Anti-idiotypic antibodies bear the internal image of a human tumor antigen.

Goat antibodies to idiotypes (anti-idiotypic antibodies; Ab2) that recognize an idiotype associated with the combining site of a BALB/c mouse IgG2a monoclonal antibody (Ab1) to human gastric carcinoma were used to immunize BALB/c mice and rabbits. A monoclonal anti-anti-idiotypic antibody (Ab3) of IgG1 isotype was obtained after immunization of mice. The Ab3 and the Ab1 showed identical binding specificities and bound with similar avidities to the same tumor antigen. The induction of Ab1-like Ab3 by Ab2 was not restricted to mice, since Ab3 could also be induced in rabbits. Both the mouse- and the rabbit-derived Ab3 bound the same gastrointestinal cancer-associated antigen as Ab1. These findings indicate that Ab2 induced the formation of antigen-specific Ab3, probably because it bears the internal image of the tumor-associated antigen. This Ab2 may therefore have potential for modulating the immune response of cancer patients to their tumors.

Levels of disialogangliosides in sera of melanoma patients monitored by sensitive thin-layer chromatography and immunostaining.

Levels of GD2, GD3, and 9-O-acetyl GD3 were monitored in sera of patients with melanoma and healthy adults with two monoclonal antibodies that specifically detect these gangliosides. By direct measurement of radioactivity in the immunolabeled chromatogram, GD2 could be detected in normal sera at 2 ng/mL. Serum levels of GD2 and GD3 were increased approximately sixfold and fivefold, respectively, in patients with disseminated melanoma, compared with those of healthy adults. The acetylated derivative of GD3, which is highly specific for melanoma cells, was not detected in serum. This sensitive assay allows the quantitation of tumor-associated gangliosides that are circulating in sera of melanoma patients.

Colorectal carcinoma invasion inhibition by CO17-1A/GA733 antigen and its murine homologue.

BACKGROUND: The gastrointestinal carcinoma antigen GA733 is a potential target for passive and active immunotherapy for patients with colorectal carcinoma. This antigen has been characterized previously as a homophilic adhesion (i.e., adhesion to self) protein, but the functional consequences of homophilic adhesion for tumor growth and invasion are unknown. The availability of a murine homologue of GA733, i.e., murine epithelial glycoprotein (mEGP), allows for functional analysis of cell adhesion as it relates to tumor growth and invasion, both in vitro and in vivo. METHODS: CT-26 murine colorectal carcinoma cells were transfected with complementary DNAs encoding either the human or the murine antigen. GA733- or mEGP-producing cells were evaluated for homophilic adhesion, growth on plastic surfaces, colony formation in soft agar, and invasion through a reconstructed basement membrane (Matrigel). mEGP-producing cells were also examined for their capacity to metastasize in mice. Reported P values are two-sided. RESULTS: Compared with control cells, mEGP-producing cells showed significantly lower growth rates, colony formation, and invasion through Matrigel in vitro (all P values <.05). Compared with vector-only transfected cells and parental cells, mEGP-producing cells showed a reduction in metastatic potential in syngeneic immunodeficient and immunocompetent mice (all P values <.05). In contrast to mEGP-transfected cells, GA733-transfected cells did not exhibit significantly reduced growth or colony formation in vitro (all P values >.05). However, GA733-transfected cells did show reduced invasion through Matrigel compared with vector-only transfected cells or parental cells (all P values <.05). CONCLUSION: The adhesion proteins GA733 and mEGP inhibit invasion of tumor cells.

Primary melanoma cells of the vertical growth phase: similarities to metastatic cells.

Three primary and 16 metastatic melanoma cell lines were established from primary and metastatic lesions of 4 patients with malignant melanoma. Comparison of metastatic melanoma cells with cells of the vertical growth phase (VGP) or late primary melanoma from the same individual revealed, generally, a shorter population-doubling time, growth to a higher cell density, higher tyrosinase activity, and more pigmentation in metastatic cells. Conversely, primary and metastatic melanoma cells had similar morphology, plating efficiency, and tumorigenicity in nude mice. Karyotypic analysis revealed clonality and nonrandom abnormalities in chromosomes 1, 6, and 7 in cells of the primary and metastatic lesions of the 3 patients studied. Few differences were found in the expression of melanoma-associated antigens on short-term and long-term cultured cells by tests with monoclonal antibodies in mixed hemadsorption assays, flow cytometry, and radioimmunoassays. Our results indicate that cells cultured from the VGP but not from the radial growth phase of primary melanoma are similar to metastatic melanoma cells.

ME491 melanoma-associated glycoprotein family: antigenic identity of ME491, NKI/C-3, neuroglandular antigen (NGA), and CD63 proteins.

BACKGROUND: Numerous monoclonal antibodies (MAbs) have been produced to antigens found in human melanomas. Three of the best characterized melanoma antigens include the melanoma-associated glycoproteins (MAGs) defined by two reagent families--the ME491 family (including ME491, 8-1H, and 8-2A) and the NKI/C-3 family (including NKI/C-3 and NKI/black-13)--as well as the neuroglandular antigen (NGA) defined by MAbs LS59, LS62, and LS140. These three antigens have significant similarities in tissue distribution, biosynthesis, and structure. The ME491 MAG has been cloned, mapped, and sequenced. Numerous non-melanoma-associated proteins (Sm23, CO-029, R2, TAPA-1, CD9, CD37, CD53, and CD63) have recently been shown to have significant homology to this sequence. PURPOSE: We conducted this study to investigate the similarity between the two MAG antigens and NGA. METHODS: Several reagents defining the three different melanoma antigens were compared, using competition immunoprecipitation, immunoassay, and inhibition radioimmunoassay techniques. RESULTS: Immunoassay experiments show that MAbs defining the three melanoma antigens bind to affinity-purified ME491 antigen and inhibit each other from binding in an inhibition radioimmunoassay. Competition immunoprecipitation experiments demonstrate that the ME491 and NKI/C-3 antibodies bind to NGA. Rabbit anti-ME491 idiotype serum recognizes determinants shared by NKI/C-3 and the anti-NGA MAbs. A competition immunoprecipitation experiment also confirms the identity of CD63, as defined by MAb RUU-SP 2.28, with the three melanoma antigens. CONCLUSION: These data indicate that the MAGs defined by ME491 and NKI/C-3 as well as the anti-NGA antibodies are epitopes of the same molecule, which is identical to CD63 by both immunochemical and molecular genetic investigations. IMPLICATIONS: Our results indicate that the data obtained in studies of these three melanoma antigens may be pooled, and we propose that the molecule recognized by these reagents be classified as CD63.

Inhibition of metastases of a human melanoma xenograft by monoclonal antibody to the GD2/GD3 gangliosides.

A human melanoma variant cell line was obtained from a lung metastasis that arose spontaneously after we inoculated melanoma cells sc into a nude mouse. In this model, IgG2a monoclonal antibody (MAb) ME 36.1 defining the GD2/GD3 gangliosides inhibited melanoma growth at the primary site and metastatic spread of the cells, whereas an IgG1 variant of MAb ME 36.1 inhibited lung metastasis formation only. Possible mechanisms of antitumor effects of MAb ME 36.1 are discussed.

Phase II clinical trial of a murine monoclonal antibody cytotoxic for gastrointestinal adenocarcinoma.

A murine monoclonal antibody (MAb) which binds to human metastatic gastrointestinal adenocarcinomas can be administered safely and has tumor effects in some patients. Its therapeutic effect was assessed in 20 patients with measurable advanced colorectal carcinoma that was refractory to prior surgical resection, chemotherapy, and/or radiotherapy. All patients had agreed to receive no other therapy at the time of MAb administration and follow-up evaluation. In one patient, tumor at all known sites responded after a single i.v. injection of antibody. One other patient had a marked reduction in a hepatic metastasis where binding of 131I-labeled F(ab')2 MAb fragments was demonstrated but not in his abdominal wall metastases where no MAb binding could be demonstrated. In a third patient, stabilization persisting for 12 mo of an aggressively growing tumor was observed. The antibody was well tolerated in all patients, although 10 patients mounted an anti-murine immunoglobulin antibody response.

Inhibition of growth of colorectal carcinoma in nude mice by monoclonal antibody.

Hybridoma-derived monoclonal anti-colorectal carcinoma antibodies suppressed the growth of colorectal carcinoma in nude mice as evidenced by a lower incidence of tumors, a longer latency period, and a smaller volume of tumors in antibody-treated than in control animals. The growth-inhibiting properties of monoclonal anti-colorectal antibodies seem to be specific for colorectal carcinoma cells. This is indicated by the lack of effect of the antibodies on the growth of melanomas or bronchogenic carcinomas and by the binding of the antibodies in vivo to colorectal carcinoma cells but not to lung or kidney cells from tumor-bearing animals or to other tumor cells implanted in other animals. Inhibition of tumor growth was most probably mediated by antibody-dependent cell-mediated cytotoxicity. The results of these studies could provide an approach to the study of immunotherapeutic possibilities for anti-colorectal carcinoma antibodies in humans.

Radioimmunodetection of human glioma xenografts by monoclonal antibody to epidermal growth factor receptor.

Murine IgG2a monoclonal antibody (MAb) 425 specifically detects epidermal growth factor receptor, which is expressed on human gliomas and tumors of other tissue origin but rarely on normal brain tissues, and not at all on bone marrow and peripheral blood cells. 131I-labeled F(ab')2 fragments of this MAb injected into nude mice grafted with U-87 MG glioma cells preferentially localized in tumor tissue compared to normal mouse tissues, as determined by differential tissue counting of radioactivity. The mean tumor-to-tissue ratios of radioactivity ranged between 8.2 (blood) and 55.8 (muscle) at 2 days after the injection of 15 muCi of 131I-425 F(ab')2/mouse. Radiolabeled fragments of an anti-hepatitis virus IgG2a MAb did not localize in tumors. The localization index derived from the ratios of specific antibody to indifferent antibody in tumor tissue relative to blood was 9.94 at 2 days following the MAb injection. The labeled MAb did not localize in a xenograft of colorectal cancer tumor, which does not express the epidermal growth factor receptor. Tumors could be located by whole-body gamma-scintigraphy without background subtraction following the injection of 100 muCi of radiolabeled MAb 425 F(ab')2 fragments. The data suggest that MAb 425 is a likely candidate for clinical diagnostic and radioimmunotherapy trials.

Radioimmunodetection of human tumor xenografts by monoclonal antibodies.

Mouse IgG2a monoclonal antibodies with specific binding reactivity in vitro to human tumors of the gastrointestinal tract were radioiodinated and injected into immunosuppressed mice xenografted with human colon carcinoma tumors. The antibodies preferentially localized in tumor tissue compared to normal mouse tissue, as determined by differential tissue counting of radioactivity. Preferential antibody localization in tumor tissue was greatly enhanced when F(ab')2 fragments of the antibodies were used, and the fragments localized specifically only in those tumors that bind the antibodies in vitro and not in unrelated tumors. Radiolabeled fragments of an anti-hepatitis virus monoclonal antibody of the same isotype as the specific antibody did not localize in tumors. Tumors could be located by whole-body gamma-scintigraphy with radiolabeled specific antibody F(ab')2 fragments without background subtraction.

Tumor growth modulation by a monoclonal antibody to the epidermal growth factor receptor: immunologically mediated and effector cell-independent effects.

A monoclonal antibody of IgG2a isotype (425) is described that reacts with the epidermal growth factor receptor on human cells of different tissue origins. Monoclonal antibody 425 mediates tumor cytotoxicity in vitro using mouse and human effector cells and suppresses in vivo tumor cell growth of epidermoid (A 431) and colorectal (SW 948) carcinoma-derived cell lines. The tumoricidal effects in vitro are proportional to the antigen density on target cells. At concentrations higher than 1 nM, monoclonal antibody 425 inhibits growth of epidermal growth factor receptor-bearing A 431 cells, showing an epidermal growth factor-like agonist activity on the growth properties of these cells. A 431 cultures grown in the presence of growth-inhibiting doses of antibody or epidermal growth factor reveal a clear decrease of the relative number of cells in S phase. Additionally, cells treated with the antibody show a decrease of G2-M-phase cells in some, but not all, cultures tested.

Human cutaneous nevi transplanted onto nude mice: a model for the study of the lesional steps in tumor progression.

Normal human cutaneous nevi were transplanted to the skin of nude mice and some of the grafts were treated topically with 7,12-dimethylbenz[a]anthracene (DMBA, 1.6 mumol weekly). Histologically proven human skin was present in 22 grafts. In the 9 untreated control grafts, the tendency of nevic cells to form nests and the number of nevomelanocytes decreased with time; the melanocytic cells showed no signs of hypertrophy or atypia. In most of the 14 specimens treated with DMBA, the nevomelanocytes showed distinct signs of hypertrophy. The cells were enlarged and often dendritic and were filled with melanin granules for which the transfer to keratinocytes appeared to be blocked. The nevomelanocytes of 4 of the 9 specimens treated with DMBA for greater than or equal to 82 days (9-16 DMBA applications), showed atypical enlarged nuclei with mitotic figures. Since atypia is one criterion for identifying precursors of transformed cells, the model of human nevi grafted onto nude mouse skin may be useful for studying the various steps involved in the progression of benign melanocytic nevi to malignant melanoma.

Phenotypic characteristics of cells derived from precursors of human melanoma.

Of 66 specimens from benign melanocytic nevi, including common acquired and congenital nevi, Spitz tumors (epithelioid cell nevi), and melanocytic nevi with dysplasia, 57 could be grown in tissue culture. The cultured cells were identified as melanocytes by the presence of premelanosomes and melanosomes. Cells from 28 of 32 nevus cultures grew in an anchorage-independent way in soft agar with a colony-forming efficiency between 0.001 and 1%. Clones derived from single cells and soft agar-selected colonies showed marked phenotypic heterogeneity, but all had a limited life span and did not undergo transformation in culture. These cells were nontumorigenic in nude mice. Cultured nevus cells expressed antigens present on melanoma but absent on normal fibroblasts and/or melanocytes as tested with monoclonal anti-melanoma antibodies. The anti-melanoma antibodies bound equally well to dysplastic, congenital, and common acquired nevi. Antigens are released by nevus cells similar to melanoma cells.

Identification of melanoma-associated antigens using fixed tissue screening of antibodies.

Early culture supernatants from hybridomas that were obtained through fusions of mouse myeloma cells with lymphocytes of melanoma-immunized mice were screened for their reactivity with a paraffin-embedded cell block of a melanoma cell line, using a biotin:avidin immunoperoxidase procedure. Eleven monoclonal antibodies were derived that define several new melanoma-associated antigens. The antigens include a neutral glycolipid, gangliosides, membrane-associated proteins, cytosolic proteins, and strongly secreted proteins. These antibodies, which detect antigens that withstand tissue fixation and embedding procedures, were tested for reactivity in fixed cell lines, as well as in melanoma biopsies. These antibodies may provide powerful tools in diagnostic studies of human malignant melanoma biopsy material.

In vitro properties of human melanoma cells metastatic in nude mice.

We have developed a human melanoma metastasis model in nude mice. In this model, a human variant cell line (451-LU) was obtained that spontaneously metastasized in nude mice. This variant cell line was selected from the lung of a nude mouse after several in vivo passages of human melanoma WM164 cells previously isolated from a melanoma metastasis of a patient. The WM164 cells were not competent for metastasis in nude mice prior to this selection. We compared the phenotypes of the parental nonmetastatic cell line and the metastatic variant with respect to growth at clonal seeding densities in protein-free medium (growth factor independence), in vitro invasion through reconstructed basement membranes, secretion of proteolytic enzymes, expression of tumor-associated antigens, and chromosomal abnormalities. Metastatic 451-LU cells showed significantly increased growth factor independence when grown at clonal seeding densities as compared to the parental cells. In in vitro chemoinvasion assays, metastatic 451-LU cells were significantly more invasive than the parental cells. The metastatic variant secreted collagenase and tissue type plasminogen activator at levels 10- and 3-fold higher than the parental WM164 cells, respectively. Polyclonal antibodies to tissue type plasminogen activator significantly inhibited invasion through reconstructed basement membranes. In metastatic 451-LU cells, expression of nerve growth factor receptor was elevated, both at the protein and transcriptional level. Metastatic cells were aneuploid with a mode of 97 chromosomes, whereas the parental nonmetastatic cells had a mode of 52 chromosomes. Our studies suggest that metastatic melanoma cell variants selected in vivo show increased independence of exogenous growth factors when grown at clonal cell densities, enhanced invasiveness in vitro, greater secretion of proteolytic enzymes, and increased chromosome mode as compared to the nonmetastatic parental cells. The data further suggest that melanoma cells isolated from metastatic lesions and maintained in vitro have an unstable invasive phenotype but that metastatic variant cells can readily be selected.

Baculovirus recombinant expressing a secreted form of a transmembrane carcinoma-associated antigen.

GA733-2 is a monoclonal antibody-defined, 40-kDa glycoprotein antigen that is associated with carcinomas of various origins. Hydrophobicity analysis of the protein sequence predicted by complementary DNA (cDNA) has suggested that the GA733-2 antigen is a type I membrane protein. In this study, the polymerase chain reaction was used in a strategy to omit cDNA sequences for the transmembrane and cytoplasmic domains, thereby converting the extracellular domain into a secretory protein. Full-length and truncated cDNAs were cloned into the baculovirus transfer vector pVL1392 and introduced into Autographa californica nuclear polyhedrosis virus by homologous recombination. The full-length cDNA baculovirus recombinant directed the expression of a 40-kDa glycoprotein that was confined to infected Spodoptera frugiperda cells, whereas cells infected with the truncated cDNA baculovirus recombinant abundantly secreted a 31-kDa glycoprotein into the culture medium. Recombinant secretory antigen displayed an in vitro immunoreactivity to monoclonal antibody and an in vivo immunogenicity in mice that were similar to native antigen. The facile purification of mg quantities of carcinoma-associated antigen will enable an evaluation of its immunogenicity in cancer patients.

Immunotherapy of human glioma xenografts with unlabeled, 131I-, or 125I-labeled monoclonal antibody 425 to epidermal growth factor receptor.

Monoclonal antibody (mAb) 425 (IgG2a) binds to the external domain of the epidermal growth factor receptor. This determinant is highly expressed by human glioma tissues but rarely by normal brain tissues, and is absent on peripheral blood lymphocytes and bone marrow cells. The mAb exerts variable cytotoxic effects against cultured human glioma cells in conjunction with human and murine effector cells. Inhibition of growth of s.c. glioma xenografts in nude mice by the mAb may be mediated by murine macrophages or may be related to the capacity of the mAb to antagonize growth stimulation of glioma cells by epidermal growth factor. In approaches to radioimmunotherapy of human glioma with mAb 425, the 125I-labeled mAb 425 exhibited more significant antitumor effects than the 131I-labeled mAb both in vitro and in vivo in xenotransplanted nude mice. These differences may be due to enhanced nuclear damage caused by 125I-labeled versus 131I-labeled fragments following their internalization into the glioma cells. Our studies provide the rationale for immunotherapy of glioma patients with either unlabeled or 125I-labeled anti-epidermal growth factor receptor mAb 425.

Characteristics of cultured human melanocytes isolated from different stages of tumor progression.

Normal melanocytes and melanocytes of normal nevi, primary melanoma in the radial (RGP) and vertical (VGP) growth phases, and metastatic melanoma exhibited and maintained phenotypic differences when grown in tissue culture or in experimental animals. Only metastatic and VGP primary melanoma cells were tumorigenic in athymic nude mice and had nonrandom chromosomal abnormalities involving chromosomes 1, 6, and 7. The colony-forming efficiency in soft agar was also highest in these two cell types. A cell line of RGP primary melanoma had characteristics of both benign and malignant cells: nevus-like morphology; nontumorigenicity in nude mice; but karyotypic abnormality of chromosome 6. It also had a ganglioside pattern similar to that of normal melanocytes but not melanomas, i.e., a high GM3 ganglioside content compared to the amounts of GM2, GD2, and GD3 gangliosides. Binding of monoclonal antibodies secreted by hybridomas generated by immunization of mice with VGP primary and metastatic melanoma was highest with cells and supernatants of cultures from advanced melanoma and least with nevus cells. There was no binding to normal melanocytes except with the monoclonal antibodies specific for nerve growth factor receptor or 9-O-acetyl-GD3 ganglioside. On the other hand, monoclonal anti-nevus antibodies bound to melanocytes, nevus cells, and RGP primary melanoma cells but not to VGP primary or metastatic melanoma cells. Cultured human melanocytic cells appear to be a unique model for the study of tumor progression.

Antibody-dependent cytotoxicity mediated by natural killer cells is enhanced by castanospermine-induced alterations of IgG glycosylation.

Inhibitors of glycosylation and carbohydrate processing were used to probe the functional consequences of specific, differential alterations in glycosylation of monoclonal IgG secreted by hybridoma clones. Neither the absence of glycosylation nor the presence of atypical oligosaccharides significantly influenced binding of the monoclonal antibody to the cell surface antigen recognized. However, lymphocyte-mediated antibody-dependent cytotoxicity was enhanced significantly, as compared to native (unmodified) IgG-sensitized target cells, when target cells were sensitized with IgG bearing the atypical oligosaccharides induced metabolically by castanospermine, N-methyldeoxynojirimycin, deoxymannojirimycin or monesin, but not by swainsonine. The enhanced cytotoxicity was mediated by natural killer cells but not by monocytes or interferon-activated polymorphonuclear leukocytes. By contrast, antibody-dependent cytotoxicity mediated by activated polymorphonuclear leukocytes against target cells sensitized with the IgG glycosylation phenotypes induced by swainsonine and tunicamycin, but not by castanospermine, was decreased in comparison to cytotoxicity against target cells sensitized with native IgG. The enhanced lymphocyte-mediated cytotoxicity was Fc receptor-dependent. A panel of monoclonal antibodies directed against different human tumor target cells was used to demonstrate that the castanospermine-induced IgG phenotype generally enhanced antibody-dependent tumoricidal activity mediated by natural killer cells. However, differences in lymphocyte response to an alteration in IgG glycosylation were observed.

Monoclonal anti-idiotypic antibodies that mimic the epitope on gp120 defined by anti-HIV-1 monoclonal antibody 0.5 beta.

OBJECTIVE: To develop effective, specific and safe anti-idiotypic antibody (Ab2) vaccines against HIV-1. DESIGN: Murine monoclonal Ab2 were generated against anti-HIV-1 antibody 0.5 beta (Ab1), which binds to gp120, neutralizes HIV-1 and inhibits virus-induced syncytia formation. METHODS: Mice were immunized with Ab1, and Ab2 were produced from immunized mice by the hybridoma technique. The Ab2 were characterized in vitro, injected into rabbits, and the anti-anti-idiotypes (Ab3) induced in the rabbits were analyzed for binding and antiviral reactivities by enzyme-linked immunosorbent assay, p24gag release and syncytia formation assays. RESULTS: Seven Ab2 bound to the antigen-combining site of Ab1, one of which (UD7) induced Ab3 in rabbits that were Ab1-like in their binding reactivities to PB1 (recombinant gp120 fragment) or peptides of gp120, and shared idiotypes with the Ab1. Crude Ab3-containing sera specifically and effectively neutralized the virus. CONCLUSION: Monoclonal Ab2 UD7 has potential as a vaccine against HIV-1.