T cell receptor interactions with human leukocyte antigen govern indirect peptide selectivity for the cancer testis antigen MAGE-A4.

T cell-mediated immunity is governed primarily by T cell receptor (TCR) recognition of peptide-human leukocyte antigen (pHLA) complexes and is essential for immunosurveillance and disease control. This interaction is generally stabilized by interactions between the HLA surface and TCR germline-encoded complementarity-determining region (CDR) loops 1 and 2, whereas peptide selectivity is guided by direct interactions with the TCR CDR3 loops. Here, we solved the structure of a newly identified TCR in complex with a clinically relevant peptide derived from the cancer testis antigen melanoma antigen-A4 (MAGE-A4). The TCR bound pHLA in a position shifted toward the peptide's N terminus. This enabled the TCR to achieve peptide selectivity via an indirect mechanism, whereby the TCR sensed the first residue of the peptide through HLA residue Trp-167, which acted as a tunable gateway. Amino acid substitutions at peptide position 1 predicted to alter the HLA Trp-167 side-chain conformation abrogated TCR binding, indicating that this indirect binding mechanism is essential for peptide recognition. These findings extend our understanding of the molecular rules that underpin antigen recognition by TCRs and have important implications for the development of TCR-based therapies.

Engineering soluble T-cell receptors for therapy.

Immunotherapy approaches that target peptide-human leukocyte antigen (pHLA) complexes are becoming highly attractive because of their potential to access virtually all foreign and cellular proteins. For this reason, there has been considerable interest in the development of the natural ligand for pHLA, the T-cell receptor (TCR), as a soluble drug to target disease-associated pHLA presented at the cell surface. However, native TCR stability is suboptimal for soluble drug development, and natural TCRs generally have weak affinities for pHLAs, limiting their potential to reach efficacious receptor occupancy levels as soluble drugs. To overcome these limitations and make full use of the TCR as a soluble drug platform, several protein engineering solutions have been applied to TCRs to enhance both their stability and affinity, with a focus on retaining target specificity and selectivity. Here, we review these advances and look to the future for the next generation of soluble TCR-based therapies that can target monomorphic HLA-like proteins presenting both peptide and nonpeptide antigens.

Molecular Rules Underpinning Enhanced Affinity Binding of Human T Cell Receptors Engineered for Immunotherapy.

Immuno-oncology approaches that utilize T cell receptors (TCRs) are becoming highly attractive because of their potential to target virtually all cellular proteins, including cancer-specific epitopes, via the recognition of peptide-human leukocyte antigen (pHLA) complexes presented at the cell surface. However, because natural TCRs generally recognize cancer-derived pHLAs with very weak affinities, efforts have been made to enhance their binding strength, in some cases by several million-fold. In this study, we investigated the mechanisms underpinning human TCR affinity enhancement by comparing the crystal structures of engineered enhanced affinity TCRs with those of their wild-type progenitors. Additionally, we performed molecular dynamics simulations to better understand the energetic mechanisms driving the affinity enhancements. These data demonstrate that supra-physiological binding affinities can be achieved without altering native TCR-pHLA binding modes via relatively subtle modifications to the interface contacts, often driven through the addition of buried hydrophobic residues. Individual energetic components of the TCR-pHLA interaction governing affinity enhancements were distinct and highly variable for each TCR, often resulting from additive, or knock-on, effects beyond the mutated residues. This comprehensive analysis of affinity-enhanced TCRs has important implications for the future rational design of engineered TCRs as efficacious and safe drugs for cancer treatment.