterologously crosslinking two antigens that are coexpressed on many tumor cells, this bispecific antibody is able to inhibit tumor growth with enhanced selectivity.