Abstract 5598: Engineered hypoimmune allogeneic CAR T cells as potential off-the-shelf CAR T cell immunotherapies
Authors: Hu Xiaomeng , White Kathy , Gattis Corie , Clarke Ryan , Landry Sam , Basco Ron , Tham Eleonore , Luo Emily , Tucker Andrew , Bandoro Christopher , Chu Elaine , Young Chi , Manner Karl , Nho Priscilla , Lam Ben , Beauchesne Pascal , Foster Aaron , Dowdle William E. , Rebar Edward J. , Fry Terry J. , Schrepfer Sonja 
Published: June 2022

Abstract
Off-the-shelf CAR T cells may offer advantages over autologous strategies, including ease of manufacturing, improved quality control with avoidance of malignant contamination and T cell dysfunction, and the ability to generate a final product from healthy T cells. However, host-versus-graft immune response against histoincompatible T cells prevents the 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. While T cells express CD47, we demonstrate here that CD47 expression above endogenous levels is important for immune evasion. We describe here the engineering of human immune evasive CAR T cells building on our previously described hypoimmune technology (Nat Biotechnol 2019;37(3):252-258 and Proc Natl Acad Sci U S A 2021;118(28):e2022091118). The goal is to achieve improved rates of durable complete remissions by improving allogeneic CD19CAR persistence, since it has been shown that autologous CAR T cells have greater durability over years than allogeneic CAR T cells. Human T cells from healthy donors were obtained by leukapheresis. To generate hypoimmune CD19CAR T cells, gene editing was used to eliminate HLA-I/II and TCR expression and lentiviral transduction was used to express CD47 and CD19CAR containing a 4-1BB costimulatory domain to generate hypoimmune CD19CAR T cells. Control CD19CAR T cells were unmanipulated, i.e., unedited, except for lentiviral transduction used to express CD19CAR. Hypoimmune CD19CAR T cells persist in allogeneic humanized mice and lack T cell activation measured using bioluminescence imaging and ELISPOT analysis, respectively. In contrast, transplantation of control CD19CAR T cells generated from the same human donor resulted in rejection (ELISPOT mean 59 and 558 spot frequencies for hypoimmune CD19CAR T cells and control CD19CAR T cells, respectively; p&lt;0.0001 unpaired t-test). Innate immune cell assays show that CD47 overexpression protects hypoimmune CD19CAR T cells from NK cell and macrophage killing. A blocking antibody against CD47 made the hypoimmune CD19CAR T cells susceptible to macrophage and NK cell killing, confirming the importance of CD47 overexpression to evade innate immune clearance. Importantly, CD47 seemed to provide protection from all NK cell populations while other tested NK cell inhibitory molecules (such as HLA-E/G, PD-L1) seemed to prevent NK cell killing of only certain subpopulations rather than primary NK cells in total. Hypoimmune CD19CAR T cells retain their antitumor activity in the Nalm-6 B cell leukemia model in vitro and in vivo comparable to control CD19CAR T cells derived from various donors. Thus, hypoimmune edits seem to not impact CD19CAR T cell activity and have the potential to provide universal CAR T cells that are able to persist without immunosuppression.
Citation Format: Xiaomeng Hu, Kathy White, Corie Gattis, Ryan Clarke, Sam Landry, Ron Basco, Eleonore Tham, Emily Luo, Andrew Tucker, Christopher Bandoro, Elaine Chu, Chi Young, Karl Manner, Priscilla Nho, Ben Lam, Pascal Beauchesne, Aaron Foster, William E. Dowdle, Edward J. Rebar, Terry J. Fry, Sonja Schrepfer. Engineered hypoimmune allogeneic CAR T cells as potential off-the-shelf CAR T cell immunotherapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5598.