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BackgroundRecent studies have demonstrated that T cells can induce vasodilation in a choline-acetyltransferase dependent manner, leading to an increase in T cell migration to infected tissues in response to viral infection, but its role in cancer is unclear. Choline acetyltransferase catalyzes the production of acetylcholine from choline and acetyl-CoA, however, acetylcholine is challenging to quantify due to its extremely short half-life while choline is stable. This study aims to correlate serum choline levels in patients with advanced solid tumors receiving pembrolizumab with treatment outcomes.MethodsBlood samples were collected at baseline and at week 7 (pre-cycle 3) in patients treated with pembrolizumab in the INvestigator-initiated Phase 2 Study of Pembrolizumab Immunological Response Evaluation phase II trial (NCT02644369). Samples were analyzed for choline and circulating tumor DNA (ctDNA). Multivariable Cox models were used to assess the association between choline and overall survival (OS) and progression-free survival (PFS) when including ΔctDNAC3 (the change in ctDNA from baseline to cycle 3), cohort, PD-L1 expression and tumor mutation burden (TMB). An independent validation cohort from the LIBERATE study (NCT03702309) included patients on early phase trials treated with a PD-1 inhibitor.ResultsA total of 106 pts were included in the analysis. With a median follow-up of 12.6 months, median PFS and OS were 1.9 and 13.7 months, respectively. An increase in serum choline level at week 7 compared with baseline (ΔcholineC3) in 81 pts was significantly associated with a better PFS (aHR 0.48, 95% CI 0.28 to 0.83, p=0.009), and a trend toward a better OS (aHR 0.64, 95% CI 0.37 to 1.12, p=0.119). A combination of ΔctDNAC3 and ΔcholineC3 was prognostic for both OS and PFS. Multivariable analyses show ΔcholineC3 was a prognostic factor for PFS independent of ΔctDNAC3, cohort, PD-L1 and TMB. In the independent validation cohort (n=51), an increase in serum choline at cycle 2 was associated with a trend to improved PFS.ConclusionsThis is the first exploratory report of serum choline levels in pan-cancer patients receiving pembrolizumab. The association between improved PFS and ΔcholineC3 suggests a possible role for the cholinergic system in the regulation of antitumor immunity. Further pre-clinical and clinical studies are required to validate this finding.Trial registration numberNCT03702309.

BackgroundThe combination of antiangiogenic agents with immune checkpoint inhibitors could potentially overcome immune suppression driven by tumor angiogenesis. We report results from a phase IB study of ziv-aflibercept plus pembrolizumab in patients with advanced solid tumors.MethodsThis is a multicenter phase IB dose-escalation study of the combination of ziv-aflibercept (at 2–4 mg/kg) plus pembrolizumab (at 2 mg/kg) administered intravenously every 2 weeks with expansion cohorts in programmed cell death protein 1 (PD-1)/programmed death-ligand 1(PD-L1)-naïve melanoma, renal cell carcinoma (RCC), microsatellite stable colorectal cancer (CRC), and ovarian cancer. The primary objective was to determine maximum tolerated dose (MTD) and recommended dose of the combination. Secondary endpoints included overall response rate (ORR) and overall survival (OS). Exploratory objectives included correlation of clinical efficacy with tumor and peripheral immune population densities.ResultsOverall, 33 patients were enrolled during dose escalation (n=3) and dose expansion (n=30). No dose-limiting toxicities were reported in the initial dose level. Ziv-aflibercept 4 mg/kg plus pembrolizumab 2 mg/kg every 2 weeks was established as the MTD. Grade ≥3 adverse events occurred in 19/33 patients (58%), the most common being hypertension (36%) and proteinuria (18%). ORR in the dose-expansion cohort was 16.7% (5/30, 90% CI 7% to 32%). Complete responses occurred in melanoma (n=2); partial responses occurred in RCC (n=1), mesothelioma (n=1), and melanoma (n=1). Median OS was as follows: melanoma, not reached (NR); RCC, 15.7 months (90% CI 2.5 to 15.7); CRC, 3.3 months (90% CI 0.6 to 3.4); ovarian, 12.5 months (90% CI 3.8 to 13.6); other solid tumors, NR. Activated tumor-infiltrating CD8 T cells at baseline (CD8+PD1+), high CD40L expression, and increased peripheral memory CD8 T cells correlated with clinical response.ConclusionThe combination of ziv-aflibercept and pembrolizumab demonstrated an acceptable safety profile with antitumor activity in solid tumors. The combination is currently being studied in sarcoma and anti-PD-1-resistant melanoma.Trial registration numberNCT02298959.

BackgroundAngiogenic factors play a role in regulating immune suppression in the tumor microenvironment and driving resistance to immune checkpoint inhibitor therapy.1 Ziv-aflibercept is a soluble decoy receptor that ”traps” endogenous vascular endothelial growth factor (VEGF) with 100-fold increased binding affinity compared to Bevacizumab.2 The combination of ziv-aflibercept with either cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) or programmed cell death protein 1 (PD-1) blockade has shown promising antitumor efficacy in mouse models.3 4 We hypothesized that a novel combination of ziv-aflibercept and anti-PD-1 would be tolerable and lead to clinical benefits in tumors that traditionally do not respond to checkpoint blockade.MethodsThis is a multicenter phase 1B dose escalation study (NCT02298959) of the combination of ziv-aflibercept (at 2–4 mg/kg) plus pembrolizumab (at 2 mg/kg) administered intravenously every 2 weeks with expansion cohorts in PD-1/PD-L1 naïve melanoma, renal cell carcinoma (RCC), microsatellite stable colorectal cancer (MSS CRC), and ovarian cancer (figure 1). The primary objective was to determine the maximum tolerated dose (MTD) and recommended dose of the combination. Secondary endpoints included overall response rate (ORR) and overall survival (OS). Exploratory objectives included correlation of clinical efficacy and immune population densities in the tissue and periphery.ResultsOverall, 33 patients were enrolled during dose escalation (n=3) and dose expansion (n=30). No dose-limiting toxicities (DLTs) were reported in the initial dose level. Ziv-aflibercept 4 mg/kg plus pembrolizumab 2 mg/kg every 2 weeks was established as the MTD. Grade ≥3 treatment-related adverse events occurred in 19/33 patients (58%), the most common being hypertension (36%) and proteinuria (18%). ORR in the dose expansion cohort was 16.7% (5/30; 95% CI, 7–32%). Complete responses occurred in melanoma (n=2), partial responses occurred in RCC (n=1), mesothelioma (n=1), and melanoma (n=1). Efficacy outcomes by tumor type are shown in table 1 and figure 2. Median OS was as follows: melanoma, not reached; RCC, 15.7 months (90% CI, 2.5–15.7); CRC, 3.3 months (90% CI, 0.6–3.4), ovarian, 12.5 months (90% CI, 3.8–13.6), other solid tumors, not reached (figure 3). Activated tumor infiltrating CD8 T cells at baseline (CD8+PD1+), high CD40L expression (figure 4), and increased memory CD8 T cells in the periphery (figure 5) correlated with clinical response to the combination therapy.Abstract 374 Figure 1Study Schema of dose escalation and dose expansion. (A) Cohort 1 included the following tumors: clear cell sarcoma, triple negative breast cancer (TNBC), and mesothelioma. (B) Other solid tumors in Cohort 2 were: epithelioid mesothelioma (2) and TNBC (1).Abstract 374 Table 1Efficacy outcomes by dose level and tumor typea. Solid tumors included clear cell sarcoma (1), breast cancer (2), mesothelioma (3).b. Both patients with CR had melanoma and were in Cohort 2 (DL2).Abstract 374 Figure 2Waterfall plot of best RECIST response. Waterfall plot of maximum change from baseline in sum of target lesions for 28 patients with tumor measurements over time. Plot is color-coded by tumor type. Triangles indicate patients who developed new lesions, yellow circles indicate the 3 patients who received DL1.Abstract 374 Figure 3Progression and overall survival by tumor type. Kaplan-Meier curves for (A) progression-free survival and (B) overall survival based on tumor type.Abstract 374 Figure 4Luminex assay analysis of clinical response. Luminex analysis of baseline biomarkers. Patients were analyzed by clinical response (complete response [CR] and partial response [PR]) and durable clinical benefit (DCB) which includes patients with CR, PR, and stable disease (SD). (A) Baseline levels of IL-6 were lower in responders vs. non-responders (median 2.605 vs. 9.847 pg/mL, p = 0.009). Baseline CD40L was increased in responders (median 2,840 vs. 2,267 pg/mL, p = 0.06). (B) Baseline levels of GroB (median 1,272 vs. 592 pg/mL, p = 0.006), CXCL5 (median 547.5 vs. 296.2 pg/mL, p = 0.02), and CD40L (median 2,807 vs. 1,595 pg/mL, p = 0.001) were higher in patients with DCB vs. no DCB. P-values for baseline comparisons were obtained through Wilcoxon rank-sum test. The solid black line indicates median. Violins show range and kernel density estimate distributions of each group. (*) p < 0.05, (**) p < 0.01.Abstract 374 Figure 5Flow cytometry analysis. Flow cytometry analysis comparing T cell populations and monocytes between patients with clinical response (CR or PR, n = 5) and non-responders (n = 18) and patients with disease control (CR, PR, or SD, n = 12) and no disease control (n = 11). (A) CD4+ populations were increased in responders vs. non-responders at all time points. (B) CD8+ populations were decreased in responders vs. non-responders at all time points. (C) Treg CD4+/CD25+/FoxP3+ was decreased at baseline in responders. (D) Treg CD4+/CD25+/FoxP3+ were decreased at baseline and 1-month in patients with disease control vs. no disease control. (E) TCM CD8+/CD45RO+/CCR7+ was increased at all time points in responders vs. non-responders. (F) TEMRA CD8+/CD45RO-/CCR7- was decreased in responders at baseline. (G) Non-classical TIE2 was increased at baseline in non-responders. (H) Classical monocytes were increased at all time points in non-responders.ConclusionsThe combination of ziv-aflibercept and pembrolizumab demonstrated an acceptable safety profile with antitumor activity in solid tumors. The combination is currently being studied in sarcoma and anti-PD-1 resistant melanoma.AcknowledgementsThis trial was supported by the National Cancer Institute (NCI) and by Merck, Sharpe, and Dohme and Sanofi via Cooperative Research and Development Agreements with the NCI. PLB was supported by NCI UM1 Grant CA186644.Trial RegistrationNCT02298959ReferencesRahma OE, Hodi FS. The intersection between tumor angiogenesis and immune suppression. Clin Cancer Res September 15 2019;25(18):5449–5457. doi:10.1158/1078-0432.CCR-18-1543Holash J, Davis S, Papadopoulos N, et al. VEGF-trap: a VEGF blocker with potent antitumor effects. Proceedings of the National Academy of Sciences 2002;99(17):11393–11398. doi:10.1073/pnas.172398299Burova E, Ioffe E, Taduriyasas C, et al. Abstract 5035: blockade of VEGF with ziv-aflibercept (VEGF Trap) enhances anti-tumor efficacy of CTLA-4 blocking antibody in an Fc dependent manner. Cancer Research 2014;74(19 Supplement):5035–5035. doi:10.1158/1538-7445.am2014-5035Di Tacchio M, Macas J, Weissenberger J, et al. Tumor vessel normalization, immunostimulatory reprogramming, and improved survival in glioblastoma with combined inhibition of PD-1, angiopoietin-2, and VEGF. Cancer Immunology Research 2019;7(12):1910–1927. doi:10.1158/2326-6066.cir-18-0865Ethics ApprovalThis trial (NCT02298959) was approved by all participating IRBs.

BackgroundGWN323 is an IgG1 monoclonal antibody (mAb) against the glucocorticoid-induced tumor necrosis factor receptor-related protein. This first-in-human, open-label phase I/Ib study aimed to investigate the safety and tolerability and to identify the recommended doses of GWN323 with/without spartalizumab, an anti-programmed cell death receptor-1 agent, for future studies. Pharmacokinetics, preliminary efficacy and efficacy biomarkers were also assessed.MethodsPatients (aged ≥18 years) with advanced/metastatic solid tumors with Eastern Cooperative Oncology Group performance status of ≤2 were included. GWN323 (10–1500 mg) or GWN323+spartalizumab (GWN323 10–750 mg+spartalizumab 100–300 mg) were administered intravenously at various dose levels and schedules during the dose-escalation phase. Dose-limiting toxicities (DLTs) were assessed during the first 21 days in a single-agent arm and 42 days in a combination arm. Adverse events (AEs) were graded per National Cancer Institute-Common Toxicity Criteria for Adverse Events V.4.03 and efficacy was assessed using Response Evaluation Criteria in Solid Tumors V.1.1.ResultsOverall, 92 patients (single-agent, n=39; combination, n=53) were included. The maximum administered doses (MADs) in the single-agent and combination arms were GWN323 1500 mg every 3 weeks (q3w) and GWN323 750 mg+spartalizumab 300 mg q3w, respectively. No DLTs were observed with single-agent treatment. Three DLTs (6%, all grade ≥3) were noted with combination treatment: blood creatine phosphokinase increase, respiratory failure and small intestinal obstruction. Serious AEs were reported in 30.8% and 34.0%, and drug-related AEs were reported in 82.1% and 77.4% of patients with single-agent and combination treatments, respectively. Disease was stable in 7 patients and progressed in 26 patients with single-agent treatment. In combination arm patients, 1 had complete response (endometrial cancer); 3, partial response (rectal cancer, adenocarcinoma of colon and melanoma); 14, stable disease; and 27, disease progression. GWN323 exhibited a pharmacokinetic profile typical of mAbs with a dose-dependent increase in the pharmacokinetic exposure. Inconsistent decreases in regulatory T cells and increases in CD8+ T cells were observed in the combination arm. Gene expression analyses showed no significant effect of GWN323 on interferon-γ or natural killer-cell signatures.ConclusionsGWN323, as a single agent and in combination, was well tolerated in patients with relapsed/refractory solid tumors. The MAD was 1500 mg q3w for single-agent and GWN323 750 mg+spartalizumab 300 mg q3w for combination treatments. Minimal single-agent activity and modest clinical benefit were observed with the spartalizumab combination.Trial registration numberNCT02740270.

PurposeTo evaluate whether administration of the oral DNA hypomethylating agent CC-486 enhances the poor response rate of immunologically ‘cold’ solid tumors to immune checkpoint inhibitor durvalumab.Experimental designPD-L1/PD-1 inhibitor naïve patients with advanced microsatellite stable colorectal cancer; platinum resistant ovarian cancer; and estrogen receptor positive, HER2 negative breast cancer were enrolled in this single-institution, investigator-initiated trial. Two 28 day regimens, regimen A (CC-486 300 mg QD Days 1–14 (cycles 1–3 only) in combination with durvalumab 1500 mg intravenous day 15) and regimen B (CC-486 100 mg QD days 1–21 (cycle 1 and beyond), vitamin C 500 mg once a day continuously and durvalumab 1500 mg intravenous day 15) were investigated. Patients underwent paired tumor biopsies and serial peripheral blood mononuclear cells (PBMCs) collection for immune-profiling, transcriptomic and epigenomic analyzes.ResultsA total of 28 patients were enrolled, 19 patients treated on regimen A and 9 on regimen B. The combination of CC-486 and durvalumab was tolerable. Regimen B, with a lower dose of CC-486 extended over a longer treatment course, showed less grade 3/4 adverse effects. Global LINE-1 methylation assessment of serial PBMCs and genome-wide DNA methylation profile in paired tumor biopsies demonstrated minimal changes in global methylation in both regimens. The lack of robust tumor DNA demethylation was accompanied by an absence of the expected ‘viral mimicry’ inflammatory response, and consequently, no clinical responses were observed. The disease control rate was 7.1%. The median progression-free survival was 1.9 months (95% CI 1.5 to 2.3) and median overall survival was 5 months (95% CI 4.5 to 10).ConclusionsThe evaluated treatment schedules of CC-486 in combination with durvalumab did not demonstrate robust pharmacodynamic or clinical activity in selected immunologically cold solid tumors. Lessons learned from this biomarker-rich study should inform continued drug development efforts using these agents.Trial registration numberNCT02811497.