Related Experiment Video
Updated: Jul 3, 2025
Production of Human CRISPR-Engineered CAR-T Cells
Published on: March 15, 2021
High-Specificity CRISPR-Mediated Genome Engineering in Anti-BCMA Allogeneic CAR T Cells Suppresses Allograft
Émilie Degagné1, Paul D Donohoue1, Suparna Roy1
1Caribou Biosciences, Inc., Berkeley, California.
Abstract:
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 graft-vs-host disease (GvHD) and suppression of allograft rejection. Here, we describe preclinical data supporting the ongoing first-in-human clinical study, the CaMMouflage trial (NCT05722418), evaluating CB-011 in patients with relapsed/refractory multiple myeloma. CB-011 is 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 HLA class I complex expression and overexpress a single-chain polyprotein complex composed of beta-2 microglobulin (B2M) tethered to HLA-E. In addition, T-cell receptor (TCR) expression was disrupted at the TCR 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 cocultures derived from patients with multiple myeloma. In addition, CB-011 cells demonstrated suppressed recognition by and cytotoxicity from HLA-mismatched T cells. CB-011 cells were protected from natural killer 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. See related Spotlight by Caimi and Melenhorst, p. 385.
Insights
Allogeneic chimeric antigen receptor (CAR) T cell therapy, CB-011, shows promise for multiple myeloma by reducing rejection and graft-versus-host disease. Preclinical data support its ongoing first-in-human trial.
Area of Science:
- Immunotherapy
- Genomic Medicine
- Hematology Oncology
Background:
- Allogeneic chimeric antigen receptor (CAR) T cell therapies offer an alternative to autologous CAR T cells, but face challenges like graft-vs-host disease (GvHD) and allograft rejection.
- Developing off-the-shelf CAR T cell products requires strategies to mitigate immune responses against the infused cells.
Purpose of the Study:
- To present preclinical data for CB-011, an allogeneic anti-BCMA CAR T cell therapy candidate, supporting its first-in-human trial (CaMMouflage, NCT05722418) in relapsed/refractory multiple myeloma.
- To evaluate the hypoimmunogenic engineering of CB-011 designed to overcome allograft rejection and GvHD.
Main Methods:
- CB-011 was engineered using Cas12a CRISPR hybrid RNA-DNA (chRDNA) technology from healthy donor T cells, incorporating four genomic alterations.
- Engineered to prevent HLA class I expression, overexpress a beta-2 microglobulin (B2M)-HLA-E complex, and disrupt T-cell receptor (TCR) expression.
- Site-specifically inserted a humanized BCMA-specific CAR and evaluated in vitro and in vivo models, including mixed lymphocyte reactions and co-cultures with multiple myeloma cells.
Main Results:
- CB-011 demonstrated robust in vitro cytotoxicity against multiple myeloma plasmablasts.
- Engineered hypoimmunogenicity suppressed recognition and cytotoxicity from HLA-mismatched T cells.
- CB-011 showed protection from natural killer cell-mediated cytotoxicity in vitro and in vivo due to B2M-HLA-E expression.
Conclusions:
- CB-011's hypoimmunogenic design, featuring immune-cloaking strategies, offers potential to dampen allograft rejection and enhance therapeutic efficacy in multiple myeloma.
- Preclinical findings support the clinical investigation of CB-011 as a potentially off-the-shelf allogeneic CAR T cell therapy for multiple myeloma.
