A p53^R273H-selective bispecific T cell engager: Computational design and functional validation

TP53 mutations occur in over 50% of human cancers and generate shared neoantigens, including p53^R273H, which represents a compelling target for precision immunotherapy. However, HLA allele-specific presentation and the risk of off-target toxicity remain major challenges. Here, we developed a group of bispecific T cell engagers (TCEs), namely, TCE01 and its optimized versions, based on a stimulated patient-derived TCR that specifically recognizes p53^R273H presented by HLA-C∗01:02. The immunodominant 9-mer epitope SFEVHVCAC was identified as selectively presented by this allele. Incorporation of the stapled single-chain TCE formats reduced molecular weight by ∼24% compared with full-length constructs, while fully preserving binding affinity and markedly enhancing in vitro T cell activation and cytotoxicity (>90% at 0.625 nM). Machine learning-guided directed evolution using Boltz-2 and EvotProtGrad yielded TCE01Cr3, exhibiting a K _d of 2.51 nM and a subpicomolar EC₅₀. Alanine scanning combined with combinatorial peptide library screening and structural modeling pinpointed peptide residues 2 and 4 as critical for TCR recognition in context of TCE01. TCE01Cr3 exhibited remarkable in vitro functional stability following 128 h of incubation in human serum and demonstrated a prolonged in vivo half-life in mice (t _1/2 = 39.40 ± 10.50 h). In 3D model, monotherapy with TCE01Cr3 eliminated >80% of p53^R273H-expressing colorectal cancer cells within 48 h while sparing non-malignant stroma, independent of chemotherapy potentiation (oxaliplatin or irinotecan). This off-the-shelf TCE provides a framework for precision immunotherapy in HLA-C∗01:02-positive patients with p53^R273H tumors.