Structure of WT1-TCB, mode of action, binding motif, and crystallography. (A) Scheme of WT1-TCB–mediated T-cell engagement. (B) Binding motif as determined by alanine scanning and pMHC array. Anchor residues Met2 and Leu9 (marked in blue) can be replaced by few amino acids, whereas Arg1, Phe3, Asn5, and Ala6 (red) are important for recognition of the RMF–pMHC complex by 11D06. Residues Pro4, Pro7, and Tyr8 (black) are less critical for binding. (C) Single-substitution plot for RMF peptide in the context of HLA-A*02–β2m heterodimer bound by 11D06 antibody. Each bar corresponds to substitution with 1 of 20 amino acids, and a deletion is shown by the last bar. The height of each bar indicates the signal intensity. Bars are ordered as follows: the 8 nonpolar residues (AFILMVWP; orange); the 7 polar/neutral residues (GSYCQTN; green); the 3 positively charged residues (RKH; blue); and the 2 negatively charged residues (DE; pink). (D) Crystal structures of the 11D06–RMF–pMHC (pdb entry 7BBG) and ESK1–RMF-pMHC (pdb entry 4WUU) complexes. (E) View of the peptide-binding site of pMHC in the Fab 11D06–HLA-A*02 RMF–pMHC complex. Heavy and light chains of Fab 11D06 are colored in blue and cyan, respectively, and the RMF peptide is colored in magenta. The HLA-A*02 pMHCI is shown as a transparent surface in white, with selected side chains highlighted in green. (F) Quantification of endogenous RMF peptide purified from a primary AML patient sample. Mass spectrometry chromatogram overlay of dioxidized endogenous RMF peptide precursor ion [m/z = 570.7790 (2+); red] and dioxidized internal standard ¹³C₆, ¹⁵N₄-labeled peptide precursor ion [m/z = 575.7831 (2+); blue]. Endogenous RMF peptide was immunoprecipitated by using WT1-TCB. Figure 2A created with BioRender.com.