Identification of tumor-specific neo-antigens is crucial to developing new immunotherapeutic approaches, e.g. personalized vaccines. While computer algorithms can predict Class I or II HLA specific neoantigens, experimental confirmation of these potentially immunogenic neoantigens remains challenging, primarily due to the complexity associated with understanding the MHC-antigen and T cell receptor interaction. Here, we developed a systematic pipeline to efficiently identify immunogenic neoantigens and evaluate their immunogenicity in healthy donors.

Whole-exome sequencing(WES) of a melanoma was used to identify non-synonymous mutations. MHC presentation was predicted with digital HLA typing and MHC class I allele-restricted binding affinity/stability algorithms (netMHCpan/netMHCstab). The tumor clonal architecture was identified for each peptide to prioritize clonal over subclonal neoantigens using BubbleTree ( Immunogenicity testing was done using a novel in-vitro assay with blood from healthy HLA-A201 donors. Naïve CD8 T cells were stimulated with autologous dendritic cells pulsed initially with peptide pools followed by individual peptides. The presence of antigen-specific T cells was determined via MHC-multimer staining and function was assessed by intracellular staining and IFNy EliSpot.

Nonsynonymous somatic mutations (N = 205) were identified by WES from the melanoma tumor biopsy. From a library of mutated tumor-specific peptides (N = 7512), 18 candidates predicted to be Class I neo-antigens were selected for immunogenicity testing. A new in-vitro assay using blood from healthy HLA-A201 donors identified one peptide that expanded antigen-specific T cells. Analysis of this mutation showed a VAF of 29.3% and TCGA revealed that the mutation identified was unique. IFNy secretion indicated that these antigen-specific T cells produced 6-fold higher IFNy to the mutant peptide vs the wildtype peptide. Direct binding of peptide to HLA was confirmed in vitro. Interrogation of the peptide expanded T cells by TCRseq showed clonal T cell expansion in response to the peptide. Unique TCR sequences identified in these T cells were present in the original melanoma tumor biopsy but not in adjacent tissue, confirming that naïve T cell donors can be used to validate neoantigens identified in cancer patients. The same TCR sequences were also identified in the peptide pool stimulated cells, suggesting that deconvolution may not be necessary to monitor neoantigen responses.

Using a newly developed pipeline combining next generation sequencing, epitope and clonal prediction algorithms, as well as an in-vitro assay to screen and evaluate putative neoantigens as targets of antitumor immunity, we confirmed one predicted neo-antigen from a melanoma patient. This pipeline could allow for the efficient and rapid identification of personalized neoantigens.

Citation Format: Nicholas M. Durham, Yelena Lazdun, Han Si, Todd Creasy, Brandon W. Higgs, Katie Streicher. A new pipeline to predict and confirm tumor neo-antigens [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4677.