Primary human activated T lymphocytes were genetically grafted with chimeric T cell receptors (TCR). Three domain single chain (sc-) TCR as well as two chain (tc-) TCR gene constructs were derived from the melanoma-specific cytotoxic human T cell (CTL) clone 82/30, and linked to the CD3-signaling element. Chimeric TCR ␣ and ␤ receptor genes were structurally designed to prevent pairing with endogenous TCR ␣ and ␤ chains in order to prevent the generation of unpredictable immune specificities. After transduction of polyclonally activated human peripheral blood lymphocytes

TCR with known antitumor reactivity can be genetically introduced into primary human T lymphocytes and provide promising tools for immunogene therapy of tumors. We molecularly characterized two distinct TCRs specific for the same HLA-A2-restricted peptide derived from the melanocyte differentiation Ag gp100, yet exhibiting different stringencies in peptide requirements. The existence of these two distinct gp100-specific TCRs allowed us to study the preservation of peptide fine specificity of native TCRαβ when engineered for TCR gene transfer into human T lymphocytes. Retroviral transduction of primary human T lymphocytes with either one of the two sets of TCRαβ constructs enabled T lymphocytes to specifically kill and produce TNF-α when triggered by native gp100pos/HLA-A2pos tumor target cells as well as gp100 peptide-loaded HLA-A2pos tumor cells. Peptide titration studies revealed that the cytolytic efficiencies of the T lymphocyte transductants were in the same range as those of the parental CTL clones. Moreover, primary human T lymphocytes expressing either one of the two engineered gp100-specific TCRs show cytolytic activities in response to a large panel of peptide mutants that are identical with those of the parental CTL. The finding that two gp100-specific TCR, derived from two different CTL, can be functionally introduced into primary human T lymphocytes without loss of the Ag reactivity and peptide fine specificity, holds great promise for the application of TCR gene transfer in cancer immunotherapy.

No abstract

Genetically engineered expression of tumor-specific single chain antibody chimeric receptors (ch-Rec) on human T lymphocytes endow these cells with the parental monoclonal antibody (mAb) dictated tumor specificity and may be useful for clinical immuno-genetherapy. Therefore it was of importance to assess how the densities of tumor-specific receptors and tumor associated antigens (TAA), respectively, affect primary human T lymphocyte functions in relation to target cell susceptibilities to lysis. We therefore studied the functional balance between ch-Rec densities on human T lymphocytes and TAA on tumor cells. The gene construct encoding a ch-Rec derived from (1) a renal carcinoma cell (RCC) specific mouse mAb (G250), and (2) the human signal transducing Fc(⑀)RI ␥-chain was used. To obtain chRec HIGH-POS and ch-Rec LOW-POS T lymphocytes, two distinct retroviral vectors were used to introduce the gene constructs into primary human T lymphocytes. Levels of ch-Rec-

No abstract