Monoclonal antibodies to different epitopes on a prostate tumor-associated antigen

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Abstract
Mouse monoclonal antibodies alpha Pro3 and alpha Pro5 bind to different epitopes on an antigen (p54) of 54 kD reduced and 175 kD nonreduced MW. p54 antigen has been characterized previously with regard to tissue distribution using alpha Pro3 monoclonal antibody; the p54 antigen is present in substantially greater quantities in malignant prostatic tissue extracts than in benign prostatic and nonmalignant nonurogenital tissue extracts. In this report, we have established that alpha Pro5 and alpha Pro3 monoclonal antibodies exhibit same molecule-different epitope recognition. That both antibodies recognize the same molecular entity has been established by partial physiochemical characterization of the antigen recognized by the two antibodies and by sequential immunoprecipitation experiments. Different determinant recognition was established by lack of competitive surface binding between alpha Pro3 and alpha Pro5 to a prostatic carcinoma cell line. The p54 antigen can be labeled with glucosamine and immunoprecipitated from urea-solubilized membrane proteins; however, p54 cannot be detected by immunoprecipitation in a glycosylated form in spent culture medium removed from glucosamine-labeled cells. Experiments using indirect cellular immunoassays and directly radioiodinated monoclonal antibody have shown that both alpha Pro3 and alpha Pro5 form stable complexes with p54 antigen on the prostatic carcinoma cell surface. To the extent that modulation occurs upon interaction of p54 with alpha Pro3 and alpha Pro5; endocytosis of the immune complex appears to be the primary route of modulation. Furthermore, modulation by endocytosis is more intense when alpha Pro3 and alpha Pro5 are used in combination than when either monoclonal antibody is used alone. Although in vivo biologic behavior does not invariably correlate with in vitro behavior, careful in vitro analysis of monoclonal antibodies with respect to cell surface behavior, nevertheless, should precede in vivo evaluation. The data presented in this report indicate that preliminary in vitro analyses will expedite the effectiveness of in vivo immunotherapeutic trials; preliminary in vitro evaluations are absolutely essential if monoclonal-toxic agent (e.g., ricin A) conjugates, which must be internalized by the tumor cell to achieve cytotoxicity, are employed as immunotherapeutic agents.