Recurrent Frameshift Neoantigen Vaccine Elicits Protective Immunity With Reduced Tumor Burden and Improved Overall Survival in a Lynch Syndrome Mouse Model

Abstract

Background & aims: DNA mismatch repair deficiency drives microsatellite instability (MSI). Cells with MSI accumulate numerous frameshift mutations. Frameshift mutations affecting cancer-related genes may promote tumorigenesis and, therefore, are shared among independently arising MSI tumors. Consequently, such recurrent frameshift mutations can give rise to shared immunogenic frameshift peptides (FSPs) that represent ideal candidates for a vaccine against MSI cancer. Pathogenic germline variants of mismatch repair genes cause Lynch syndrome (LS), a hereditary cancer syndrome affecting approximately 20-25 million individuals worldwide. Individuals with LS are at high risk of developing MSI cancer. Previously, we demonstrated safety and immunogenicity of an FSP-based vaccine in a phase I/IIa clinical trial in patients with a history of MSI colorectal cancer. However, the cancer-preventive effect of FSP vaccination in the scenario of LS has not yet been demonstrated.

Methods: A genome-wide database of 488,235 mouse coding mononucleotide repeats was established, from which a set of candidates was selected based on repeat length, gene expression, and mutation frequency. In silico prediction, in vivo immunogenicity testing, and epitope mapping was used to identify candidates for FSP vaccination.

Results: We identified 4 shared FSP neoantigens (Nacad [FSP-1], Maz [FSP-1], Senp6 [FSP-1], Xirp1 [FSP-1]) that induced CD4/CD8 T cell responses in naïve C57BL/6 mice. Using VCMsh2 mice, which have a conditional knockout of Msh2 in the intestinal tract and develop intestinal cancer, we showed vaccination with a combination of only 4 FSPs significantly increased FSP-specific adaptive immunity, reduced intestinal tumor burden, and prolonged overall survival. Combination of FSP vaccination with daily naproxen treatment potentiated immune response, delayed tumor growth, and prolonged survival even more effectively than FSP vaccination alone.

Conclusions: Our preclinical findings support a clinical strategy of recurrent FSP neoantigen vaccination for LS cancer immunoprevention.

Keywords: Colorectal Cancer; Frameshift Neoantigens; Lynch Syndrome; Mouse Model; Preventive Cancer Vaccine.

Figure 1.. Experimental Strategy for Developing a Murine FSP Vaccine

Figure 2.. Distribution of coding mononucleotide repeats (cMNRs) in the mouse genome.

The occurrence of cMNRs is shown according to repeat type and length(≥ 8 nucleotides).

Figure 3.. FSP Immunogenicity.

(A) FSP vaccination scheme of C57BL/6 mice. FSP mixes or OVA mix (50μg each) were injected subcutaneously biweekly for four times using CpG ODN 1826 (20μg) as an adjuvant. The vaccine schedule was chosen to ensure life-long robust FSP-specific immune responses, including a starting point in early adulthood and booster vaccinations after the initial priming phase. (B) IFN-γ ELISpot analysis. The average number of spot forming units (SFUs) for each mouse is shown for each peptide. OVA mix corresponds to the mixture of the OVA peptide CD4 and CD8 epitope, which was used as a control in this experimental setup. (C) CD4 and CD8 T cell responses against four FSPs. Average SFU numbers are presented for each peptide. (D) Epitope mapping of immunogenic peptides. Wildtype peptides (grey) as well as FSPs with overlapping N/C-terminal sequences (blue boxes, red amino acids) were synthesized and immunogenicity for the regions where epitopes might be located was determined by IFN-γ ELISpot. The average number of SFUs per mouse are shown in the graph. (E) Endogenous FSP reactivity in Lynch Mice. IFN-γ ELISpot analysis. Each dot represents the number of spot forming units (SFUs) of splenocytes per mouse. VCMsh2 mice were pulsed with a mixture of 4 peptides (FSPs). C57BL/6 (B6) mice pulsed with either FSPs or OVA peptide served as control. The median, interquartile range/standard deviation and significance level is indicated.

Figure 4.. Cancer-Immunoprevention in Lynch Mice.

Reduced tumor burden from combination NSAID and recurrent FSP neoantigen vaccination. (A) Recurrent FSP neoantigen vaccination in combination with NSAID prolongs overall survival of VCMsh2 mice. Kaplan-Meier survival curves of VCMsh2 mice treated with control (untreated), ASA, NAP, FSP vaccine, FSP vaccine with ASA or FSP vaccine with NAP as described in the methods. (B) FSP vaccination and combination with NSAID treatment decreases tumor burden in VCMsh2 mice. Scatter dot plot representing tumor burden (sum of all intestinal tumor weights per mouse, [mg]) per mouse in cohorts of control (untreated), treated with ASA, NAP, FSP vaccine, FSP vaccine plus ASA or FSP vaccine plus NAP.Both panels present data updated from that originally presented in Figure S3 of PMID: 32641470, here including a larger cohort of control and naproxen treated VCMsh2 mice.

Figure 5.. Quantitative evaluation of immune cell subtypes.

(A) Representative IHC stainings. (B) Quantitative evaluation. Immune cell densities are shown for the antibodies detecting CD4, CD8, Foxp3, PD-1. Black lines indicate median values. P values are provided for all comparisons.