Abstract SY42-01: Targeting T cell malignancies with gene edited CAR-T
Authors: Dipersio John F. , Cooper Matthew 
Published: August 2018

Abstract
T-cell malignancies represent a class of devastating hematologic cancers with high rates of relapse and mortality in both children and adults for which there are currently no effective or targeted therapies. Despite intensive multiagent chemotherapy regimens, fewer than 50% of adults and 75% of children with T-ALL survive beyond five years. For those who relapse after initial therapy, salvage chemotherapy regimens induce remissions in 20-30% of cases. Allogeneic stem cell transplant, with its associated risks and toxicities, is the only curative therapy. Targeted therapy against T-cell malignancies represents a significant unmet medical need. Such targeted therapies have shown great potential for inducing both remissions and even long-term relapse-free survival in patients with B-cell leukemia and lymphoma. Engineered T cells that express a chimeric antigen receptor (CAR) directed against T-cell malignancies are limited by several significant obstacles, but are a promising cancer immunotherapy. First, the shared expression of target antigens between T effector cells and T-cell malignancies results in fratricide, or self-killing, of CAR-T cells. Second, harvesting adequate numbers of autologous T cells without contamination by malignant cells is, at best, technically challenging and prohibitively expensive. Third, the use of genetically modified CAR-T cells from allogeneic donors may result in life-threatening graft-vs.-host disease (GvHD) when infused into immune-compromised HLA-matched or mismatched recipients. We hypothesized that deletion of CD7 and the T-cell receptor alpha chain (TRAC) using CRISPR/Cas9 in CAR-T targeting CD7 (UCART7) would result in the efficient targeting and killing of malignant T cells without significant effector T-cell fratricide or induction of GvHD. We chose to target CD7 on malignant T cells because it is overexpressed on the vast majority of T-cell and NK-cell malignancies. Second, germline biallelic deletion of CD7 resulted in mice with normal T-cell numbers, T-cell subsets, and T-cell function. We generated a CD7 CAR using a anti-CD7 single chain variable fragment (scFv) created using commercial gene synthesis and cloned into the backbone of a 3rd-generation CAR with CD28 and 4-1BB internal signaling domains. The construct was modified to express CD34 via a P2A peptide to enable detection of CAR following viral transduction. Human primary T cells were activated using anti-CD3/CD28 beads for 48 hours prior to bead removal and electroporation with CD7 gRNA, TRAC gRNA, and Cas9 mRNA. On day three, T cells were transduced with lentivirus particles encoding either CD7 CAR or CAR CD19 control and allowed to expand for a further 6 days. Transduction efficiency and ablation of CD7 and TRAC was confirmed by flow cytometry. Multiplex CRISPR/Cas9 gene-editing resulted in the simultaneous deletion of both CD7 and TRAC in 72.8%±1.92 of cells, as determined by FACS analysis. To prevent alloreactivity, CD3+ CAR-T were removed from the product by magnetic depletion. UCART7 effectively killed T-ALL cell lines (CCRF-CEM, MOLT3, and HSB2) and human primary T-ALL blasts in vitro. Next, we tested the capacity of UCART7 to kill primary T-ALL in vivo without xenogeneic GvHD. Considerable expansion of alloreactive T cells, severe GvHD (mean clinical GvHD score = 5.66), and a robust graft vs. leukemia effect were observed in recipients of WT T cells. In contrast, GvHD was completely absent, T cells were undetectable, and considerable tumor burden was observed in mice receiving TRACΔ T cells. Mice receiving UCART7 had no GvHD and unlike UCART19 controls, effectively cleared T-ALL blasts. Fratricide-resistant and allo-tolerant "off-the-shelf" UCART7 signifies a novel strategy for treatment of relapsed and refractory T-ALL and non-Hodgkin's T-cell lymphoma.
Citation Format: John F. Dipersio, Matthew Cooper. Targeting T cell malignancies with gene edited CAR-T [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr SY42-01.