Computational Design of a TCR-like Human V_H Domain Antibody That Specifically Recognizes a WT1-Derived Peptide...

T cell receptor (TCR)-like antibodies that recognize a peptide-major histocompatibility complex (pMHC) can enable precise targeting of cancer cells, but developing single-domain binders with high specificity and affinity is challenging. Here, we report the computational design and experimental validation of a TCR-like human heavy chain variable (V_H) domain antibody that specifically recognizes the Wilms' tumor 1 protein (WT1)-derived peptide RMFPNAPYL presented by HLA-A*02:01/β2-microglobulin (RMF/HLA-A*02). Starting from the crystal structure of a Fab antibody bound to RMF/HLA-A*02, we repurposed the soluble trastuzumab-derived V_H scaffold to bind RMF/HLA-A*02. The resulting V_H domain, αRMF_V_H, spans all 9 peptide residues and showed no binding to a control pMHC with a different peptide. Fusion of 1 or 2 αRMF_V_H molecules to human serum albumin domain III yielded monovalent and bivalent constructs with good biophysical properties. The monovalent construct bound selectively to RMF/HLA-A*02 with 81 nM affinity, and when expressed as part of a chimeric antigen receptor on T cells, it functioned as an antigen-recognition domain to selectively activate T cells in the presence of RMF/HLA-A*02-positive cells. The bivalent construct exhibited a dramatically increased apparent binding affinity of 0.37 nM. When both constructs were engineered into the bispecific T cell engager (TCE) format containing an anti-CD3 Fv, the bivalent TCE induced antigen-dependent cytotoxicity much more effectively than the monovalent TCE. These results demonstrate a viable strategy to develop high-specificity, peptide-focused TCR-like V_H domain antibodies that can be readily extended into potent bivalent TCEs for pMHC-targeted therapeutics.