(4) and normal and malignant tissues (6, 7) showed that R24 reacts with melanocytes, astrocytes, melanomas, astrocytomas, and a subset of sarcomas. R24 also mediates a variety of biological effector functions, including tumor cell aggregation, human complement-mediated cytotoxicity, and antibody-dependent cell-mediated cytotoxicity with human effector cells (refs. 8-10; unpublished work).In the present study, we have investigated the response of melanoma patients to R24 with regard to different dose levels, toxicity, serological parameters, and tumor response. MATERIALS AND METHODSPatients. Patients' tumors were shown to express GD3 by indirect immunofluorescence tests on frozen sections prior to treatment. All patients had objectively measurable disease, a performance status (Karnofsky scale) of at least 60, and had been off anticancer therapy for at least 4 weeks. No concurrent anticancer therapy was given during evaluation. Patients were considered evaluable 6 weeks after initiation of therapy; 10 patients are evaluable and 2 have not yet reached the 6-week mark.Preparation and Administration of R24. R24 was prepared from ascites of (BALB/c x C57BL/6)F1 mice and purified by ammonium sulfate precipitation, chromatography over protein A-Sepharose and Sephadex G-25 columns, and filtration. Each R24 batch was tested for antibody reactivity and assayed for nucleic acids, 16 mouse viruses, bacteria, fungi, and mycoplasma. Preparations underwent standard safety testing in mice and guinea pigs and pyrogenicity testing in rabbits.R24 was administered by i.v. infusion in 100-200 ml of 0.9% saline/5% human serum albumin. Skin tests with 0.1 ,ug of R24 were done before the first treatment. The schedule of treatment was 1 or 10 mg/M2 every other day for eight treatments or 30 mg/M2 per day by continuous infusion on days 1-5 and 8-12.Serological Tests. R24 antibody titers were determined by testing serum samples in protein A mixed hemadsorption assays (11) against the melanoma target cell line SK-MEL-28. R24 concentrations were measured by an enzyme-linked immunoassay. Falcon 3034 plates were precoated with purified R24 at 125 ,g/ml. Rabbit anti-mouse IgG3 (Bionetics, Kensington, MD) diluted 1:100 was mixed (1:1, vol/vol) with patients' serum samples diluted 1:4 and incubated for 120 min. The mixture was transferred to the precoated wells and incubated for 60 min, and wells were washed with phosphatebuffered saline. Wells were incubated with goat anti-rabbit IgG linked to alkaline phosphatase (Sigma) for 60 min. Alkaline phosphatase activity was determined using p-nitrophenyl disodium phosphate substrate (12). R24 concentrations were determined by comparison with standards using different concentrations of purified R24 diluted in a pretreatment serum sample from the patient.Human IgG antibody against mouse Ig was detected by enzyme-linked immunoassays. Falcon 3034 plates precoated with R24 at 50 ug/ml were incubated with patients' serum samples diluted 1:50 for 60 min and washed with'phosphatebuffered saline. Anti-human I...
The FAP tumor fibroblast antigen is highly expressed in primary and metastatic colorectal carcinomas and shows limited expression in normal adult tissues. This highly selective expression pattern allows imaging of colorectal carcinoma lesions as small as 1 cm in diameter on 131I-mAbF19 scans. Because of the consistent presence of FAP in the stroma of epithelial cancers and the accessibility of FAP-positive tumor stromal fibroblasts to circulating monoclonal antibodies (mAbs), this study suggests possible diagnostic and therapeutic applications of humanized mAbF19 and mAbF19 constructs with novel immune and nonimmune effector functions.
The mAb A33 detects a membrane antigen that is expressed in normal human colonic and small bowel epithelium and >95% of human colon cancers. It is absent from most other human tissues and tumor types. The murine A33 mAb has been shown to target colon cancer in clinical trials, and the therapeutic potential of a humanized antibody is currently being evaluated. Using detergent extracts of the human colon carcinoma cell lines LIM1215 and SW1222, in which the antigen is highly expressed, the molecule was purified, yielding a 43-kDa protein. The N-terminal sequence was determined and further internal peptide sequence obtained following enzymatic cleavage. Degenerate primers were used in PCRs to produce a probe to screen a LIM1215 cDNA library, yielding clones that enabled us to deduce the complete amino acid sequence of the A33 antigen and express the protein. The available data bases have been searched and reveal no overall sequence similarities with known proteins. Based on a hydrophilicity plot, the A33 protein has three distinct structural domains: an extracellular region of 213 amino acids (which, by sequence alignment of conserved residues, contains two putative immunoglobulin-like domains), a single hydrophobic transmembrane domain, and a highly polar intracellular tail containing four consecutive cysteine residues. These data indicate that the A33 antigen is a novel cell surface receptor or cell adhesion molecule in the immunoglobulin superfamily.
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