Abstract 1705: Identification and validation of neoantigens for developing a preventive vaccine for mismatch-repair deficient cancers
Authors: Charles Bowen, Ana Bolivar, Kyle Chang, Wendy Wu, Laura Reyes-Uribe, Gregory A. Lizee, Krishna Sinha, Eduardo Vilar
Journal: Cancer Research (2021)

Abstract Background: Lynch Syndrome (LS) is the most common cause of hereditary colorectal cancer (CRC). LS arises when normal colorectal cells become DNA mismatch repair deficient (dMMR) upon acquiring a somatic hit in the alternative allele of the same MMR gene that harbors the germline mutation. This ‘second hit' manifests into insertion-deletion (indel) mutations in microsatellite sequences, which generates frameshift peptides (FSP) that become neoantigens (neoAg).Different system biology platforms have used next-generation sequencing (NGS) to rapidly screen the mutational landscape of LS cancers, which have identified a variety of nonsynonymous mutations that may be recognized as foreign antigens (neoantigens) to the host immune system. Putative neoantigens characterized by NGS data in LS patients provide the groundwork for developing immunoprevention strategies based on different neoantigen vaccination platforms. Methods: We utilized paired whole-exome sequencing and mRNAseq in LS CRC (stage I-III) and pre-cancers to catalog and identify the most frequently recurrent neoAg present in LS patients, and used in-silico metrics such as HLA genotype, mutational frequency, HLA binding affinity, and expression levels to predict immunogenicity. To validate our computational predictions, we harvested cytotoxic lymphocytes from a total of 3 LS patients and generated neoAg-loaded tetramers to mimic MHC-I presentation of 10 different neoAg from our prediction list. After neoAg-specific CTLs were enumerated and isolated using tetramer stains, ELISpots, and a 15-plex cytokine profiling ELISA assay were used to ascertain the immunogenic potential of each neoAg. Results: MHC-tetramer staining revealed that neoAg-specific CTLs comprised approximately 0.5-1% of total peripheral CTL population, which is consistent with previous studies. ELISpots performed using CTLs showed significant secretion of IFNγ (spot forming units) upon overnight stimulation with neoAg-loaded tetramers compared to controls. A 15-plex cytokine profile using CTLs from one patient identified significant activation of proinflammatory (IL-1a, IL-1b, IL-12, IL-17, IL-23) and proliferative (IL-2, IL-15) cytokines upon neoAg stimulation compared to the unstimulated control. Conclusion: Our results provide strong evidence to suggest our in silico computational pipelines accurately predict the immunogenicity of LS neoAg and that these neoAgs have the potential to mount an immune response consistent with previously published work performed in other cancers. Our study provides the foundation for developing an immunoprevention vaccine for LS carriers. Citation Format: Charles Bowen, Ana Bolivar, Kyle Chang, Wendy Wu, Laura Reyes-Uribe, Gregory A. Lizee, Krishna Sinha, Eduardo Vilar. Identification and validation of neoantigens for developing a preventive vaccine for mismatch-repair deficient cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1705.