EMD Serono, Inc. (United States)

PURPOSE: We report results from two clinical trials of the cyclic dinucleotide stimulator of IFN genes (STING) agonist ulevostinag. PATIENTS AND METHODS: In a phase I study (NCT03010176) with an accelerated titration design/modified toxicity probability interval method, participants with advanced/metastatic solid tumors or lymphomas received intratumoral ulevostinag (±intravenous pembrolizumab). In an expansion phase, participants with head and neck squamous cell carcinoma (HNSCC) or triple-negative breast cancer received the combination. Primary objectives were safety/tolerability and identifying the recommended phase II dose; biomarkers were exploratory. In a randomized phase II study (NCT04220866), participants with untreated metastatic or unresectable, recurrent HNSCC received intravenous pembrolizumab (±ulevostinag 540 µg). The primary objective was antitumor activity. Pembrolizumab 200 mg was administered every 3 weeks in both studies. RESULTS: In the phase I study (NCT03010176; N = 156), the most common adverse event was pyrexia (70%). Plasma ulevostinag concentrations increased dose-dependently. Circulating levels of C-X-C motif chemokine 10, IFNγ, and IL-6 showed elevation at 2 to 4 hours, peak at 6 to 8 hours, and plateau/partial resolution at 24 hours but, beyond the 540 µg dose, did not show a clear dose-effect relationship. Ten participants experienced dose-limiting toxicities; the recommended phase II dose for intratumoral ulevostinag was 540 µg. In the phase II study (NCT04220866), 4 of 8 participants treated with combination therapy and 1 of 10 treated with pembrolizumab monotherapy had a complete or partial response. The most common adverse event was pyrexia (n = 5). CONCLUSIONS: Intratumoral ulevostinag (±pembrolizumab) had manageable toxicity, dose-dependent pharmacokinetics, and evidence of STING activation and target engagement. Combination therapy showed antitumor activity in participants with untreated metastatic or unresectable, recurrent HNSCC.

PURPOSE: We evaluated the noncyclic dinucleotide stimulator of IFN genes agonist MK-2118 ± pembrolizumab in participants with advanced solid tumors or lymphomas. PATIENTS AND METHODS: This first-in-human study (NCT03249792) enrolled participants with refractory, advanced solid tumors or lymphomas. Participants received intratumoral (IT) MK-2118 100 to 20,000 μg (arm 1), IT MK-2118 900 to 15,000 μg plus intravenous (IV) pembrolizumab 200 mg every 3 weeks (arm 2), or subcutaneous (SC) MK-2118 5,000 to 150,000 μg plus IV pembrolizumab 200 mg every 3 weeks (arm 4); arm 3 (visceral injection of MK-2118) was not pursued. IT dosing used an accelerated titration design and modified toxicity probability interval method; SC dosing (arm 4) was started subsequent to arms 1 and 2. The primary objectives were safety/tolerability. MK-2118 pharmacokinetics was a secondary endpoint; objective responses and biomarkers were exploratory endpoints. RESULTS: A total of 140 participants were enrolled (arm 1, n = 27; arm 2, n = 57; arm 4, n = 56). Grade 3/4 treatment-related adverse events occurred in 22%, 23%, and 11% of participants, respectively, but no maximum tolerated dose was identified up to MK-2118 20,000, 15,000, and 150,000 μg across the three arms. Dose-dependent increases in MK-2118 systemic exposure were observed following IT and subcutaneous administration. Objective responses were seen in 0%, 6%, and 4% of participants, respectively. IT MK-2118 led to dose-dependent changes in stimulator of interferon genes-based blood RNA expression levels, IFNγ, IFNγ-induced protein 10, and IL6; SC MK-2118 did not generate dose-related immune responses. CONCLUSIONS: IT MK-2118 ± pembrolizumab and SC MK-2118 plus pembrolizumab had manageable toxicity and limited antitumor activity. IT but not SC administration demonstrated systemic immune effects.

While A2A adenosine receptor (AR) was considered as a major contributor to adenosine-mediated immunosuppression, A2B, having the lowest affinity to adenosine, has also emerged as a potential contributor to tumor promotion. Therefore, in adenosine-rich tumor microenvironment (TME), where A2B could be complementary and/or compensatory to A2A, simultaneous targeting of A2A and A2B ARs can provide higher potential for cancer immunotherapy. We developed M1069-a highly selective dual antagonist of the A2A and A2B AR. In assays with primary human and murine immune cells, M1069 rescued IL2 production from T cells (A2A dependent) and inhibited VEGF production by myeloid cells (A2B dependent) in adenosine-high settings. M1069 also demonstrated superior suppression of the secretion of protumorigenic cytokines CXCL1, CXCL5, and rescue of IL12 secretion from adenosine-differentiated dendritic cells compared to an A2A-selective antagonist (A2Ai). In a one-way mixed lymphocyte reaction (MLR) assay, adenosine-differentiated human and murine dendritic cells treated with M1069 demonstrated superior T-cell stimulatory activity compared to dendritic cells differentiated in presence of A2Ai. In vivo, M1069 decreased tumor growth as a monotherapy and enhanced antitumor activity of bintrafusp alfa (BA) or cisplatin in syngeneic adenosinehi/CD73hi 4T1 breast tumor model, but not in the CD73 knockout 4T1 tumor model or in adenosinelow/CD73low MC38 murine colon carcinoma model. In summary, our dual A2A/A2B AR antagonist M1069 may counteract immune-suppressive mechanisms of high concentrations of adenosine in vitro and in vivo and enhance the antitumor activity of other agents, including BA and cisplatin.

PURPOSE: Avelumab (anti-PD-L1) became the first approved treatment for metastatic Merkel cell carcinoma (mMCC) based on results from the phase II JAVELIN Merkel 200 trial. In this study, we report exploratory biomarker analyses from the trial. PATIENTS AND METHODS: Patients with mMCC (n = 88) with or without prior first-line chemotherapy received avelumab 10 mg/kg every 2 weeks. We conducted analyses on somatic mutations, mutational signatures, and tumor mutational burden using paired whole-exome sequencing. Additionally, we examined gene and gene set expression, immune content from RNA sequencing profiles, as well as tumor PD-L1 and CD8 statuses from IHC and CD8 status from digital pathology. RESULTS: Tumors positive for Merkel cell polyomavirus (MCPyV) were characterized by an absence of driver mutations and a low tumor mutational burden, consistent with previous studies. A novel MCPyV-specific host gene expression signature was identified. MCPyV+ tumors had increased levels of immunosuppressive M2 macrophages in the tumor microenvironment, which seemed to correlate with PD-L1 expression; high CD8+ T-cell density in these tumors did not predict response to avelumab. Conversely, in patients with MCPyV- tumors, higher CD8+ T-cell density seemed to be associated with response to avelumab. Mutations in several genes were associated with treatment outcomes. Compared with tumors sampled before chemotherapy, tumors sampled after chemotherapy had downregulated gene signatures for immune responses, including reduced expression of IFNγ-related pathways. Levels of activated dendritic cells in responding patients were higher in patients assessed after versus before chemotherapy. CONCLUSIONS: Exploratory analyses provide insights into mMCC biology and potential associations with response to avelumab. Chemotherapy seems to negatively modulate the immune microenvironment.

Abstract Background: A randomized multicenter trial (BMT CTN 1703) demonstrated that after reduced intensity conditioning HCT, PT-Cy-based GVHD prophylaxis led to significant reductions in acute and chronic GVHD, but was associated with significantly more Grade 2 (moderate) infections and similar overall survival vs Tac/MTX. A linked mechanistic study (BMT CTN 1801) found that PT-Cy led to pan-T cell depletion and reduced T cell receptor (TCR) repertoire diversity vs Tac/MTX. To discover the molecular mechanisms driving the association of PT-Cy with increased infections, we examined pathogen-specific T cell reconstitution in BMT CTN 1801 patients (n=165 PT-Cy, n=159 Tac/MTX). Methods: We performed deep TCR sequencing on 2,369 blood samples collected pre-HCT (recipient baseline) and on Days +7, 14, 28, 63, 100, 180, 270, 365, 730, and from the graft infusion product. We then applied a novel set of TCR classifiers (Adaptive Biotechnologies) capable of identifying pathogen-specific clones associated with CMV, EBV, SARS-CoV-2, HSV-1, HSV-2, parvovirus, RSV, influenza A, rhinovirus, adenovirus, HHV-6B, norovirus and Toxoplasma. Single-cell (sc) TCR/RNA-sequencing on a sample subset (n=26) was performed to determine the phenotype of pathogen-specific T cells. Results. By linking scRNA-seq with pathogen-specific TCR barcoding, we identified CD4 and CD8 memory T cells as their predominant T cell reservoir. We found that the total number of pathogen-specific singleton TCRs was significantly lower with PT-Cy, starting early post-HCT and persisting through 2 years. For example, at Day +14, PT-Cy patients had 7.8-fold fewer pathogen-specific singleton TCRs vs Tac/MTX (mean of 6.9 vs 54.1, p<0.001). Consistent differences between PT-Cy and Tac/MTX were observed for TCRs specific for each pathogen individually. Patients who developed Grade 3 (severe) viral infections had significantly fewer pathogen-specific singleton TCRs at Day +14 compared to those who had no viral infections (mean of 4.0 vs 32.5, p=0.03). Leveraging intrinsic TCR barcoding of the HCT infusion (“graft”) and of the patient's baseline samples (“recipient”), we could assign the graft/recipient origin of the pathogen-specific cells with high fidelity. This analysis revealed pronounced differences in the origin of pathogen-specific T cells with PT-Cy vs Tac/MTX, especially prior to Day +100. The absolute number of pathogen-specific TCRs originating from the graft was significantly higher with Tac/MTX vs PT-Cy. This difference was observed from day +7 through 6 months post-HCT, with up to 7.1-fold more graft-derived pathogen-specific TCRs in Tac/MTX vs PT-Cy (p<0.001 at each timepoint before day +100). Within patients who received Tac/MTX, the balance of graft vs recipient pathogen-specific clones significantly favored graft clones (for example, a mean of 14.9 graft vs 8.6 recipient pathogen-specific TCRs at Day+14, p=0.02). By contrast, with PT-Cy, in addition to having substantially fewer pathogen-specific TCRs overall, the proportion of these TCRs was not significantly different between graft and recipient clones (mean of 2.1 graft vs 3.3 recipient pathogen-specific TCRs at Day+14, p=0.42). These data uncover that, in addition to the reduction in pathogen-specific T cells with PT-Cy, there was a relative skewing of the anti-microbial T cell reservoir towards recipient cells that persisted after conditioning. Conclusions: PT-Cy led to in vivo T cell depletion across all T cell subsets, which prominently included clones tracked from the allograft itself into the HCT recipient, with a relative sparing of recipient T cell clones that survived transplant conditioning. While the mechanism for relative sparing of recipient cells has not yet been determined, these conditioning-exposed cells may be less proliferative than newly infused graft T cells. The depletion of graft T cells included CD4 and CD8 memory cells specific for infections, which may have contributed to relative deficits in protective immunity against infection in patients receiving PT-Cy. Importantly, these data suggest that with PT-Cy, persisting recipient TCRs may play a key role in preserving anti-infectious immunity. Taken together, these results provide a mechanistic basis for key post-transplant clinical outcomes of the landmark BMT CTN 1703 study. They also underscore the importance of recipient immune status with PT-Cy, creating the opportunity for targeted infection risk assessment in these patients.

Normal human uterine stromal cells undergo decidualization in a cyclical manner, a critical event in successful implantation. IL1b & Th2-cytokines are compatible with implantation while TNFa & Th1 cytokines in endometrium are associated with inflammation and implantation failure. In vitro, isolated human uterine stromal fibroblasts (Huf6 cells) can be differentiated to a decidual phenotype by various treatments including cAMP in the presence of steroid hormones. Treatment of Huf6 cells with IL-1b induces the expression of a number of genes up regulated during decidualization and at the implantation site. Earlier studies from our group have demonstrated that IL17 can antagonize the effect of TNFa on stromal cytokine production during in vitro biochemical decidualization. Our hypothesis was that local elevations of TNF-a in eutopic endometrium due to inflammatory uterine disease would adversely impact decidualization and implantation. Thus we tested if IL-17 can redirect the effect of TNF-a on decidualization towards implantation and more closely resemble the effect of IL-1b. We carried out genome wide gene expression analysis in HuF6 cells to identify novel targets for implantation therapy. Gene expression analysis was performed on RNA isolated from cells treated with TNFa(10ng/ml), or IL-1b(5ng/ml) alone or in the presence of IL-17(10ng/ml) for 24h using Affymetrix gene chip. Differential expression (p<0.001; fold change >2 or <-2) of 750 probe sets resulted from treatment of cells with IL1b, while TNFa and IL-17 differentially expressed 960 & 24 transcripts respectively from the untreated cells. Combined treatment of TNF-a and IL-17 altered expression of 964 probe sets. These cytokines uniquely modulated 159 transcripts that were not affected by either treatment alone. Heat maps of the differentially expressed genes suggest the combination treatment was closer to IL-1b modulated gene expression profile than TNF-a alone. Gene expression of a number of up and down regulated genes was confirmed by qPCR. In addition, ELISA confirmed the protein expression levels of a number of cytokines including IL11, a marker of decidualization. Our results led to the identification of IL-17 as an important candidate to promote decidualization of human uterine stromal cells in the presence of TNFa similar to that observed with IL-1b. Further gene expression analysis led to identification of several genes up regulated during decidualization that may be important targets to promote implantation.