Human CD8+ cytotoxic T lymphocytes can mediate tumor regression in melanoma through the specific recognition of HLA-restricted peptides. Because of the relatively weak affinity of most anti-cancer T-cell receptors (TCRs), there is growing emphasis on immunizing melanoma patients with altered peptide ligands in order to induce strong anti-tumor immunity capable of breaking tolerance toward these self-antigens. However, previous studies have shown that these immunogenic designer peptides are not always effective. The melanocyte differentiation protein, glycoprotein 100 (gp100), encodes a naturally processed epitope that is an attractive target for melanoma immunotherapies, in particular peptide-based vaccines. Previous studies have shown that substitutions at peptide residue Glu 3 have a broad negative impact on polyclonal T-cell responses. Here, we describe the first atomic structure of a natural cognate TCR in complex with this gp100 epitope and highlight the relatively high affinity of the interaction. Alanine scan mutagenesis performed across the gp100 280–288 peptide showed that Glu 3 was critically important for TCR binding. Unexpectedly, structural analysis demonstrated that the Glu 3 → Ala substitution resulted in a molecular switch that was transmitted to adjacent residues, abrogating TCR binding and T-cell recognition. These findings help to clarify the mechanism of T-cell recognition of gp100 during melanoma responses and could direct the development of altered peptides for vaccination.
Cytotoxic T-cells can mediate a specific response against autologous melanoma cells by recognizing tumor-derived peptides presented at the cell surface by human leukocyte antigen (pHLA).6 In particular, epitopes encoded by differentiation melanocyte proteins may represent shared melanoma-associated antigens targeted by T-cell receptors (TCRs) on patients' lymphocytes. Glycoprotein 100 (gp100) has been a widely studied target for melanoma immunotherapy. This 661-amino acid long melanoma differentiation antigen is a melanosome matrix protein involved in melanosome maturation and melanin synthesis. In vivo, the protein has significantly differential expression between tumor cells, being often overexpressed in all stages of melanoma progression, compared with normal melanocytes.
Previous studies showed that gp100 encoded epitopes are recognized by tumor-infiltrating lymphocytes and circulating T-cells, associated with tumor regression in metastatic melanoma patients after adoptive therapy. Among these, the nonamer epitope gp100 280–288 (YLEPGPVTA) was originally shown to be recognized by HLA-A*0201+ tumor-infiltrating lymphocytes from melanoma patients and subsequently eluted from HLA-A*0201 molecules on melanoma cells. Immunization with gp100 280–288 peptide has been shown to stimulate an in vitro polyclonal T-cell response in the context of HLA-A*0201, present in 49% of Caucasian individuals. These findings generated renewed interest in developing gp100-based anti-melanoma vaccines. However, we and others have previously shown, through direct biophysical measurements, that anti-cancer TCRs bind to their cognate pHLA with affinities that are approximately 1 order of magnitude weaker than those of pathogen-specific TCRs. Thus, altered peptide ligands, with improved primary HLA anchor residues (heteroclitic peptides), have been designed for a few melanoma-associated antigens in order to increase immunogenicity. Among these, the heteroclitic version of gp100 280–288 (in which a valine replaces alanine at anchor position 9 to improve pHLA stability) enhanced the induction of melanoma-reactive cytotoxic T lymphocytes in vitro and has been successfully used in clinical trials. Another heteroclitic form of gp100 280–288, in which peptide residue Glu 3 was substituted to Ala, abrogated recognition by a polyclonal population of gp100 280–288-specific T-cells. Thus, a more complete understanding of the molecular mechanisms underlying gp100 280–288 targeting by specific TCRs is needed to direct the design of improved altered peptide ligands.
Previous studies using another HLA-A*0201-restricted melanoma-derived epitope have demonstrated that even minor changes in peptide anchor residues can substantially alter T-cell recognition in unpredictable ways. In order to aid in the future design of enhanced peptide vaccines based on gp100 280–288, we solved the ternary atomic structure of a human TCR in complex with the heteroclitic gp100 280–288 peptide. We then used a peptide scanning approach to demonstrate the impact of peptide substitutions on TCRs from two different T-cell clones by performing in depth biophysical and functional experiments. These data demonstrate that modification of peptide residues outside of the TCR binding motif can have unpredictable knock-on effects (a modification to a residue that affects an adjacent residue indirectly) on adjacent peptide residues that abrogate TCR binding and T-cell recognition. Indeed, even conservative peptide substitutions can have unexpected consequences for T-cell recognition due to knock-on structural changes in the HLA-bound peptide. Our findings provide a molecular explanation for the sensitivity to substitutions at gp100 280–288 peptide residue Glu 3 and represent the first example of the structural mechanisms underlying T-cell recognition of this important therapeutic target for melanoma.
The PMEL17 TCR (TRAV21 TRBV7-3) and gp100 TCR (TRAV17 TRBV19) are both specific for the human HLA-A*0201 restricted YLE epitope (gp100 280–288, sequence YLEPGPVTA). For each TCR, a disulfide-linked construct was used to produce the soluble α- and β-chain domains (variable and constant). The HLA*0201 α-chain and β2m sequences were generated by PCR cloning. All sequences were confirmed
