Abstract LB144: Overexpression of CD47 protects hypoimmune CAR T cells from innate immune cell killing

Abstract

Off-the-shelf CAR T cells could offer advantages over autologous strategies, including ease of manufacturing, quality control and avoidance of malignant contamination and T cell dysfunction. TCR editing can effectively prevent graft-versus-host reactions. However, the vigorous host-versus-graft immune response against histoincompatible T cells prevents expansion and persistence of allogeneic CAR T cells and mitigates the efficacy of this approach. A major challenge is that, while HLA deletion can result in adaptive immune evasion, innate reactivity is enhanced. CD47 overexpression can block both NK cell and macrophage killing (J Exp Med (2021) 218 (3): e20200839), and we hypothesized that T cells would lose their immunogenicity when human leukocyte antigen (HLA) class I and II genes are inactivated and CD47 is over-expressed. We describe here the engineering of human immune evasive CAR T cells based on our previously described hypoimmune technology. Human T cells from healthy donors were obtained by leukapheresis. CRISPR/Cas9 technology was used to delete b2m, CIITA, and TCR and lentiviral transduction to overexpress CD47 and CD19CAR. Control T cells were unmanipulated except for overexpression of CD19CAR containing a 41BB costimulatory domain. When transplanted into allogeneic humanized mice, hypoimmunogenic HLA-I/II- TCR- CD47+ CD19CAR+ T cells evade immune recognition by T and B cells compared to CD19CAR+ T cells generated from the same human donor using ELISPOT and flow cytometry analysis. Innate immune cell assays show that CD47 overexpression protects HLA-I/II deficient CAR T cells from NK cell and macrophage killing in vitro and in vivo. Relative CD47 expression levels were analyzed to understand the relevance of CD47 for protection from macrophage and NK cell killing. A blocking antibody against CD47 made the hypoimmunogenic CAR T cells susceptible to macrophage and NK cell killing, confirming the importance of CD47 overexpression to evade innate immune clearance. The use of CD47 blocking could additionally be envisioned as a safety strategy for our hypoimmunogenic CAR T cells. Neither isolated CD47 overexpression nor all three hypoimmune modifications or knockout of the TCR showed any effect on the cytotoxic potential of CAR+ T cells. Hypoimmune CAR+ T cells retain their antitumor activity in the Nalm-6 B cell leukemia model in vitro and clear leukemic cells in NSG mice across a range of tumor cell: CAR T cell ratios comparable to unmodified CAR T cells. These findings show that hypoimmunogenic CAR T cells are functionally immune evasive in allogeneic recipients with cytotoxic anti-tumor capacity and suggest they could provide universal CAR T cells that is able to persist without immunosuppression. Blocking CD47 could additionally serve as safety strategy for our hypoimmunogenic CAR T cells. Citation Format: Xiaomeng Hu, Mo Dao, Kathy White, Ryan Clarke, Sam Landry, Ron Basco, Corie Gattis, Eleonore Tham, Emily Luo, Andrew Tucker, Christopher Bandoro, Elaine Chu, Junmo Kim, Chi Young, William E. Dowdle, Edward J. Rebar, Terry J. Fry, Sonja Schrepfer. Overexpression of CD47 protects hypoimmune CAR T cells from innate immune cell killing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB144.

CD47 expression is critical for CAR T-cell survival in vivo

“…48 49 There is also growing evidence supporting a role for overexpressing CD47 to enhance the survival of adoptively transferred immune cells. 50 Our data supports the notion that overexpressing CD47 in immune cells could be protective in the context of cell therapy for cancer.…”

Genetic engineering strategies to enhance antitumor reactivity and reduce alloreactivity for allogeneic cell-based cancer therapy

“…To prevent macrophage killing, exploitation of CD47/SIRPα pathway provides a potential remedy; akin to CD47 hi tumors, graft cells engineered to overexpress CD47 evade macrophage-mediated phagocytosis ( 163 ). By combining lentivirus transduction of CD47 with gRNA guided CRISPR-Cas 9 knockdown of B2M and CIITA, it is feasible to engineer HLA − CD47 hi effector cells that are immune to both T cell and macrophage mediated HvG effect ( 163 , 164 ). Compared to non-engineered allogeneic grafts, CD47 modified cells showed enhanced survival and persistence within in vivo mouse xenograft models ( 163 ).…”