Abstract LB009: A BCMA-specific allogeneic CAR-T cell therapy (CB-011) genome-engineered to express an HLA-E fusion transgene to prevent immune cell rejection

The approval and commercial launch of multiple first-generation CD19- or BCMA-directed, autologous CAR-T cell products have laid the foundation and opened a path for the development of more advanced cellular therapeutics, including CAR-T cell products with next-generation capabilities. Among these newer designs, allogeneic cell therapies are positioned to unlock the broad potential of engineered immune cells as a leading therapeutic modality. However, expansion, persistence, armoring, and trafficking of allogeneic CAR-T cells are critical to achieving long-term efficacy. Caribou Biosciences is advancing a BCMA-specific allogeneic CAR-T cell product candidate, CB-011, with an immune cloaking approach that includes both the removal of the endogenous B2M protein and the insertion of a B2M-HLA-E-peptide (B2M-HLA-E) fusion protein transgene. This strategy is designed to blunt CAR-T cell rejection by both recipient T cells and NK cells, and CB-011 is in preclinical development for relapsed or refractory multiple myeloma (r/r MM). We used Cas12a chRDNA guides in the manufacture of CB-011 to make four edits, including site-specific insertion of a proprietary humanized anti-BCMA CAR into the TRAC locus with high specificity and efficiency, thus eliminating TCR expression to prevent graft-versus-host disease (GvHD). In addition, we inserted a gene encoding a B2M-HLA-E fusion protein into the native B2M gene locus. This method simultaneously prevents the expression of the native B2M protein and expresses a minor HLA class I antigen HLA-E to blunt both T- and NK-mediated rejection of the CAR-T cells by the recipient’s immune system. B2M is a protein that stabilizes all HLA class I antigens on the cell surface, therefore its knockout eliminates endogenous HLA class I presentation on the surface of the CAR-T cells. To demonstrate that the B2M-HLA-E fusion protein expression protects CB-011 from NK-mediated cell killing, we developed an in vitro competition assay in which CB-011 cells are co-incubated with NK cells. We observed that CAR-T cells expressing the B2M-HLA-E fusion have a survival advantage over cells that do not express the fusion in the presence of NK cells in vitro, indicating that they could resist killing by a recipient’s NK cells and potentially circulate longer. The BCMA-specific CAR leads to long-term survival in mice bearing established orthotopically-engrafted MM tumor cells. Furthermore, mice treated with high doses of CB-011 did not experience symp