Abstract B104: Post-translationally modified homocitrulline induced by MDSCs can be an effective antitumor target for CD4 T-cells
Authors: Cook Katherine W. , Xue Wei , Symonds Peter , Gijon Mohamed , Shah Sabaria , Atabani Suha , Vaghela Poonam , Choudhury Ruhul , Metheringham Rachael L. , Daniels Ian , Brentville Victoria , Durrant Lindy 
Published: February 2019

Abstract
Post-translational modifications such as citrullination and phosphorylation have been shown to provide novel target for cancer vaccines. Epitopes from modified proteins allow the generation of cytotoxic CD4 cells capable of targeting tumor. In this study we show that homocitrulline, an amino acid produced when lysine is post translationally modified by the process of carbamylation, can be a target for effective tumor immunity. Homocitrullinated epitopes from the cytoskeletal protein vimentin and the glycolytic enzyme aldolase were selected based upon MHC-II binding algorithms. Immunization of HLA transgenic mice with three homocitrullinated peptides, one from vimentin and two from aldolase A, induced potent IFNγ responses restricted via HLA-DR4, DR1 or DP4. Responses are specific to the homocitrullinated peptides and did not cross react with unmodified peptides. The responses have been characterized as CD4-mediated, Th1-type responses with minimal IL-10 or IL-17 production. In humans, we have also shown that healthy donors and cancer patients both have a CD4 T-cell repertoire recognizing these homocitrullinated peptides. In B16 melanoma tumor models, vaccination with homocitrullinated peptides increases survival in transgenic HLA-DR4 (survival 70%, p=0.0042), HHDII/DR1 (survival 50%, p&lt;0.0001) and HHDII/DP4 (survival 90%, p&lt;0.0001) mouse strains. Carbamylation is known to increase in the presence of high levels of isocyanic acid, which is in turn increased by the enzyme myeloperoxidase (MPO). Tumor cells do not express MPO; however, MPO+ cells within the tumor environment express markers associated with myeloid-derived suppressor cells (MDSCs). Depletion of these cell populations reduces MPO expression in the tumor microenvironment and attenuates the antitumor effect of the homocitrullinated peptide vaccine (p=0.0480). However, the antitumor effect is specifically directed against tumor cells as the antitumor effect is reduced in mice implanted with tumor lacking human MHC-II, in the mouse strains HLA-DR4 (survival 20%, p=0.0010), HHDII/DR1 (survival 0%) and HHDII/DP4 (survival 0%) mice. These results suggest that the MPO produced by MDSCs catalyses the build-up of isocyanic acid, which diffuses into the tumor and carbamylates cytoplasmic proteins, which are then degraded and presented by MHC-II. Together these data suggest that MDSCs can mediate carbamylation of proteins within tumor cells, which become targets for cytotoxic CD4 T-cells.
Citation Format: Katherine W. Cook, Wei Xue, Peter Symonds, Mohamed Gijon, Sabaria Shah, Suha Atabani, Poonam Vaghela, Ruhul Choudhury, Rachael L. Metheringham, Ian Daniels, Victoria Brentville, Lindy Durrant. Post-translationally modified homocitrulline induced by MDSCs can be an effective antitumor target for CD4 T-cells [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B104.