SRP475734 PRJNA1048509 GSE249316 High-specificity CRISPR-mediated genome engineering in CB-011, an anti-BCMA allogeneic CAR-T cell, suppresses allograft rejection in preclinical models Allogeneic chimeric antigen receptor (CAR)-T cell therapies hold the potential to overcome many of the challenges associated with patient-derived (autologous) CAR-T cells. Key considerations in the development of allogeneic CAR-T cell therapies include prevention of GvHD and suppression of allograft rejection. Here we describe preclinical data supporting the ongoing first-in-human clinical trial (CaMMouflage) in relapsed/refractory multiple myeloma patients evaluating CB-011, a hypoimmunogenic, allogeneic anti–B cell maturation antigen (BCMA) CAR-T cell therapy candidate. CB-011 cells feature 4 genomic alterations and were engineered from healthy donor-derived T cells using a Cas12a CRISPR hybrid RNA-DNA (chRDNA) genome-editing technology platform. To address allograft rejection, CAR-T cells were engineered to prevent endogenous human leukocyte antigen (HLA) class I complex expression and overexpress a single-chain polyprotein complex composed of beta-2 microglobulin (B2M) tethered to HLA-E. Additionally, T cell receptor expression was disrupted at the T cell receptor alpha constant locus in combination with the site-specific insertion of a humanized BCMA-specific CAR. CB-011 cells exhibited robust plasmablast cytotoxicity in vitro in a mixed lymphocyte reaction in cell co-cultures derived from patients with multiple myeloma. Additionally, CB-011 cells demonstrated suppressed recognition by and cytotoxicity from HLA-mismatched T cells. CB-011 cells were protected from natural killer (NK) cell–mediated cytotoxicity in vitro and in vivo due to endogenous promoter-driven expression of B2M–HLA-E. Potent antitumor efficacy, when combined with an immune-cloaking armoring strategy to dampen allograft rejection, offers optimized therapeutic potential in multiple myeloma. Overall design: For profiling of NK and T cells following infusion in vivo, 6 different cohorts were established in NOG-Tg (Hu-IL15) mice according to the protocol described above: (i) 2 cohorts of CB-011 cells without pNK cells (CB-011 only, n = 2 mice/cohort), (ii) 2 cohorts of CB-011 cells with NK cells (CB-011+NK, n = 3 mice/cohort), (iii) 1 cohort of B2M KO CAR-T cells without NK cells (B2M KO only, n = 2 mice), and (iv) 1 cohort of B2M KO CAR-T cells with NK cells (B2M KO+NK, n = 3 mice). Additionally, NK and CB-011 cells were isolated for single-cell RNA sequencing (scRNA-Seq; NK IP and CB-011 IP, n = 1/group) prior to infusion. NK and CAR-T cells from either the CB-011 or the CAR KI B2M KO cohorts were isolated from the spleens of NOG-Tg (Hu-IL15 mice) mice 4 days post CAR-T cell infusion by FACS using a human CD45-specific antibody. Cells were resuspended in PBS + 0.4% BSA at a concentration of 1x106 cells/mL and single-cell RNA sequencing libraries were generated (Supplemental methods). GSE249316 pubmed 38345397