Safety of Anti-NKG2A Blocking Antibody Monalizumab As Maintenance Therapy after Allogeneic Hematopoietic Stem Cell Transplantation: A Phase I Study

Background Following allogeneic hematopoietic stem cell transplantation (Allo-HSCT), the phenotype and function of circulating NK Cells are altered; notably NK cells display an immature (CD56-bright/KIR -/CD57 -/NKG2A +) phenotype and a low cytotoxic activity. Since NK cells have demonstrated anti-leukemic activity in the context of Allo-HCT while less prone to induce GVHD than T cells, we hypothesized that NKG2A inhibition using the checkpoint inhibitor monalizumab (humanized IgG4 blocking monoclonal antibody) could improve NK cell mediated graft-versus-tumor effect, without triggering graft-versus-host disease. We thus performed the first clinical trial evaluating the safety of monalizumab as a prophylactic treatment after Allo-HSCT (NCT02921685). Method The objective was to determine the maximal tolerated dose (MTD) of a single infusion of monalizumab administered on day+75 or thereafter following Allo-HSCT. Inclusion criteria were: 1) adult patients who underwent first Allo-HSCT for hematological malignancy; 2) no prior history of GVHD, 3) reduced toxicity conditioning regimen, 4) ATG or PT-Cy based GVHD prophylaxis, and 5) GVHD prophylaxis with cyclosporine A ongoing at the time of monalizumab infusion. Four dose levels were investigated in this dose escalation study (0.04, 0.1, 0.3, and 1.0 mg/kg). Dose limiting toxicities (DLT) were defined by the occurrence of any grade 3 to 4 monalizumab-related adverse event within 28 days after infusion, including grade 3 to 4 acute GVHD and moderate to severe chronic GVHD. In case of DLT, a Bayesian continuous reassessment model (CRM) was used to predict the maximal tolerated dose (MTD) after each series of 3 patients treated at a given dose level. It was planned to treat 18 patients. Beyond clinical safety, blood samples were collected for NK and T cell immunomonitoring and receptor saturation assay. Results Fifteen patients received monalizumab at a median time of 83 days (range: 75-103) after Allo-HSCT (3 at 0.04 mg/kg, 3 at 0.1 mg/kg, 3 at 0.3 mg/kg, and 6 at 1 mg/kg). Patients were transplanted for hematological diseases (9 AML, 3 MDS, 1 lymphoma, 1 CLL and 1 myelofibrosis), from a matched sibling (n=8), matched unrelated (n=6) or haploidentical donor (n=1). No DLT was observed during the dose escalation process or among the 6 patients treated at the highest dose level, justifying study early termination after 15 patients. Two patients developed transient low grade GVHD that spontaneously resolved without treatment (1 grade I acute GVHD on day+8 and 1 mild chronic GVHD on day+118). Systemic steroid requiring GVHD occurred in 3 patients (20%): 1 severe overlap GVHD (day+42, CR after treatment), 1 severe chronic GVHD (day+80, death from GVHD) and 1 late severe chronic GVHD after cyclosporine tapering for mixed chimerism (day+149, complete remission [CR] after treatment). No disease recurrence was observed, except for one patient with myelofibrosis who relapsed 3 years after monalizumab infusion. At a median follow up of 22 months (3-48) after monalizumab infusion, 13 out of 15 patients are in CR from their underlining malignancy without GVHD and immunosuppressive treatment at last contact. At the dose of 1 mg/kg, receptor saturation assays (RSA) showed persistent binding of monalizumab on peripheral blood NK cells. Indeed, more than 90% of NKG2A-positive NK cells were positive for monalizumab detection (Anti-IgG4-PE) until at least 14 weeks post treatment. Conclusions Monalizumab can be safely administered after Allo-HSCT, with prolonged persistence on circulating NK cells. In the absence of DLT, MTD was not reached. Future trials will aim at measuring the clinical efficacy of monalizumab in this context, alone or in combination therapy. Disclosures boyer Chammard: Innate Pharma: Current Employment. Chabannon: Sanofi SA: Other: Travel Support, Research Funding, Speakers Bureau; Bellicum Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees, Other: Travel Support, Speakers Bureau; Novartis: Speakers Bureau; BMS/Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Terumo BCT: Speakers Bureau; Miltenyi Biotech: Research Funding; Fresenius Kabi: Research Funding; EBMT: Membership on an entity's Board of Directors or advisory committees. Vey: Amgen: Honoraria; BMS: Honoraria; BIOKINESIS: Consultancy, Research Funding; NOVARTIS: Consultancy, Honoraria, Research Funding; SERVIER: Consultancy; JAZZ PHARMACEUTICALS: Honoraria; JANSSEN: Consultancy. Olive: ImCheck Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Alderaan Biotechnology: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Emergence Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees. Blaise: Jazz Pharmaceuticals: Honoraria.

Natalizumab Pharmacokinetics and -Dynamics and Serum Neurofilament in Patients With Multiple Sclerosis

Background: Natalizumab (NAT) is a high-efficacy treatment for relapsing remitting multiple sclerosis (RRMS). However, it is associated with an increased risk of progressive multifocal leukoencephalopathy that sometimes requires treatment cessation with a risk of returning disease activity. The aim of this study was to characterize the pharmacokinetics and -dynamics as well as neurodestruction marker serum neurofilament light chain (sNfL) in patients with RRMS and secondary progressive MS (SPMS) stopping NAT in correlation to clinical data.Methods: In this study, 50 RRMS and 9 SPMS patients after NAT cessation were included. Five RRMS patients on NAT treatment holiday were evaluated. Clinical and radiological disease activity were systemically assessed by frequent exams after NAT stop. Free NAT concentration, cell bound NAT, α4-integrin expression and α4-integrin-receptor saturation as well as immune cell frequencies were measured for up to 4 months after NAT withdrawal. Additionally, sNfL levels were observed up to 12 months in RRMS and up to 4 months in SPMS patients.Results: NAT cessation was associated with a return of disease activity in 38% of the RRMS and 33% of the SPMS patients within 12 and 7 months, respectively. Concentration of free and cell bound NAT as well as α4-integrin-receptor saturation decreased in the RRMS and SPMS patients whereas α4-integrin expression increased over time. NAT induced increase of lymphocytes and its subsets normalized and a non-significant drop of NK and Th17 T-cells counts could be detected. All RRMS patients showed physiological sNfL levels <8pg/ml 1 month after last NAT infusion. During follow-up period sNfL levels peaked up to 16-fold and were linked to return of disease activity in 19 of the 37 RRMS patients. Treatment holiday was also associated with a return of disease activity in 4 of 5 patients and with an increase of sNfL at an individual level.Conclusions: We demonstrate the reversibility of NAT pharmacodynamic and -kinetic markers. sNfL levels are associated with the recurrence of disease activity and can also serve as an early marker to predict present before onset of clinical or radiological disease activity on the individual level.

Combinatorial nanodot stripe assay to systematically study cell haptotaxis

Haptotaxis is critical to cell guidance and development and has been studied in vitro using either gradients or stripe assays that present a binary choice between full and zero coverage of a protein cue. However, stripes offer only a choice between extremes, while for gradients, cell receptor saturation, migration history, and directional persistence confound the interpretation of cellular responses. Here, we introduce nanodot stripe assays (NSAs) formed by adjacent stripes of nanodot arrays with different surface coverage. Twenty-one pairwise combinations were designed using 0, 1, 3, 10, 30, 44 and 100% stripes and were patterned with 200 × 200, 400 × 400 or 800 × 800 nm2 nanodots. We studied the migration choices of C2C12 myoblasts that express neogenin on NSAs (and three-step gradients) of netrin-1. The reference surface between the nanodots was backfilled with a mixture of polyethylene glycol and poly-d-lysine to minimize nonspecific cell response. Unexpectedly, cell response was independent of nanodot size. Relative to a 0% stripe, cells increasingly chose the high-density stripe with up to ~90% of cells on stripes with 10% coverage and higher. Cell preference for higher vs. lower netrin-1 coverage was observed only for coverage ratios >2.3, with cell preference plateauing at ~80% for ratios ≥4. The combinatorial NSA enables quantitative studies of cell haptotaxis over the full range of surface coverages and ratios and provides a means to elucidate haptotactic mechanisms.

17-BI-1206-02 Phase 1/2a Clinical Trial of BI-1206, a Monoclonal Antibody to Fcgriib, in Combination with Rituximab in Subjects with Indolent B-Cell Non-Hodgkin Lymphoma That Has Relapsed or Is Refractory to Rituximab

Introduction BI-1206 is a fully-human IgG1 monoclonal antibody that exquisitely recognizes and blocks FcγRIIB. BI-1206 enhances the activity of anti-CD20 antibodies such as rituximab by preventing interaction with FcγRIIB and may thus overcome resistance to those treatments. BI-1206 is currently in clinical investigation in combination with rituximab for the treatment of indolent NHL. This report presents a preliminary pharmacokinetic evaluation of the data generated in the trial. Methods The safety and tolerability profile of BI-1206 in combination with rituximab is currently investigated in the Phase 1/2a clinical trial 17-BI-1206-02. The study population includes patients with follicular lymphoma (FL), marginal zone lymphoma (MZL), and mantle cell lymphoma (MCL) who have relapsed or are refractory to rituximab. BI-1206 and rituximab are administered as i.v. infusions once per week for four weeks. In Phase 1a, a 3+3 study design is used, with escalating doses of BI-1206 and a fixed dose of rituximab (375 mg/m2), with the aim of selecting the RP2D of BI-1206 for an expansion cohort in Phase 2a. Patients showing clinical benefit, are eligible for continued maintenance therapy with dosing of BI-1206 and rituximab every 8 weeks. The assessment of the pharmacokinetics (PK) of BI-1206 included non-compartmental analysis (NCA), and the assessment of the pharmacodynamics (PD) included receptor occupancy (RO%). PK modelling was conducted to further characterize the PK behavior and to provide predictions of upcoming dose levels. In addition, the effect of BI-1206 on the PK of rituximab was investigated by comparing PK parameters of rituximab to literature values of rituximab monotherapy. Results Up to 100 mg BI-1206 has been administered in combination with rituximab (375 mg/m2). Increasing doses of BI-1206 from 30 mg to 70 or 100 mg gave rise to a supra-proportional increase in Cmax as well as an increase in the half-life of BI-1206. A trend of accumulation after consecutive doses was also seen. When comparing the serum-concentrations against the associated RO% of FcγRIIB there is a trend that higher doses almost fully saturate the receptors immediately after dosing and for up to 72 hours. It is likely that increasing the dose further, will give rise to full receptor saturation, which should be maintained for an extended period. Clinical response, assessed by reduction of tumour size, has been observed at the 70 mg cohort. This at a dose which typically does not saturate receptors for the entire dose interval. It may therefor be speculated that doses which enable full RO% over the entire dosing interval, may show additional clinical benefit for patients. PK modelling showed that there was a significant contribution of a non-linear component on the elimination of BI-1206, which may be attributed to receptor binding. A two-compartment model with linear (non-saturating) and non-linear (saturating) elimination best describes the data. Using the model for predictions of upcoming doses revealed that doses close to the ones already administered may be sufficient for full receptor saturation during the entire dosing interval. Finally, the Cmax after one dose of rituximab was in the same range as previously reported values, indicating no substantial effect on the PK of rituximab. Consequently, at the current dose levels, there is no apparent need for dose-adjustments for rituximab. Conclusions This report presents preliminary data of the clinical trial 17-BI-1206-02, where BI-1206 is combined with rituximab. The presented data is encouraging, both in terms of first clinical response against tumors, as well as showing signs of overcoming target-mediated drug disposition, which may allow weekly or even less frequent dosing at clinically relevant dose levels. Disclosures Jerkeman: Roche:Research Funding;Celgene:Research Funding;Gilead:Research Funding;Abbvie:Research Funding;Janssen:Research Funding.McAllister:BioInvent International AB:Current Employment, Current equity holder in publicly-traded company.Roos:BioInvent International AB:Current Employment.Lindell Andersson:BioInvent International AB:Current Employment.Karlsson:BioInvent International AB:Current Employment.Borggren:BioInvent International AB:Current Employment.Abrisqueta:Celgene:Consultancy, Honoraria;Janssen:Consultancy, Honoraria, Speakers Bureau;AbbVie:Consultancy, Honoraria, Speakers Bureau;Roche:Consultancy, Honoraria, Speakers Bureau.Teige:BioInvent International AB:Current Employment, Current equity holder in publicly-traded company.Frendéus:BioInvent International AB:Current Employment, Current equity holder in publicly-traded company.

Modulation of Sex Pheromone Discrimination by a UDP-Glycosyltransferase in Drosophila melanogaster

The detection and processing of chemical stimuli involve coordinated neuronal networks that process sensory information. This allows animals, such as the model species Drosophila melanogaster, to detect food sources and to choose a potential mate. In peripheral olfactory tissues, several classes of proteins are acting to modulate the detection of chemosensory signals. This includes odorant-binding proteins together with odorant-degrading enzymes (ODEs). These enzymes, which primarily act to eliminate toxic compounds from the whole organism also modulate chemodetection. ODEs are thought to neutralize the stimulus molecule concurrently to its detection, avoiding receptor saturation thus allowing chemosensory neurons to respond to the next stimulus. Here, we show that one UDP-glycosyltransferase (UGT36E1) expressed in D. melanogaster antennal olfactory sensory neurons (OSNs) is involved in sex pheromone discrimination. UGT36E1 overexpression caused by an insertion mutation affected male behavioral ability to discriminate sex pheromones while it increased OSN electrophysiological activity to male pheromones. Reciprocally, the decreased expression of UGT36E1, controlled by an RNAi transgene, improved male ability to discriminate sex pheromones whereas it decreased electrophysiological activity in the relevant OSNs. When we combined the two genotypes (mutation and RNAi), we restored wild-type-like levels both for the behavioral discrimination and UGT36E1 expression. Taken together, our results strongly suggest that this UGT plays a pivotal role in Drosophila pheromonal detection.

Pharmacodynamics of natalizumab extended interval dosing in MS

ObjectiveTo determine if the concentration and saturation of natalizumab (NTZ) administration at extended interval dosing (EID; every 5–8 weeks) over 18 months is able to be maintained in the range considered adequate to sustain the clinical efficacy of NTZ.MethodsIn a cross-sectional assessment of patients with multiple sclerosis (MS) who received standard interval dosing (every 4 weeks) or EID, serum NTZ concentrations were measured using ELISA, and α4-integrin receptor saturations were analyzed via cytometry, in blood samples obtained at trough timepoints.ResultsTrough serum concentration was above the “therapeutic” concentration of 2.0 μg/mL in 72% of EID patients. Trough saturation was above the “therapeutic” 50% threshold in 79% of EID-treated patients. Our model predicted that at least 9 NTZ infusions/year are required to maintain adequate trough saturation and concentration levels. Higher body mass index (BMI) was a predictor of suboptimal trough saturation on EID NTZ.ConclusionsTrough α4-integrin receptor saturation >50% correlated with high clinical efficacy of NTZ in previous studies. A continual treatment with EID maintains receptor saturation and concentration that are in the “therapeutic range” for most patients. This finding provides biological plausibility for the clinical efficacy of NTZ EID. Patients with higher BMI may require closer clinical and MRI follow-up.

Antibodies for the Treatment of Brain Metastases, a Dream or a Reality?

The incidence of brain metastases (BM) in cancer patients is increasing. After diagnosis, overall survival (OS) is poor, elicited by the lack of an effective treatment. Monoclonal antibody (mAb)-based therapy has achieved remarkable success in treating both hematologic and non-central-nervous system (CNS) tumors due to their inherent targeting specificity. However, the use of mAbs in the treatment of CNS tumors is restricted by the blood–brain barrier (BBB) that hinders the delivery of either small-molecules drugs (sMDs) or therapeutic proteins (TPs). To overcome this limitation, active research is focused on the development of strategies to deliver TPs and increase their concentration in the brain. Yet, their molecular weight and hydrophilic nature turn this task into a challenge. The use of BBB peptide shuttles is an elegant strategy. They explore either receptor-mediated transcytosis (RMT) or adsorptive-mediated transcytosis (AMT) to cross the BBB. The latter is preferable since it avoids enzymatic degradation, receptor saturation, and competition with natural receptor substrates, which reduces adverse events. Therefore, the combination of mAbs properties (e.g., selectivity and long half-life) with BBB peptide shuttles (e.g., BBB translocation and delivery into the brain) turns the therapeutic conjugate in a valid approach to safely overcome the BBB and efficiently eliminate metastatic brain cells.

P012 α4β7 Receptor occupancy by vedolizumab at trough time-point occurs at the membranal level and is not associated with endoscopic healing in inflammatory bowel disease patients

Background Previous studies have shown inconclusive associations between vedolizumab trough levels and therapy outcomes. A near-complete occupancy of blood and intestinal α4β7 was exerted by vedolizumab through a wide-range of drug levels, regardless of clinical outcomes. However, these observations were not conducted specifically at the time of trough drug levels, did not include α4β7 highly expressing eosinophils and did not establish whether occupancy is in fact due to fully internalised VDZ- α4β7 complex in the target T-cell. Methods Peripheral blood (PB) and intestinal lamina propria (LP) were collected from IBD patients undergoing lower endoscopies on the day of vedolizumab infusion (trough time-point). Clinical and endoscopic scores were prospectively recorded. FACS analysis was performed using fluorescent conjugated-vedolizumab to investigate α4β7 occupancy on effector-memory T cells (CD3+CD45RO+ cells) and on eosinophils (CD49d+CD294+) in PB. Using a newly-developed dissociation assay, we also assessed the degree of membrane-bound VDZ-mediated blocked- α4β7. Results Forty-five IBD patients (16, 35% Crohn’s patients, 18, 40% in endoscopic remission) underwent clinical and endoscopic assessment just before their scheduled vedolizumab infusions. The median percentage of free α4β7 receptors at trough was 1.3% (IQR 0.5–3.7%) for PB T cells and 7.2% (IQR 3.6–23.4%) for LP T cells. No statistically significant difference was detected in α4β7 receptor saturation between patients with and without mucosal healing, both for PB (free α4β7 receptors: median 0.5%, 1.46%, IQR 0.3–1.4%, 0.9–3.5% respectively, p = 0.15) and for LP (free α4β7 receptors, median: 11.5%, 7%, IQR 3.5–24.2%, 5.8–20.9%, p = 0.88). Similarly, almost complete α4β7 receptor saturation at trough was detected among PB eosinophils (1.5% free α4β7 receptors, IQR 0.9–2.6%). Upon dissociation assay, a significant number of membrane-bound α4β7 receptors became detectable both on PB and LP T cells, with medians of 31% (IQR 24–41%), 22.2% (IQR 15–31%) respectively, and also for eosinophils, with a median of 83% (IQR 67–89%). When compared with values before dissociation, the proportion of unbound receptors was considerably lower (p < 0.001 for all analyses, respectively). Conclusion Even at trough, almost complete α4β7 receptor saturation is reached, both for PB and LP T cells, as well as for PB eosinophils, regardless of endoscopic activity status. This receptor occupancy is unrelated to receptor-internalisation and reflects a complete and continuous antigen-binding at the membranal level.