Novel `xTRACeRs` system enables generalizable antigenic peptide targeting across diverse HLA-I allotypes

The extreme polymorphism of the Human Leucocyte Antigen class I (HLA-I) locus presents a significant barrier to developing broad-spectrum immunotherapies. T cell receptors (TCRs) and TCR-mimicking antibodies recognize peptide antigens presented by specific HLA-I allotypes, but the vast genetic diversity in HLA proteins and peptide structures limits therapeutic coverage across patients. Current modalities struggle to confer therapeutic benefit across divergent genetic backgrounds, leaving many patients without effective HLA-targeted treatment options. This research addresses this critical gap by developing a system to engineer binders compatible with multiple HLA allotypes.

Researchers developed a generalized framework for engineering cross-HLA binders, termed xTRACeRs, by integrating a peptide conformational prediction tool, PepPred, with a cross-HLA binding protein engineering system, TRACeR-I. This system was used to design and validate xTRACeRs against established, clinically relevant peptide antigens. Validation occurred across common alleles within five HLA-A/B/C supertypes. Cryo-electron microscopy (cryo-EM) was employed to determine the structures of xTRACeR-pHLA complexes for an oncofetal antigen from PRAME and a neuroblastoma-specific peptide from PHOX2B. Finally, these two xTRACeRs were implemented as Chimeric Antigen Receptor (CAR) T cells to assess their killing efficacy and specificity.

The developed xTRACeRs system successfully generated binders capable of targeting antigenic peptides across multiple HLA-I allotypes, demonstrating compatibility across common alleles within five HLA-A/B/C supertypes. Cryo-EM structures of xTRACeR-pHLA complexes for PRAME and PHOX2B antigens revealed the molecular mechanisms enabling this broad targeting. These structures demonstrated that xTRACeRs effectively navigate polymorphic HLA surface residues while maintaining extensive interactions with the peptide antigen itself. This dual interaction strategy ensures both broad HLA coverage and high peptide specificity. When implemented as CAR T cells, the PRAME- and PHOX2B-specific xTRACeRs exhibited potent killing efficacy and high specificity towards their target cells. This indicates the system's potential to overcome the HLA restriction that has historically limited TCR-based immunotherapies.

The xTRACeRs system successfully developed binders with compatibility across HLA allotypes, maintaining high specificity towards peptide antigens and demonstrating potent CAR T-cell killing efficacy.