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We have generated an immunoglobulin G1 (IgG1) murine monoclonal anti-idiotype antibody (Ab2) designated ACA125, which mimics a specific epitope on the tumor-associated antigen CA125. This antigen is expressed by most of malignant ovarian tumors. Patients with CA125-positive tumors are immunologically tolerant to CA125. We used ACA125 as a surrogate for the tumor-associated antigen CA125 for vaccine therapy of 16 patients with advanced epithelial ovarian cancer or recurrences. Each of the patients received a minimum of 3 injections up to 19 injections of the complete anti-idiotype MAb ACA125 at a dosage of 2 mg per injection. Nine of 16 patients developed anti-anti-idiotypic (Ab3) responses to the ACA125. All 9 patients generated specific anti-CA125 antibody demonstrated by reactivity with purified CA125. Nine of 16 patients developed a CA125-specific cellular immune response by their peripheral blood lymphocytes (PBL) and 3 of 16 showed an increase in gamma-interferon concentrations accompanied by Ab3 responses. Toxicity was limited to abdominal pain in one case, which led to the withdrawal of further immunizations. The median progression free survival in those patients, who showed a specific immune response to the tumor-associated antigen CA125, was 11.0 +/- 5.6 months without any other therapy, in contrast to 8.0 +/- 4.2 months in the anti-anti-idiotype negative group. This is the first report of the induction of a specific active immunity to the tumor-associated antigen CA125 in patients with advanced ovarian cancer treated with an anti-idiotype antibody that "mimics" CA125. Patients showed the development of a specific humoral and cellular immune response to an otherwise nonimmunogenic tumor antigen. The immune responses in patients treated with this anti-idiotype vaccine, the low rate of side effects, and the improved time to progression after the induction of a specific immune response against the tumor-associated antigen CA125 justify follow-up clinical trials in advanced ovarian cancer patients with minimal residual disease in an adjuvant approach.

A new concept of oncological immunotherapy comprises the attempt to trigger the immune system of the host into a response against tumor cells. Antiidiotypic antibodies bearing the internal image of an antigen expressed on the surface of the tumor seem to be most suited for this purpose. We have generated a murine antiidiotypic antibody (ACA 125) functionally imitating the tumor-associated antigen CA 125, which can be detected in about 80% of ovarian carcinomas. The hybridoma cell was adapted to serum-free medium and antibody was produced in a hollow fiber cell culture system (Technomouse). ACA 125 (Ab2) shows high affinity for the paratope of Ab1 (affinity constant: 2.3 x 10(9) liters/mol) and binding of Ab2 to Ab1 is completely inhibited by the nominal antigen. Application of F(ab')2 fragments of ACA 125 to rats lead to an anti-CA 125 immunity by production of IgG and IgM antiantiidiotypic antibodies (Ab3) that bind to both ACA 125 and CA 125. Furthermore the induction of a non-MHC-restricted cell-mediated cytotoxicity for human ovarian adenocarcinoma cell line NIH-OVCAR3 (expressing CA 125 on its surface) could be proved; additionally complement-dependent cytotoxicity (CDC) as well as an antibody-dependent cellular cytotoxicity (ADCC) was observed. Thus, monoclonal antiidiotypic antibody ACA 125 fulfills recent criteria for an antibody, which might be successful in immunotherapy using the anti-idiotypic network approach.

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The F(ab')2 fragment of the murine monoclonal anti-idiotypic antibody ACA125 mimicking the tumor-associated antigen CA125 is used as a vaccine for the induction of an anti-tumoral immunity in patients with ovarian carcinoma. We tried to generate a single-chain fragment (ScFv) composed of ACA125 heavy- and light-chain variable domains connected by a polypeptide linker as an alternative to the corresponding F(ab')2 fragment. Heavy- and light-chain genes of antibody-producing mouse hybridoma cell line were amplified separately and assembled into a ScFv gene with linker DNA by the polymerase chain reaction (PCR). The ScFv gene was ligated into the phagemid vector pCANTAB5E, which allows the production of both phage-displayed and soluble ScFv. Transformed Escherichia coli TG1 cells were infected with M13K07 helper phage to yield recombinant phage, which display ScFv fragments as a g3p fusion protein on the surface of the filamentous phage M13. Recombinant phages could be selected by binding to the idiotypic antibody OC125 after one round of panning and directly used to reinfect E. coli TG1 cells. The E. coli nonsuppressor strain HB2151 was infected with an antigen-positive phage clone, previously screened by enzyme-linked immunosorbent assay (ELISA), to express soluble ScFv fragments. Functional soluble ScFv binding to the idiotypic antibody OC125 F(ab')2 could be detected in the bacterial periplasm by Western blot and ELISA. The variable heavy- and light-chain genes of the ACA125 ScFv fragment were further sequenced and compared with known antibody sequences.