Ben K. Seon - US grants

Our overall objectives remain to be the immunological and biochemical characterization of human leukemia-lymphoma (HLL)-associated cell membrane antigens and the preparation of specific anti-HLL antibody reagents for clinical use as well as for immunochemical studies of human malignant lymphoreticular cells. In addition to our previously described monoclonal antibody (mAb) SN1 directed to a unique human T-cell leukemia antigen, termed TALLA, we have generated monoclonal antibodies, termed SN2, SN2a, and SN2b, that define a human T-cell leukemia-associated cell surface glycoprotein, GP37, with an approximate Mr = 37,000. These antibodies were generated by using a human leukemia antigen preparation. The reactivity and specificity of these monoclonal antibodies (mAbs) were characterized by a sensitive radioimmunoassay against a variety of cultured and uncultured human cells. In selected cases, the cell specimens were further tested by indirect immunofluorescence-staining. Among the various cultured malignant and nonmalignant human cell lines tested, these mAbs reacted only with leukemic T-cell lines with one exception. Results consistent with the above were obtained from studies in which uncultured malignant cell specimens from different cancer patients were tested against these mAbs; they reacted only with T leukemia cells. Among various uncultured normal cell specimens tested, these mAbs did not react with thymocytes, bone marrow cells, peripheral blood lymphocytes containing B and T cells, purified T cells, monocytes, granulocytes, or erythrocytes. However, they reacted with platelets. It should be noted that mAb SN1 does not react with platelets. SN2 and SN2a immunoprecipitate a Mr = 37,000 antigen from both a [unreadable]125[unreadable]I-labeled leukemia antigen preparation and from [unreadable]125[unreadable]I-labeled glycoproteins of MOLT-4 cells. These mAbs will be useful in diagnosis and perhaps also in therapy of human T-cell leukemia, which is known to be associated with poor prognosis. It is important to note that immunotoxins prepared by conjugating ricin A chain with mAbs SN1 and SN2 showed specific killing of human T leukemia cells in vitro. (2)

The primary objective of this proposal is to define, through in vitro and in vivo studies, the various factors important in developing protocols for therapeutic utilization of monoclonal antibodies (McAbs) directed toward human tumor associated cell surface antigens. We will utilize both McAbs and immunotoxins which are prepared by covalently conjugating McAbs with ricin A chain. As the tumor model, human leukemic-lymphoma (HLL) will be used. Thus, the in vivo studies will be carried out using HLL cells transplanted in irradiated or splenectomized-irradiated nude mice. We hope that these studies may lead to means by which we can circumvent or overcome some critical barriers in the therapeutic use of McAbs. Our approach to the generation of anti-HLL McAbs differs from the conventional one in that we are using isolated HLL cell membrane antigen preparations rather than whole cells to immunize mice in order to obtain the appropriate McAbs. The generated hybridomas are being characterized by means of a radioimmunoassay, an immunofluorescence-staining test and/or an immunoperoxidase-staining test using various human cells and tissues as the targets. By this approach we have recently generated and cloned several mouse hybridomas that produce anti-HLL McAbs. Some of these antibodies showed strikingly high specificity for HLL and appear to define unique HLL associated cell surface antigens. Further, colony-forming unit (CFU) assays are being carried out to confirm the absence of reactivity of these McAbs with hematopoietic stem cells. We will place our emphasis on killing of HLL cells by multiple-targeting, i.e., by combined use of multiple McAbs or of immunotoxins containing multiple McAbs. In order to develop protocols for the therapeutic utilization of these McAbs and immunotoxins we will carry out the following studies: (1) detailed studies of the specificity of our generated McAbs, (2) comparison of McAbs of different isotypes and IgG subclasses with respect to the efficacy of specific killing of HLL cells in vivo, (3) comparison of the intact IgG antibodies and their univalent antigen-binding derivatives with respect to their specific cytotoxic activity against HLL cells in vivo, (4) establishment of the experimental design for using McAbs and immunotoxins in the in vitro eradication of tumor cells in the bone marrow from HLL patients, and (5) investigation of various factors important for in vivo therapeutic utilization of immunotoxins.