A randomized, double-blind phase 1b study evaluating the safety, tolerability, pharmacokinetics and pharmacodynamics of the NLRP3 inhibitor selnoflast in patients with moderate to severe active ulcerative colitis.

BACKGROUND: The NLRP3 inflammasome drives release of pro-inflammatory cytokines including interleukin (IL)-1ß and IL-18 and is a potential target for ulcerative colitis (UC). Selnoflast (RO7486967) is an orally active, potent, selective and reversible small molecule NLRP3 inhibitor. We conducted a randomized, placebo-controlled Phase 1b study to assess the safety, tolerability, pharmacokinetics (PK) and pharmacodynamics (PD) of selnoflast. METHODS: Nineteen adults with previous diagnosis of UC and current active moderate to severe disease were randomized 2:1 to selnoflast or placebo for 7 days. A dose of 450 mg QD (once daily) was selected to achieve 90% IL-1ß inhibition in plasma and colon tissue. Consecutive blood, sigmoid colon biopsies and stool samples were analyzed for a variety of PD markers. Safety and PK were also evaluated. RESULTS: Selnoflast was well-tolerated. Plasma concentrations increased rapidly after oral administration, reaching Tmax 1 h post-dose. Mean plasma concentrations stayed above the IL-1ß IC90 level throughout the dosing interval (mean Ctrough on Day 1 and Day 5: 2.55 µg/mL and 2.66 µg/mL, respectively). At steady state, post-dose selnoflast concentrations in sigmoid colon (5-20 µg/g) were above the IC90 . Production of IL-1ß was reduced in whole blood following ex vivo stimulation with lipopolysaccharide (LPS) (in the selnoflast arm). No changes were observed in plasma IL-18 levels. There were no meaningful differences in the expression of an IL-1-related gene signature in sigmoid colon tissue, and no differences in the expression of stool biomarkers. CONCLUSIONS: Selnoflast was safe and well-tolerated. Selnoflast 450 mg QD achieved plasma and tissue exposure predicted to maintain IL-1ß IC90 over the dosing interval. However, PD biomarker results showed no robust differences between treatment arms, suggesting no major therapeutic effects are to be expected in UC. The limitations of this study are its small sample size and indirect assessment of the effect on IL-1ß in tissue. TRIAL REGISTRATION: ISRCTN16847938.

Deciphering molecular and cellular ex vivo responses to bispecific antibodies PD1-TIM3 and PD1-LAG3 in human tumors.

BACKGROUND: Next-generation cancer immunotherapies are designed to broaden the therapeutic repertoire by targeting new immune checkpoints including lymphocyte-activation gene 3 (LAG-3) and T cell immunoglobulin and mucin-domain containing-3 (TIM-3). Yet, the molecular and cellular mechanisms by which either receptor functions to mediate its inhibitory effects are still poorly understood. Similarly, little is known on the differential effects of dual, compared with single, checkpoint inhibition. METHODS: We here performed in-depth characterization, including multicolor flow cytometry, single cell RNA sequencing and multiplex supernatant analysis, using tumor single cell suspensions from patients with cancer treated ex vivo with novel bispecific antibodies targeting programmed cell death protein 1 (PD-1) and TIM-3 (PD1-TIM3), PD-1 and LAG-3 (PD1-LAG3), or with anti-PD-1. RESULTS: We identified patient samples which were responsive to PD1-TIM3, PD1-LAG3 or anti-PD-1 using an in vitro approach, validated by the analysis of 659 soluble proteins and enrichment for an anti-PD-1 responder signature. We found increased abundance of an activated (HLA-DR+CD25+GranzymeB+) CD8+ T cell subset and of proliferating CD8+ T cells, in response to bispecific antibody or anti-PD-1 treatment. Bispecific antibodies, but not anti-PD-1, significantly increased the abundance of a proliferating natural killer cell subset, which exhibited enrichment for a tissue-residency signature. Key phenotypic and transcriptional changes occurred in a PD-1+CXCL13+CD4+ T cell subset, in response to all treatments, including increased interleukin-17 secretion and signaling toward plasma cells. Interestingly, LAG-3 protein upregulation was detected as a unique pharmacodynamic effect mediated by PD1-LAG3, but not by PD1-TIM3 or anti-PD-1. CONCLUSIONS: Our in vitro system reliably assessed responses to bispecific antibodies co-targeting PD-1 together with LAG-3 or TIM-3 using patients' tumor infiltrating immune cells and revealed transcriptional and phenotypic imprinting by bispecific antibody formats currently tested in early clinical trials.

A human receptor occupancy assay to measure anti-PD-1 binding in patients with prior anti-PD-1.

Receptor occupancy (RO) assessment by flow cytometry is an important pharmacodynamic (PD) biomarker in the clinical development of large molecules such as monoclonal therapeutic antibodies (mAbs). The total-drug-bound RO assay format directly assesses mAb binding to cell surface targets using anti-drug detection antibodies. Here, we generated a flow cytometry detection antibody specifically binding to mAbs of the IgG1 P329GLALA backbone. Using this reagent, we developed a total-drug-bound RO assay format for RG7769, a bi-specific P329GLALA containing mAb targeting PD-1 and TIM3 on T cells. In its fit-for-purpose validated version, this RO assay has been used in the Phase-I dose escalation study of RG7769, informing on peripheral T cell RO and RG7769 antibody binding capacity (ABC). We assessed RG7769 RO in checkpoint-inhibitor (CPI) naïve patients and anti-PD-1 CPI experienced patients using our novel assay. Here, we show that in both groups, complete T cell RO can be achieved (~100%). However, we found that the maximum number of T cell binding sites for RG7769 pre-dosing was roughly twofold lower in patients recently having undergone anti-PD-1 treatment. We show that this is due to steric hindrance exerted by competing mAbs masking the available drug binding sites. Our findings highlight the importance of quantitative mAb assessment in addition to relative RO especially in the context of patients who have previously received anti-PD-1 treatment.

Aberrant Lck Signal via CD28 Costimulation Augments Antigen-Specific Functionality and Tumor Control by Redirected T Cells with PD-1 Blockade in Humanized Mice.

Purpose: Combination therapy of adoptively transferred redirected T cells and checkpoint inhibitors aims for higher response rates in tumors poorly responsive to immunotherapy like malignant pleural mesothelioma (MPM). Only most recently the issue of an optimally active chimeric antigen receptor (CAR) and the combination with checkpoint inhibitors is starting to be addressed.Experimental Design: Fibroblast activation protein (FAP)-specific CARs with different costimulatory domains, including CD28, Δ-CD28 (lacking lck binding moiety), or 4-1BB were established. CAR-T cells were characterized in vitro and antitumor efficacy was tested in vivo in a humanized mouse model in combination with PD-1 blockade. Finally, the Δ-CD28 CAR was tested clinically in a patient with MPM.Results: All the three CARs demonstrated FAP-specific functionality in vitro Gene expression data indicated a distinct activity profile for the Δ-CD28 CAR, including higher expression of genes involved in cell division, glycolysis, fatty acid oxidation, and oxidative phosphorylation. In vivo, only T cells expressing the Δ-CD28 CAR in combination with PD-1 blockade controlled tumor growth. When injected into the pleural effusion of a patient with MPM, the Δ-CD28 CAR could be detected for up to 21 days and showed functionality.Conclusions: Overall, anti-FAP-Δ-CD28/CD3ζ CAR T cells revealed superior in vitro functionality, better tumor control in combination with PD-1 blockade in humanized mice, and persistence up to 21 days in a patient with MPM. Therefore, further clinical investigation of this optimized CAR is warranted. Clin Cancer Res; 24(16); 3981-93. ©2018 AACR.

Treatment of malignant pleural mesothelioma by fibroblast activation protein-specific re-directed T cells.

INTRODUCTION: Malignant pleural mesothelioma (MPM) is an incurable malignant disease, which results from chronic exposition to asbestos in at least 70% of the cases. Fibroblast activation protein (FAP) is predominantly expressed on the surface of reactive tumor-associated fibroblasts as well as on particular cancer types. Because of its expression on the cell surface, FAP is an attractive target for adoptive T cell therapy. T cells can be re-directed by retroviral transfer of chimeric antigen receptors (CAR) against tumor-associated antigens (TAA) and therefore represent a therapeutic strategy of adoptive immunotherapy. METHODS: To evaluate FAP expression immunohistochemistry was performed in tumor tissue from MPM patients. CD8+ human T cells were retrovirally transduced with an anti-FAP-F19-∆CD28/CD3ζ-CAR. T cell function was evaluated in vitro by cytokine release and cytotoxicity assays. In vivo function was tested with an intraperitoneal xenograft tumor model in immunodeficient mice. RESULTS: FAP was found to be expressed in all subtypes of MPM. Additionally, FAP expression was evaluated in healthy adult tissue samples and was only detected in specific areas in the pancreas, the placenta and very weakly for cervix and uterus. Expression of the anti-FAP-F19-∆CD28/CD3ζ-CAR in CD8+ T cells resulted in antigen-specific IFNγ release. Additionally, FAP-specific re-directed T cells lysed FAP positive mesothelioma cells and inflammatory fibroblasts in an antigen-specific manner in vitro. Furthermore, FAP-specific re-directed T cells inhibited the growth of FAP positive human tumor cells in the peritoneal cavity of mice and significantly prolonged survival of mice. CONCLUSION: FAP re-directed CD8+ T cells showed antigen-specific functionality in vitro and in vivo. Furthermore, FAP expression was verified in all MPM histotypes. Therefore, our data support performing a phase I clinical trial in which MPM patients are treated with adoptively transferred FAP-specific re-directed T cells.