Fig. 1: Overview of the TRACeR-I platform design scheme.

a. Summary of TCR and TCR-mimicking antibodies with pMHC-binding modes (PDB identifiers in Supplementary Appendix 1). TCRs, white ribbons; antibodies, magenta. Two edge-case TCRs are rendered as tan and purple surfaces. b, TRACeR-II platform. The N-terminal flexible region on TRACeR-II binders is defined as the ARE. The ARE loop and the scaffold form a concave surface for MHC II engagement. c, Comparison of the different peptide conformations and TRACeR (in yellow) orientations on MHC I and II. The bulged conformation of MHC I peptides requires a parallel binding mode while MHC II peptides can be engaged in a perpendicular orientation relative to the MHC. d, Schematic of our TRACeR-I development approach. In step 1, global docking was performed with PatchDock-seeded RifDock (Supplementary Appendix 2). In step 2, iterative RosettaDock and FastDesign cycles were performed to generate sequence diversity on the binding interface (Supplementary Appendices 3 and 4). Output models were evaluated by binding energy, binding energy to antigen, shape complementarity, buried unsatisfied hydrogen bonds and contact area (Supplementary Appendix 5). In step 3, the diverse sequence suggested from step 2 was implemented into a combinatorial library (library 1) for screening with FACS. In step 4, library 2 enabled the development of specific TRACeRs for different targets. Peptide-focused MHC I binders bind their cognate antigens but have minimal cross-reactivity with irrelevant pMHCs. Staining concentration, 50 nM tetramer concentration. e, Titration curves of TRACeRs with different monomeric pMHC targets under a concentration range from 0.1 nM to 50 μM. Binding signals are shown as the MFI ± s.d. (n = 3 technical replicates). f, Binding kinetics of TRACeRs determined by BLI. Binding kinetics and fitting quality are summarized in Supplementary Table 2.