A neoepitope derived from a novel human germline APC gene mutation in familial adenomatous polyposis shows selective immunogenicity

Abstract

Familial adenomatous polyposis (FAP) is an inherited condition arising from genetic defects in the Adenomatous polyposis coli (APC) gene. Carriers with mutations in the APC gene develop polyps in the colon and rectum which if not managed, transition into colon cancer. In this study, we identified a novel germline mutation in the APC gene in members of an FAP-affected (Familial adenomatous polyposis) family. This unique heterozygous variant (c.735_736insT; p.Ser246PhefsTer6) was identified in ten out of twenty six family members, ranging in age from 6 to 60 years. Polyps were detected in six of the ten individuals (35-60 years) carrying this mutation. The remaining four members (6-23 years) remain polyp free. A significant fraction of FAP affected individuals eventually develop colon cancer and therapeutic interventions to prevent cancer progression remain elusive. To address this issue, we sought to determine if peptides derived from the novel APC mutation could induce a cytotoxic T cell response, thereby qualifying them as vaccine candidates. Peptides harboring the variant amino acids were first interrogated in silico for their immunogenicity using a proprietary neoepitope prioritization pipeline, OncoPeptVAC. A single 9-mer peptide was predicted to be immunogenic. Remarkably, CD8+ T cells isolated from either an FAP+/ APCmut individual, or from a FAP-/ APCmut individual, failed to respond to the peptide, whereas those from either an unaffected family member (FAP-/ APCwt) or from healthy unrelated donors, showed a robust response, suggesting that CD8+ T cells from individuals carrying this germline APC mutation have been tolerized to the mutation. Furthermore, experimental testing of six additional reported APC gene mutation-derived peptides revealed one of the six to be immunogenic. While not all APC mutant peptides are inmmunogenic, a few qualify as vaccine candidates offering novel treatment opportunities to patients with somatic APC gene mutations to delay/treat colorectal cancer.

Fig 1. Pedigree of the FAP affected family.

The proband (indicated by the red arrow) was diagnosed with FAP and surgically treated for polyp removal and was demonstrated to have a novel APC gene mutation. Five additional members of the proband’s extended family were diagnosed with FAP. Biopsy results showed that polyps detected in all affected family members were nonmalignant. Four family members had no polyps but had the APC gene mutation. The remaining family members were normal. Color Key: black box: APC gene mutation; green box: diagnosed with polyps; and orange box: diagnosed with oral cancer and polyps. A slash through the shape indicates a deceased member. Roman numerals indicate generations. * indicates donors whose PBMC samples have been used for the in vitro T cell activation assay. Note: Sample for individual V.4 could not be collected.

Fig 2

A. Structure of the 2842 amino acid APC protein (drawn to scale). The frame-shift mutation (S246Ffs6X) is indicated and the resultant mutant amino acids highlighted in yellow. A number of color coded functional motifs have been illustrated, including the mutation cluster region (MCR) harboring the β-catenin binding domains which are critical for the tumor suppressor function of the APC protein. B. Sanger sequencing chromatograms revealing the wildtype and mutant APC gene sequences. The mutation involving the insertion of a T between nucleotide 735 and 736 was found in two individuals tested (indicated by a black arrow), IV.8 and IV.6 (middle and bottom panels), but absent in the unaffected individual (VI.4, top panel). C. The nucleotide and protein sequences of both wildtype and mutant APC genes are indicated and the mutant sequences highlighted in yellow. D. In silico prediction of 9 mer-peptides generated from the wildtype and mutant protein sequences. Residues indicated in red are amino acids that are unique to the mutant APC protein.

Fig 3. The mutant APC peptide is selectively immunogenic.

A. Purified CD8⁺ T cells and monocyte-derived DCs from each of the three individuals was tested for antigen-specific T cell activation using wildtype and mutant APC peptides (panels A-C). Donor IV.9 (FAP^-/APC^wt) and unrelated healthy donor 1 demonstrated a robust CD8⁺T cell response to the mutant APC peptide compared to the wildtype peptide as measured by IFNγ⁺ positivity (panel 3 and 4 respectively). Donor IV.2 (FAP^-/APC^mut, panel 2) and donor III.7 (FAP⁺/APC^mut, panel 1) showed no increase in IFNγ⁺ cells. Red arrows indicate an upregulation of IFNγ⁺ cells. Positive control CEF pool peptides (FLU) showed upregulation of IFNγ⁺ in all four donors validating cells and reagents used in the assay. B. Graphical representation of the Flow cytometry data of FAP family members III.7, IV.2 and IV.9 in Fig 3A (*p = 0.003). C. Graphical representation of the Flow cytometry data of unrelated healthy donor 1 in Fig 3A bottom panel. The data plotted are the means of triplicates +/- SD (*p = 0.0009).

Fig 4. HLA A11:01 specific peptide (Mut1) derived from APC mutation p.P2018fsX26 is immunogenic.

A. Data represents flow cytometry analysis indicating CD8⁺ IFNγ⁺ positive T cells from healthy donor 3 (HLA type in S3 Table) following exposure to Mut 1 peptide. Flu and MART1 peptides served as positive controls. Red arrows indicated an increase in CD8⁺ IFNγ⁺ positive cells B. Graphical representation of the Flow cytometry data in Fig 4A (*p = 0.04).