Abstract 2937: Editing central memory T lymphocyte specificity for safe and effective adoptive immunotherapy of leukemia
Authors: Genovese Pietro , Provasi Elena , Magnani Zulma , Lombardo Angelo , Pello Oscar Muniz , Kuball Jurgen , Bondanza Attilio , Gregory Philip D. , Bordignon Claudio , Holmes Michael C. , Greenberg Philip D. , Naldini Luigi , Bonini Chiara 
Published: February 2011

Abstract
The transfer of high-avidity T cell receptors (TCR) isolated from tumor-specific lymphocytes into polyclonal T cells is an attractive strategy for the adoptive immunotherapy of tumors. However, three major hurdles hinder the effectiveness of this strategy: i) inefficient gene transfer and unstable transgene expression, ii) exhaustion of gene-modified cells, and iii) mispairing of endogenous and exogenous TCR chains. To solve these issues, we used a bi-directional lentiviral vector (LV) expressing a codon-optimized, cysteine-modified TCR for the Wilms tumor antigen 1 (WT1) to genetically modified human central memory T lymphocytes (TCM). TCM have been reported superior to effectors in mediating long-lasting anti-tumor responses in vivo. Transduced cells showed high (∼16%) and stable (70 days in culture) levels of WT1-pentamer binding and cytotoxic activity towards WT1+ primary AML leukemic blasts. To further enhance the safety profile and potency of modified TCM, we developed a platform to genome edit TCM based on Zinc Finger Nucleases (ZFN). In order to ensure stable and consistent transgene expression, we demonstrated the targeted insertion of the transgene into a predefined genomic locus (CCR5) by homologous recombination in up to 6% of treated TCM, with concurrent inactivation of 18% of the targeted CCR5 gene by non-homologous end joining (NHEJ). These findings prompted us to address the major technological hurdle of TCR gene transfer: the co-expression of endogenous and tumor specific TCRs in the same cell. Indeed, this result in competition for the CD3 co-receptor molecules, leading to TCR dilution, and in TCR mispairing, giving rise to unpredictable specificities that are potentially autoreactive. To solve these issues, we developed ZFN specific for the endogenous TCR β chain gene to promote the disruption of TCR β by NHEJ. We obtained functional inactivation of this gene in up to 7% of TCM. Targeted lymphocytes failed in expressing the CD3/TCR complex on cell surface, and could be sorted as CD3neg. CD3neg proved stable in culture, maintained a TCM phenotype and were resistant to further TCR-mediated triggering. For a complete editing of T cell specificity, the WT1-specific TCR was transferred with high efficiency (&gt;20%) by LV on CD3neg cells. WT1-TCR rescued CD3 surface expression and was expressed in edited lymphocytes at higher levels to that observed upon conventional TCR gene transfer. Accordingly, TCR-edited cells proved superior to cells undergoing conventional TCR gene transfer in promoting killing of relevant targets, and in recognizing low (10 nM) peptide concentrations. Overall, these data show the feasibility of rapid and efficient editing of TCM to generate safe and functional tumor-specific T cells and may open new perspectives to cancer adoptive immunotherapy. (PG and EP: equal contribution)
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2937.