Nivolumab reaches brain lesions in patients with recurrent glioblastoma and induces T-cell activity and upregulation of checkpoint pathways.

Glioblastoma (GBM) is an aggressive brain tumor with poor prognosis. Although immunotherapy is being explored as a potential treatment option for patients with GBM, it is unclear whether systemic immunotherapy can reach and modify the tumor microenvironment in the brain. We evaluated immune characteristics in patients receiving the anti-PD1 immune checkpoint inhibitor Nivolumab one week prior to surgery, compared to control patients receiving salvage resection without prior Nivolumab treatment. We observed saturating levels of Nivolumab bound to intratumorally- and tissue-resident T cells in the brain, implicating saturating levels of Nivolumab reaching brain tumors. Following Nivolumab treatment, significant changes in T-cell activation and proliferation were observed in the tumor resident T-cell population, and peripheral T cells upregulated chemokine receptors related to brain homing. A strong Nivolumab-driven upregulation in compensatory checkpoint inhibition molecules, TIGIT, LAG-3, TIM-3 and CTLA-4 was observed, potentially counteracting the treatment effect. Finally, tumor-reactive tumor-infiltrating lymphocytes (TILs) were found in a subset of Nivolumab-treated patients with prolonged survival, and neoantigen-reactive T cells were identified in both TILs and blood. This indicates a systemic response towards GBM in a subset of patients, which was further boosted by Nivolumab, with T-cell responses towards tumor-derived neoantigens. Our study demonstrates that Nivolumab does reach the GBM tumor lesion and enhances antitumor T-cell responses both intratumorally and systemically. However, various anti-inflammatory mechanisms mitigate the clinical efficacy of the anti-PD1 treatment.

Dose escalation study of a personalized peptide-based neoantigen vaccine (EVX-01) in patients with metastatic melanoma.

BACKGROUND: Neoantigens can serve as targets for T cell-mediated antitumor immunity via personalized neopeptide vaccines. Interim data from our clinical study NCT03715985 showed that the personalized peptide-based neoantigen vaccine EVX-01, formulated in the liposomal adjuvant, CAF09b, was safe and able to elicit EVX-01-specific T cell responses in patients with metastatic melanoma. Here, we present results from the dose-escalation part of the study, evaluating the feasibility, safety, efficacy, and immunogenicity of EVX-01 in addition to anti-PD-1 therapy. METHODS: Patients with metastatic melanoma on anti-PD-1 therapy were treated in three cohorts with increasing vaccine dosages (twofold and fourfold). Tumor-derived neoantigens were selected by the AI platform PIONEER and used in personalized therapeutic cancer peptide vaccines EVX-01. Vaccines were administered at 2-week intervals for a total of three intraperitoneal and three intramuscular injections. The study's primary endpoint was safety and tolerability. Additional endpoints were immunological responses, survival, and objective response rates. RESULTS: Compared with the base dose level previously reported, no new vaccine-related serious adverse events were observed during dose escalation of EVX-01 in combination with an anti-PD-1 agent given according to local guidelines. Two patients at the third dose level (fourfold dose) developed grade 3 toxicity, most likely related to pembrolizumab. Overall, 8 out of the 12 patients had objective clinical responses (6 partial response (PR) and 2 CR), with all 4 patients at the highest dose level having a CR (1 CR, 3 PR). EVX-01 induced peptide-specific CD4+ and/or CD8+T cell responses in all treated patients, with CD4+T cells as the dominating responses. The magnitude of immune responses measured by IFN-γ ELISpot assay correlated with individual peptide doses. A significant correlation between the PIONEER quality score and induced T cell immunogenicity was detected, while better CRs correlated with both the number of immunogenic EVX-01 peptides and the PIONEER quality score. CONCLUSION: Immunization with EVX-01-CAF09b in addition to anti-PD-1 therapy was shown to be safe and well tolerated and elicit vaccine neoantigen-specific CD4+and CD8+ T cell responses at all dose levels. In addition, objective tumor responses were observed in 67% of patients. The results encourage further assessment of the antitumor efficacy of EVX-01 in combination with anti-PD-1 therapy.

Personalized therapy with peptide-based neoantigen vaccine (EVX-01) including a novel adjuvant, CAF®09b, in patients with metastatic melanoma.

The majority of neoantigens arise from unique mutations that are not shared between individual patients, making neoantigen-directed immunotherapy a fully personalized treatment approach. Novel technical advances in next-generation sequencing of tumor samples and artificial intelligence (AI) allow fast and systematic prediction of tumor neoantigens. This study investigates feasibility, safety, immunity, and anti-tumor potential of the personalized peptide-based neoantigen vaccine, EVX-01, including the novel CD8+ T-cell inducing adjuvant, CAF®09b, in patients with metastatic melanoma (NTC03715985). The AI platform PIONEERTM was used for identification of tumor-derived neoantigens to be included in a peptide-based personalized therapeutic cancer vaccine. EVX-01 immunotherapy consisted of 6 administrations with 5-10 PIONEERTM-predicted neoantigens as synthetic peptides combined with the novel liposome-based Cationic Adjuvant Formulation 09b (CAF®09b) to strengthen T-cell responses. EVX-01 was combined with immune checkpoint inhibitors to augment the activity of EVX-01-induced immune responses. The primary endpoint was safety, exploratory endpoints included feasibility, immunologic and objective responses. This interim analysis reports the results from the first dose-level cohort of five patients. We documented a short vaccine manufacturing time of 48-55 days which enabled the initiation of EVX-01 treatment within 60 days from baseline biopsy. No severe adverse events were observed. EVX-01 elicited long-lasting EVX-01-specific T-cell responses in all patients. Competitive manufacturing time was demonstrated. EVX-01 was shown to be safe and able to elicit immune responses targeting tumor neoantigens with encouraging early indications of a clinical and meaningful antitumor efficacy, warranting further study.

Vγ9Vδ2 T Cells Concurrently Kill Cancer Cells and Cross-Present Tumor Antigens.

The human Vγ9Vδ2 T cell is a unique cell type that holds great potential in immunotherapy of cancer. In particular, the therapeutic potential of this cell type in adoptive cell therapy (ACT) has gained interest. In this regard optimization of in vitro expansion methods and functional characterization is desirable. We show that Vγ9Vδ2 T cells, expanded in vitro with zoledronic acid (Zometa or ZOL) and Interleukin-2 (IL-2), are efficient cancer cell killers with a trend towards increased killing efficacy after prolonged expansion time. Thus, Vγ9Vδ2 T cells expanded for 25 days in vitro killed prostate cancer cells more efficiently than Vγ9Vδ2 T cells expanded for 9 days. These data are supported by phenotype characteristics, showing increased expression of CD56 and NKG2D over time, reaching above 90% positive cells after 25 days of expansion. At the early stage of expansion, we demonstrate that Vγ9Vδ2 T cells are capable of cross-presenting tumor antigens. In this regard, our data show that Vγ9Vδ2 T cells can take up tumor-associated antigens (TAA) gp100, MART-1 and MAGE-A3 - either as long peptide or recombinant protein - and then present TAA-derived peptides on the cell surface in the context of HLA class I molecules, demonstrated by their recognition as targets by peptide-specific CD8 T cells. Importantly, we show that cross-presentation is impaired by the proteasome inhibitor lactacystin. In conclusion, our data indicate that Vγ9Vδ2 T cells are broadly tumor-specific killers with the additional ability to cross-present MHC class I-restricted peptides, thereby inducing or supporting tumor-specific αßTCR CD8 T cell responses. The dual functionality is dynamic during in vitro expansion, yet, both functions are of interest to explore in ACT for cancer therapy.

Expansion With IL-15 Increases Cytotoxicity of Vγ9Vδ2 T Cells and Is Associated With Higher Levels of Cytotoxic Molecules and T-bet.

Cancer immunotherapy has shown great advances during recent years, but it has yet to reach its full potential in all cancer types. Adoptive cell therapy (ACT) is now an approved treatment option for certain hematological cancers and has also shown success for some solid cancers. Still, benefit and eligibility do not extend to all patients. ACT with Vγ9Vδ2 T cells is a promising approach to overcome this hurdle. In this study, we aimed to explore the effect of different cytokine conditions on the expansion of Vγ9Vδ2 T cells in vitro. We could show that Vγ9Vδ2 T cell expansion is feasible with two different cytokine conditions: (a) 1,000 U/ml interleukin (IL)-2 and (b) 100 U/ml IL-2 + 100 U/ml IL-15. We did not observe differences in expansion rate or Vγ9Vδ2 T cell purity between the conditions; however, IL-2/IL-15-expanded Vγ9Vδ2 T cells displayed enhanced cytotoxicity against tumor cells, also in hypoxia. While this increase in killing capacity was not reflected in natural killer (NK) cell marker or activation marker expression, we demonstrated that IL-2/IL-15-expanded Vγ9Vδ2 T cells were characterized by an increased expression of perforin, granzyme B, and granulysin compared to IL-2-expanded cells. These cytotoxic molecules were not only increased in a resting state, but also released to a greater extent upon target recognition. In contrast, CD107a and cytokine expression did not differ between expansion conditions. However, IL-2/IL-15-expanded Vγ9Vδ2 T cells showed higher levels of transcription factor T-bet expression, which could indicate that T-bet and cytotoxic molecule levels confer the increased cytotoxicity. These results advocate the inclusion of IL-15 into ex vivo Vγ9Vδ2 T cell expansion protocols in future clinical studies.

Chemokine receptor engineering of T cells with CXCR2 improves homing towards subcutaneous human melanomas in xenograft mouse model.

Adoptive cell therapy (ACT) using in vitro expanded tumor infiltrating T lymphocytes (TILs) from biopsy material represents a highly promising treatment of disseminated cancer. A crucial prerequisite for successful ACT is sufficient recruitment of transferred lymphocytes to the tumor site; however, despite infusion of billions of lymphocytes, T cell infiltration into the tumor post ACT is limited. By PCR and Luminex analyses we found that a majority of malignant melanoma (MM) cell lines expressed chemokines CXCL1/Groα, CXCL8/IL-8, CXCL12/SDF-1 and CCL2. Concerning expression of the corresponding receptors on T cells, only the IL-8 receptor, CXCR2, was not expressed on T cells. CXCR2 was therefore expressed in T cells by lentiviral transduction, and shown to lead to ligand specific transwell migration of engineered T cells, as well as increased migration towards MM conditioned medium. In vivo homing was assessed in a xenograft NOG mouse model. Mice with subcutaneous human melanoma were treated with MAGE-A3 specific T cells transduced with either CXCR2 or MOCK. Transducing T cells carrying the MAGE-A3a3a high affinity T cell receptor with CXCR2 increased tumor infiltration. Flow cytometry analysis 7 days after ACT showed a doubling in CD3+ T cells in tumor digest of mice receiving CXCR2 transduced T cells compared to MOCK treated mice, a finding confirmed by immunohistochemistry. In conclusion, our CXCR2 transduced T cells are functional in vitro and transduction with CXCR2 increases in vivo homing of T cells to tumor site.

Improved migration of tumor ascites lymphocytes to ovarian cancer microenvironment by CXCR2 transduction.

Chemokines are essential mediators of cellular trafficking, interactions and tumor development. Though adoptive cell therapy (ACT) has been a tremendous success in the treatment of metastatic melanoma (MM), a major obstacle for successful ACT, is limited homing of effector T cells to immune suppressive tumor sites. We hypothesized that equipping T cells with chemokine receptors matching the chemokines of the tumor microenvironment, could improve tumor homing of T cells. T cells from malignant ascites (n = 13); blood from ovarian cancer (OC) patients (n = 14); and healthy donors (n = 13) were analyzed by flow cytometry. We found that FoxP3+ regulatory T cells accumulation in patients with OC associates with CCR4 expression. We characterized a chemokine profile of ascites chemokines, and expression of corresponding receptors on circulating T cells and tumor ascites lymphocytes (TALs). CCL22, CXCL9, CXCL10 and CXCL12 associated with enrichment of CCR4+, CCR5+, CXCR3+ and CXCR4+ T cells in ascites. Circulating T cells and TALs however did not express CXCR2, identifying CXCR2 as candidate for chemokine receptor transduction. TALs readily expressed IFNγ and TNFα upon stimulation despite the frequency decreasing with in vitro expansion. Lentiviral transduction of TALs (n = 4) with chemokine receptor CXCR2 significantly increased transwell migration of TALs towards rhIL8 and autologous ascites. The majority of expanded and transduced TALs were of a T effector memory subtype. This proof of concept study shows that chemokine receptor engineering with CXCR2 is feasible and improves homing of transduced TALs towards the OC microenvironment.