Study introduces TRACeR-I, a protein platform with broad HLA compatibility, paving the way for advanced immune response engineering and disease-specific targeting.

Study: Targeting peptide antigens using a multiallelic MHC I-binding system. Image Credit: Nemes Laszlo / Shutterstock.com

In a recent study published in Nature Biotechnology, researchers describe the molecular structure of targeted recognition of antigen-MHC complex reporter for MHC I (TRACeR-I), a protein platform that can be used to engineer immune responses.

Importance of MHC I-peptide presentation

In diseased cells, multiple aberrant proteins accumulate over time, including tumor-associated antigens or neoantigens, as well as pathogen-derived antigens, which are ultimately degraded within proteosomes and lysosomes. Some of these antigenic peptide fragments, which are between eight and 12 residues in length, are presented on the cell surface by class I major histocompatibility complex (MHC I) proteins.

MHC 1 presentation is crucial to the immune response, as it allows innate immune cell-mediated killing of diseased cells and stimulates adaptive immunity. Adaptive immunity allows T-cells to recognize antigens and undergo activation, thereby triggering cell-mediated killing and antibody production.

The potential of T-cell receptors

T-cell receptors (TCRs) bind to peptide-MHC complexes (pMHC) through a combination of six flexible complementarity-determining region (CDR) loops. The diversity introduced by the combinatorial mechanism, as well as variations in docking angles and binding orientations, allows for significant specificity of antigen recognition.

Currently, scientists are engineering TCR CDRs to produce MHC I binding molecules with binding specificity against disease antigens. However, there are several challenges associated with these studies.

To this end, the development of TCRs from cells possessing low affinity for the antigen is very slow. Furthermore, TCRs are associated with inherent polyspecificity, which facilitates immune surveillance for many pathogen-derived epitopes with a relatively restricted TCR repertoire while also limiting their specificity as therapeutic agents.

The HLA genes encoding MHC I peptides have over 38,000 allotypes across populations and genetic groups. TCRs are restricted to pMHC targets across only a few HLA versions, thus limiting their utility in divergent genetic contexts.

What is TRACeR-I?

The authors of the current study exploited the limited number of backbone conformations on MHC I antigens to produce a platform compatible with a wide range of HLA allotypes.

A single docking orientation can be used by most binders to interact with MHC I if it covered the full length of the antigen. This pMHC I binder scaffold has a surface that can be adapted to specifically bind to multiple disease-related peptides, which has the potential to be a highly cost-effective and rapid system.  

The scaffold of TRACeR-I uses a modified form of their earlier peptide-focused pMHC II-binding platform, TRACeR-II, to create an MHC I counterpart. TRACeR-II has a concave surface feature that normally binds perpendicular to the peptide-binding groove on extended peptide structures of MHC III.

This platform was adapted by introducing a directed mutation to engage the bulged peptide conformation on MHC I in a parallel orientation. Using computational modelling, a sequence compatible with the MHC I surface was identified. Thereafter, variations were introduced within the sequence in the concave feature to optimize the binding mode.

Binding specificity

To test the platform, three distinct pMHC I targets including peptides derived from esophageal squamous cell carcinoma, Epstein-Barr virus, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were presented on HLA targets and across both HLA A*02 and B*08 allotypes.

All three targets were specifically bound by TRACeR, the peptide-focused binding interface binder, thus suggesting a generalized binder-generating platform capable of specifically targeting multiple HLA alleles.

The specific binding ability for divergent pMHC I targets indicates that this platform can bind molecules over a wide range of targets and across populations for multiple applications. Despite their bacterial origin, TRACeRs do not provoke robust immune or cytotoxic responses in mice.

This simplistic approach allows the rapid and facile creation of peptide-focused pMHC binders for a wide range of antigens.”

Investigating the efficacy of TRACeR I

The TRACeR I platform was incorporated into a humanized antibody fragment in a bispecific T-cell engager (BiTE) format. When tested against patient-derived cancer cells, effective T-cell activation was observed with on-target killing at nanomolar concentration.

Molecular mechanism

X-ray crystallography demonstrated that TRACeR engages the pMHC I target along its whole length by shape complementarity. The interface possesses a set of eight residues that remain constant across different HLAs, thereby eliminating the need for variable pMHC I recognition modes by its invariant binding mode.

Further variation among the eight-residue set allowed binding to a wide range of epitopes across multiple diseases presented by divergent HLA allotypes. This point-substitution resolution indicates that TRACeRs can be developed for divergent antigens without losing specificity.

A monomeric form of the TRACeR was subsequently generated to be compatible with chimera antigen receptor T-cells (CAR-T). This form was found to effectively bind CARs at high affinity and induce on-target killing of cancer cells.

Conclusions

Our platforms have high peptide-focused specificity, broad compatibility with a variety of antigens and simpler development that significantly expand the accessibility of targetable MHC biomarkers.”

Further development of TRACeR-I could help develop more targetable antigens. However, more research is required to optimize cell killing efficacy, prevemt unwanted immunogenicity, and examine the long-term performance of this platform in vivo.