De novo design and structure of a peptide-centric TCR mimic binding module

Abstract

T cell receptor (TCR) mimics offer a promising platform for tumor-specific targeting of peptide-MHC in cancer immunotherapy. Here, we designed a de novo α-helical TCR mimic (TCRm) specific for the NY-ESO-1 peptide presented by HLA-A*02, achieving high on-target specificity with nanomolar affinity (K_d = 9.5 nM). The structure of the TCRm/pMHC complex at 2.05 A resolution revealed a rigid TCR-like docking mode with an unusual degree of focus on the up-facing NY-ESO-1 side chains, suggesting the potential for reduced off-target reactivity. Indeed, a structure-informed in silico screen of 14,363 HLA-A*02 peptides correctly predicted two off-target peptides, yet our TCRm maintained a wide therapeutic window as a T cell engager. These results represent a path for precision targeting of tumor antigens with peptide-focused α-helical TCR mimics.

Fig. 1.. Design and experimental validation of de novo mini-TCR mimics.

(A) Schematic of RFdiffusion fold-conditioning pipeline to generate four scaffolds over the NY-ESO-1 HLA-A*02 input (red and gray). (B) Schematic of pipeline using ProteinMPNN to design sequences for Scaffold #1 (purple) and predict binders with AlphaFold2. (C) Yeast display screening of top de novo designs with NY-ESO-1 (red) vs. MART-1 HLA-A*02 (black) tetramers. (D) Binders staining NY-ESO-1 but not MART-1 HLA-A*02 tetramer by flow cytometry. (E) SPR analysis of mini-TCRm 1.1 analyte with immobilized NY-ESO-1 HLA-A*02 (red) vs. MART-1 HLA-A*02 (black). Dissociation constants indicated on sensograms (K_d ); n.d., not determined; RU, response unit. (F) SEC profiles of mini-TCRm 1.1 (purple) vs. co-eluting complex (red) of mini-TCRm 1.1, NY-ESO-1 HLA-A*02, and AD01 nanobody. SDS-Page gel showing fractions of co-eluting complex pooled for crystal screens. mAU, milli-Absorbance Units; kDa, kilodaltons.

Fig. 2.. High-resolution crystal structure reveals peptide-specific interactions.

(A) Front view of mini-TCRm/pMHC complex (PDB ID 9MIN). A1-A4 alpha-helices of mini-TCRm (purple); α1 and α2 helices of HLA-A*02 and full chain (light blue); NY-ESO-1 peptide (gold); β2M (dark blue); AD01 nanobody (white). (B) Side view of complex with Met4-Trp5 bulge on the NY-ESO-1 peptide. (C) Top-down view of complex. (D) Bottom-up view of peptide-specific interactions with surface of mini-TCRm (purple). Box zooming in on NY-ESO-1’s Met4-Trp5 motif (gold) fitting into mini-TCRm pockets formed between the A2 and A3 helices (key residue labels in white). (E) Hydrogen bonds (dashed lines) and key peptide-centric residues between mini-TCRm and NY-ESO-1 peptide. (F) Hydrogen bonds and salt bridges (dashed lines) between mini-TCRm and HLA-A*02.

Fig. 3.. Comparison to existing antibody TCR mimic and natural TCR.

(A) Front view comparison between mini-TCRm (purple), 3M4E5 Fab (magenta and salmon), and 1G4 TCR (brown and green) bound to NY-ESO-1 peptide (gold) and HLA-A*02 (light blue). Height and width of binders shown in Angstroms (Å). (B) Top-down comparison of docking footprints of each binder with MHC contact residues (red orange) and NY-ESO-1 peptide side chains (gold) (C) Bottom-up comparison of interactions between the NY-ESO-1 peptide (gold) and binding pockets, with the Met4-Trp5 motif labeled. Dissociation constants (Kd) for each complex is shown at the bottom. nanomolar, nM; micromolar, μM.

Fig. 4.. Structure-guided identification of off-target peptides reveals a robust therapeutic window.

(A) Mutation scanning the NY-ESO-1 peptide (sequence shown above plot; mutated residues in red) and pulsing on T2 cells. Variants are named by original residue, position, followed by the mutation. Measured mini-TCRm MFI (Mean Fluorescence Intensity) by flow cytometry. (B) Table of candidate off-targets from the MHC Motif Atlas. (C) Ranking of candidate off-targets based on ProteinMPNN and the crystal structure. (D) Pulsing candidate off-target peptides on T2 cells and staining with mini-TCRm. (E) Schematic of a mini-TCRm T cell engager (TCE; purple) activating T cells against a cancer cell (salmon) with an on-target peptide (red) but not a healthy cell (gray) with an off-target peptide (black). (F) T2-Jurkat co-culture assay with pulsed peptides and mini-TCRm TCE. T cell activation shown as percentage of CD69+ NFAT-eGFP+ Jurkat cells versus no-pulse control from flow cytometry analysis. TCE dose shown in nanomolar on the log scale (log(nM)).