Monoclonal antibody (mAb) blockade of immune checkpoints such as PD-1 and CTLA-4 can stimulate therapeutic, T cell-dependent anti-tumor activity in mice and humans. We asked whether exome sequencing and epitope prediction algorithms could identify antigens in progressively growing tumors that render them susceptible to anti-PD-1- or anti-CTLA-4-induced immune elimination. Expressed mutations in progressively growing, T3 methylcholanthrene (MCA)-sarcomas were identified by exome sequencing and prioritized based primarily on potential MHC class I binding affinity and predicted peptide processing. The two best candidate tumor-specific mutant antigens (TSMA) were identified from mutations in Laminin α subunit 4 (Lama4) and a glucosyltransferase called Alg8. When tested in vitro, mutant Lama4 (mLama4) and mutant Alg8 (mAlg8) peptides were the only top predicted TSMA that stimulated T3-specific T cells. The relevance of these findings was validated by four in vivo studies: (i) mLama4 and mAlg8 peptides bound to H-2Kb were biochemically detected on the surface of T3 tumors, (ii) as shown by tetramer staining, CD8+ tumor infiltrating lymphocytes (TIL) expressing TCRs for mLama4 and mAlg8 accumulated over time in T3 tumors in checkpoint blockade treated, tumor-bearing mice, reaching maximum levels just prior to tumor rejection, (iii) vaccination of naïve wild type mice with mutant but not WT forms of Lama4 or Alg8 induced strong CD8+ T cell responses and (iv) prophylactic or therapeutic vaccination with mLama4 plus mAlg8 synthetic long peptides (SLP) resulted in rejection of T3 cells. The therapeutic protection provided by the SLP vaccine was equal to that afforded by checkpoint blockade therapy. Since we did not see evidence of T cell responses to any other TSMA, we then explored whether the immunodominant antigens masked responses to other TSMA. Using CRISPR/Cas9, we introduced point mutations to revert mLama4 and mAlg8 in T3 back to their nonimmunogenic wild type counterparts. T3 tumor cells with either mLama4 or mAlg8 reverted to wild type were still susceptible to anti-PD-1 or anti-CTLA-4 immune-mediated control with skewing of the T cell response in the TIL towards the remaining TSMA. Additionally, we have obtained T3 tumor cells with both TSMA reverted to wild type and are currently testing whether these “fixed” T3 tumor cells are still subject to anti-PD-1 or anti-CTLA-4 and whether SLP vaccines comprised of subdominant TSMA still present in fixed T3 tumor cells can protect mice against tumor growth. This system will allow us to address the many questions surrounding poorly understood phenomenon of immunodominance and how this may influence patient-specific personalized cancer immunotherapy directed against TSMA.
Citation Format: Matthew M. Gubin, Jeffrey P. Ward, Takuro Noguchi, Xiuli Zhang, Cora Arthur, Willem-Jan Krebber, Gwenn E. Mulder, Cornelis J.M. Melief, William E. Gillanders, Maxim Artyomov, Elaine R. Mardis, Robert D. Schreiber. Tumor-specific mutant antigens in cancer immunotherapy [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A001.