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Clinical-grade T cells are genetically modified ex vivo to express a chimeric antigen receptor (CAR) to redirect specificity to a tumor associated antigen (TAA) thereby conferring antitumor activity in vivo. T cells expressing a CD19-specific CAR recognize B-cell malignancies in multiple recipients independent of major histocompatibility complex (MHC) because the specificity domains are cloned from the variable chains of a CD19 monoclonal antibody. We now report a major step toward eliminating the need to generate patient-specific T cells by generating universal allogeneic TAA-specific T cells from one donor that might be administered to multiple recipients. This was achieved by genetically editing CD19-specific CAR+ T cells to eliminate expression of the endogenous αβ T-cell receptor (TCR) to prevent a graft-versus-host response without compromising CAR-dependent effector functions. Genetically modified T cells were generated using the Sleeping Beauty system to stably introduce the CD19-specific CAR with subsequent permanent deletion of α or β TCR chains with designer zinc finger nucleases. We show that these engineered T cells display the expected property of having redirected specificity for CD19 without responding to TCR stimulation. CAR+TCRneg T cells of this type may potentially have efficacy as an off-the-shelf therapy for investigational treatment of B-lineage malignancies.

Abstract 667 Transfer of high-avidity T-cell receptor (TCR) genes isolated from rare tumor-specific lymphocytes into polyclonal CD8+ T cells is an attractive strategy for targeted cancer immunotherapy. However, the successful implementation of this approach is limited by technical and safety issues, including inefficient gene transfer, unstable transgene expression, exhaustion of gene-modified cells and most importantly, the unpredictable results of mispairing between the endogenous and exogenous TCR chains. Indeed, co-expression of the endogenous and exogenous TCR in the same cell not only reduces cell-surface expression of the introduced tumor-specific TCR, but also drives the potential for these gene-modified T cells to acquire autoreactive specificities. Such TCR mispairing has been shown to result in autoreactive T cells in animal models [Bendle et al., Nat Med. 2010]. To partially overcome these limitations, we have developed a novel strategy based on zinc finger nucleases (ZFNs) that permits editing of T cell specificity at the DNA level, combining the disruption of the endogenous TCR chain genes with the transfer of a tumor-specific TCR. Two sets of ZFNs were designed targeting the constant regions of the α (TRAC-ZFN) and β (TRBC-ZFN) TCR chain genes, respectively. We transiently delivered these ZFNs into primary T lymphocytes activated with anti-CD3 and anti-CD28 antibody-conjugated beads and cultured with low doses of IL-7 and IL-15, to promote the survival and expansion of the ZFN-modified cells. ZFN delivery into activated T lymphocytes abrogated expression of the CD3/TCR complex on the cell surface (% of CD3neg cells with TRAC-ZFN: 34%±11 and with TRBC-ZFN: 30%±9). No phenotypic differences were observed in CD3pos and CD3neg lymphocytes, which displayed a similar CD4/CD8 ratio while displaying an early T-cell differentiation phenotype, as evidenced by high expression of CD62L, CD27, CD28 and IL-7Rα markers. Sorted CD3neg cells proved stable in culture (demonstrating that ZFN exposure was well tolerated), and did not respond to TCR-dependent stimulation with the mitogen PHA, as expected for cells carrying a disrupted TCR gene. CD3neg cells were efficiently transduced with a lentiviral vector encoding a tumor-specific exogenous TCR chain, resulting in the restoration of cell surface translocation of CD3, thus facilitating the selective expansion of TCR-transduced cells by polyclonal stimulation. To demonstrate the antitumor activity of these modified cells we selected an HLA-A2 restricted, codon-optimized cysteine-modified TCR specific for the Wilms' tumor antigen 1 (WT1). To achieve complete editing of T cell specificity, we established a protocol that sequentially disrupted the endogenous TCR α and β chains with high efficiency (averages: 36% and 18%), followed by lentiviral transfer of the tumor-specific TCR α and β chains (average efficiencies: 65% and 25%). This procedure resulted in a population of TCR-edited lymphocytes encoding only the tumor-specific TCR that, in the absence of competition from the endogenous receptor, was expressed at high physiological levels. Accordingly, TCR-edited lymphocytes were superior to conventional TCR-transferred cells in promoting specific recognition of WT1-expressing targets, including primary leukemias, and most importantly, were devoid of residual endogenous TCR reactivity including alloreactivity. Finally, in a humanized GvHD model, we showed that, at a variance with 100% of mice infused with unmanipulated T cells, and 80% of mice receiving TCR-transferred lymphocytes developing lethal GvHD, no GvHD was observed upon infusion of matched TCR-edited cells, despite robust T cell engraftment rates across all groups. These data demonstrate that the successful genetic re-programming of T cell specificity in primary lymphocytes results in a functionally superior target specific killing activity and thus has the potential to greatly improve the safety and therapeutic benefit of cancer immunotherapy, without triggering its potentially negative effects. (Provasi and Genovese: equal contribution). Disclosures: Liu: Sangamo Biosciences: Employment. Reik:Sangamo Biosciences: Employment. Chu:Sangamo Biosciences: Employment. Paschon:Sangamo Biosciences: Employment. Zhang:Sangamo Biosciences: Employment. Bordignon:Molmed: Employment. Holmes:Sangamo Biosciences: Employment. Gregory:Sangamo Biosciences: Employment. Bonini:Molmed: Consultancy.

The potency of cellular adoptive immunotherapy against cancer has been revealed by persistent and complete clinical responses obtained with allogeneic hemopoietic cell transplantation (allo-HSCT) followed by the adoptive transfer of donor T lymphocytes and by initial clinical responses observed with the adoptive transfer of tumor specific cytotoxic T lymphocytes (CTLs) in cancer patients. Major hurdles limiting adoptive T cell therapy relate to toxicity (i.e. graft-versus-host disease-GvHD in allo-HSCT) and efficacy (i.e. difficulty in expanding rare, high-avidity tumor-specific CTLs in conditions that can preserve their function and prevent exhaustion). The transfer of the T cell receptor (TCR) from high-avidity tumor-specific CTLs to polyclonal lymphocytes may overcome these difficulties, but is still limited by low and transient transgene expression, unpredictable pairing of the exogenous and endogenous TCR chains and poor survival and expansion potential of gene-modified effector lymphocytes. To overcome these limitations, we cloned genes encoding a high-avidity TCR specific for an HLA-A2-restricted peptide from the oncogenic Wilms tumor antigen 1 (WT1126-135), in a third generation lentiviral vector under the control of a bi-directional PGK or a bi-directional EF1α promoter. To increase TCR expression and facilitate appropriate TCR pairing, we used a codon-optimized TCR, modified with point mutations to introduce cysteines into the constant regions of the α and 7β chains. Human T lymphocytes were efficiently transduced by both vectors, following activation with anti-CD3 and anti-CD28 antibody-conjugated beads (bCD3/CD28) and culture with low doses of IL-7/IL-15. However, the PGK promoter was superior to EF1α in sustaining stochiometric expression of WT1-specific TCR chains, at levels appropriate for efficient HLA-A2/WT1 pentamer binding (16%), for up to 50 days, in the absence of further T cell stimulation. Additionally, we observed that a phenotype consistent with early (naïve and central memory) T cell differentiation (CD45RA−/+CD62L+, CD28+CD27+, IL7Ra+, IL-2+ γIFN±) was preserved in TCR-modified lymphocytes generated in these culture conditions. Sorted naïve (CD45RA+/CD62L+) and central memory (CD45RA−/CD62L+) lymphocytes were efficiently transduced by TCR-LV and maintained the original T cell phenotype. Accordingly, TCR-modified lymphocytes showed excellent survival and expansion capacity, and, upon antigenic stimulation mediated high WT1-specific γIFN production and cytotoxic activity. To improve the safety of the strategy, we attempted sitespecific integration of transgenes using of the ZFN technology. Adenoviral transfer of a set of ZFN specific for the putative safe-harbor locus CCR5 coupled with integrase defective lentiviral vectors carrying the donor DNA flanked transgene, enables efficient site-specific integration in human lymphocytes resulting in stable transgene expression. Analyses of sorted gene-modified cells is currently ongoing. Site-specific integration of an optimized, leukemia-specific TCR into both naïve and central memory lymphocytes may represent an effective method for the generation of robust engineered tumor-specific CTLs.