TCRs Used in Cancer Gene Therapy Cross-React with MART-1/Melan-A Tumor Antigens via Distinct Mechanisms

Abstract

T cells engineered to express T cell receptors (TCRs) specific for tumor antigens can drive cancer regression. The first TCRs used in cancer gene therapy, DMF4 and DMF5, recognize two structurally distinct peptide epitopes of the melanoma-associated MART-1/Melan-A protein, both presented by the class I MHC protein HLA-A*0201. To help understand the mechanisms of TCR cross-reactivity and provide a foundation for the further development of immunotherapy, we determined the crystallographic structures of DMF4 and DMF5 in complex with both of the MART-1/Melan-A epitopes. The two TCRs use different mechanisms to accommodate the two ligands. Whereas DMF4 binds the two with a different orientation, altering its position over the peptide/MHC, DMF5 binds them both identically. The simpler mode of cross-reactivity by DMF5 is associated with higher affinity towards both ligands, consistent with the superior functional avidity of DMF5. More generally, the observation of two diverging mechanisms of cross-reactivity with the same antigens and the finding that TCR binding orientation can be determined by peptide alone extend our understanding of the mechanisms underlying TCR cross-reactivity.

How structural adaptability exists alongside HLA-A2 bias in the human αβ TCR repertoire

“…However, it is clear that any intrinsic bias TCRs have toward MHC proteins must exist alongside the need for TCRs to structurally adapt to different ligands or alterations in the TCR-pMHC interface (12). For example, changes to a peptide alone are sufficient to alter how the same TCR sits over the same MHC protein, altering TCR-MHC atomic contacts (13,14). Similar results are seen with changes to TCR variable domains or hypervariable loops, or even MHC mutations (15)(16)(17)(18).…”

“…For analysis of D26α-Arg65 pair, the L98βW single mutant was used. We note that, excluding the sites of the two mutations, the structures of the high-affinity and wild-type DMF5 TCR complexes are essentially identical (13,65). The utilization of affinity-enhancing mutations is unlikely to impact the double-mutant cycle experiments, owing to the structural similarities and the robust nature of double-mutant cycle analysis (40,41).…”

“…interaction we have previously studied in detail (13,40). The shape complementarity between Tyr48β and Arg65 in the DMF5 interface is high, with an interresidue Sc statistic of 0.8.…”

How an alloreactive T-cell receptor achieves peptide and MHC specificity

“…Data were indexed, integrated, and scaled with the program XDS22. The complexes were solved by molecular replacement with Phaser (57) using the Protein Database (PDB) ID codes 3QEU and 3MRM as search models (58). Rigid body refinement followed by translation/libration/screw refinement was performed with Phenix and Refmac5 (59,60).…”

Peptide Modulation of Class I Major Histocompatibility Complex Protein Molecular Flexibility and the Implications for Immune Recognition*

“…␣␤ T cell receptors (TCRs) 4 on the surfaces of CD8ϩ T cells recognize antigenic peptides bound and presented by class I major histocompatibility complex proteins (class I pMHC complexes) to initiate and propagate an antigen-dependent immune response. Both TCR and peptide conformational properties play key roles in antigen recognition and discrimination.…”