Bispecific NK‐Cell Engager Targeting TP53 Mutation Through Integration of a TCR‐mimic Antibody

ABSTRACT Natural killer (NK) cells are valuable for cancer immunotherapy because they kill tumor cells without prior sensitization or human leukocyte antigen (HLA) restriction. However, their efficacy is often limited by immune‐evasion, especially major histocompatibility complex I (MHC‐I)‐mediated NK cell inhibition. NK cell engagers (NKCEs) can redirect NK cells to tumor for more effective immunotherapy, but most current NKCEs target tumor‐associated antigens (TAAs), risking on‐target, off‐tumor toxicity in clinical applications. Neoantigens, as tumor‐specific antigens (TSAs) arising from somatic mutations and presented by MHC‐I on tumor cell surfaces, represent ideal tumor‐specific target. Here, we engineered NKCEs built on neoantigen‐targeting TCR‐mimic antibodies (TCRm‐Abs) to enhance tumor‐specific NK cell‐mediated cytotoxicity. Using a high‐throughput hydrogel‐based microwell single‐cell chip (HMSC‐chip) coupled with T2 cell‐based de‐selection, we identified C33‐Ab, a functional TCRm‐Ab specific for p53 R175H /HLA‐A*02:01, from ∼500,000 hybridoma cells within 8 weeks. Based on this TCRm‐Ab, we constructed a bispecific NKCE (C33×CD16), a single‐chain diabody (scDb) linking C33‐Ab to an anti‐CD16 scFv. C33×CD16 specifically bridges NK cells with p53 R175H /HLA‐A*02:01‐positive cancer cells. It potently enhanced NK cell‐mediated cytotoxicity in vitro and antitumor efficacy in vivo upon adoptive NK‐cell transfer. This TCRm‐Ab‐based bispecific NKCE offers a promising strategy for tumor‐specific NK cell‐mediated immunotherapy by targeting intracellular oncogenic hotspot mutations.