Previous studies in our laboratory have demonstrated that both CD8+ and CD4+ T cells infiltrating melanomas and others types of cancers can manifest MHC-restricted recognition of tumor-associated antigens (Ag). MHC class I-restricted Ag recognized by CD8+ cytolytic T cells have been targeted by experimental immunotherapies, with limited clinical success. Current projects in our laboratory focus on identifying tumor-associated proteins recognized by CD4+ helper T cells for developing more effective cancer vaccines, as well as on extending this work to prostate cancer. 1) CHARACTERIZATION OF TUMOR-ASSOCIATED PROTEINS RECOGNIZED BY CD4+ T CELLS. A number of strategies have been pursued to develop methods for identifying MHC class II-resticted tumor Ag recognized by """"""""helper"""""""" T cells,including evaluation of candidate Ag, a biochemical approach for isolating immunogenic tumor proteins, and a molecular cloning strategy. Molecular cloning, by far the most efficient and sensitive of these techniques, depends on targeting proteins expressed by a tumor-derived cDNA library to the endosomal processing compartment,using invariant chain fusion. The first tumor Ag to be identified with this system was a mutated CDC27, derived from a melanoma. More recently, a novel RNA processing enzyme, neo-poly(A) polymerase (neo-PAP), was discovered from a melanoma library through molecular cloning. By Northern blotting, neo-PAP appears to be overexpressed in a variety of human cancers including melanomas, prostate cancers and colon cancers. Current studies use real-time quantitative RT-PCR and antibody detection to better define the tissue expression profile of neo-PAP. Immune responses against this protein are being assessed by testing the activity of lymphocytes from cancer patients against candidate neo-PAP peptides, and against the full length neo-PAP protein expressed in antigen presenting cells by a recombinant lentivirus. These studies are designed to assess the suitability of neo-PAP as a target for clinical immunotherapy. 2) CLINICAL EVALUATION OF TYROSINASE AS AN IMMUNOGEN AGAINST MALIGNANT MELANOMA. Our laboratory has identified the melanoma associated protein, tyrosinase, as a tumor Ag recognized by both cytolytic (CD8+) T cells and helper (CD4+) T cells. Tyrosinase was tested as an immunogen against melanoma in a recently completed NCI clinical protocol #99-C-0095, """"""""Immunization of patients with metastatic melanoma using recombinant vaccinia and fowlpox viruses encoding the tyrosinase antigen"""""""". This was one of the first trials to use two different vaccine vectors in a heterologous prime/boost format. The observed clinical responses did not achieve the targeted response rate, nevertheless evidence of tumor regressions as well as in vitro evidence of immunization against tyrosinase have led to the design of a follow-up trial. A new method for in vitro monitoring of T cell responses against the full length tyrosinase protein was developed, which depends on real-time quantitative RT-PCR analysis of cytokine mRNA expression in stimulated T cells. This method will be generally useful for monitoring immune responses in patients receiving whole protein/gene vaccines, as well as for investigating immune responses against candidate protein Ag. 3) DEFINING IMMUNE RESPONSES AGAINST PROSTATE CANCER. Limited information is available on the human immune response to prostate cancer, in part due to a scarcity of cultured prostate cancer lines for testing. We previously developed an innovative method for generating immortal cultures from human prostatic epithelium which has proved uniformly successful in establishing over 20 new cell lines from benign and malignant prostatic tissue. Loss of allelic heterozygosity on chromosome 8p, the potential site of a suppresser gene related to prostate cancer, was used to characterize these lines. Using these lines as in vitro stimulants to raise tumor-reactive T cells from prostate cancer patients, we have identified prostate cancer-specific CD8+ T cells resticted in one case by HLA-B or -C, and in another case by a non-polymorphic MHC-like molecule. We are in the process of cloning the latter T cell receptor associated with non-classical recognition of a variety of prostate cancers as well as colon cancers, to further define the recognized Ag and presenting molecule.The ultimate goal of these studies is to develop novel prostate cancer immunotherpies.
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