TCR-mimic bispecific nanobody-based T cell engager targeting intracellular tumor antigens for cancer immunotherapy.

T cell engager (TCE) immunotherapies have revolutionized the landscape of cancer treatment; however, their efficacy remains limited by the inaccessibility of intracellular tumor antigens. Conventional bispecific T cell engagers, typically constructed from aggregation-prone single-chain variable fragments (scFvs), suffer from structural instability and an antigenic scope restricted to extracellular targets. To overcome these critical limitations, we presented a proof-of-concept study establishing a modular bispecific VHH‑VHH immunotherapeutic platform. Specifically, we developed a first-in-class TCR-mimic bispecific nanobody (Nb)-based T cell engager (TCRm Bi-NbTE) platform that simultaneously engages CD3ε on T cells and tumor-specific peptide-MHC class I (pMHC I) complexes, exemplified by HLA-A2/WT1126-134 or HLA-A2/GPC3144-152. Functional analyses in vitro and in vivo studies demonstrated that TCRm Bi-NbTE exhibits exceptional specificity, potently induces antigen-restricted T cell activation, and mediates selective lysis of pMHC I⁺ tumor cells while sparing antigen-negative cells. In multiple mouse xenograft models, including both cell-derived xenograft (CDX) and patient-derived xenograft (PDX) models, TCRm Bi-NbTE significantly suppressed tumor growth, prolonged survival, and enhanced T cell infiltration without treatment-related adverse effects. By redirecting T cell against intracellular antigens in an HLA-restricted manner, TCRm Bi-NbTE establishes a modular, scalable, and clinically translatable platform for next-generation cancer immunotherapy across a broad spectrum of solid and hematologic malignancies.