Endowing Universal CAR-T cell with Immune-evasive Properties Using TALEN-gene Editing

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Summary

Universal CAR-T is produced from healthy donor T cells and is engineered to kill tumor cells in allogeneic situations. To achieve this aim, TAL Effector Nucleases (TALEN) technology combined with recombinant adeno-associated virus particles (AAV6) are used to engineer T cells. An allogeneic CAR-T is developed by knocking out TRAC and beta-2 microglobulin (B2M) to inactivate HLA-I molecules on CAR-T cells; CAR and HLA-E genes are inserted into the respective loci. The generated universal CAR-T cell showed significant anti-tumor function and allogeneic survival capability.

Endowing Universal CAR T-cell with Immune-evasive Properties Using TALEN-gene Editing

Universal CAR-T is produced from healthy donor T cells and engineered to kill tumor cells in allogeneic situations. The donor T cells should be engineered to avoid immune depletion by host T and NK cells and deviate from killing host normal cells To function in allogeneic situations. An allogeneic CAR-T production strategy is developed by knocking out beta-2 Microglobulin (B2M) to inactivate hla-i molecules on CAR-T cells, aiming to attenuate host T cell attack. This strategy is independent of lymphodepletion drugs but increases the possibility of host nk cell depletion by the missing-self mechanism.

To resolve this problem, this article provides an innovative strategy to introduce HLA-E molecular on CAR-T cells to resist NK cell attack. TAL Effector Nucleases (TALEN) technology combined with recombinant adeno-associated-virus particle (AAV6) is used to engineer T cells. HLA-E is introduced to the B2M locus using B2M-specific TALEN and AAV6 vectors, encompassing the HLA-E gene and a pair of homology arms. In combination with TRAC-specific TALEN and AAV6 vector, the CAR fragment is also inserted into the T cell genome. The double knockout system is efficient, and the HLA-ABC- TRAC- rate could reach 96%. The AAV6-mediated car and HLA-E insertion is efficient and get a high gene knock-in level, as much as 68%. Oligo capture assay and sequence analysis revealed that the CAR and HLA-E fragments are precisely integrated into correct sites dependent on homology recombination insertion.

Fig.1 Universal CAR-T engineering strategy and production efficiency.¹

In this article, the CD22 and CD123 targeted universal CAR-T cells are successfully developed with specific CARs and HLA-E molecular. These engineered CAR-T cells showed significant cytotoxicity against cancer cell lines in vitro and leukemia tumors in mouse models and prolonged the survival period of treated mice.

Fig.2 Antitumor efficacy of CD22 targeted and CD123 targeted universal CAR-T cells.¹

Besides significant anti-tumor activity, they verified the invasion features of the engineered universal CAR-T cells to resist host depletion. Data shows that the HLA-ABC disrupted CAR-T cells are of low immunogenicity and could resist elimination from allogeneic T cells, proliferating 23 times more than HLA-ABC positive control T cells while co-culturing. Moreover, exogenous HLA-E is introduced to the B2M locus to reduce HLA-ABC absent induced attack from NK cells. Results demonstrated that HLA-E can protect most HLA-ABC deficient CAR-T cells from depletion by allogeneic NK cells. HLA-ABC- HLA-E- T cells are efficiently killed by missing-self induced cytolytic function; conversely, HLA-ABC- HLA-E+ cells showed minimal increase in numbers. Moreover, the most prevail NKG2A than NKG2Cphenotype, inhibits and activates receptors of NK cells, among populations endows natural protection on HLA-ABC- HLA-E+ CAR-T cells.

Fig.3 HLA-E expression at disrupted B2M loci endowed CAR-T cells improved antitumor functions in vivo.¹

The antitumor activity of the universal CAR-T cells is also studied by in vitro challenge with Raji and NK cells to mimic the physiological conditions in patients. The inhibition function of HLA-E on NK cell cytolytic activity is also monitored on NK cells from AML and ALL patients. hIL-15 NOG mouse model is used as a complex in vivo model, and the similar protection on HLA-ABC- HLA-E+ cells are observed in vivo.

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Reference

Jo, S.; et al. Endowing universal CAR T-cell with immune-evasive properties using TALEN-gene editing. Nat Commun. 2022, 13, 3453.