1092 Leveraging CRISPR/Cas9 and HDR to create an engineered CD33 CAR-T to treat AML

Background Acute Myeloid Leukemia (AML) has a 5-year survival rate of <30%. The heterogenous expression of the disease coupled with the lack of cancer specific antigens has made the treatment of AML difficult. To unlock the full potential of AML therapies, we used CRISPR-Cas9 to genetically ablate the myeloid protein CD33 from healthy donor hematopoietic stem and progenitor cells (HSPCs), creating tremtelectogene empogeditemcel (trem-cel). Trem-cel is a HSPC transplant product designed to provide a reconstituted hematopoietic compartment that is resistant to anti-CD33 drug cytotoxicity. Empowered by the ability to create and target ‘cancer specific antigens’, we leveraged our genome engineering approaches to develop a more sensitive CD33-targeting therapy to address the range of target antigen expression found on patient cells. Recent advances in genome engineering demonstrated the feasibility of directed chimeric antigen receptor (CAR) insertion into T cells to enable next generation CAR-T therapies.1 2 Site-specific insertion of CAR into the TRAC locus has the potential to yield a more uniform CAR-T product, minimize graft-versus-host disease, and enhance CAR potency. Methods CD33-directed CAR constructs were integrated into specific sites within the genome using CRISPR/Cas9 and homology-directed repair (HDR). Several different donor template formats were assessed. CAR frequency, phenotype, and cytokine production were characterized using flow cytometry and Luminex; CAR knock in was validated by nanopore long-read sequencing. Results We achieved consistent CAR+ efficiencies while maintaining high cell viability and CAR-T cell expansion in vitro across several insertion sites. Notably, when CD33-CAR was directed into the TRAC locus (TRAC-CD33-CAR) we observed an average of 38% CD33-CAR+ cells, >95% total TRAC knockout, >85% cell viability and >40x total cell expansion (N=6–13). Cells were predominantly central memory and effector T cells. Molecular characterization by Nanopore confirmed full length sequence insertion and targeted integration into the TRAC locus. When assessed for function, TRAC-CD33-CAR-T cells show target-specific and dose-dependent killing of CD33+ MOLM13 cells in vitro (N=6). To assess the sensitivity of TRAC-CD33-CAR we evaluated cytotoxicity in an in vitro CD33 antigen density modeling platform (0.4k-50k molecules, N=3). TRAC-CD33-CAR-T cells demonstrated greater sensitivity when compared to the standard of care FDA-approved antibody-drug conjugate gemtuzumab ozogamicin (GO) in this assay system. Conclusions This work establishes a genome engineered CAR-T platform that enables development of immunotherapies with potent cytolytic activity, either as a stand-alone treatment or in combination with Vor’s platform of engineered transplant, to eliminate on-target, off-tumor toxicity of CAR-T cells to fully benefit high-risk AML patients. References Eyquem J, Mansilla-Soto J, Giavridis T, et al. Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection. Nature. 2017;543(7643):113–117. doi:10.1038/nature21405. Oh Soyoung A, et al. ‘High-efficiency nonviral CRISPR/Cas9-mediated gene editing of human T cells using plasmid donor DNA.’ The Journal of Experimental Medicine 2022;219(5):e20211530. doi:10.1084/jem.20211530.