Structural and functional characterization of T and NK cell receptors

National Institute Of Allergy And Infectious Diseases

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Abstract

We primarily focused our effort in the structural understanding of T cell development and T cell mediated immunotherapy. Adoptive T cell immunotherapy (ACT) treats cancer by the injection of large numbers of autologous tumor specific T cells. While promising, ACT is far from effective and understanding to the mechanism is missing. One successful treatment to metastatic colorectal cancer where the tumor carried the common oncogenic KRAS-G12D mutation depended on T cell recognition of peptides derived from mutant the G12D neoantigen presented by MHC on tumors. Several TCRs specific for this mutation were identified to be restricted by HLA-C*08:02 recognizing two G12D peptide epitopes, a 9mer (GADGVGKSA) and a 10mer (GADGVGKSAL). We solved the high resolution crystal structures of HLA-C*08:02 presenting the 9mer and 10mer peptides alone and in complex with their cognate T cell receptors (TCRs). The TCR free HLA-C structures demonstrated a bulge in the 10mer peptide compared to the 9mer, peaking at K7. In the TCR:HLA-C-10mer structure, this bulge in the peptide shifted from K7 to V5 and is accommodated through hydrophobic contacts with the CDR3 chain, while the main peptide contact is through a Y97 in the CDR3. This conformation dependent recognition of the 10mer provides a structural explanation for why the 10mer specific TCR is unable to recognize the highly similar 9mer peptide. The TCR:HLA-C-9mer structure revealed that the 9mer peptide was recognized primarily through a E51 and Y50 of the CDR2, while the CDR3 did not contribute to peptide recognition, and interacted with the HLA-C heavy chain. The CDR3 chain through Q98 contacted both the peptide at position D3 and HLA-C at R156. Additionally, our structures demonstrated that both epitopes are similarly presented by HLA-C*08:02 through a direct contribution of the G12D mutation, which forms a salt bridge between peptide D3 and R156 of HLA-C*08:02. This interaction is essential for antigen presentation of the G12D KRAS peptides. Since the wild type KRAS peptides cannot be presented by HLA-C*08:02, they are not recognized by these TCRs. Collectively, our data provide a structural basis for effective ACT to mutant KRAS G12D and offer a blueprint for rational design of peptide vaccines and TCR based immunotherapies. Further, our biochemical studies on the affinities of these TCR binding to their HLA ligands showed that all 9-mer recognizing TCRs exhibited high affinities (10-100 nM) and their affinities appear to correlate with their persistence in patient during ACT therapy. We conclude that the success of T cell transfer immunotherapy depends on the use of oligoclonal high affinity tumor specific T cells with variant peptide-HLA affinities to ensure both tumor killing and therapeutic persistence. We continue to collaborate with Dr. Rosenberg at NCI to further investigate the structural mechanism of tumor antigen recognition by tumor infiltrating TCR. We also initiated a collaborative research agreement (CRADA) recently with Dr. Robert Holt, Professor of Medical Genetics, at University of British Columbia on structural characterization of tumor-specific TCR and their recognition of tumor antigens.