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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-zeta signaling element. Chimeric TCR alpha and beta receptor genes were structurally designed to prevent pairing with endogenous TCR alpha and beta chains in order to prevent the generation of unpredictable immune specificities. After transduction of polyclonally activated human peripheral blood lymphocytes with retroviral vectors harboring the chimeric receptor genes, genetically engineered cells specifically recognized and responded to MAGE-A1POS/HLA-A1POS cells. Importantly, each type of transduced T lymphocytes that bound specifically to peptide/MHC complexes also showed specific antitumor reactivity as well as lymphokine production. Genetically engineered primary human T lymphocytes expressing chimeric sc- or tc-TCR therefore hold promise for disease-specific therapies.

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(epsilon)RI gamma-chain was used. To obtain ch-RecHIGH-POS and ch-RecLOW-POS T lymphocytes, two distinct retroviral vectors were used to introduce the gene constructs into primary human T lymphocytes. Levels of ch-Rec-redirected T lymphocyte mediated tumor cell lysis, as well as lymphokine production were determined using RCC lines as target/stimulator cells, which express either no or increasing densities of the TAA. A functional and dynamic balance between ch-Rec densities on cytotoxic T lymphocytes (CTLs) on the one hand and TAA densities on RCCs on the other, was found. In short, ch-RecHIGH-POS CTLs are triggered by TAAHIGH-POS as well as TAALOW-POS RCCs to lyse tumor cells and produce (IFN-gamma and TNF-alpha) lymphokine. In contrast, ch-RecLOW-POS T lymphocytes are only triggered for cytolysis and lymphokine production by relatively TAAHIGH-POS RCCs. In conclusion, (1) the activation of T lymphocyte responses is co-determined by the expression levels of the ch-Rec on T lymphocytes and the TAA on tumor cells and (2) at relatively high T lymphocyte ch-Rec expression levels the CTLs lyse tumor cells with a wide range of TAA densities. Gene Therapy (2000) 7, 35-42.

Genetic engineering of T lymphocytes for adoptive clinical immunotherapy calls for efficient gene transduction methods. Therefore, a transient retroviral gene transduction system 'STITCH' was developed comprising pSTITCH retroviral vector encoding the transgene, plasmids encoding Moloney murine leukemia virus gag/pol and gibbon ape leukemia virus envelope, and the human kidney cell line 293T as a packaging line. Cotransfection of retroviral vector and packaging plasmids in 293T cells results in the production of GALV env pseudotyped viral particles with a titer of 10(7) infectious units per milliliter. The 'STITCH' gene transduction system efficiently transduces genes into activated human T lymphocytes derived from healthy donors and cancer patients. The efficacy of gene transduction is donor-independent. A direct application of the 'STITCH' gene transduction system is the genetic engineering of activated human T lymphocytes to induce expression of antibody based chimeric receptors in their membrane. Introduction of these chimeric receptors into activated human T lymphocytes graft these cells with specificity for, for example, renal cell carcinoma. In order to study the effect of the chimeric receptor gene structure on the processes ultimately leading to functional membrane expression, we designed a number of different chimeric receptor gene structures and subsequently compared their membrane expression on 293T cells and activated human T lymphocytes. Distinct membrane expression densities were observed on 293T cells and human T lymphocytes for the different chimeric receptor gene constructs. Gene transduction of activated human T lymphocytes with four out of five chimeric receptor gene constructs resulted in functional expression of chimeric receptor as demonstrated by specific recognition and cytolysis of renal cell carcinoma.