Abstract 4091: Engineered hypoimmune CAR T cells provide lasting tumor control in immunocompetent allogeneic humanized mice even with re-challenge
Authors: Hu Xiaomeng , Beauchesne Pascal , Manner Karl , Gattis Corie , Ngo Priscilla , De Jesus Rowena , Ankala Ramya , Young Chi , Wells Frank , Weng Lindong , White Kathy , Dowdle William E. , Foster Aaron , Fry Terry J. , Schrepfer Sonja 
Published: April 2023

Abstract
Off-the-shelf CAR T cells potentially offer advantages over autologous strategies such as ease of manufacturing, quality control, off-the-shelf availability, and lack of T cell dysfunction, as well as the ability to generate a more consistent CAR T product from healthy T cells. 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 with this approach. 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 disrupted and CD47 is over-expressed. 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).Human T cells from healthy donors were obtained by leukapheresis. CRISPR/Cas12b technology was used to disrupt the B2M, CIITA, and TCR genes, and lentiviral transduction was used to overexpress CD47 and to express a CD19 CAR to generate hypoimmune (HIP) CD19 CAR T cells. Control T cells were unmanipulated except for overexpression of the CD19 CAR (unmodified).For 3 months persistence studies, allogeneic SGM3 humanized mice were injected with 1 × 106 Luc+ Nalm6 cells and received 7 × 106 control CD19 CAR T cells or HIP CD19 CAR T cells.In the mice treated with either unmodified CD19 CAR T cells and HIP CD19 CAR T cells, tumor control was initially rapidly achieved. However, unmodified CD19 CAR T cells were eventually rejected by the host and the loss of these cells resulted in re-growth of tumor. By contrast, in HIP CD19 CAR T injected mice, tumor control was maintained throughout the study, including following a rechallenge at day 83 with NALM6 cells without further administration of HIP CD19 CAR T cell. Flow cytometry at endpoint from bone marrow and spleen confirmed persistence of HIP CD19 CAR T cells.These findings show that HIP CD19 CAR T cells are immune evasive in allogeneic recipients and data suggest that HIP CD19 CAR T cells are able to persist and maintain efficacy without immunosuppression.
Citation Format: Xiaomeng Hu, Pascal Beauchesne, Karl Manner, Corie Gattis, Priscilla Ngo, Rowena De Jesus, Ramya Ankala, Chi Young, Frank Wells, Lindong Weng, Kathy White, William E. Dowdle, Aaron Foster, Terry J. Fry, Sonja Schrepfer. Engineered hypoimmune CAR T cells provide lasting tumor control in immunocompetent allogeneic humanized mice even with re-challenge. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4091.