CD7 CAR for the Treatment of Acute Myeloid and Lymphoid Leukemia
Authors: Silva Diogo , Tashiro Haruko , Srinivasan Madhuwanti , Brenner Malcolm K. , Mamonkin Maksim 
Published: October 2019

Abstract
CD7 is a T- and NK-cell marker highly expressed in T cell leukemia and in 30% of AML cases, where its presence correlates with chemoresistance and worse prognosis. We created CD7-directed chimeric antigen receptors (CARs) using single chain variable fragment (scFv) sequences derived from three different CD7-specific mAb clones and evaluated the feasibility and potency of using CD7 CAR T cells for the treatment of the CD7+ hematologic malignancies. Since normal T cells themselves express CD7, their transduction with CD7 CARs limited their expansion in vitro, resulting in approximately 30-fold fewer T cells after 14 days of culture compared to control CAR-transduced cells. To circumvent this problem of fratricide, we used the CRISPR/Cas9 system to disrupt the CD7 gene in T cells prior to retroviral transduction with CD7 CAR. The CD7 gene was successfully disrupted in 90% of T cells, minimizing fratricide of CD7 CAR T cells, and leading to a robust T cell expansion, similar to that of control T cells transduced with an irrelevant CAR (310-fold vs 380-fold expansion after 14 days, respectively). Lack of CD7 did not compromise CAR T cell effector function, as we observed equal cytotoxicity and expansion in CD7+ and CD7- CD19 CAR T cells upon coculture with a CD19+ cell line Raji (P=0.99 and P=0.86, respectively).
Expanded CD7-deficient CD7 CAR T cells demonstrated robust cytotoxicity against CD7+ AML cell lines KG-1a and Kasumi-3 and T-ALL cell line CCRF-CEM, resulting on average in a 19-fold expansion of CAR T cells and elimination of malignant cells (92-99.9%) after 7 days of co-culture at a 1:4 initial effector-to-target ratio. This cytotoxicity was CD7-specific, as CD7 CAR T cells showed no activity against a CD7-negative cell line Raji. We also observed potent activity of CD7 CAR T cells against primary AML cells, leading to an 80% reduction in blast counts after 48 hours of co-culture at a 1:1 effector-to-target ratio, compared with control CAR T cells. Moreover, a 5-hour co-culture of CD7 CAR T cells with primary AML blasts and subsequent culture on a methylcellulose medium for 12 days resulted on average in a 26-fold reduction in leukemic colonies; these data suggest that the CD7 CAR T cells can eliminate primitive leukemic progenitors.
Because CD7 is expressed on some myeloid progenitors, we assessed the activity of CD7 CAR T cells against normal myeloid precursors. After coculturing cord blood cells with CD7 CAR T cells at a 10:1 effector-to-target ratio and subsequently expanding myeloid colonies on a methylcellulose medium for 14 days, we observed no significant differences in the numbers of monocytic, erythrocytic and granulocytic colonies compared to a coculture with control CAR T cells. Hence, CD7 CAR T cells appear non-toxic to normal myeloid progenitor cells.
In summary, we show that CD7 CAR T cells can be efficiently generated and expanded following genomic disruption of the CD7 gene by CRISPR/Cas9. Expanded CD7 CAR T cells produce robust cytotoxic activity against AML and T-ALL cell lines as well as primary AML blasts, and show no toxicity against normal myeloid progenitors. These results demonstrate the potency and support the feasibility of using CD7 CAR T cells for the targeted therapy of acute myeloid and lymphoid leukemia.

Disclosures
Brenner: Viracyte: Equity Ownership; Cell Medica: Patents & Royalties.