Editing Human Lymphocyte Specificity for Safe and Effective Adoptive Immunotherapy of Leukemia.
Authors: Provasi Elena , Genovese Pietro , Lombardo Angelo , Magnani Zulma , Reik Andreas , Liu Pei-Qi , Chu Victoria , Kuball Jurgen , Bondanza Attilio , Casorati Giulia , Ciceri Fabio , Bordignon Claudio , Greenberg Philip D. , Holmes Michael C. , Gregory Philip D. , Naldini Luigi , Bonini Chiara 
Published: October 2019

Abstract
Abstract 3764
T cell receptor (TCR) gene-transfer is an attractive strategy for the adoptive immunotherapy of tumors. However, the full potential of this approach is limited by a number of technical hurdles including inefficient gene transfer, unstable transgene expression, exhaustion of gene-modified cells and most importantly, co-expression of the endogenous and exogenous tumor-specific TCR in the same cell. The co-expression of endogenous and exogenous TCR causes not only reduced cell-surface expression of the introduced tumor-specific TCR, but also the potential for T cells to acquire autoreactive specificities due to mispairing between the different TCR chains. Mispaired TCR have been shown to be autoreactive and potentially harmful in animal models. To address these limitations, we developed a novel strategy based on zinc finger nucleases (ZFNs) that allows for the first time the editing of T cell specificity at the DNA level, by combining the disruption of the endogenous TCR chain genes with the transfer of a tumor-specific TCR. We first stimulated PBL with anti-CD3 and anti-CD28 antibody-conjugated beads, and cultured these cells in low dose IL-7/IL-15 to preserve early differentiated T cells. We transiently expressed ZFNs targeting the constant region of the TCR alpha or beta chain genes in activated T lymphocytes using a chimeric Ad5/F35 vector, thus promoting the genetic disruption of the endogenous TCR. Lymphocytes targeted by each set of ZFNs abrogated expression of the CD3/TCR complex on the cell surface. CD3neg cells could be expanded in culture with IL7 and IL15 and expressed differentiation markers typical of central memory T cells (CD62L, CD127, CD27 and CD28), indistinguishably from unmodified T cells. Sorted CD3neg cells proved stable in culture and permissive to lentiviral transduction. Indeed, introduction of exogenous TCR chains on a Lentivirus restored the expression and functionality of the CD3/TCR complex and allowed selective expansion of TCR-transduced cells by stimulation via the TCR complex. As a model TCR, we selected an HLA-A2 restricted, codon-optimized cysteine-modified TCR specific for the Wilms' tumor antigen 1 (WT1), which is expressed by several solid tumors and leukemias and contributes to the uncontrolled proliferation of cancer cells. For a complete editing of T cell specificity, we established a protocol that sequentially disrupted the endogenous TCR chains followed by lentiviral transfer of the WT1-specific TCR. This procedure resulted in a population of TCR-edited lymphocytes encoding only the tumor-specific TCR that, in the absence of competition, was expressed at high and 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 reactivity including alloreactivity. 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. (Provasi and Genovese: equal contribution).

Disclosures:
Reik: Sangamo: Employment. Liu:Sangamo: Employment. Chu:Sangamo: Employment. Bordignon:Molmed: Employment. Holmes:Sangamo: Employment. Gregory:Sangamo: Employment. Bonini:Molmed: Consultancy.