Evidence for a novel hybrid immunotoxin recognizing ricin A-chain by one antigen-combining site and a prostate-restricted antigen by the remaining antigen-combining site: potential for immunotherapy

Abstract

We have used cell fusion technology to produce second-generation hybridomas which secrete a population of murine monoclonal antibodies (MABs), a portion of which are bifunctional antibodies. The bifunctional hybrid MABs produced are capable of recognizing ricin A-chain (RAC) via one antigen-combining site and a prostate-restricted antigen via the other antigen-combining site of the IgG molecule. The second-generation hybridoma described in this report resulted from the fusion of spleen cells from mice immunized with purified RAC to hybridoma cells which secrete prostate-directed alpha Pro 15 monoclonal antibody. We have demonstrated that the MAB population secreted by the second-generation hybridoma can be physicochemically separated into distinct populations exhibiting differential binding to the cultured prostatic carcinoma cell surface and to RAC immobilized in a solid phase; specifically, a subset of the monoclonal antibody population which exhibits high binding to both prostatic carcinoma cells and to solid-phase RAC can be enriched by physicochemical methods. Binding of hybrid immunotoxin (HIT) MAB population to RAC can be quantitatively reduced by prior adsorption of the antibody population with prostate carcinoma cells; conversely, hybrid MAB binding to prostate carcinoma cells can be quantitatively reduced by prior adsorption with RAC. The biologic impact of the HIT has been evaluated by the ability of the HIT-RAC conjugate to reduce the uptake of 14C-amino acids into cellular protein. This effect is selective, since HIT-RAC conjugates do not exert an effect on labeled amino acid uptake by a cell line that does not express the target antigen recognized by the prostate-directed component of the hybrid MAB. Furthermore, depression of labeled amino acid uptake by prostate carcinoma cells exhibits a stoichiometric relationship with respect to both the concentration of HIT-MAB and to RAC to which the cells are exposed. The biologic impact of HIT-RAC conjugates on prostate carcinoma cells is enhanced markedly in the presence of lysosomotropic amines.