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).

Fusion of P3 X 63 Ag8 mouse myeloma cells with splenocytes obtained from mice immunized with cells derived from human colorectal carcinomas resulted in the production of antibody-secreting hybridomas. Two hybridomas (1083-17 and 1116-56) and their clones secreted antibodies binding specifically to human colorectal carcinoma cells either grown in culture or obtained from patients, but did not bind to normal colonic mucosa or other normal and malignant human cells. The binding specificity was consistent in three assays: radioimmunoassay, mixed hemadsorption, and immunofluorescence. Adsorption of these antibodies to colorectal carcinoma cell lines totally eliminated their specific binding.Despite a large number of reports, there is no evidence available that tumor-specific antigens are present on cells of human colorectal carcinoma (1, 2). For instance, the carcinoembryonic antigen(s) present on colorectal carcinoma are also expressed by other human tumors and by normal tissues (1). In chemically induced colon carcinomas of the rat, on the other hand, there is evidence of common tumor-specific antigens (3-5).-In a previous publication (6), we reported the presence on human melanoma cells of tumor-specific antigens detected by monoclonal antibodies. The present paper extends these observations to antigen(s) expressed by human colorectal carcinomas either maintained in tissue culture or obtained directly from patients.

The binding of monoclonal antibody specific for colon carcinoma was inhibited by serum from patients with adenocarcinoma of the colon but not by serum from patients with other bowel diseases or from healthy volunteers. Of other malignancies studied, serum from two patients with gastric carcinoma and two patients with pancreatic carcinoma also inhibited the specific binding of monoclonal antibody. The levels of carcinoembryonic antigen in these serum samples were not correlated with their levels of binding inhibition. Such monoclonal antibodies may prove useful for the detection of colorectal carcinoma.

Tumor-associated carbohydrate (TAC) antigens are important targets in cancer vaccine efforts.Carbohydrates are, however, frequently poor immunogens, in that they are T-cell-independent antigens. Molecular mimicry of TAC by peptides is an alternative approach to generating anti-carbohydrate immune responses. Here we demonstrate that peptide mimotopes can elicit antibody responses that cross-react with representative human TAC antigens. Primary immunization with such a multiple antigenic peptide, along with QS-21 as adjuvant, elicits cytotoxic antibodies reactive with naturally occurring forms of TAC expressed on tumor cells, and vaccination of mice with peptide mimotopes reduced tumor growth and prolonged host survival in a murine tumor model.

Defined by monoclonal antibody GA733, the GA733-2 antigen is a cell surface 40-kDa glycoprotein associated with human carcinomas of various origins. Molecular clones for the GA733-2 antigen were isolated from a colorectal carcinoma cell line cDNA library using the high-efficiency COS cell expression system. A 1.4-kilobase cDNA species was enriched by immunoselection with monoclonal antibody. The authenticity of individual clones was established by immunologic and sequence criteria. At the amino acid sequence level, GA733-2 was found to be >99% identical to the previously described KSA antigen defined by monoclonal antibody KS1/4. The amino acid sequence derived from the previously described GA733-related gene, GA733-1, was found to be 49% identical to GA733-2. The positions of 12 cysteine residues in the extracellular domains of the two GA733 antigens are conserved, as is the overall distribution of hydrophobic and hydrophilic residues. A 1.45-kilobase transcript of the GA733-2/KSA gene was found to be expressed in cell lines derived from colorectal and pancreatic carcinoma.in a colorectal carcinoma cell line. Both GA733-2 and GA733-1 sequences have significant homology to an exon-encoded repeat unit in thyroglobulin and the HLA-DR-associated invariant chain.Here we report the isolation of cDNA clones for the GA733-2 antigen using the high-efficiency COS cell expression system (7-9) and the sequence of the GA733-2 cDNA.t Molecular clones for the GA733-2 antigen were enriched from a colorectal carcinoma cell line cDNA library by iterating cycles of expression of the plasmid library in COS cells, immunoselection of antigen-positive cells by "panning" with the mAb GA733, and transfer of plasmid DNA from COS cells into Escherichia coli. The sequence of GA733-2 was compared to that of the carcinoma-associated antigen KSA (10, 11) and to the related GA733-1 antigen (6). A group of related mAbs (1, 12, 13) were assayed for reactivity to the antigen expressed by the GA733-2 clone. The size and expression of the GA733-2 mRNA in tumor cell lines were also studied.Monoclonal antibodies (mAbs) defining tumor-associated cell surface antigens are currently being evaluated for use in the diagnosis and therapy ofhuman cancer. Initial studies of mAbs C017-1A and GA733 (1), demonstrating cytotoxic activity in vitro and tumoricidal activity in experimental animals (for review, see ref.2), led to an evaluation of their immunotherapeutic potential in cancer patients. Idiotypic cascades have been demonstrated in cancer patients treated with mAb C017-1A, providing support to the hypothesis that antigen-specific anti-anti-idiotypic antibody (Ab3) responses may explain some of the observed delayed clinical responses (3).The independently derived mAbs GA733 and C017-1A define the same 40-kDa cell surface glycoprotein (4), hereafter referred to as the GA733-2 antigen. It is expressed by carcinomas of several histologic types and to some extent by normal tissues (5). The molecular cloning of cDNA for the tumor-associated GA733-2 an...