We have generated a mouse monoclonal antibody (H23) against the retrovirus-like particles (human mammary tumor virus) released in vitro by the human breast adenocarcinoma cell line T47D. This antibody reacts specifically with a glycoprotein with an apparent molecular mass of 68 kDa (gp68) that is detected in the growth medium of T47D cells as well as in pleural effusion fluids from breast adenocarcinoma patients. No detectable levels of this antigen could be observed in pleural effusions of patients with cancers other than of breast origin. The H23-related antigen was localized in the cytoplasm of breast tumor cells as well as on the cell surface of both T47D cells and metastatic cells from breast cancer patients. A survey oftissue from 812 patients was performed by using H23 in an indirect immunoperoxidase assay. The results showed that the antigen was detectable in 91% of all breast tumors tested. No cytoplasmic staining was observed in either normal tissues or nonbreast carcinomas. Only one ofthe benign breast tissues tested (out of a total of 56 samples of tissue) was positive for this antigen. Given the ability of this antibody to specifically detect breast tumor cells, H23 may be of importance in diagnosis and in clinical follow-up of patients for the detection of metastatic lesions by imaging and for therapy. (13). To further investigate the relationship of these viral particles with human breast cancer, we generated mouse monoclonal antibodies (mAbs) to HuMTV.We report here the production and characterization of these mAbs against HuMTV. One antibody was shown not only to be very specific to breast carcinomas but also to be able to recognize a very high percentage of breast malignancies. MATERIALS AND METHODSProduction of Mouse mAbs. Immunization. Threemonth-old BALB/c mice were injected three times subcutaneously at weekly intervals with 20 Ag of purified HuMTV (14). The first injection was given in complete Freund's adjuvant and the subsequent two in incomplete Freund's adjuvant. Three days before hybridization, the mice were injected intravenously with 10 ,ug of HuMTV in isotonic phosphate-buffered saline (PBS).Hybridization. Approximately 108 splenocytes from the immunized mice were fused with 2.5 x 107 mouse myeloma x63-Ag8.653 cells by using 50% (wt/vol) polyethylene glycol (Mr, 1000; Sigma) as described by Kohler and Milstein (15 To obtain large amounts of mAbs, hybridoma cells were suspended in PBS and injected i.p. into pristane-sensitized BALB/c mice. The hybrid cells grew as ascitic tumors producing mAb-containing ascitic fluids. The tumor cells from these fluids were removed by centrifugation, immunoglobulins were purified (6), and their titers were determined by ELISA on HuMTV.

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Tumor cells obtained from leukemia and lymphoma patients were investigated for specific insulin receptors. Using radioactive 125I- labeled insulin, specific insulin binding sites were demonstrated on most acute lymphocytic leukemia (ALL) and acute myelocytic leukemia (AML) cells, including acute promyelocytic leukemia (APL), chronic myelocytic leukemia (CML), and acute monocytic leukemia (AMoL) cells. Insulin receptors were not found on chronic lymphocytic leukemia (CLL) and malignant lymphoma (ML) cells. Specific insulin binding sites were also found on monocytes and thymocytes after treatment with phytohemagglutinin (PHA-P), but not on inactivated tonsil cells, peripheral blood lymphocytes, or thymocytes. There was no inverse correlation between the content of insulin receptors and the basal level of circulating insulin. These data suggest that the insulin receptor may be a new marker of acute leukemia and chronic myelocytic leukemia.