A Recurrent Mutation in Anaplastic Lymphoma Kinase with Distinct Neoepitope Conformations

Abstract

The identification of recurrent human leukocyte antigen (HLA) neoepitopes driving T cell responses against tumors poses a significant bottleneck in the development of approaches for precision cancer therapeutics. Here, we employ a bioinformatics method, Prediction of T Cell Epitopes for Cancer Therapy, to analyze sequencing data from neuroblastoma patients and identify a recurrent anaplastic lymphoma kinase mutation (ALK R1275Q) that leads to two high affinity neoepitopes when expressed in complex with common HLA alleles. Analysis of the X-ray structures of the two peptides bound to HLA-B*15:01 reveals drastically different conformations with measurable changes in the stability of the protein complexes, while the self-epitope is excluded from binding due to steric hindrance in the MHC groove. To evaluate the range of HLA alleles that could display the ALK neoepitopes, we used structure-based Rosetta comparative modeling calculations, which accurately predict several additional high affinity interactions and compare our results with commonly used prediction tools. Subsequent determination of the X-ray structure of an HLA-A*01:01 bound neoepitope validates atomic features seen in our Rosetta models with respect to key residues relevant for MHC stability and T cell receptor recognition. Finally, MHC tetramer staining of peripheral blood mononuclear cells from HLA-matched donors shows that the two neoepitopes are recognized by CD8+ T cells. This work provides a rational approach toward high-throughput identification and further optimization of putative neoantigen/HLA targets with desired recognition features for cancer immunotherapy.

Emerging Roles of ALK in Immunity and Insights for Immunotherapy

“…In addition to functional approaches, a bioinformatics approach called Prediction of T Cell Epitopes for Cancer Therapy (ProTECT) has been recently developed to identify ALK neoepitopes from recurrent ALK R1275Q mutation [37]. ProTECT allows the prediction of high affinity binders (epitopes) to common HLA alleles, with which a nonamer (AQDIYRASY) and two decamer sequences of ALK (MAQDIYRASY and AQDIYRASYY) with high affinities to HLA-B*15:01 were identified.…”

Molecular determinants of chaperone interactions on MHC-I for folding and antigen repertoire selection

“…Each MHC-I heavy chain was expressed in E. coli, and refolded together with a high-affinity peptide and human β2m (hβ2m) (13,19). We utilized well-characterized epitopes: the p29 peptide (YPNVNIHNF) for H2-L d (41), the HIV gp120 P18-I10 peptide (RGPGRAFVTI) for H2-D d (42), the neuroblastoma related NRAS Q61K peptide (ILDTAGKEEY) for HLA-A*01:01 (43), and the HTLV-1 TAX peptide (LLFGYPVYV) for HLA-A*02:01 (44). Using a differential scanning fluorimetry assay (45), we further confirmed that all purified samples displayed thermal stabilities that were characteristic of properly conformed, peptide-bound molecular species with T m values ranging from 51 to 63 °C (Table S1).…”

Structure Based Prediction of Neoantigen Immunogenicity

“…Yet for a peptide bound to an MHC protein, the impact of features such as exposed hydrophobic surface and charges are determined by the peptide's conformation within the binding groove, as well as the size and position of the various amino acid side chains. Thus, efforts to predict peptide immunogenicity should be strengthened by approaches that account for the structural properties of peptide/MHC complexes (8, 16, 35).…”