Integrating Multisector Molecular Characterization into Personalized Peptide Vaccine Design for Patients with Newly Diagnosed Glioblastoma.

PURPOSE: Outcomes for patients with glioblastoma (GBM) remain poor despite multimodality treatment with surgery, radiation, and chemotherapy. There are few immunotherapy options due to the lack of tumor immunogenicity. Several clinical trials have reported promising results with cancer vaccines. To date, studies have used data from a single tumor site to identify targetable antigens, but this approach limits the antigen pool and is antithetical to the heterogeneity of GBM. We have implemented multisector sequencing to increase the pool of neoantigens across the GBM genomic landscape that can be incorporated into personalized peptide vaccines called NeoVax. PATIENTS AND METHODS: In this study, we report the findings of four patients enrolled onto the NeoVax clinical trial (NCT0342209). RESULTS: Immune reactivity to NeoVax neoantigens was assessed in peripheral blood mononuclear cells pre- and post-NeoVax for patients 1 to 3 using IFNγ-ELISPOT assay. A statistically significant increase in IFNγ producing T cells at the post-NeoVax time point for several neoantigens was observed. Furthermore, a post-NeoVax tumor biopsy was obtained from patient 3 and, upon evaluation, revealed evidence of infiltrating, clonally expanded T cells. CONCLUSIONS: Collectively, our findings suggest that NeoVax stimulated the expansion of neoantigen-specific effector T cells and provide encouraging results to aid in the development of future neoantigen vaccine-based clinical trials in patients with GBM. Herein, we demonstrate the feasibility of incorporating multisector sampling in cancer vaccine design and provide information on the clinical applicability of clonality, distribution, and immunogenicity of the neoantigen landscape in patients with GBM.

Glioblastoma-Infiltrating CD8+ T Cells Are Predominantly a Clonally Expanded GZMK+ Effector Population.

Recent clinical trials have highlighted the limited efficacy of T cell-based immunotherapy in patients with glioblastoma (GBM). To better understand the characteristics of tumor-infiltrating lymphocytes (TIL) in GBM, we performed cellular indexing of transcriptomes and epitopes by sequencing and single-cell RNA sequencing with paired V(D)J sequencing, respectively, on TILs from two cohorts of patients totaling 15 patients with high-grade glioma, including GBM or astrocytoma, IDH-mutant, grade 4 (G4A). Analysis of the CD8+ TIL landscape reveals an enrichment of clonally expanded GZMK+ effector T cells in the tumor compared with matched blood, which was validated at the protein level. Furthermore, integration with other cancer types highlights the lack of a canonically exhausted CD8+ T-cell population in GBM TIL. These data suggest that GZMK+ effector T cells represent an important T-cell subset within the GBM microenvironment and may harbor potential therapeutic implications. SIGNIFICANCE: To understand the limited efficacy of immune-checkpoint blockade in GBM, we applied a multiomics approach to understand the TIL landscape. By highlighting the enrichment of GZMK+ effector T cells and the lack of exhausted T cells, we provide a new potential mechanism of resistance to immunotherapy in GBM. This article is featured in Selected Articles from This Issue, p. 897.

TCR-engineered adoptive cell therapy effectively treats intracranial murine glioblastoma.

BACKGROUND: Adoptive cellular therapies with chimeric antigen receptor T cells have revolutionized the treatment of some malignancies but have shown limited efficacy in solid tumors such as glioblastoma and face a scarcity of safe therapeutic targets. As an alternative, T cell receptor (TCR)-engineered cellular therapy against tumor-specific neoantigens has generated significant excitement, but there exist no preclinical systems to rigorously model this approach in glioblastoma. METHODS: We employed single-cell PCR to isolate a TCR specific for the Imp3D81N neoantigen (mImp3) previously identified within the murine glioblastoma model GL261. This TCR was used to generate the Mutant Imp3-Specific TCR TransgenIC (MISTIC) mouse in which all CD8 T cells are specific for mImp3. The therapeutic efficacy of neoantigen-specific T cells was assessed through a model of cellular therapy consisting of the transfer of activated MISTIC T cells and interleukin 2 into lymphodepleted tumor-bearing mice. We employed flow cytometry, single-cell RNA sequencing, and whole-exome and RNA sequencing to examine the factors underlying treatment response. RESULTS: We isolated and characterized the 3×1.1C TCR that displayed a high affinity for mImp3 but no wild-type cross-reactivity. To provide a source of mImp3-specific T cells, we generated the MISTIC mouse. In a model of adoptive cellular therapy, the infusion of activated MISTIC T cells resulted in rapid intratumoral infiltration and profound antitumor effects with long-term cures in a majority of GL261-bearing mice. The subset of mice that did not respond to the adoptive cell therapy showed evidence of retained neoantigen expression but intratumoral MISTIC T cell dysfunction. The efficacy of MISTIC T cell therapy was lost in mice bearing a tumor with heterogeneous mImp3 expression, showcasing the barriers to targeted therapy in polyclonal human tumors. CONCLUSIONS: We generated and characterized the first TCR transgenic against an endogenous neoantigen within a preclinical glioma model and demonstrated the therapeutic potential of adoptively transferred neoantigen-specific T cells. The MISTIC mouse provides a powerful novel platform for basic and translational studies of antitumor T-cell responses in glioblastoma.

The Conventional Dendritic Cell 1 Subset Primes CD8+ T Cells and Traffics Tumor Antigen to Drive Antitumor Immunity in the Brain.

The central nervous system (CNS) antigen-presenting cell (APC) that primes antitumor CD8+ T-cell responses remains undefined. Elsewhere in the body, the conventional dendritic cell 1 (cDC1) performs this role. However, steady-state brain parenchyma cDC1 are extremely rare; cDCs localize to the choroid plexus and dura. Thus, whether the cDC1 play a function in presenting antigen derived from parenchymal sources in the tumor setting remains unknown. Using preclinical glioblastoma (GBM) models and cDC1-deficient mice, we explored the presently unknown role of cDC1 in CNS antitumor immunity. We determined that, in addition to infiltrating the brain tumor parenchyma itself, cDC1 prime neoantigen-specific CD8+ T cells against brain tumors and mediate checkpoint blockade-induced survival benefit. We observed that cDC, including cDC1, isolated from the tumor, the dura, and the CNS-draining cervical lymph nodes harbored a traceable fluorescent tumor antigen. In patient samples, we observed several APC subsets (including the CD141+ cDC1 equivalent) infiltrating glioblastomas, meningiomas, and dura. In these same APC subsets, we identified a tumor-specific fluorescent metabolite of 5-aminolevulinic acid, which fluorescently labeled tumor cells during fluorescence-guided GBM resection. Together, these data elucidate the specialized behavior of cDC1 and suggest that cDC1 play a significant role in CNS antitumor immunity.

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.