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Chemotherapy combined with immunotherapy has improved the treatment of certain solid tumors, but effective regimens remain elusive for pancreatic ductal adenocarcinoma (PDAC). We conducted a randomized phase 2 trial evaluating the efficacy of nivolumab (nivo; anti-PD-1) and/or sotigalimab (sotiga; CD40 agonistic antibody) with gemcitabine/nab-paclitaxel (chemotherapy) in patients with first-line metastatic PDAC ( NCT03214250 ). In 105 patients analyzed for efficacy, the primary endpoint of 1-year overall survival (OS) was met for nivo/chemo (57.7%, P  = 0.006 compared to historical 1-year OS of 35%, n  = 34) but was not met for sotiga/chemo (48.1%, P  = 0.062, n  = 36) or sotiga/nivo/chemo (41.3%, P  = 0.223, n  = 35). Secondary endpoints were progression-free survival, objective response rate, disease control rate, duration of response and safety. Treatment-related adverse event rates were similar across arms. Multi-omic circulating and tumor biomarker analyses identified distinct immune signatures associated with survival for nivo/chemo and sotiga/chemo. Survival after nivo/chemo correlated with a less suppressive tumor microenvironment and higher numbers of activated, antigen-experienced circulating T cells at baseline. Survival after sotiga/chemo correlated with greater intratumoral CD4 T cell infiltration and circulating differentiated CD4 T cells and antigen-presenting cells. A patient subset benefitting from sotiga/nivo/chemo was not identified. Collectively, these analyses suggest potential treatment-specific correlates of efficacy and may enable biomarker-selected patient populations in subsequent PDAC chemoimmunotherapy trials. In a randomized phase 2 trial, sotigalimab, a CD40 agonist, did not significantly improve overall survival in patients with previously untreated metastatic pancreatic cancer when combined with chemotherapy or with nivolumab and chemotherapy. Multi-omic exploratory analyses provide insights into immunologic features associated with clinical benefit.

Efficacy of cancer vaccines remains low and mechanistic understanding of antigen presenting cell function in cancer may improve vaccine design and outcomes. Here, we analyze the transcriptomic and immune-metabolic profiles of Dendritic Cells (DCs) from 35 subjects enrolled in a trial of DC vaccines in late-stage melanoma (NCT01622933). Multiple platforms identify metabolism as an important biomarker of DC function and patient overall survival (OS). We demonstrate multiple immune and metabolic gene expression pathway alterations, a functional decrease in OCR/OXPHOS and increase in ECAR/glycolysis in patient vaccines. To dissect molecular mechanisms, we utilize single cell SCENITH functional profiling and show patient clinical outcomes (OS) correlate with DC metabolic profile, and that metabolism is linked to immune phenotype. With single cell metabolic regulome profiling, we show that MCT1 (monocarboxylate transporter-1), a lactate transporter, is increased in patient DCs, as is glucose uptake and lactate secretion. Importantly, pre-vaccination circulating myeloid cells in patients used as precursors for DC vaccine generation are significantly skewed metabolically as are several DC subsets. Together, we demonstrate that the metabolic profile of DC is tightly associated with the immunostimulatory potential of DC vaccines from cancer patients. We link phenotypic and functional metabolic changes to immune signatures that correspond to suppressed DC differentiation. Efficacy of dendritic cell (DC)-based vaccines remains unsatisfactory. Here the authors analyse the transcriptomic and immune-metabolic profiles of DCs from patients enrolled in a DC vaccine trial in late-stage melanoma, suggesting that the metabolic profile of DC is associated with the immunostimulatory potential of the cancer vaccine.

To accelerate the development of T cell–based immunotherapies that are effective for more patients with cancer, there is an urgent need to decipher the precise attributes of the ideal therapeutic T cell. In March 2021, the Parker Institute of Cancer Immunotherapy and 10x Genomics partnered to bring together a group of T cell immunotherapy researchers and single-cell-technology innovators for a day’s workshop. Participants evaluated the current cutting edge of knowledge, identified areas for focused technology development, and put forward a call to action to the field. Insights were provided on how to best leverage single-cell technologies and key areas for future development were proposed — with the goal of facilitating a better understanding of T cell research and translation of this research into effective cancer immunotherapies. The key points of discussion that emerged from this workshop are summarized here.

While high circulating tumor DNA (ctDNA) levels are associated with poor survival for multiple cancers, variant-specific differences in the association of ctDNA levels and survival have not been examined. Here we investigate KRAS ctDNA (ctKRAS) variant-specific associations with overall and progression-free survival (OS/PFS) in first-line metastatic pancreatic ductal adenocarcinoma (mPDAC) for patients receiving chemoimmunotherapy (“PRINCE”, NCT03214250), and an independent cohort receiving standard of care (SOC) chemotherapy. For PRINCE, higher baseline plasma levels are associated with worse OS for ctKRAS G12D (log-rank p = 0.0010) but not G12V (p = 0.7101), even with adjustment for clinical covariates. Early, on-therapy clearance of G12D (p = 0.0002), but not G12V (p = 0.4058), strongly associates with OS for PRINCE. Similar results are obtained for the SOC cohort, and for PFS in both cohorts. These results suggest ctKRAS G12D but not G12V as a promising prognostic biomarker for mPDAC and that G12D clearance could also serve as an early biomarker of response. ctDNA is a known poor prognostic factor for multiple cancer types, but variant-specificity is unknown. Here, the authors show variant-specific association of ctKRAS levels with survival in previously untreated metastatic pancreatic ductal adenocarcinoma patients in multiple cohorts.

Background Various proinflammatory cytokines can be detected within the melanoma tumor microenvironment. Interleukin 32 (IL32) is produced by T cells, NK cells and monocytes/macrophages, but also by a subset of melanoma cells. We sought to better understand the biology of IL32 in human melanoma. Methods We analyzed RNA sequencing data from 53 in-house established human melanoma cell lines and 479 melanoma tumors from The Cancer Genome Atlas dataset. We evaluated global gene expression patterns associated with IL32 expression. We also evaluated the impact of proinflammatory molecules TNFα and IFNγ on IL32 expression and dedifferentiation in melanoma cell lines in vitro. In order to study the transcriptional regulation of IL32 in these cell lines, we cloned up to 10.5 kb of the 5′ upstream region of the human IL32 gene into a luciferase reporter vector. Results A significant proportion of established human melanoma cell lines express IL32, with its expression being highly correlated with a dedifferentiation genetic signature (high AXL/low MITF). Non IL32-expressing differentiated melanoma cell lines exposed to TNFα or IFNγ can be induced to express the three predominant isoforms (α, β, γ) of IL32. Cis -acting elements within this 5′ upstream region of the human IL32 gene appear to govern both induced and constitutive gene expression. In the tumor microenvironment, IL32 expression is highly correlated with genes related to T cell infiltration, and also positively correlates with high AXL/low MITF dedifferentiated gene signature. Conclusions Expression of IL32 in human melanoma can be induced by TNFα or IFNγ and correlates with a treatment-resistant dedifferentiated genetic signature. Constitutive and induced expression are regulated, in part, by cis -acting sequences within the 5′ upstream region.

Background Cancer vaccines are designed to promote systemic antitumor immunity and tumor eradication. Cancer vaccination may be more efficacious in combination with additional interventions that may build on or amplify their effects. Methods Based on our previous clinical and in vitro studies, we designed an antigen-engineered DC vaccine trial to promote a polyclonal CD8 + and CD4 + T cell response against three shared melanoma antigens. The 35 vaccine recipients were then randomized to receive one month of high-dose IFNα or observation. Results The resulting clinical outcomes were 2 partial responses, 8 stable disease and 14 progressive disease among patients with measurable disease using RECIST 1.1, and, of 11 surgically treated patients with no evidence of disease (NED), 4 remain NED at a median follow-up of 3 years. The majority of vaccinated patients showed an increase in vaccine antigen-specific CD8 + and CD4 + T cell responses. The addition of IFNα did not appear to improve immune or clinical responses in this trial. Examination of the DC vaccine profiles showed that IL-12p70 secretion did not correlate with immune or clinical responses. In depth immune biomarker studies support the importance of circulating Treg and MDSC for development of antigen-specific T cell responses, and of circulating CD8 + and CD4 + T cell subsets in clinical responses. Conclusions DC vaccines are a safe and reliable platform for promoting antitumor immunity. This combination with one month of high dose IFNα did not improve outcomes. Immune biomarker analysis in the blood identified several predictive and prognostic biomarkers for further analysis, including MDSC. Trial registration NCT01622933 .

BackgroundA first-in-human, randomized pilot phase II clinical trial combining vaccines targeting overexpressed, non-mutated tumor blood vessel antigens (TBVA) and tyrosine kinase inhibitor dasatinib was conducted in human leukocyte antigen (HLA)-A2+ patients with advanced melanoma.MethodsPatient monocyte-derived type-1-polarized dendritic cells were loaded with HLA-A2-presented peptides derived from TBVA (DLK1, EphA2, HBB, NRP1, RGS5, TEM1) and injected intradermally as a vaccine into the upper extremities every other week. Patients were randomized into one of two treatment arms receiving oral dasatinib (70 mg two times per day) beginning in week 5 (Arm A) or in week 1 (Arm B). Trial endpoints included T cell response to vaccine peptides (interferon-γ enzyme-linked immunosorbent spot), objective clinical response (Response Evaluation Criteria in Solid Tumors V.1.1) and exploratory tumor, blood and serum profiling of immune-associated genes/proteins.ResultsSixteen patients with advanced-stage cutaneous (n=10), mucosal (n=1) or uveal (n=5) melanoma were accrued, 15 of whom had previously progressed on programmed cell death protein 1 (PD-1) blockade. Of 13 evaluable patients, 6 patients developed specific peripheral blood T cell responses against ≥3 vaccine-associated peptides, with further evidence of epitope spreading. All six patients with specific CD8+ T cell response to vaccine-targeted antigens exhibited evidence of T cell receptor (TCR) convergence in association with preferred clinical outcomes (four partial response and two stabilization of disease (SD)). Seven patients failed to respond to vaccination (one SD and six progressive disease). Patients in Arm B (immediate dasatinib) outperformed those in Arm A (delayed dasatinib) for immune response rate (IRR; 66.7% vs 28.6%), objective response rate (ORR) (66.7% vs 0%), overall survival (median 15.45 vs 3.47 months; p=0.0086) and progression-free survival (median 7.87 vs 1.97 months; p=0.063). IRR (80% vs 25%) and ORR (60% vs 12.5%) was greater for females versus male patients. Tumors in patients exhibiting response to treatment displayed (1) evidence of innate and adaptive immune-mediated inflammation and TCR convergence at baseline, (2) on-treatment transcriptional changes associated with reduced hypoxia/acidosis/glycolysis, and (3) increased inflammatory immune cell infiltration and tertiary lymphoid structure neogenesis.ConclusionsCombined vaccination against TBVA plus dasatinib was safe and resulted in coordinating immunologic and/or objective clinical responses in 6/13 (46%) evaluable patients with melanoma, particularly those initiating treatment with both agents.Trial registration numberNCT01876212.

BackgroundThe therapeutic efficacy of Dendritic cells (DC) vaccines remains low and there is an unmet need for more effective vaccine design to achieve durable clinical outcomes. Our study analyzed the transcriptomic and energetic metabolism profile of an adenoviral-based DC vaccine targeted against three commonly shared melanoma antigens: Tyrosinase, MART-1 and MAGE-A6 from 35 subjects enrolled in a Phase I study of autologous DC vaccines in late-stage melanoma.1 To further investigate the immuno-metabolic features of monocyte-derived DC vaccines, we are employing a novel flow cytometry-based method, called SCENITH2 to integrate functional metabolic states with multiparametric DC immune phenotypes.Methods iDC were generated from HD and patient monocytes using GM-CSF+IL-4 for 5d. DC were matured (mDC) using IFN?+LPS for additional 24 hrs. Tolerogenic DC (Tol DC) were generated using vitamin-D3 and dexamethasone. Seahorse® was used to measure DC metabolic profile. Cytek/Aurora spectral flow cytometry was used for multiparametric-phenotypic and metabolic analysis by SCENITH™.ResultsMelanoma patient mDC used for autologous vaccine generation showed significantly altered metabolic gene signatures associated with enhanced oxidative phosphorylation (OXPHOS) and lipid metabolism pathways as compared to HD mDC. Furthermore, increased enrichment for mitochondrial respiration genes involved in the TCA cycle, electron transport chain and fatty acid oxidation (FAO) correlated with inferior tumor antigen-specific T cell responses and clinical outcome in patients. Seahorse analyses confirmed that HD and good outcome patient DC demonstrated the highest maturation-induced reduction in maximal oxygen consumption rate/OXPHOS and exogenous FAO. Interestingly, while the glycolytic rate of non-responding patient DC was the lowest, overall, we observe only a moderate increase in glycolytic capacity during DC maturation. SCENITH analysis showed that unlike monocytes, which are primarily glycolytic, differentiated mono-derived iDC and mDC utilize both glycolysis and mitochondrial respiration. Interestingly, under tolerogenic (Tol) differentiation conditions Tol iDC shift from glucose dependence into FAO and/or glutaminolysis while Tol mDC strongly depend on OXPHOS. Consistent with dependence on mitochondrial respiration, Tol mDC exhibit reduced HIF1a levels together with enhanced p-AMPK:p-mTOR ratio. Additionally, we show that the altered metabolism of Tol mDC is linked to retention of CD14-monocyte antigen with reduced DC markers HLA-DR, CD86, CD206, CD11c, CD33, with increased PD-L1 and ILT3 expression. Furthermore, we show that unlike HD mDC, tolerogenic and melanoma patient-derived mDC populations exhibit similar metabolic and immune characteristics.ConclusionsWe demonstrate that metabolic profile of DCs is tightly associated to the immuno-stimulatory potential of DC vaccines from cancer patients. Using SCENITH, we linked phenotypic and functional metabolic changes associated to immune signatures that correspond to heathy and immuno-suppressed DC differentiation.Ethics ApprovalThe clinical trial reported was fully approved by the Univ. Pittsburgh PRC and IRB (PRO12010416, #09–021) and had FDA IND #15044 and NCT01622933.ReferencesButterfield LH, Vujanovic L, Santos PM, Maurer DM, Gambotto A, Lohr J, Li C, Waldman J, Chandran U, Lin Y, et al. Multiple antigen-engineered DC vaccines with or without IFNalpha to promote antitumor immunity in melanoma. Journal for immunotherapy of cancer 2019;7:113.Argüello RJ, Combes AJ, Char R, Bousiquot E, Gigan JP, Camosseto V, Samad B, Tsui J, Yan P, et al. SCENITH: A flow cytometry based method for functional profiling energy metabolism with single cell resolution. BioRxiv 2020.03.10.985796; doi: https://doi.org/10.1101/2020.03.10.985796. Cell Metabolism. (Sneak and Peek: under review 10.2139/ssrn.3565001)

Efficacy of cancer vaccines remains low and mechanistic understanding of antigen presenting cell function in cancer may improve vaccine design and outcomes. Here, we analyze the transcriptomic and immune-metabolic profiles of Dendritic Cells (DCs) from 35 subjects enrolled in a trial of DC vaccines in late-stage melanoma (NCT01622933). Multiple platforms identify metabolism as an important biomarker of DC function and patient overall survival (OS). We demonstrate multiple immune and metabolic gene expression pathway alterations, a functional decrease in OCR/OXPHOS and increase in ECAR/glycolysis in patient vaccines. To dissect molecular mechanisms, we utilize single cell SCENITH functional profiling and show patient clinical outcomes (OS) correlate with DC metabolic profile, and that metabolism is linked to immune phenotype. With single cell metabolic regulome profiling, we show that MCT1 (monocarboxylate transporter-1), a lactate transporter, is increased in patient DCs, as is glucose uptake and lactate secretion. Importantly, pre-vaccination circulating myeloid cells in patients used as precursors for DC vaccine generation are significantly skewed metabolically as are several DC subsets. Together, we demonstrate that the metabolic profile of DC is tightly associated with the immunostimulatory potential of DC vaccines from cancer patients. We link phenotypic and functional metabolic changes to immune signatures that correspond to suppressed DC differentiation.