Oral Presentation CD1-MR1 Workshop 2025

A protein scaffold for the facile generation of ligand-specific MR1 binders (#66)

Jingjia Liu 1 , Photis Rotsides 2 3 , Haotian Du 4 , Yi Sun 2 3 , Hongli Yang 1 , Daria Passow 5 , Nikolaos G. Sgourakis 2 3 , Po-Ssu Huang 1 5
  1. Department of Bioengineering, Stanford University, Stanford, CA, USA
  2. Center for Computational and Genomic Medicine and Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia, Philadelphia, PA, USA
  3. Department of Biochemistry and Biophysics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA
  4. Department of Chemistry, Stanford University, Stanford, CA, USA
  5. Biophysics Program, Stanford University, Stanford, CA, USA

Various ligands presented by MR1 are characteristic of certain metabolically disordered cells, such as infected cells and cancer cells, making MR1-ligand complexes a promising handle to target these diseased cells. However, there haven’t been any engineered modalities that recognize MR1 with ligand specificity beyond a few TCRs identified from patient samples.

Recently, we established modular platforms TRACeR-I and TRACeR-II to efficiently generate peptide-specific protein binders for MHC-I and MHC-II, respectively. The TRACeR platforms use a constant scaffold to anchor the conserved antigen-presenting cleft and a combinatorial library at the antigen recognition element (ARE) positions to allow selection towards specificity. This strategy enables the generation of antigen-specific binders from a yeast-displayed TRACeR library in a matter of weeks, which is much faster than any antibody or TCR development campaign.

The structural similarity between MR1 and MHCs inspired us to adapt the TRACeR platform for MR1 targeting. we explored two strategies. (1) The TRACeR scaffold was docked to MR1 structures, and the interface residues were redesigned computationally to be compatible with MR1. The resulting designs were built into a yeast-displayed library, from which we successfully enriched binders specific for 5OPRU and 6FP, respectively. SPR measurements using the recombinant binders revealed sub-micromolar binding affinities. (2) Hypothesizing that the MHC-I binding scaffold might be promiscuous for MR1, we conducted direct selection from the TRACeR-I platform and discovered a binder specific to 6FP with micromolar affinity when measures by SPR.

We are now working to uncover the binding modes of the discovered binders through structural studies. We will optimize and finalize the molecular design based on the structural insights and aim to deliver a molecular platform that can efficiently generate a spectrum of ligand-specific MR1 binders. Eventually, we hope to develop binders for therapeutically relevant MR1 targets to facilitate research and MR1-targeting therapeutics.

  1. Haotian Du, Jingjia Liu, Kevin M. Jude, Xinbo Yang, Ying Li, Braxton Bell, Hongli Yang et al. "A general system for targeting MHC class II–antigen complex via a single adaptable loop." Nature Biotechnology (2024): 1-10.
  2. Haotian Du, Leena Mallik, Daniel Hwang, Yi Sun, Chengzi Kaku, Daniel Hoces, Shirley M. Sun et al. "Targeting peptide antigens using a multiallelic MHC I-binding system." Nature Biotechnology (2024): 1-11.