The objective of this project is to utilize a monoclonal antibody, MAG-1, to develop new, rational, and successful treatment of breast cancer. The hypothesis being tested is that Glycopeptide-Related cell-Surface Antigen (GRSA) will provide a sensitive and reliable target for the effective treatment of breast cancer by MAG-1 and related antibodies. The data from the studies of Phase 1 of the project clearly show that short-term (6 day) MAG-1 treatment of both estrogen-responsive and estrogen-resistant (triple-negative) human breast tumor xenografts, can not only prevent growth of the tumors but produce large decreases in size. This was found for the unmodified antibody as well as for 90Yttrium-labeled antibody. More significantly, longer-term (16 day) treatment with unmodified antibody prevents tumor re-growth by inducing massive necrosis. Our earlier data show GRSA expression is a feature common to all, or most, breast cancers and DCIS. It also shows GRSA-like antigens can be targeted in patients with antibodies, and that our MAG-1 monoclonal antibody not only recognizes GRSA in all breast tumors but also targets GRSA in vitro. Phase 2 is directed at advancing treatment of breast cancer with MAG-1 by developing a human chimeric form (cMAG-1) of the mouse monoclonal antibody, and then a humanized form (hMAG-1). The ability of cMAG-1 and hMAG-1 to target and treat human breast cancer xenografts in mice will then be tested. Phase 2 goals are directed towards (i) generating a chimeric form (cMAG-1) of MAG-1with the constant regions of human IgG;(ii) establishing that the targeting, recognition, and treatment profiles of mMAG-1 are retained by cMAG-1;(iii) modeling a humanized form (hMAG-1) of MAG-1 from the cMAG-1 with genetically grafting CDRs from the VH and VL regions of mMAG-1 into the DNA framework of a human antibody;(iv) establishing that the targeting, recognition, and treatment profiles of mMAG-1 are retained by hMAG-1. These investigations will employ, RT-PCR, ligation, and cloning, DNA recombinance, DNA sequencing, immunohistochemistry, antibody modification, Northern and Western analysis with densiometric quantitation, ELISA, RIA, tumor-directed targeting, whole-body scintigraphy for 99mTechnetium, cytofluorographic and radiometric quantitation, radioligand binding, flow cytometry, CDC assay, ADCC assay, TUNEL assay, and cell and tumor growth assessments in nu/nu mice. A successful end-point of our Phase 2 studies would be the generation of cMAG-1and/or hMAG-1 forms of our antibody that show a similar binding affinity as mMAG-1, recognize all or most breast cancers, do not react with normal tissues, can reduce the size of tumors, and/or prevent their growth in vivo. The proposed research is expected to rapidly lead to new and successful therapeutic approaches for managing breast cancer. PUBLIC HEALTH RELEVANCE: With 217,440 new breast cancer patients per year, the identification of a novel cancer marker affords a unique opportunity to develop both diagnostic and therapeutic products to treat this disease. The proposed research is expected to lead to new and successful therapeutic approaches for managing breast cancer, thereby leading to a higher survival rate of the patients.