BCMA-Targeted, Hypoimmune Allogeneic CAR T Cells Exhibit Potent Anti-Tumor Activity Together with the Ability to Evade Innate and Adaptive Immune Rejection in Pre-Clinical Tumor Models

Introduction: Treatment with autologous B cell maturation antigen (BCMA) targeted chimeric antigen receptor T cells (CAR T cells) has yielded robust and durable responses among patients with refractory multiple myeloma. Off-the-shelf CAR T cell therapy using cells from healthy donors provides potential advantages over the current autologous strategy through streamlined manufacturing in addition to allowing more quality control and consistency of the T cell product. However, successful treatment with allogeneic CAR T cell products requires averting rejection by the patient’s innate and adaptive immune responses. To this end, we have engineered human CAR T cells that demonstrate the ability to avoid both adaptive and innate immune rejection. This is accomplished through disruption of the T cell receptor (TCR) and major histocompatibility complexes (HLAI [B2M] and HLAII [CIITA]) preventing rejection through recognition of “non-self.” In addition, “missing-self” recognition is achieved through lentiviral transduction that drives overexpression of CD47 that we have previously shown to repress NK and macrophage cell killing. Additionally, BCMA CAR expression occurs as part of the same lentiviral construct in a bi-cistronic format. These hypoimmune allogeneic BCMA CAR T cells aim to enhance CAR T cell persistence through immune evasion with the goal of providing better clinical efficacy in patients with refractory multiple myeloma.

Methods: T cells, obtained from healthy donor leukapheresis, were transduced with lentivirus containing a second-generation BCMA CAR. Clinically validated BCMA CARs were produced in a similar fashion and used as benchmark controls for initial characterization studies. Transduction efficiency of BCMA CARs was assessed via flow cytometry. BCMA CAR efficacy was analyzed in vitro via luciferase, and Incucyte cytotoxicity assays against RPMI8226 and MM.1S multiple myeloma tumor lines. Systemic MM.1S in vivo tumor models (IV injection) were performed in NSG mice. Hypoimmune allogeneic CAR T cells were produced using lentiviral vectors packaging both CD47 and BCMA CAR in combination with gene editing to disrupt the B2M, CIITA, and TCR genes using electroporation and CRISPR-Cas12b. TCR depleted CAR T cells were subjected to in vitro and in vivo efficacy analyses as mentioned above, using unedited BCMA CAR T cells as controls.

Results: In MM.1S in vivo tumor models, BCMA CAR T cells significantly reduced flux and prolonged survival relative to Mock (untransduced) and tumor only controls, demonstrating tumor clearance that is in line with other clinically validated BCMA CARs. Transduction with lentivirus containing both CD47 and BCMA CAR generates a population of CAR+ T cells that simultaneously overexpress CD47 compared to non-CAR T cells. In Incucyte cytotoxicity assays, CD47 overexpressing BCMA CAR T cells expand and reduce tumor cell counts at low E:T ratios of 1:8 while uncontrolled tumor growth was observed in wells treated with the same total number of Mock, untransduced T cells.

Summary: Our BCMA CAR T cells control MM.1S tumor cells in vivo with efficacy on par to other clinically validated BCMA CARs. BCMA CAR T cells overexpressing CD47 exhibit superior in vitro efficacy against BCMA+ tumor cells compared to treatment with the same total number of Mock untransduced T cells. Studies to characterize immune evasion properties and in vivo efficacy of our hypoimmune-edited BCMA CAR T cells that overexpress CD47 are ongoing. However, our team has previously demonstrated the ability of hypoimmune-edited CD19 CAR T cells to evade both innate and adaptive immune cell recognition. Together these data indicate hypoimmune CD47-BCMA CAR T cells may provide an improved alternative to current autologous CAR T cell therapies.