Immunotherapy for recurrent colorectal cancers with human monoclonal antibody SK-1.

BACKGROUND: The human monoclonal antibody SK-1 recognizes a glycoprotein expressed on the majority of colon cancer tissues. In the current study, we evaluated the safety, toxicity and preliminary efficacy of escalating dosages of SK-1 in patients with advanced colon cancer. PATIENTS AND METHODS: SK-1 was administered intravenously at 2, 4 or 10 mg three times to three groups of patients with recurrent colon cancer. The clinical outcome and the induction of serum anti-idiotypic antibody (Ab2) were assessed periodically. RESULTS: The mean rate of serum CEA level increase declined significantly during the eight weeks following the treatment. In four patients, serum titer of anti-idiotypic IgG antibodies to SK-1 (Ab2) continued to increase following the treatment. CONCLUSION: HuMAb SK-1 was well-tolerated and can be safely administered. It was suggested that SK-1 natural antibody not only possessed direct cytostatic activity against colon carcinoma, but may also have induced carcinoma-related, anti-idiotypic antibody responses.

Identification of the immunoreactive peptide sequence for AgSK1, an adenocarcinoma-restricted antigen.

SK1, a human immunoglobulin M (IgM) monoclonal antibody was derived from regional nodal lymphocytes of a Dukes B colon carcinoma patient. The antigen recognized by the human monoclonal antibody (HuMab) SK1, termed AgSK1, was shown to be a two-chain glycoprotein with an apparent molecular weight range of 42-46 kDa and preferentially expressed by human adenocarcinomas, particularly human gastrointestinal malignancies. To identify the gene encoding the AgSK1 antigenic epitope, a cDNA expression library constructed in lambda gt22A using mRNA from the colon carcinoma cell line HT29 was screened and one of the isolated clones encoding a 1.5-kb cDNA, which showed strong immunoreactivity with HuMab SK1, was selected for further analysis. This clone consisted of an amino terminal open reading frame of 54 amino acids and the carboxyl terminal 20 amino acids of this protein coding region contained the antigenic epitope recognized by HuMab SK1.

Co-expression of tumor antigens and their modulation by pleiotrophic modifiers enhance targeting of human monoclonal antibodies to pancreatic carcinoma.

Cocktails of human monoclonal antibodies (HuMAbs) have been used to increase the likelihood of identifying heterogeneous cancer cells. We utilized 3 HuMAbs termed SK-1, GM4, and GMA1, which recognized a 42-46 kDa two chain structure, a 57 kDa antigen, and the ganglioside GD3, respectively. An estimated two dozen cell lines were tested for the coexpression of these antigens and this was found to be present only on pancreatic carcinoma cell line, PANC-1; A 24 hr treatment of PANC-1 cells with interferon gamma (IFN gamma; 100 units), interferon beta (IFN beta; 1000 units), as well as interferon alpha (IFN alpha; 1000 units) resulted in roughly a four fold increase in the co-expression of the 42-46 kDa/GD3 antigens as well as the 42-46 kDa/57 kDa antigens. After a 4 day incubation the co-expression of these antigens progressed and IFN alpha treatment had the most pronounced effect, which was 8 fold higher than background for the 42-46 kDa/57 kDa antigens, whereas IFN beta resulted in a five fold antigen upregulation. The pronounced effect of vinblastine on the co-expression of the 42-46 kDa/GD3 antigens (4 fold on day 1 and, 10 fold on day 4) and 42-46 kDa/57 kDa antigens on PANC-1 (5 fold on day 1 and 7 fold over background on day 4) cells can be seen at concentrations as low as 10(-7)M. Colchicine and vincristine dramatically enhanced co-expression of these tumor antigens on day 4 but not on day 1 PANC-1 cells. The expression of these antigens was also found to be cell cycle dependent.

Lessons learned about the therapeutic potential of the natural human immune response to lung cancer.

Lung cancer is the leading cause of cancer deaths; patients with this disease often develop antibodies to their own tumour antigens. TB94 is one such human antibody, and was obtained from the natural immune response from a patient with lung cancer. The data generated so far on TB94 suggest that this human antibody may have clinical applications in the diagnosis and treatment of lung cancer. Since humans make antibodies to tumour antigens, the intelligence of the human immune response can be exploited for the discovery of potential novel antigens. These novel antigens can then be developed for vaccine applications. Creating effective strategies to analyse systematically the natural human immune response to tumour antigens will open up new areas of immunotherapy for the control and eradication of disease.

A human natural antibody to adenocarcinoma that inhibits tumour cell migration.

We characterized a natural human antibody to adenocarcinomas and investigated the biological role of this Ab/Ag complex in cancer expansion. Human monoclonal antibodies (HuMAbs) were generated with hybridoma fusion methods using regional nodal lymphocytes of colon carcinoma patients. Among 1036 HuMAbs, only one, termed SK1, an IgM, was adenocarcinoma specific in the immunohistochemical study. The antigen recognized by SK1 (Ag-SK1) was a glycoprotein with a molecular weight of 42-46 kDa. The expression of Ag-SK1 on carcinoma cells varied according to the cell growth periods but was independent of cell cycle state as elucidated by two-colour fluorescence-activated cell sorter (FACS) analysis. A dot-blot analysis showed that the concentration of Ag-SK1 per total protein differed considerably among eight colon carcinoma cells examined and that the difference was closely correlated with the invasion capacity of the cells as assessed by a microchemotaxis assay. Furthermore, up to 87% of cell migration was inhibited by SK1 in a dose-dependent manner. These data suggested that Ag-SK1 is metabolized and expressed on highly invasive carcinoma cells. In addition, it appears that, although rare, some patients do mount an anti-cancer antigen response in their draining lymph nodes. A HuMAb such as SK1 may be a good candidate for the treatment of cancer invasion and metastasis.

Immunoscreening protocols for the identification of clinically useful antibodies and antigens.

The antigen-antibody interaction is a powerful tool for the immuno-screening of several diseases, including cancer and genetic disorders. The high specificity of monoclonal antibodies (mAbs) enables them to target antigens and form complexes that can be detected with enzymes, radionuclides, fluorescent dyes or other markers. The antibody molecule, which has an antigen binding site, can be used as an intact molecule or as a fragment, for example, F(ab)(2), Fab, Fv or scFv. Similarly, the antigen can also be varied. In this review, immuno-screening techniques that can be used to detect clinically relevant antibody-antigen interactions will be discussed.

Cloning and expression of the human tumor-specific antibody GM4.

The human monoclonal antibody GM4 was generated by fusing pooled lymphocytes from cancer patients with the lymphoblastoid cell line SHFP-1. Immunohistochemical staining of tumor and normal tissue indicated that this human IgG4 antibody preferentially reacted with melanomas and neuroblastomas. In this study, we demonstrate that GM4 recognizes a "vimentin-like" peptide sequence that we have termed AgGM4. To generate a recombinant derivative of this human antibody, we isolated and expressed the complete heavy and light chain genes. The entire coding sequence for both the heavy and light chains was isolated by RT-PCR using a set of degenerate 5' signal sequence specific primers and a 3' constant region primer. High level antibody synthesis and secretion was achieved in Chinese hamster ovary (CHO) cells using a vector designed to maximize expression. Western blot and FACS analysis indicated recombinant GM4 reacted with human tumor cell lines and AgGM4 in a manner similar to the antibody produced by the hybridoma cell line, demonstrating that the specificity of the antibody was not altered during molecular cloning.

Isotype-directed enrichment of B cells by magnetic beads in the generation of immunoreactive human monoclonal antibodies.

Pooled lymphocytes collected from cancer patients were mixed with a biotinylated murine MAb specific to human IgG4. To this were added streptavidin-conjugated magnetic beads. After magnetically separating the bead-lymphocyte complex, the B cells were washed and fused with the WIL-2 derived human fusion partner, SHFP-1. Subsequently derived human-human hybridomas were screened for IgG4 immunoreactivity to target tumor cell lines. Several hybridomas reacted with a variety of malignant cell types, including melanoma, neuroblastoma, and pancreatic tumor cells. One hybridoma in particular, designated SC-GM4, recognized an antigen by Western blot with an apparent molecular weight of 57 kDa. This facile approach of magnetically separating selected populations of lymphocytes should be relatively simple to apply to other antigens and antibodies to preselect the type, class, and properties of the desired MAb.

Tumor-associated antigens recognized by human monoclonal antibodies.

BACKGROUND: Nonhuman monoclonal antibodies (MoAbs) of desired specificities have been studied in cancer treatment and tumor targeting with minimal success. Attempts of using humanized chimeric antibodies have not improved significantly their clinical applications. We have engaged in the development of human MoAbs by incorporating the in vitro immunization protocols to the nodal lymphocytes of cancer patients. Three human MoAbs thus generated were found to be strongly reactive with various human malignancies. The antigens recognized by the three antibodies were selected for immunochemical and biochemical characterizations. METHODS: The antigens investigated were AgSK1, PA 1-2 and PA 3-1. The patterns of each antigen expression in various human cancer cell lines were studied by the immunocytochemical staining technique. The expression of AgSK1 in association with cellular proliferation was examined by the flow cytometry analysis. In studying the biochemical natures of these antigens, their sensitivities toward various chemical and physical treatments were determined. The antigens that were shown to be proteins were subjected to SDS-PAGE and Western blot for estimations of molecular weights. RESULTS: The AgSK1 was detected in 10 human carcinoma cell lines but in none of the melanoma cell lines. This suggests that SK1 may be an epithelial or carcinoma marker. The phenotypic expressions of AgSK1 were shown to be associated with proliferation of carcinoma cells. Biochemically AgSK1 was a sialophycoprotein with an estimated molecular weight of 42-44 kilodaltons (kDa). HuMAb PA1-2 demonstrated a unique staining pattern at both the cytoplasmic and intercellular interface. The stained filamentlike structures extending from cell to cell indicated that Ag PA1-2 might play a role in cellular interactions. Biochemically, Ag PA1-2 appeared to be an asialocarbohydrate. The Ag PA3-1 was a cytoplasmic glycoprotein expressed by all 13 cell lines. The estimated molecular weights of PA3-1 were 164, 104, and 40 kDa. CONCLUSIONS: Tumor-associated antigens recognized by the human MoAbs may be more relevant clinically than those recognized by the mouse immune system. Carcinoma-specific human MoAbs are desirable for cancer treatment and tumor localization.

Use of severe combined immunodeficient (SCID) mice to produce human hybridoma ascites.

The use of the severe combined immunodeficient mouse (SCID), CB-17/Icr//Imd-SCID, was investigated for the production of human hybridoma ascites containing human antibody. Human-human hybridomas, generated from the fusion of lymphocytes isolated from regional draining lymph nodes of cancer patients with the SHFP-1 fusion partner, were injected i.p. at various cell concentrations into pristane-primed SCID mice. Ascites growth was typically observed at 7-14 days postinoculation. No significant differences in ascites yield or production were observed between IgG- and IgM-secreting hybridomas. Yields of immunoreactive human immunoglobulin ranged from approximately 0.5 to 3 mg/ml of harvested ascites. The ease and relatively low cost suggest that the use of SCID mice is preferred over conventional and costly large-scale industrial procedures.

Generation of human monoclonal antibodies against colon cancer.

Lymphocytes from regional lymph nodes of patients with colon cancer were fused with a human lymphoblastoid cell line with or without in vitro immunization. The efficacy of these two protocols for the generation of human monoclonal antibodies against colon cancer was investigated. The hyperplastic lymph nodes adjacent to the tumor were the best source of B lymphocytes. Fusion frequency and the number of tumor-reactive clones were markedly increased when the in vitro immunization protocol was applied prior to fusion. As a stimulant in in vitro immunization, the supernatant of pokeweed mitogen-stimulated T lymphocytes was superior to the supernatant of mixed lymphocytes culture. Carcinoembryonic antigen at 20 micrograms/L seemed to be the optimal dose for in vitro immunization. The reactivities of human monoclonal antibodies thus generated were measured by enzyme-linked immunosorbent assay and confirmed by immunoperoxidase staining. Combining in vitro immunization with lymphocytes of cancer patients may lead to the successful production of clinically useful human monoclonal antibodies.

Human monoclonal antibodies to nuclear antigens.

Human lymph node lymphocytes from cancer patients were fused with either the UC 729-6 or SHFP-1 human fusion partners. Resulting human-human hybridomas were tetraploid, expressed markers from both parent cells, and secreted approximately 1 microgram Ig/10(6) cells/ml/day. Immunofluorescence analysis of some of the human MAbs with a panel of normal and malignant cell lines revealed a staining pattern of only the nuclear region. One IgM secreting hybridoma, TLN1F4, derived from a teratocarcinoma lymph node, predominantly stained the nuclear regions of adherent tumor cell lines and no hematopoietic cell lines or normal fibroblasts. PLN3C8, an IgG1 secreting hybridoma, derived from a prostate carcinoma lymph node, predominantly stained the nucleolus of LnCap, a a carcinoma of the prostate cell line. CLN2E5, an IgM secreting human hybridoma, derived from a carcinoma of the cervix lymph node, predominantly stained both cytoplasmic and nuclear components to tumor cell lines and not normal fibroblasts or hematopoietic cell lines. These data suggest that the immune response occurring within regional draining lymph nodes is capable of recognizing nuclear-associated antigens.

Serum-free media in hybridoma culture and monoclonal antibody production.

The replacement of serum in hybridoma cultures is considered. The focus is on the effects of serum-free media on hybridoma growth and monoclonal antibody secretion. Comparative literature data with serum supplemented cultures are discussed with an analysis of serum-free formulations and selection rules for the serum-free ingredients. In general, serum-free media which are "lipid rich" can sustain cell growth rates approaching that of serum supplemented cultures. Specific antibody secretion rate, however, is usually higher in serum-free media, irrespective of the lipid content.

Molecular biotherapy with human monoclonal antibodies.

Human monoclonal antibodies are theoretically superior to murine monoclonal antibodies for many reasons. However, there are many hurdles in their preparation and clinical use, including the source of B lymphocytes, immortalization techniques, screening methods, scale-up limitations, purification steps, and the problems associated with conjugating to cytotoxic substances. Chimeric mouse/human antibodies are an alternative approach. Optimum clinical utilization will require a better understanding of target antigens and the ability to formulate cocktails.

Growth of human-human hybridomas in serum-free media enhances antibody secretion.

Human-human hybridomas derived from fusing lymph node lymphocytes with UC 729-6 were adapted to grow in commercially available serum-free medium and were compared with serum-supplemented [10% fetal bovine serum (FBS)] cultures. Over a 6-d period, no significant changes occurred in the growth of the cells in 10% FBS or serum-free medium. In cultures supplemented with 10% FBS more total proteins were secreted than in serum-free cultures. However, there was an enhanced secretion of three- to four-fold of both immunoreactive human IgG and IgM under serum-free conditions compared to serum-supplemented conditions. Serum-free conditions may provide the appropriate milieu for the increased level of Ig secretion from human hybridomas derived from UC 729-6 in that there are no inhibitors that may be present in serum.

The generation of Ig-secreting UC 729-6 derived human hybridomas by electrofusion.

UC 729-6, a 6-thioguanine resistant human lymphoblastoid B cell line, was fused with human lymphocytes by electrofusion. Resulting human-human hybridomas were tetraploid, expressed markers derived from both fusion parents, and secreted approximately 1 microgram Ig/10(6) cells/ml/day. Cells to be used for electrofusion were washed in 0.3M mannitol, and fusions were performed with glass slides, 1.0 ml, and 50.0 ml chambers. Fusion sequences consisted of alignment, compression, and the fusion event itself. The optimal cell concentration for electrofusion was 5 X 10(6)/ml. Fusion efficiencies of human lymphocytes were ranked as follows: lymphoma cells greater than lymph node lymphocytes greater than PBL. 80% of the lymphoma hybridomas, 60% of the lymph node hybridomas, and 40% of the PBL hybridomas were Ig secretors. These data demonstrate that the UC 729-6 cell line is a suitable vector for generating human-human hybridomas by electrofusion.

Immortalization of human lymphocytes from a tumor-involved lymph node.

Lymphocytes isolated from a regional draining lymph node of a patient with a carcinoma of the vulva were fused with human UC 729-6 cells. The generated human-human hybridomas were tetraploid with the 21p+ chromosomal marker of UC 729-6, expressed HLA derived from the patient's cells, and have been stable in culture for 2 yr. Supernatants from six immunoglobulin G (IgG)-secreting hybridomas were found to have broad reactivity profiles with human carcinoma cells and no reactivity with hematopoietic cell lines (B- and T-cells, myelomas, leukemias, lymphomas), red blood cells, peripheral blood lymphocytes, and normal fibroblasts. All six human IgG monoclonal antibodies reacted with the A431 cell line, an epidermoid carcinoma of the vulva, similar to the patient's cancer. The supernatant of one hybridoma, which phenotyped as having T-cell parentage, enhanced the cloning efficiency of human hybrids, suggesting the presence of a growth factor(s). In serum-supplemented cultures these hybrids secreted 200 ng to 3 micrograms of IgG/ml/10(6) cells/24 h and in serum-free medium, an enhanced production of 1 to 9 micrograms of IgG/ml/10(6) cells/24 h. One IgG monoclonal antibody, VLN3G2, precipitated a single chain protein with an apparent molecular weight of 48,000 from Nonidet P-40 extracts of A431 cells. Altogether, the data suggest that regional draining lymph nodes of cancer patients are highly immunoreactive and contain B-cells whose immunoglobulin recognizes putative tumor-associated antigens.

Genetically stable human hybridomas secreting tumor-reactive human monoclonal IgM.

Human lymphocytes obtained from regional draining lymph nodes of patients with cancers of the cervix, kidney, prostate, and vulva were immortalized by polyethylene glycol-mediated somatic cell hybridization with either human UC 729-6 or murine P3-NS-1-Ag4-1. Four reactive human IgM-secreting hybridomas, termed CLNH5, MHG7, VLN1H12, and WLNA67 were isolated and characterized. Hybrids obtained by fusions with UC 729-6 have remained tetraploid for over 18 months, have doubling times from 25-35 hours, and have continuously secreted approximately 0.5-5.0 micrograms IgM/10(6) cells/ml per day. MHG7, a mouse-human hybrid, required subcloning every 4-6 months to maintain human IgM secretion. Binding of these human monoclonal antibodies (MoAbs) against a panel of cell lines was assessed by an enzyme-linked immunoassay (EIA). CLNH5 reacted with carcinomas of the cervix, lung, and vulva. MHG7 reacted with carcinomas of the prostate, stomach, and vulva. VLN1H12 reacted with carcinomas of the cervix, lung, prostate, stomach, and vulva. WLNA6 reacted strongly with a carcinoma of the lung. All four human MoAbs failed to react by EIA with hematopoietic cells or normal fibroblast cell lines. The data suggest that regional draining lymph nodes of cancer patients have been primed to produce antibodies against antigens associated with tumor cells and that UC 729-6 served as a genetically suitable vector for the capture and immortalization of these Ig-secreting B lymphocytes.