Treatment of an aggressive orthotopic murine glioblastoma model with combination checkpoint blockade and a multivalent neoantigen vaccine.

BACKGROUND: Although clinical trials testing immunotherapies in glioblastoma (GBM) have yielded mixed results, new strategies targeting tumor-specific somatic coding mutations, termed "neoantigens," represent promising therapeutic approaches. We characterized the microenvironment and neoantigen landscape of the aggressive CT2A GBM model in order to develop a platform to test combination checkpoint blockade and neoantigen vaccination. METHODS: Flow cytometric analysis was performed on intracranial CT2A and GL261 tumor-infiltrating lymphocytes (TILs). Whole-exome DNA and RNA sequencing of the CT2A murine GBM was employed to identify expressed, somatic mutations. Predicted neoantigens were identified using the pVAC-seq software suite, and top-ranking candidates were screened for reactivity by interferon-gamma enzyme linked immunospot assays. Survival analysis was performed comparing neoantigen vaccination, anti-programmed cell death ligand 1 (αPD-L1), or combination therapy. RESULTS: Compared with the GL261 model, CT2A exhibited immunologic features consistent with human GBM including reduced αPD-L1 sensitivity and hypofunctional TILs. Of the 29 CT2A neoantigens screened, we identified neoantigen-specific CD8+ T-cell responses in the intracranial TIL and draining lymph nodes to two H2-Kb restricted (Epb4H471L and Pomgnt1R497L) and one H2-Db restricted neoantigen (Plin2G332R). Survival analysis showed that therapeutic neoantigen vaccination with Epb4H471L, Pomgnt1R497L, and Plin2G332R, in combination with αPD-L1 treatment was superior to αPD-L1 alone. CONCLUSIONS: We identified endogenous neoantigen specific CD8+ T cells within an αPD-L1 resistant murine GBM and show that neoantigen vaccination significantly augments survival benefit in combination with αPD-L1 treatment. These observations provide important preclinical correlates for GBM immunotherapy trials and support further investigation into the effects of multimodal immunotherapeutic interventions on antiglioma immunity. KEY POINTS: 1. Neoantigen vaccines combined with checkpoint blockade may be promising treatments.2. CT2A tumors exhibit features of human GBM microenvironments.3. Differential scanning fluorimetry assays may complement in silico neoantigen prediction tools.

High-Throughput Stability Screening of Neoantigen/HLA Complexes Improves Immunogenicity Predictions.

Mutated peptides (neoantigens) from a patient's cancer genome can serve as targets for T-cell immunity, but identifying which peptides can be presented by an MHC molecule and elicit T cells has been difficult. Although algorithms that predict MHC binding exist, they are not yet able to distinguish experimental differences in half-lives of the complexes (an immunologically relevant parameter, referred to here as kinetic stability). Improvement in determining actual neoantigen peptide/MHC stability could be important, as only a small fraction of peptides in most current vaccines are capable of eliciting CD8+ T-cell responses. Here, we used a rapid, high-throughput method to experimentally determine peptide/HLA thermal stability on a scale that will be necessary for analysis of neoantigens from thousands of patients. The method combined the use of UV-cleavable peptide/HLA class I complexes and differential scanning fluorimetry to determine the Tm values of neoantigen complexes. Measured Tm values were accurate and reproducible and were directly proportional to the half-lives of the complexes. Analysis of known HLA-A2-restricted immunogenic peptides showed that Tm values better correlated with immunogenicity than algorithm-predicted binding affinities. We propose that temperature stability information can be used as a guide for the selection of neoantigens in cancer vaccines in order to focus attention on those mutated peptides with the highest probability of being expressed on the cell surface.