Employing a technology called differential immunization for antigen and antibody discovery (DIAAD), we aimed to generate monoclonal antibodies (mAbs) specific to human multiple myeloma (MM) cells. The fundamental principles of DIAAD rely on the induction of high zone tolerance to the "wild type" (normal) antigen. followed by immunization with the modified (diseased) antigen. Because chronic myelogenic leukemia (CML) cells are derived from a lineage closely related to MM, we immunized mice by contrasting a pool of MM cells with CML cells. Monoclonal antibody VAC69 reacted exclusively with MM cells, identifying a membrane molecule composed of a single-chain glycoprotein with a molecular weight of 78-120 kd. This antigen exhibited narrow tissue specificity and was not found on human cancers such as prostate, breast, or cervical carcinoma; leukemia; or lymphoma, nor was it seen on normal human peripheral lymphocytes or on Epstein-Barr virus-transformed B-cell lines. By immunohistochemistry, mAb VAC69 showed no binding to antigens expressed on normal human ovary, breast, prostate, lung or colon tissue, nor did it bind to human breast or prostate cancer. Conversely, mAb VAC69 bound strongly to human MM, although showing only slight binding to histiocytes or inflamed cells in human lymph nodes and human tumors of the colon, lung, and ovary. Monoclonal antibody VAC69 also triggered cancer-specific cytotoxicity in vitro (in the presence of complement) as well as in vivo using a sever combined immunodeficiency model transplanted with human MM. Further studies showed the ability of mAb VAC69 to be specifically internalized by human MM cells, indicating its potential use for therapeutic intervention in MM by delivering drugs into cancer cells.