Although substantial regressions of certain tumors have occurred in advanced cancer patients following adoptive immunotherapy with LAK cells or TIL plus IL-2, several limitations exist in this therapeutic approach. These include: 1. Altered trafficking/homing patterns of ex vivo activated and expanded effector cells (e.g., the vast majority of LAK cells or TIL do not traffic to tumor deposits upon adoptive transfer), which may be due to loss of critical adhesion/homing molecules/receptors as a direct outcome of tissue culture. The transferred cells often may become trapped in (and cleared by) the lungs, liver and/or spleen. 2. Very high costs of supplies and labor are associated with ex vivo expansions of effector cells for adoptive transfer into patients (e.g., expansions to at least 10(11) cells); the methodologies employed are cumbersome and time consuming. 3. Since ablative chemotherapy or radiation therapy is not generally administered as a standard regimen in most adoptive immunotherapy protocols, tumor-induced, active immune suppression and/or defects in T cell signaling are known to exist in cancer patients receiving transferred effector cells, which may down regulate the functional activity of the latter. 4. Low incidence of antigen-reactive T cells in cancer patients (less than 1 preCTL in 10(5)-10(6) circulating PBL) has resulted in difficulty to often achieve large scale ex vivo expansions (e.g., cells cease to grow and/or lose functionality after """"""""long-term"""""""" culture). 5. Known, significant difficulties exists in achieving stable, high gene transfer efficiency and expression in """"""""shorter"""""""" lines of mature effector cells (e.g., LAK cells and TIL). In an attempt to overcome those limitations defined above and to substantially increase the precursor frequency of tumor antigen-reactive T cells attempts will be made to introduce genes (via retroviral vectors) encoding """"""""classic"""""""" T cell receptors (TcR) with specificity to defined shared or common tumor antigens into hematopoietic stem/progenitor cells (denoted HSC). The applicant has unique experience in the isolation, gene-modification, and functional testing both in vitro and in vivo of human HSC. He has been successful in introducing report (LacZ and NeoR) genes into HSC as well as in establishing functional assays of their biologic activity.
The Specific Aims of this application are to: 1. Insert the genes encoding the alpha and beta chains of a functional TcR recognizing the MART-1 peptide expressed by human HLA-A2 melanoma into a highly enriched population of human HSC; 2. Determine whether TcR gene transfer adversely affects the ability of HSC to self-renew and to give rise to progeny myeloid and lymphoid cells by in vitro (in methylcellulose, LTBMC, and TOC) and in vivo (in SCID-hu mice) assay; 3. Determine the function (by cytokine release and cytotoxicity) of the TcR transgenes in progeny T cells introduced at the HSC level following in vitro stimulation of these T cells with MART-1 expressing human tumor cells or peptide-pulsed T2 cells. This strategy is an attempt to """"""""arm"""""""" a bone marrow transplant with antitumor reactivity to target residual disease (e.g., in cases of breast, ovarian, or lung cancer in adults, or of neuroblastoma in children).
