Methods for production of monoclonal antibodies with specificity for human lung cancer cells

Summary We have developed a screening strategy and technology to produce monoclonal antibodies with specificity for human lung cancer cells. Mice and rats were immunized with well-characterized tissue culture lines of human small cell lung cancer (SCLC), mouse myeloma x spleen hybrids formed by the technique of Kohler and Milstein, and the resulting culture fluids were screened for antibody binding phenotype using a radioimmunoassay. To facilitate testing large numbers of culture fluids, a 96-well, microtiter based, resuable, replicating device was designed. Using this, many hybridoma culture fluids were replica plated for antibody binding tests on a series of human target cell plates. Hybrids producing antibodies that reacted with the immunizing SCLC line and another independent SCLC line, but not with autologous B-lymphoblastoid cells derived from one of the patients, were identified, selected, and then repeatedly recloned using the same screening strategy. With this technology, hybridomas representing less than 0.5% of all hybrids generated could be isolated and stable antibody producing cultures derived. Such antibodies reacted with a panel of well-characterized SCLC lines and SCLC samples taken directly from patients but not with a variety of normal tissues. Using these antibodies we can demonstrate: tumor cell contamination of bone marrow specimens, marked heterogeneity of antigen expression on cells within individual SCLC lines and individual patients, and inhibition of clonal growth of SCLC lines in soft agarose assays. All of these findings have potential clinical and cell biologic application. Presented in the symposium on The Biology of Hybridomas at the 32nd Annual Meeting of the Tissue Culture Association, Washington, D.C., June 7–11, 1981. This symposium was supported in part by the following organizations: Bethesda Research Laboratories, Cetus Corporation, Hybritech Incorporated, MAB-Monoclonal Antibodies, Inc., National Capital Area Branch of the Tissue Culture Association, New England Nuclear Corporation, and Ortho Pharmaceutical Corporation.     

Hybridomas: A new dimension in biological analyses

Summary Since the first report of hybridomas producing monoclonal antibodies by Kohler and Milstein in 1975, this technique has spread to nearly all areas of biological, biochemical, and biomedical research. Watching the use of these methods spread from immunologists to cell biologists, developmental biologists, biochemists and to other biological disciplines and observing the nearly logarithmic increase in publications using these reagents has been in itself fascinating and informative. An overview of the development of this technology and its applications is presented including the use of monoclonal antibodies to study cell surface molecules, differentiation antigens, receptors, and histocompatibility antigens. The use of these antibodies to analyze microorganisms and parasitic antigens as well as their use in the genetic analysis of human cell surface antigens and the detection of polymorphic variation in enzymes and other proteins is discussed. Examples of the application of monoclonal reagents to the study of tumor cell biology including the labeling of metastatic tumor cells and the detection of cell surface molecules implicated in the regulation of growth control and cell division are provided. Presented in the symposium on The Biology of Hybridomas at the 32nd Annual Meeting of the Tissue Culture Association. Washington, D.C., June 7–11. 1981. This symposium was supported in part by the following organizations: Bethesda Research Laboratories, Cetus Corporation, Hybritech Incorporated, MAB-Monoclonal Antibodies, Inc., National Capital Area Branch of the Tissue Culture Association, New England Nuclear Corporation, and Ortho Pharmaceutical Corporation.     

Hybridoma monoclonal antibodies in the analysis fo human cell surface antigens

Summary In the present paper, we will summarize studies we have performed on two distinct human lymphocyte cell surface antigens defined by monoclonal antibodies: Leu-1 and HLA-DR. Presented in the symposium on The Biology of Hybridomas at the 32nd Annual Meeting of the Tissue Culture Association, Washington, D.C., June 7–11, 1981. This work was supported by USPHS-NIH Grants CA-21223, AI-11313, and CA-09302. This symposium was supported in part by the following organizations: Bethesda Research Laboratories, Cetus Corporation, Hybritech Incorporated, MAB-Monoclonal Antibodies, Inc., National Capital Area Branch of the Tissue Culture Association, New England Nuclear Corporation, and Ortho Pharmaceutical Corporation.     

Monoclonal antibody specific for T cell-derived human IgE binding factors

A B cell hybridoma secreting monoclonal antibody against human IgE binding factors was obtained by immunization of BALB/c mice with partially purified IgE binding factors, and fusion of their spleen cells with SP-2/0-AG14 cells. The monoclonal antibody bound all of the 60,000, 30,000, and 15,000 dalton IgE binding factors from two T cell hybridomas and those from activated T cells of a normal individual. The antibody bound both IgE-potentiating factors, which had affinity for lentil lectin, and IgE-suppressive factors, which had affinity for peanut agglutinin. However, the monoclonal anti-IgE-binding factor bound neither Fc epsilon R on RPMI 8866 cells nor IgE binding factors from the B lymphoblastoid cells. A monoclonal antibody against Fc epsilon R on B cells (H107) bound the 60,000 and 30,000 dalton IgE binding factors from both T cell hybridomas and RPMI 8866 cells but did not bind the 15,000 dalton IgE binding factors from either T cells or B cells. The results indicate that T cell-derived IgE binding factors have a unique antigenic determinant that is lacking in both Fc epsilon R on B cells and B cell-derived IgE binding factors. The anti-IgE binding factor and anti-Fc epsilon R monoclonal antibodies both failed to stain cell surface components of IgE binding factor-producing T cell hybridomas. However, both antibodies induced the T cell hybridoma to form IgE binding factors. The results suggest that the T cell hybridomas bear low numbers of Fc epsilon R that share antigenic determinants with IgE binding factors secreted from the cells.     

Hepatitis C virus (HCV)-induced immunoglobulin hypermutation reduces the affinity and neutralizing activities of antibodies against HCV envelope protein

Hepatitis C virus (HCV) often causes persistent infection despite the presence of neutralizing antibodies against the virus in the sera of hepatitis C patients. HCV infects both hepatocytes and B cells through the binding of its envelope glycoprotein E2 to CD81, the putative viral receptor. Previously, we have shown that E2-CD81 interaction induces hypermutation of heavy-chain immunoglobulin (V(H)) in B cells. We hypothesize that if HCV infects antibody-producing B cells, the resultant hypermutation of V(H) may lower the affinity and specificity of the HCV-specific antibodies, enabling HCV to escape from immune surveillance. To test this hypothesis, we infected human hybridoma clones producing either neutralizing or non-neutralizing anti-E2 or anti-E1 antibodies with a lymphotropic HCV (SB strain). All of the hybridoma clones, except for a neutralizing antibody-producing hybridoma, could be infected with HCV and support virus replication for at least 8 weeks after infection. The V(H) sequences in the infected hybridomas had a significantly higher mutation frequency than those in the uninfected hybridomas, with mutations concentrating in complementarity-determining region 3. These mutations lowered the antibody affinity against the targeting protein and also lowered the virus-neutralizing activity of anti-E2 antibodies. Furthermore, antibody-mediated complement-dependent cytotoxicity with the antibodies secreted from the HCV-infected hybridomas was impaired. These results suggest that HCV infection could cause some anti-HCV-antibody-producing hybridoma B cells to make less-protective antibodies.     

Immunization with Leishmania-specific T cell not B cell lines or hybridomas can modulate the response of susceptible mice infected with viable parasites

Monoclonal antibodies (mAb), T cell lines, and T or B cell hybridomas were prepared from BALB/c, CBA, or E1 mice infected with Leishmania mexicana. Various mAb were produced which inhibited the growth and motility of parasites in vitro. T cell lines (hybridomas) were screened for their ability to release interleukin 2 on specific antigen exposure. Passive transfer of mAb or T cell lines to infected adult mice caused little perturbation of parasite growth. Recipient naive mice were immunized with purified Ig or irradiated cells from these sources and were subsequently infected with viable parasites. Only preimmunization with T cell lines (hybridomas) led to exacerbation of parasite growth, although enzyme-linked immunosorbent assays could detect the production of anti-idiotype antibodies in mAb (B cell hybridoma)-immunized mice. Either nylon wool-purified T cells or serum Ig from T cell-immunized mice could be used to immunize further naive recipients for protection against parasite growth. These data have implications for the development of anti-idiotype vaccines for Leishmania antigens.     

Use of I region-restricted, antigen-specific T cell hybridomas to produce idiotypically specific anti-receptor antibodies

Murine T cell hybridomas bearing receptors for antigen plus I region gene products were used as immunogens in mice in an effort to raise anti-receptor antisera. The antisera were assayed for anti-receptor activity by the ability to inhibit interleukin 2 production by the T cell hybridomas stimulated by antigen and I region expressing antigen-presenting cells. The T cell hybridomas used in these experiments were made by fusing antigen-specific, I region-restricted BALB/c T cell blasts to the AKR thymoma, BW5147. Three groups of mice were immunized with the T cell hybridomas: (BALB/c X AKR)F1 animals, syngeneic to the hybridoma; (BALB.B X aKR)F1 animals, differing from the hybridomas at H2; and (C.B20 X AKR)F1 animals, differing from the hybridomas at Igh. Mice were immunized multiple times and sera from individual animals were assayed for anti-receptor antibodies. In all groups, some mice produced anti-receptor antibodies by the criterion that they were inhibitory in the assay mentioned above. The frequency of mice producing these inhibitory antibodies varied considerably between groups, with the (BALB.B X AKR)F1 animals producing these antibodies most frequently, and the (BALB/c X AKR)F1 animals producing them least often. All inhibitory antisera were idiotypically specific; they inhibited the response of the immunizing T cell hybridomas, but not the responses of closely related hybridomas with different specificities. Moreover, when they could be absorbed, the inhibitory antibodies could only be absorbed by the immunizing hybridoma. It is hoped that these antisera, and B cell hybridomas prepared from the immunized animals, will be useful in the elucidation of the structure of the receptors for antigen plus I region products on T cells.     

Monoclonal antibodies against Mycoplasma hyorhinis: A secondary effect of immunization with cultured cells

Monoclonal antibodies were prepared against two different human tumour cell lines, the melanoma cell line SK-Mel-25 and the acute lymphoblastic leukemia T cell line CCRF-CEM. Presence of antibodies against human tumour cells in the supernatants of hybridoma cultures was tested by binding of ¹²⁵I-F(ab′)₂ anti-mouse IgG. On two occasions a hybridoma culture, initially selected for subsequent cloning as it seemingly produced antibodies against tumour cells, was later found to produce monoclonal antibodies specific for Mycoplasma hyorhinis. In immunofluorescent staining patchy structures were visible which seemed to be attached to the cell surface. By combined staining with FITC-conjugated anti-mouse immunoglobulin for monoclonal antibody, Evans blue for cytoplasm and Hoechst compound no. 33258 for DNA, the reaction against mycoplasma could be recognized. These results demonstrate that if cultured cells are used for preparation of monoclonal antibodies, there is a good chance that the selected hybridomas may produce antibodies against ‘culture artifacts’ such as mycoplasmas, in addition to the target antigens. Thus mycoplasma contamination of cell cultures poses a serious problem in the hybridoma research and the testing system for antibody specificity should be carefully monitored.     

Mapping of SJL/J reticulum cell sarcoma tumor-associated Ia antigens by T cell hybridomas: characterization of tumor-specific and shared epitopes detected on IE+ allogeneic cells

Previous studies have suggested that reticulum cell sarcoma (RCS) tumor cells of SJL/J (IA + IE-) mice express neospecificities that are related to antigenic specificities characteristic of IE+ allogeneic cells. These neospecificities have also been suggested to play a role in the strong syngeneic antitumor proliferative response as well as in regulating RCS growth in vivo. The present studies characterize four RCS tumor-specific T cell hybridoma clones prepared from the fusion of BW5147 thymoma with T cells derived from lymph nodes of tumor-bearing mice. Upon stimulation, these hybridomas secrete IL 2 in the supernatant. Two hybridomas responded to RCS to IE+k and to IE+d allogeneic cells, respectively, and the other two hybridomas were tumor specific. The specificity of these hybridomas was assessed by response to both spontaneous and transplantable RCS lines and failure to stimulate a response by either normal or LPS-induced B cell blasts from the host SJL/J cells. The epitopes recognized by the T cell hybridomas were examined by the ability of several monoclonal antibodies to inhibit the IL 2-induced response by the T cell hybridomas. Antibodies directed against the IABs polypeptide of the IA hybrid molecule blocked the antitumor response by all four hybridomas. However, the response to allogeneic IE+ cells was not blocked by anti-IAs antibody but was blocked by antibodies directed against either the IAk,d or IEk,d hybrid molecules or the corresponding alpha- or beta-chains. The response to both RCS and allogeneic cells was blocked by monoclonal antibodies directed against L3T4 antigens on the T cells. Based on the exquisite specificity of the T cell receptors, the results here demonstrate that RCS tumor cells express on their surface both tumor-specific I-A-associated epitopes and Ia-associated antigenic specificities that are shared with IE+ allogeneic cells. The present studies of adapting T cell hybridomas and blocking antibodies proved useful to characterize and map distinct tumor-associated epitopes on the surface of tumor cells. These findings, when combined with structural studies, should help unravel the molecular complexity of tumor-associated antigens.     

Production and specificity of monoclonal antibodies and polyclonal antibodies to Escherichia coli

Monoclonal antibodies were produced to whole cells of heat-treated Escherichia coli. Balb/c mice were immunized with a pool of five strains of heat-treated E. coli , and the resulting hybridomas were screened by indirect immunoassay. E. coli strains other than those used for immunization were used for screening to detect hybridomas producing antibody that reacted with a large number of E. coli strains. Of 864 hybridomas, 32 reacted strongly with either two or all three of the strains used for screening; 15 were successfully cloned. Antibody from hybridoma 6H2 reacted with 35 of 68 (51%) E. coli ; of 13 non- E. coli tested, only Enterobacter agglomerans was weakly positive. Hybridoma 9B12 antibody reacted with all six E. coli tested. Hybridoma 9B12, however, stopped producing antibody. Five hybridomas produced antibody which reacted with a majority of the bacteria tested whereas antibodies from two other hybridomas reacted with several E. coli and non- E. coli. Polyclonal antibodies produced to two strains of E. coli varied in the numbers of E. coli with which they reacted; both antisera cross-reacted with several non- E. coli.     

Production and specificity of monoclonal antibodies and polyclonal antibodies to Escherichia coli

Monoclonal antibodies were produced to whole cells of heat-treated Escherichia coli. Balb/c mice were immunized with a pool of five strains of heat-treated E. coli, and the resulting hybridomas were screened by indirect immunoassay. E. coli strains other than those used for immunization were used for screening to detect hybridomas producing antibody that reacted with a large number of E. coli strains. Of 864 hybridomas, 32 reacted strongly with either two or all three of the strains used for screening; 15 were successfully cloned. Antibody from hybridoma 6H2 reacted with 35 of 68 (51%) E. coli; of 13 non-E. coli tested, only Enterobacter agglomerans was weakly positive. Hybridoma 9B12 antibody reacted with all six E. coli tested. Hybridoma 9B12, however, stopped producing antibody. Five hybridomas produced antibody which reacted with a majority of the bacteria tested whereas antibodies from two other hybridomas reacted with several E. coli and non-E. coli. Polyclonal antibodies produced to two strains of E. coli varied in the numbers of E. coli with which they reacted; both antisera cross-reacted with several non-E. coli.     

Deletion of the κ genes from mouse hybridomas established with NS-1 myelomas

Monoclonal antibodies (mAbs) of the IgG class produced by mouse hybridomas raised with NS-1 myelomas have been shown to contain two types of immunoglobulin light (κ) chains derived from the myelomas and antigen-stimulated spleen lymphocytes, and the hybridomas produce three mAb species with light chain heterogeneity (Abe and Inouye, 1993). In the present study, 9 hybridoma lines secreting homogeneous mAbs have been isolated from 63 lines cloned from an established hybridoma line producing three mAbs. They secrete homogeneous mAbs containing light chains derived from either myeloma or spleen cells. They contain either κ gene derived from the respective cells, and the other gene was deleted during the cultivation. The deletion frequency of the κ gene of myelomas is 3 times higher than that of spleen cells, although 80–85% of hybridomas reach the stable state containing both κ genes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Contribution of antigen processing to the recognition of a synthetic peptide antigen by specific T cell hybridomas

Most antigens recognized by T cells require unfolding or partial degradation (processing) followed by association with Major Histocompatibility Complex (MHC) molecules. We examined the processing requirements for the presentation of antigen to two T cell hybridomas which recognize the alpha-helical synthetic polypeptide antigen Poly 18, Poly [EYK(EYA)5], in association with I-Ad. Hybridoma A.1.1 responds to EYK(EYA)4 as the minimum antigenic sequence while hybridoma B.1.1 recognizes (EYA)5 sequence. It was found that these hybridomas responded to Poly 18 and to minimum peptide sequences presented by glutaraldehyde and chloroquine treated antigen presenting cells (APC), suggesting that antigen processing is not a requirement for the activation of these cells. The reactivity pattern of hybridoma B.1.1 in the presence of glutaraldehyde fixed APC revealed that antigens containing lysine were presented with much less efficiency than antigens without lysine, suggesting an interaction of these residues with the antigen presenting cell surface. We discuss the possibility that alanine residues in the alpha-helical Poly 18 form a hydrophobic ridge which may be required for appropriate interaction between antigen, the T cell receptor, and MHC molecules.     

Hybridoma anti-DNA autoantibodies from patients with rheumatoid arthritis and systemic lupus erythematosus demonstrate similar nucleic acid binding characteristics

Hybridoma anti-DNA antibodies have been generated from the fusion of the GM 4672 lymphoblastoid line with peripheral blood lymphocytes from four normal subjects, nine patients with rheumatoid arthritis (RA), and 13 patients with systemic lupus erythematosus (SLE). A total of 441 hybridoma clones were obtained, of which 37 secreted anti-DNA autoantibodies. The nucleic acid binding characteristics of the anti-DNA antibodies produced by two hybridomas from normal subjects, nine hybridomas from RA patients, and 18 hybridomas from SLE patients are reported. The hybridoma anti-DNA antibodies from all three groups showed similar antigen-binding characteristics for denatured DNA (dDNA), native DNA (nDNA), poly(I), poly(dT), and cardiolipin, by both direct binding and competitive binding analyses. One difference noted between normal-derived anti-DNA antibodies and autoimmune-derived antibodies was the inability of the former to react with z-DNA. However, this requires further substantiation with larger numbers of normal-derived clones. The broad overlap of reactivity to nucleic acid antigens among individual anti-DNA autoantibodies found in two clinically different autoimmune diseases, namely RA and SLE, suggests that the pathogenicity of anti-DNA autoantibodies may bear no relationship to their nucleic acid antigen-binding characteristics.     

Somatic mutation and clonal expansion of B cells in an antigen-driven immune response.

The variable (V) regions of three closely related monoclonal antibodies produced by hybridomas which had been isolated from a single mouse were sequenced at the level of the mRNA. The sequences and the restriction analysis of the immunoglobulin loci carried by the hybridoma cells indicate that the antibodies are derived from cells belonging to a single B cell clone. The sequence data imply a high frequency and stepwise occurrence of somatic point mutations in the expressed V region genes and substantial clonal expansion of B cells in the mouse. The mutations appear to be randomly introduced into heavy and light chain V region genes. Mutations are also seen in the complementarity determining regions which may thus have been involved in the selection of the cells producing the three antibodies.     

Ovarian substance induces steroid production in cultured granulosa cells

Summary The rat ovary produces an apparently low molecular weight substance that mimics the action of follitropin (FSH) on ovarian granulosa cells in culture. Similar to FSH action, the ovarian substance (OS) induces temporal cell rounding and, later on, intensive progestin production. However, unlike FSH, OS does not induce accumulation of cyclic AMP (cAMP) in the granulosa cells. The ovarian factor cannot be cAMP as its action is not abolished by phosphodiesterase (PDE) treatment. Neither is it a possible PDE inhibitor, as it does not augment cAMP accumulation in granulosa cells or Friend erythroleukemic cells induced by FSH or PGE₁, respectively. The factor is still active after heating for 20 min at 90° C but is rapidly inactivated by alkali treatment. In addition, treatment with various proteases did not abolish the steroidogenic activity. These findings suggest a possible novel intraovarian regulator of the granulosa cell function. Presented in the symposium on Plant and Animal Physiology in Vitro at the 33rd Annual Meeting of the Tissue Culture Association, San Diego, California, June 6–10, 1982. This work was supported by the United States-Israel Binational Science Foundation, Grant 2656/81. This symposium was supported in part by the following organizations: Bellco Glass, Inc., California Branch of the Tissue Culture Association, Collaborative Research, Hana Media, Hybridtech, K C Biological, Inc., and Millipore Corporation.     

Colorectal carcinoma antigens detected by hybridoma antibodies.

Hybridoma cells which secrete colorectal carcinoma-specific antibodies have been produced and used to study the antigenic structure of these tumor cells. Nineteen antibodies have been studied in detail, and 15 of these are colorectal carcinoma specific. Only two antibodies reactive with carcinoembryonic antigen (CEA) have been discovered and five other antibodies that react with distinct epitopes on the cell surface have been defined. Several antigens with distinct molecular characteristics have been shown to exist by use of hybridoma antibodies. Six hybridoma antibodies have been shown to mediate antibody-dependent cell-mediated cytotoxicity (ADCC).     

Ceruloplasmin,a copper-transport protein,can act as a growth promoter for some cell lines in serum-free medium

Summary Ceruloplasmin (Cp) is a copper-transport protein with ferric oxidase activity found in high concentrations in the plasma of all vertebrates. Five cell lines (TR-1, TR-M, TR-ST, TM₃, and TM₄) derived from the testis can be grown in hormone-supplemented serum-free medium. Cp stimulates the growth of four of these five cell lines in serum-free medium. The growth stimulation by Cp is not affected by the addition or deletion of free copper, nor does copper itself elicit any significant growth response. Cp can stimulate growth also in the absence of TF suggesting that it is not acting solely to promote Fe(III)-TF binding. A strong interaction is seen between Cp and high density lipoprotein (HDL), with the presence of either decreasing the growth-promoting activity of the other. It is suggested that these cell lines may provide an ideal system for studying the action of Cp at the cellular level. Presented in the symposium on Plant and Animal Physiology in Vitro at the 33rd Annual Meeting of the Tissue Culture Association, San Diego, California, June 6–10, 1982. This work was supported by NIH P50 Grant HO-13541. I am grateful to Ms. Florence Kaczorowski for her expert technical assistance. This symposium was supported in part by the following organizations: Bellco Glass, Inc., California Branch of the Tissue Culture Association, Collaborative Research, Hana Media, Hybridtech, K C Biological, Inc., and Millipore Corporation.