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

Abstract

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.

Anti-PD-1 Immunotherapy in Preclinical GL261 Glioblastoma: Influence of Therapeutic Parameters and Non-Invasive Response Biomarker Assessment with MRSI-Based Approaches

“…Nonetheless, it seems clear that this basal immunogenicity alone is not able to make C57BL/6 mice resistant to GL261 GB growth, in the absence of IMS–TMZ therapy. It may be relevant to evaluate in future work whether less immunogenic models respond to IMS combined TMZ/anti PD-1, like the CT-2A model [ 44 ]. Moreover, other authors favour genetically engineered mice (GEM) models of GB, harbouring defined driver mutations leading to tumour progression [ 45 , 46 , 47 ], which are claimed to better reproduce human GB development.…”

Combination therapies utilizing neoepitope-targeted vaccines

“…In syngeneic murine models of glioma, melanoma, sarcoma, and colorectal cancer, neoepitope vaccines (delivered with various adjuvants) induced robust antigen-specific IFNγ responses in CD4 and CD8 T cells. This resulted in decreased tumor [14,[18][19][20][21][22]. Tumor growth control was associated with increased infiltration of anti-tumor immune cells and decreased suppressive cells in the tumor microenvironment [14,19].…”

“…Additionally, it is important to rationally select agents for combination and to ensure that agents do not antagonize each other. Due to promising results from monotherapy vaccine studies targeting neoepitopes, recent preclinical studies investigated combining neoepitope vaccines with other cancer therapies, including checkpoint inhibitors [18,20,28,[30][31][32][33], other immunooncology agents [18,34], and "non-immune" therapies [35] to improve their efficacies. Additionally, numerous clinical trials, summarized in Table 1, have been proposed to investigate neoepitope vaccination in combination with checkpoint inhibitors, other immuno-oncology agents, and "non-immune" cancer therapies.…”

“…Overall, 60% of mice bearing CT2A tumors had long-term survival when treated with the neoepitope vaccine, consisting of 27-mer peptides and poly-ICLC, in combination with anti-PD-L1, compared to median overall survival of 17.5 and 25 days for mice treated with monotherapy vaccine or anti-PD-L1 alone, respectively. Additionally, compared to monotherapy treatments, combination therapy increased the number of tumor-infiltrating neoepitope-specific CD8 T cells [20].…”

Bedside formulation of a personalized multi-neoantigen vaccine against mammary carcinoma

“…T cells specific for neoantigenic peptides are not attenuated by immune tolerance mechanism and do not harm healthy tissues (1). Recent clinical trials have shown that de novo immune responses can be induced by vaccination with neoantigens in melanoma and glioblastoma patients (2)(3)(4)(5). The optimal peptide length for therapeutic cancer vaccines has been identified in preclinical and clinical studies.…”