Protein kinase C as an effector of lipid-derived second messengers.

International audienceMembers of the protein kinase C family are major effectors of lipid second messengers. We describe three protocols to assess protein kinase C activity in polymorphonuclear leukocytes (neutrophils). These methods are useful to study the activation and function of protein kinase C in these immune cells. Since neutrophils provide a ready source of human primary tissue, these methods are also useful for pharmacological studies on the protein kinase C system and for evaluation of protein kinase C activators and inhibitors in the context of human primary cells. Furthermore, since protein kinase C activity is determined by a number of lipid-generating signaling systems, the methods described here can also be employed to study the pharmacology of these "upstream" signaling systems

Protein kinase C as an effector of lipid-derived second messengers.

International audienceMembers of the protein kinase C family are major effectors of lipid second messengers. We describe three protocols to assess protein kinase C activity in polymorphonuclear leukocytes (neutrophils). These methods are useful to study the activation and function of protein kinase C in these immune cells. Since neutrophils provide a ready source of human primary tissue, these methods are also useful for pharmacological studies on the protein kinase C system and for evaluation of protein kinase C activators and inhibitors in the context of human primary cells. Furthermore, since protein kinase C activity is determined by a number of lipid-generating signaling systems, the methods described here can also be employed to study the pharmacology of these "upstream" signaling systems

Margetuximab plus pembrolizumab in patients with previously treated, HER2-positive gastro-oesophageal adenocarcinoma (CP-MGAH22-05): a single-arm, phase 1b-2 trial

Background Margetuximab, a novel, investigational, Fc-engineered, anti-HER2 monoclonal antibody, is designed to more effectively potentiate innate immunity than trastuzumab. We aimed to evaluate the safety, tolerability, and antitumour activity of margetuximab plus pembrolizumab (an anti-PD-1 monoclonal antibody) in previously treated patients with HER2-positive gastro-oesophageal adenocarcinoma. Methods CP-MGAH22-05 was a single-arm, open-label, phase 1b-2 dose-escalation and cohort expansion study done at 11 academic centres in the USA and Canada and 15 centres in southeast Asia (Korea, Taiwan, and Singapore) that enrolled men and women aged 18 years or older with histologically proven, unresectable, locally advanced or metastatic, HER2-positive, PD-L1-unselected gastro-oesophageal adenocarcinoma, with an Eastern Cooperative Oncology Group performance status of 0 or 1, who had progressed after at least one previous line of therapy with trastuzumab plus chemotherapy in the locally advanced unresectable or metastatic setting. In the dose-escalation phase, nine patients were treated: three received margetuximab 10 mg/kg intravenously plus pembrolizumab 200 mg intravenously every 3 weeks and six received the recommended phase 2 dose of margetuximab 15 mg/kg plus pembrolizumab 200 mg intravenously every 3 weeks. An additional 86 patients were enrolled in the phase 2 cohort expansion and received the recommended phase 2 dose. The primary endpoints were safety and tolerability, assessed in the safety population (patients who received at least one dose of either margetuximab or pembrolizumab) and the objective response rate as assessed by the investigator according to both Response Evaluation Criteria in Solid Tumors (RECIST), version 1.1, in the response-evaluable population (patients with measurable disease at baseline and who received the recommended phase 2 dose of margetuximab and pembrolizumab). This trial is registered with ClinicalTrials.gov, NCT02689284. Recruitment for the trial has completed and follow-up is ongoing. Findings Between Feb 11, 2016, and Oct 2, 2018, 95 patients were enrolled. Median follow-up was 19.9 months (IQR 10.7-23.1). The combination therapy showed acceptable safety and tolerability; there were no dose-limiting toxicities in the dose-escalation phase. The most common grade 3-4 treatment-related adverse events were anaemia (four [4%]) and infusion-related reactions (three [3%]). Serious treatment-related adverse events were reported in nine (9%) patients. No treatment-related deaths were reported. Objective responses were observed in 17 (18.48%; 95% CI 11.15-27.93) of 92 evaluable patients. Interpretation These findings serve as proof of concept of synergistic antitumour activity with the combination of an Fc-optimised anti-HER2 agent (margetuximab) along with anti-PD-1 checkpoint blockade (pembrolizumab). Copyright (C) 2020 Elsevier Ltd. All rights reserved

Margetuximab plus pembrolizumab in patients with previously treated, HER2-positive gastro-oesophageal adenocarcinoma (CP-MGAH22-05): a single-arm, phase 1b-2 trial.

BACKGROUND: Margetuximab, a novel, investigational, Fc-engineered, anti-HER2 monoclonal antibody, is designed to more effectively potentiate innate immunity than trastuzumab. We aimed to evaluate the safety, tolerability, and antitumour activity of margetuximab plus pembrolizumab (an anti-PD-1 monoclonal antibody) in previously treated patients with HER2-positive gastro-oesophageal adenocarcinoma. METHODS: CP-MGAH22-05 was a single-arm, open-label, phase 1b-2 dose-escalation and cohort expansion study done at 11 academic centres in the USA and Canada and 15 centres in southeast Asia (Korea, Taiwan, and Singapore) that enrolled men and women aged 18 years or older with histologically proven, unresectable, locally advanced or metastatic, HER2-positive, PD-L1-unselected gastro-oesophageal adenocarcinoma, with an Eastern Cooperative Oncology Group performance status of 0 or 1, who had progressed after at least one previous line of therapy with trastuzumab plus chemotherapy in the locally advanced unresectable or metastatic setting. In the dose-escalation phase, nine patients were treated: three received margetuximab 10 mg/kg intravenously plus pembrolizumab 200 mg intravenously every 3 weeks and six received the recommended phase 2 dose of margetuximab 15 mg/kg plus pembrolizumab 200 mg intravenously every 3 weeks. An additional 86 patients were enrolled in the phase 2 cohort expansion and received the recommended phase 2 dose. The primary endpoints were safety and tolerability, assessed in the safety population (patients who received at least one dose of either margetuximab or pembrolizumab) and the objective response rate as assessed by the investigator according to both Response Evaluation Criteria in Solid Tumors (RECIST), version 1.1, in the response-evaluable population (patients with measurable disease at baseline and who received the recommended phase 2 dose of margetuximab and pembrolizumab). This trial is registered with ClinicalTrials.gov, NCT02689284. Recruitment for the trial has completed and follow-up is ongoing. FINDINGS: Between Feb 11, 2016, and Oct 2, 2018, 95 patients were enrolled. Median follow-up was 19·9 months (IQR 10·7-23·1). The combination therapy showed acceptable safety and tolerability; there were no dose-limiting toxicities in the dose-escalation phase. The most common grade 3-4 treatment-related adverse events were anaemia (four [4%]) and infusion-related reactions (three [3%]). Serious treatment-related adverse events were reported in nine (9%) patients. No treatment-related deaths were reported. Objective responses were observed in 17 (18·48%; 95% CI 11·15-27·93) of 92 evaluable patients. INTERPRETATION: These findings serve as proof of concept of synergistic antitumour activity with the combination of an Fc-optimised anti-HER2 agent (margetuximab) along with anti-PD-1 checkpoint blockade (pembrolizumab). FUNDING: MacroGenics

Prophylactic Ruxolitinib for Cytokine Release Syndrome in Relapse/Refractory AML Patients Treated with Flotetuzumab.

2817 Prophylactic Ruxolitinib for Cytokine Release Syndrome (CRS) in Relapse/Refractory (R/R) AML Patients Treated with Flotetuzumab Program: Oral and Poster AbstractsSession: 613. Acute Myeloid Leukemia: Clinical Studies: Poster IIIHematology Disease Topics & Pathways:AML, antibodies, Biological, CRS, Adult, Diseases, Therapies, Adverse Events, Biological Processes, Study Population, Myeloid Malignancies, Clinically relevant, TKI Monday, December 7, 2020, 7:00 AM-3:30 PM Geoffrey L Uy, MD1, Michael P. Rettig, PhD2, Stephanie Christ, MS3*, Ibrahim Aldoss, MD4, Michael T. Byrne, DO5, Harry P. Erba, MD, PhD6, Martha L. Arellano, MD7, Matthew C Foster, MD8, John E. Godwin, MD9, Farhad Ravandi, MBBS10, Peter H. Sayre, MD, PhD11, Anjali S Advani, MD12, Matthew J. Wieduwilt, MD, PhD13, Ashkan Emadi, M.D., Ph.D.14, Laura C. Michaelis, MD15, Patrick J. Stiff, MD16, Martin Wermke17*, Norbert Vey, MD18, Patrice Chevalier, MD, PhD19*, Emmanuel Gyan, MD, PhD20, Christian Recher, MD, PhD21, Fabio Ciceri, MD22*, Matteo Giovanni Carrabba, MD23*, Antonio Curti, MD PhD24, Geert Huls, MD, PhD25, Max S. Topp, MD26, Mojca Jongen-Lavrencic, MD, PhD27, John Muth, MS28*, Teia Curtis29*, Mary Beth Collins30*, Erin Timmeny31*, Kuo Guo, MSc32*, Jian Zhao, PhD32*, Kathy Tran28*, Patrick Kaminker, PhD33*, Priyanka Patel, PharmD30*, Ouiam Bakkacha, MD34*, Kenneth Jacobs, MD35*, Maya Kostova, PhD32*, Jennifer Seiler, PhD, RAC30*, Bob Lowenberg, MD, PhD36, Sergio Rutella, MD, PhD, FRCPath37, Roland B. Walter, MD, PhD, MS38, Ezio Bonvini, MD33, Jan K Davidson-Moncada, MD, PhD39 and John F. DiPersio, MD1 1Washington University School of Medicine, Saint Louis, MO2Department of Internal Medicine, Division of Oncology, Washington Univ. School of Med., Saint Louis, MO3Department of Medicine, Division of Oncology, Washington University School of Medicine, Saint Louis, MO4Gehr Family Center for Leukemia Research, City of Hope, Duarte, CA5Department of Medicine, Division of Hematology-Oncology, Vanderbilt University Medical Center, Nashville, TN6University of Alabama at Birmingham, Birmingham, AL7Winship Cancer Institute, Emory University School of Medicine, Atlanta, GA8Lineberger Comprehensive Cancer Center, UNC, Chapel Hill, Chapel Hill, NC9Providence Portland Medical Center, Portland, OR10Department of Leukemia, University of Texas- MD Anderson Cancer Center, Houston, TX11University of California, San Francisco, San Francisco, CA12Cleveland Clinic, Taussig Cancer Institute, Cleveland, OH13Moores Cancer Center, University of California, San Diego, La Jolla, CA14University of Maryland Greenebaum Comprehensive Cancer Center, Baltimore, MD15Division of Hematology/Oncology, Department of Medicine, The Medical College of Wisconsin Inc., Milwaukee, WI16Loyola University Chicago Stritch School of Medicine, Maywood, IL17NCT/UCC Early Clinical Trial Unit, University Hospital Carl Gustav Carus, Dresden, Germany18Hematologie clinique, Institut Paoli Clamettes, Marseille, France19Department of Hematology and Cell Therapy, CHU Nantes, Nantes, France20CHU de Tours - Hôpital Bretonneau, Tours, France21Service d\u27Hématologie, Centre Hospitalier Universitaire de Toulouse, Institut Universitaire du Cancer de Toulouse Oncopole, Toulouse, France22Haematology and BMT Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy23Hematology and Bone Marrow Transplantation Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy24Hematology/Oncology L. e A. Seràgnoli , Sant’Orsola-Malpighi University Hospital, Bologna, Bologna, Italy25Department of Hematology, University Medical Center Groningen, Groningen, GZ, Netherlands26Medizinische Klinik Und Poliklinik II, Universitätsklinikum Würzburg, Würzburg, Germany27Erasmus University Medical Center, Rotterdam, Netherlands28MacroGenics, Inc., Rockville, MD29MacroGenics, Inc., Frederick, MD30MacroGenics, Rockville31MacroGenics, Inc., ROCKVILLE, MD32MacroGenics, Rockville, MD33Macrogenics, Rockville, MD34Macrogenics,Inc, ROCKVILLE, MD35MacroGenics, Inc, Rockville, MD36Department of Hematology, Erasmus University Medical Center, Rotterdam, Netherlands37John van Geest Cancer Centre School of Science and Technology, Nottingham Trent University, Nottingham, ENG, United Kingdom38Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA39MacroGenics, Inc., Washington, DC Introduction: CRS is a potentially life-threatening toxicity observed following T cell-redirecting therapies. CRS is associated with elevated cytokines, including IL6, IFNγ, TNFα, IL2 and GM-CSF. Glucocorticosteroids (GC) and the IL6 receptor blocking antibody tocilizumab (TCZ) can reduce CRS severity; however, CRS may still occur and limit the therapeutic window of novel immunotherapeutic agents. Disruption of cytokine signaling via Janus kinase (JAK) pathway interference may represent a complementary approach to blocking CRS. Ruxolitinib (RUX), an oral JAK1/2 inhibitor approved for the treatment of myelofibrosis and polycythemia vera, interferes with signaling of several cytokines, including IFNγ and IL6, via blockade of the JAK/STAT pathway. We hypothesized that RUX may reduce the frequency and severity of CRS in R/R AML patients (pts) undergoing treatment with flotetuzumab (FLZ), an investigational CD123 x CD3 bispecific DART® molecule. Methods: Relapse/refractory (including primary induction failure, early relapse and late relapse) AML pts were included in this study. RUX pts were treated at a single site, Washington University, St. Louis, MO. RUX was dosed at 10 mg or 20mg BID days -1 through 14. Comparator (non-RUX) pts (n=23) were treated at other clinical sites. FLZ was administered at 500 ng/kg/day continuously in 28-day cycles following multi-step lead-in dosing in week 1 of cycle 1. CRS was graded per Lee criteria1. Results: As of July 1st, 2020, 10 R/R AML pts, median age 65 (range 40-82) years, have been enrolled and treated in the RUX cohort (6 at 10mg, 4 at 20 mg of RUX). All pts had non-favorable risk by ELN 2017 criteria (8 adverse and 2 intermediate); 1 (10.0%) pt had secondary AML; pt characteristics in the RUX and non-RUX cohorts were balanced, except for median baseline BM blasts which was higher in non-RUX pts: 15% (range 5-72) vs (40% (range 7-84), RUX and non-RUX pts respectively. Cytokine analysis showed statistically significant (p\u3c0.05) lower levels of IL4, IL12p70, IL13, IL15, IL17A, IFNα2, but higher levels of GM-CSF were measured in RUX vs non-RUX pts, specifically during co-administration with FLZ (Fig. 1). However, incidence and severity of CRS events were similar. In the RUX cohort, 9 (90%) pts experienced mild to moderate (grade ≤ 2; 48.6% of events were grade 1) CRS events whereas no grade ≥ 3 CRS were reported; in the non-RUX cohort, 23 (100%) pts experienced mild to moderate (grade ≤ 2; 73.1% of events were grade 1) CRS events, 1 (4.3%) grade ≥ 3 CRS was reported. Most CRS events occurred in the first 2 weeks of FLZ administration (75% and 92%, respectively). No differences in duration of CRS events were noted. However, more CRS-directed treatment was used in the RUX cohort. Five (50%) pts received a total of 12 doses of TCZ, 1 (10%) pt received GC and 1 (10%) pts received vasopressors in the RUX cohort. In the non-RUX cohort, 5 (21.7%) pts received 8 doses of TCZ, 3 (13.0%) pts received GC and 1 (3.7%) pt received vasopressors. Dose intensity (DI) at FLZ dose of 500 ng/kg/day was comparable, with median DI of 97.6% and 98.0% in RUX and non-RUX cohorts, respectively. Time to first response (TTFR; BM \u3c 5% blasts) and time on treatment (ToT) were similar between both groups. Median TTFR was 1 cycle for both groups (range 1-2 cycles), and median ToT was 1.4 (range 0.9-5.1) and 1.8 (range 1.3-5.1) months, for RUX and non-RUX pts, respectively. Complete response rate (BM \u3c 5% blasts) was similar: 4 (40%) in RUX pts, and 8 (34.8%) in non-RUX pts; 2 RUX (50%) and 5 non-RUX (62.5%) responders transitioned to stem cell transplant. Conclusion: Prophylactic RUX produced a clear difference in cytokine profiles but no discernable improvement in clinical CRS or response rates in FLZ treated patients. A larger study may be required to determine the prophylactic role of RUX in CRS