Allogeneic cell therapies offer a scalable and off-the-shelf alternative to the autologous approach, but immune rejection, particularly by natural killer (NK) cells following human leukocyte antigen (HLA) ablation, remains a major barrier to their persistence. Here, we report an improved synthetic NKG2A engager to selectively inhibit NKG2A⁺ NK cells while avoiding activation of NKG2C⁺ subsets, thereby overcoming a key limitation of the natural ligand HLA-E. Engineered regulatory T cells (EngTregs) lacking HLA and expressing the engager were protected from in vitro NK cell-mediated cytotoxicity more effectively than previously reported NK inhibitory strategies. In humanized mouse models, EngTregs persisted for up to 12 weeks, whereas unprotected cells were rapidly rejected. Incorporation of the engager into a clinically compatible dual-AAV EngTregs preserved Treg identity and function while conferring resistance to immune rejection. Together, these findings establish the improved NKG2A engager as an effective synthetic immune-evasion strategy and provide a clinically translatable approach to enable durable persistence of off-the-shelf EngTreg therapies.
Tingxi Guo, Kaya Epstein, M. Hoover et al.· EMBO Molecular Medicine· 0 citations
Peptides presented by class-I Human Leukocyte Antigen (HLA-I) proteins provide the basis of immune surveillance. Conversely, reduced surface HLA-I expression is a hallmark of immune evasion in cancers, which confounds the identification of peptide antigens and neoantigens. Here, we outline a system (HLA-Shuttle) for in vitro manipulation of cells with engineered components of the HLA-I processing pathway that improves recovery of the immunopeptidome of immunologically “cold” tumors.
HLA-Shuttle is comprised of an engineered variant of the HLA class I chaperone tapasin that bypasses its native degradation and ER retention signals, enabling improved expression and escape from the ER. HLA-Shuttle provides a continuum of chaperoning activity for HLA-I complexes from their point of assembly in the ER to the cell surface, improving antigen presentation in those cells.
Our data suggest that HLA-Shuttle functions in a multimodal fashion, both enhancing HLA-I complex production in the ER while stabilizing the folded conformation of HLA-I molecules globally. This is evidenced by increased surface expression of HLA-I, while cellular trafficking assays and single particle tracking reveal an extension of their cell-surface lifetimes and microdomain formation, implying an enhancement in their stability. Leveraging this technology, we captured the immunopeptidomes of neuroblastoma cell lines. We observed improved immunoprecipitation of HLA-I complexes, which correlated with a significant expansion of the observable immunopeptidome in immunologically cold neuroblastoma cells. Following bioinformatics analysis to search for therapeutically relevant peptides, we identified multiple novel tumor associated antigens (TAAs) from both known and novel cancer immunotherapy targets.
In conclusion, HLA-Shuttle restores antigen presentation in immunologically cold tumor cells, facilitating identification of TAAs with favorable therapeutic potential.
T32 Fellowship
Classical and Non-Classical Antigen Presenting Cells (APC)
Daniel Hwang, Molly C. Erdman, Santosh Adhikari et al.· Journal of Immunology· 0 citations
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