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Both monoclonal and polyclonal antiidiotypic antibodies mimicking the human colorectal carcinoma (CRC) associated antigen CO17-1A/GA733 have induced antigen-specific humoral and cellular immunity in CRC patients. The immune responses may underlie the clinical responses observed in some of the treated patients. Recently, the CO17-1A/GA733 antigen has been molecularly cloned and expressed in baculo-, adeno-, and vaccinia viruses. In preclinical studies, these recombinant antigen preparations elicited specific humoral immunity (cytotoxic antibodies) and cellular immunity (DTH-reactive and proliferative T cells). Antibody titers elicited in animals by recombinant antigen were significantly higher than those elicited by antiidiotypes. The recombinant antigen has a potential as a vaccine for CRC patients.

Monoclonal antibody 17-1A was administered to 25 patients with advanced unresectable carcinoma of the pancreas. Ten patients received 17-1A alone in 400 mg doses delivered intravenously, while 15 patients received 400 mg 17-1A absorbed on to autologous peripheral blood mononuclear cells collected by leukapheresis (usual yield greater than 10(9) cells). No toxicity was observed. Twenty-three patients developed circulating anti-murine immunoglobulin within three weeks of treatment, and 11 patients developed circulating anti-idiotypic immunoglobulin. Four out of 19 clinically evaluable patients (21%) showed objective regressions of tumor. Response did not correlate with the presence or absence of anti-idiotypic antibody and did not correlate with the method of treatment with 17-1A alone or 17-1A and mononuclear cells, at the time of current analysis.

Anti-idiotypic antibodies (Ab2) raised during the course of monoclonal antibody (MAb) therapy against anti-colorectal carcinoma (CRC) MAb CO17-1A were characterized in 142 patients with carcinomas of the colon, rectum or pancreas. Ab2 comprised between 21 and 80% of the total human anti-mouse IgG antibodies in various patients, and up to 42 micrograms of Ab2 were isolated per ml serum. In one patient significant levels of Ab2 could be detected for greater than 770 days. Between 20 and 70% of Ab2 in various patients bound to the combining site of MAb CO17-1A and may therefore bear the internal image of the 17-1A tumor antigen. Furthermore, Ab2 isolated from different patients showed considerable cross-reactivities. A beneficial role of the Ab2 responses in the cancer patients who improved clinically following MAb immunotherapy is discussed in light of induction by Ab2 of anti-anti-idiotypic antibodies (Ab3) with tumor-binding activities.

We have established a solid-phase binding inhibition radioimmunoassay for the detection of colorectal carcinoma-specific antigens in tissue culture supernatants of human colorectal carcinoma cell lines and in serum and urine of colorectal carcinoma patients. Using the [3H]glucosamine-labeled cell membrane glycolipid antigen and colorectal carcinoma-specific monoclonal antibodies in this assay, we have been able to detect several human colorectal carcinoma membrane-specific antigens that are released from the cell membrane into tissue culture supernatants, and an antigen detected by antibodies 1116-NS-19-9 and 1116-NS-52a that is found only in the serum and urine of cancer patients.

Twenty-seven patients with metastatic adenocarcinoma of the colon or pancreas were treated with 400mg of monoclonal antibody 17-1A. This antibody, which binds to a cell surface glycoprotein moiety preferentially expressed by adenocarcinomas of the rectum, colon, pancreas, and stomach, is postulated to induce antibody-dependent monocyte cytotoxicity (ADMC) as a mechanism of tumor lysis. Therapy was preceded by four days of gamma interferon infusions, with the intent of activating peripheral blood monocytes, enhancing monocyte Fc receptor expression and increasing the likelihood of tumor lysis as reflected by enhanced ADMC directed against a colon carcinoma cell line (SW1116) which expresses 17-1A's target antigen. In this Phase I study patients were treated daily at one of the following gamma interferon dose levels (X 10(6) U/M2/day): 0.001, 0.01, 0.1, 1.0, 10.0, 40.0, 60.0, 80.0. Addition of 100 U/ml of rIFN-gamma in vitro to monocytes isolated from normal controls or from patients prior to treatment significantly enhanced monocyte Fc receptor expression and ADMC. in vitro tumor cell killing by monocytes and monoclonal antibody was enhanced by treatment with low doses of rIFN-gamma, while treatment with high doses of rIFN-gamma did not enhance ADMC. No objective clinical responses were noted, although serum tumor markers dropped transiently in 36% of the treated patients. Seven of 11 assayed patients developed human anti-idiotype antibodies. With better scheduling of rIFN- and 17-1A we hope to duplicate optimal in vitro conditions for antibody-mediated cytotoxicity, hopefully enhancing in vivo antibody mediated tumor lysis.

A class switch variant of hybridoma CO19-9 secreting IgG2a antibodies was shown to have the same immunoperoxidase binding pattern in human tissue as the IgG1 antibody secreted by the parental hybridoma. The IP tissue binding of GA73.3 and 17-1A monoclonal antibodies which have been suggested to bind to structurally related antigens were compared; although quite similar in distribution, some differences were noted. GA73.3 bound to 12/12 colon carcinomas compared to 17-1A which reacted with 11/12 tumors. In several cases, the percentage of cells reactive with GA73.3 (90-100%) exceeded those reactive with 17-1A (10-25%). Additionally, the intensity of reactivity for GA73.3 was consistently greater than that seen with 17-1A. The detection of murine antibody bound to human tissues following therapeutic infusion of 19-9 IgG2a or GA73.3 differed. Detection of antibody-antigen complexes was seen less often in patients who had received 19-9 IgG2a (2/6) than in patients who had received GA73.3 (5/5). Additionally, the presence of murine immunoglobulin was seen only in the extracellular mucin of the patients receiving 19-9 IgG2a, whereas strong cellular binding of murine immunoglobulin was noted following infusion with GA73.3.

Of 14 monoclonal antibodies produced in six different laboratories, 13 bound to purified preparations of carcinoembryonic antigen (CEA). All antibodies reacted to spent medium of colorectal carcinoma cell lines. Competitive binding studies indicated that 12 different antigenic determinants representing six different groups were detected on the CEA molecule(s). Six antibodies were used in double determinant radioimmunoassays (RIA) to detect CEA and CEA-related antigens in sera of 311 patients with various gastrointestinal diseases and of normal donors. None of up to 115 sera of healthy donors had elevated antigen levels with four out of the six monoclonal antibodies tested, whereas up to 9% of sera showed elevated antigen levels when tested with two antibodies. Between 1.4% and 4.4% of sera from patients with inflammatory and benign neoplastic diseases of the gastrointestinal tract were positive. Antigen levels were elevated in 56 to 75% (depending on antibody used) of sera from patients with advanced gastrointestinal tumors. These preliminary results indicate that double-determinant immunoassays with a panel of monoclonal antibodies might improve conventional CEA assays by reducing the number of false positive sera detected by polyclonal sera in patients with benign inflammatory bowel diseases.