T cells of colorectal cancer patients’ stimulated by neoantigenic and cryptic peptides better recognize autologous tumor cells

Abstract

BackgroundPatients with cancers that exhibit extraordinarily high somatic mutation numbers are ideal candidates for immunotherapy and enable identifying tumor-specific peptides through stimulation of tumor-reactive T cells (Tc).MethodsColorectal cancers (CRC) HROC113 and HROC285 were selected based on high TMB, microsatellite instability and HLA class I expression. Their HLA ligandome was characterized using mass spectrometry, compared with the HLA ligand atlas and HLA class I-binding affinity was predicted. Cryptic peptides were identified using Peptide-PRISM. Patients’ Tc were isolated from either peripheral blood (pTc) or tumor material (tumor-infiltrating Tc, TiTc) and expanded. In addition, B-lymphoblastoid cells (B-LCL) were generated and used as antigen-presenting cells. pTc and TiTc were stimulated twice for 7 days using peptide pool-loaded B-LCL. Subsequently, interferon gamma (IFNγ) release was quantified by ELISpot. Finally, cytotoxicity against autologous tumor cells was assessed in a degranulation assay.Results100 tumor-specific candidate peptides—97 cryptic peptides and 3 classically mutated neoantigens—were selected. The neoantigens originated from single nucleotide substitutions in the genesIQGAP1, CTNNB1,andTRIT1. Cryptic and neoantigenic peptides inducing IFNγ secretion of Tc were further investigated. Stimulation of pTc and TiTc with neoantigens and selected cryptic peptides resulted in increased release of cytotoxic granules in the presence of autologous tumor cells, substantiating their improved tumor cell recognition. Tetramer staining showed an enhanced number of pTc and TiTc specific for the IQGAP1 neoantigen. Subpopulation analysis prior to peptide stimulation revealed that pTc mainly consisted of memory Tc, whereas TiTc constituted primarily of effector and effector memory Tc. This allows to infer that TiTc reacting to neoantigens and cryptic peptides must be present within the tumor microenvironment.ConclusionThese results prove that the analyzed CRC present both mutated neoantigenic and cryptic peptides on their HLA class I molecules. Moreover, stimulation with these peptides significantly strengthened tumor cell recognition by Tc. Since the overall number of neoantigenic peptides identifiable by HLA ligandome analysis hitherto is small, our data emphasize the relevance of increasing the target scope for cancer vaccines by the cryptic peptide category.

Tumor-specific T cells in head and neck cancer have rescuable functionality and can be identified through single-cell co-culture

“…Second, there is no guarantee that neoantigens identified in silico will be expressed unmodified at the protein level and presented by tumor cells in their native state. Further, reliance on exomic mutational profiling omits immunogenic neoantigens derived from other tumor metabolic derangements, such as altered RNA editing, proteomic processing, or non-canonical translation [37] , [38] , [39] . This is supported by studies demonstrating TCR recognition of patient-derived xenograft tissue in the absence of reactivity against predicted peptide neoantigens [32] .…”

Peptide-stimulation of autologous T cells reverts immune checkpoint inhibitor resistance of hypermutated colorectal cancer cells

“…When comparing gene expression of proteins involved in antigen processing 14 between our two tumor models and normal colon tissue, the majority of genes showed a higher expression in tumor than in normal tissue; hinting at a preservation of functional antigen processing. Even though B2M expression was decreased, a HLA ligandome analysis revealed thousands of HLA-eluted peptides in both cell lines 20 . Thus, the presentation of tumor-specific peptides with the potential to activate T cells was maintained in these two CRC cases, contrasting the well-established correlation of MSI and diminished HLA expression 22–24 .…”

“…In both tumor cell lines, the mean expression of the selected genes resembled or even exceeded the levels in CRC samples from TCGA, but due to the small size of the cell line cohort (n=2), statistical analysis was inadmissible. Moreover, functional antigen processing was previously assured through HLA ligandome analyses enabling the characterization of several thousand HLA ligands for each CRC cell line, including tumor-specific antigens 20 .…”

Neo‐Antigen‐Reactive T Cells Immunotherapy for Colorectal Cancer: A More Personalized Cancer Therapy Approach

“…Moreover, following tetramer staining, it was revealed that most TILs and peripheral blood T cells were specific for IQGAP1 neoantigen. [98] Regarding the low tumor mutational burden in some tumors, the abundance of neoantigens in them is limited. An alternative chemical-based compound (RECTAS) to generate spliceneoantigens in CRC mice models.…”

“…Moreover, following tetramer staining, it was revealed that most TILs and peripheral blood T cells were specific for IQGAP1 neoantigen. [ 98 ]…”