Background A popular target for tumor vaccines and TCR-T cell therapy is NY-ESO-1, a cancer testis antigen (CTA) family member. NY-ESO-1 is expressed at varying degrees in several cancers such as synovial sarcoma, neuroblastoma, melanoma, and breast cancer, but its expression in normal tissues is restricted (testis), which makes it an ideal target for limiting potential off-target toxicity. NY-ESO-1 is an intracellular antigen whose nine peptides (157-165) can be presented to the cell surface by HLA-A02 for T-cell recognition. Biocytogen’s TCR-mimic (TCRm) platform is capable of developing TCR-like antibodies that target the NY-ESO-1/HLA-A02 complex to overcome limitations of low TCR affinity.
Methods Biocytogen’s unique RenMabTM mice were engineered to express HLA-A02 and immunized to obtain TCRm antibodies against NY-ESO-1/HLA-A02. To detect antibody binding to target or off-target cells, flow cytometry was performed. Surface plasmon resonance (SPR) measured the affinity between antibodies and the NY-ESO-1/HLA-A02 complex. Alanine scan substitution was performed to identify the residues critical for TCRm antibody recognition. Cytotoxicity and activation of TCRm-T cells were measured by LDH and an NFAT Jurkat reporter cell line, respectively. In vivo tumor suppression experiments tested the efficacy of NY-ESO-1 TCRm-T cell therapy in severely combined immunodeficient B-NDG mice inoculated with tumor cells.
Results Through immuno-exclusive HLA-A02/RenMab mice, combined with high-throughput Beacon screening and sequence verification, fully humanized antibody sequences of NY-ESO-1/HLA-A02 with mature affinity in vivo were obtained. The obtained antibody affinity is at the nM level (TCR is generally at the μM level). We detected key antibody-antigen binding sites and speculated potential off-target peptides following an alanine scan, then using NFAT Jurkat reporter cells, we identified that our candidate antibodies exhibited low off-target risk. The optimal sequence was applied to cell therapy, and we observed that TCRm-CD8+ T cells demonstrated specific killing of target cells in vitro, and showed rapid and obvious clearance effect on NY-ESO-1+HLA-A02+ tumors with low abundance antigen in vivo.
Conclusions Biocytogen’s TCRm antibody discovery platform combines unique immune technology with a multiplex screening method to obtain a high-affinity NY-ESO-1/HLA-A02 antibody, which can bind and specifically kill HLA-A02-positive cell lines presenting NY-ESO-1. This TCRm platform overcomes the limitation of targeting extracellular antigens and displays a wider range of applications.
Ethics Approval All animal studies were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Biocytogen Beijing Co., Ltd.
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