Abstract 1320: Engineered expression of HLA-E and HLA-G protects iPSC-derived cells from killing by primary NK cells
Authors: Lebid Andriana , Kim Dae Hwan , Greco Gabrielle , Jessup Heidi , Suarez Alyssa , Brown Shelby , Alexander Nicholas , Desai Mitan , Gurung Buddha , Morse Barry , Perry Daniel J. , Naso Michael F. , Levitsky Hyam 
Published: March 2024

Abstract
Background: A major challenge to off-the-shelf allogeneic cell therapies is the HLA-targeted elimination of genetically dissimilar cells. To combat this, HLA Class I can be deleted through transgene insertion at the beta-2 microglobulin (B2M) locus, while HLA Class II can be deleted by disruption of the gene encoding MHC Class II Transactivator (CIITA). Lack of HLA Class I expression, however, marks cells for elimination by natural killer (NK) cells, which normally receive inhibitory signals from this protein family. HLA-E and HLA-G are non-classical HLA Class I molecules that are far less polymorphic than HLA-A/B/C. They can inhibit natural killer cell-mediated activation by engaging NKG2A, KIR2DL4, ILT2, and ILT4. The objective of this study was to assess allo-evasion from NK cells by iPSC-derived cells engineered to overexpress HLA-E and -G.
Methods: Allo-evasion by HLA-E, -G or both was first assessed in the chronic myelogenous leukemia K562 cell line, which expresses very low HLA Class I and is highly susceptible to NK-mediated killing. K562 cells were engineered using CRISPR/CAS9 to express chimeric molecules encoding either HLA-E or HLA-G heavy chains tethered to a defined nominal self-peptide and B2M, or HLA-E/peptide/B2M-P2A-HLA-G/peptide/B2M. Along with parental K562, these were used as targets in co-cultures with allogeneic PBMCs (n=22 donors) at different E:T ratios. Target killing was observed using flow cytometry and normalized to parental K562. Phenotyping of effector PBMCs for NK frequency and expression of HLA-E and -G receptors (NKG2A, KIR2DL4, ILT2, and ILT4) was performed to study correlations with killing efficiency and allo-evasion. IPSCs with endogenous B2M knocked out were also engineered to overexpress HLA-E and -G with the constructs described above. These were then differentiated into NK cells and gamma delta T cells using proprietary processes. Allo-evasion of these cells from killing by the same PBMC cohort was evaluated by flow cytometry.
Results: While NK cells across donors expressed heterogeneous combinations of HLA-E and -G receptors, the strongest correlation with PBMC allo-responsiveness was NK frequency. K562 and iPSC-derived cells lacking HLA were susceptible to killing by PBMCs. Overexpression of HLA-E and -G offered protection to K562 and iPSC-derived cells against all tested donors. HLA-E offered more protection than HLA-G, and the combination of both HLA-E and -G was most potent.
Conclusion: When genetically dissimilar HLA Class I protein family must be deleted to prevent graft rejection, expression of the more-conserved HLA-E and -G can effectively protect allogeneic drug products from elimination. These Allo-evasionTM edits may increase the in vivo persistence and enable multi-dosing of our iPSC-derived allogeneic cell therapies.
Citation Format: Andriana Lebid, Dae Hwan Kim, Gabrielle Greco, Heidi Jessup, Alyssa Suarez, Shelby Brown, Nicholas Alexander, Mitan Desai, Buddha Gurung, Barry Morse, Daniel J. Perry, Michael F. Naso, Hyam Levitsky. Engineered expression of HLA-E and HLA-G protects iPSC-derived cells from killing by primary NK cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1320.