Avelumab for metastatic or locally advanced previously treated solid tumours (JAVELIN Solid Tumor): a phase 1a, multicohort, dose-escalation trial.

BACKGROUND: Avelumab (MSB0010718C) is a human IgG1 monoclonal antibody that binds to PD-L1, inhibiting its binding to PD-1, which inactivates T cells. We aimed to establish the safety and pharmacokinetics of avelumab in patients with solid tumours while assessing biological correlatives for future development. METHODS: This open-label, single-centre, phase 1a, dose-escalation trial (part of the JAVELIN Solid Tumor trial) assessed four doses of avelumab (1 mg/kg, 3 mg/kg, 10 mg/kg, and 20 mg/kg), with dose-level cohort expansions to provide additional safety, pharmacokinetics, and target occupancy data. This study used a standard 3 + 3 cohort design and assigned patients sequentially at trial entry according to the 3 + 3 dose-escalation algorithm and depending on the number of dose-limiting toxicities during the first 3-week assessment period (the primary endpoint). Patient eligibility criteria included age 18 years or older, Eastern Cooperative Oncology Group performance status 0-1, metastatic or locally advanced previously treated solid tumours, and adequate end-organ function. Avelumab was given as a 1-h intravenous infusion every 2 weeks. Patients in the dose-limiting toxicity analysis set were assessed for the primary endpoint of dose-limiting toxicity, and all patients enrolled in the dose-escalation part were assessed for the secondary endpoints of safety (treatment-emergent and treatment-related adverse events according to National Cancer Institute Common Terminology Criteria for Adverse Events version 4.0), pharmacokinetic and pharmacodynamic profiles (immunological effects), best overall response by Response Evaluation Criteria, and antidrug antibody formation. The population for the pharmacokinetic analysis included a subset of patients with rich pharmacokinetic samples from two selected disease-specific expansion cohorts at the same study site who had serum samples obtained at multiple early timepoints. This trial is registered with ClinicalTrials.gov, number NCT01772004. Patient recruitment to the dose-escalation part reported here is closed. FINDINGS: Between Jan 31, 2013, and Oct 8, 2014, 53 patients were enrolled (four patients at 1 mg/kg, 13 at 3 mg/kg, 15 at 10 mg/kg, and 21 at 20 mg/kg). 18 patients were analysed in the dose-limiting toxicity analysis set: three at dose level 1 (1 mg/kg), three at dose level 2 (3 mg/kg), six at dose level 3 (10 mg/kg), and six at dose level 4 (20 mg/kg). Only one dose-limiting toxicity occurred, at the 20 mg/kg dose, and thus the maximum tolerated dose was not reached. In all 53 enrolled patients (the safety analysis set), common treatment-related adverse events (occurring in >10% of patients) included fatigue (21 patients [40%]), influenza-like symptoms (11 [21%]), fever (8 [15%]), and chills (6 [11%]). Grade 3-4 treatment-related adverse events occurred in nine (17%) of 53 patients, with autoimmune disorder (n=3), increased blood creatine phosphokinase (n=2), and increased aspartate aminotransferase (n=2) each occurring in more than one patient (autoimmune disorder in two patients at 10 mg/kg and one patient at 20 mg/kg, increased blood creatine phosphokinase in two patients at 20 mg/kg, and increased aspartate aminotransferase in one patient at 1 mg/kg, and one patient at 10 mg/kg). Six (11%) of 53 patients had a serious treatment-related adverse event: autoimmune disorder (two [13%]), lower abdominal pain (one [7%]), fatigue (one [7%]), and influenza-like illness (one [7%]) in three patients treated at 10 mg/kg dose level, and autoimmune disorder (one [5%]), increased amylase (one [5%]), myositis (one [5%]), and dysphonia (one [5%]) in three patients who received the 20 mg/kg dose. We recorded some evidence of clinical activity in various solid tumours, with partial confirmed or unconfirmed responses in four (8%) of 53 patients; 30 (57%) additional patients had stable disease. Pharmacokinetic analysis (n=86) showed a dose-proportional exposure between doses of 3 mg/kg and 20 mg/kg and a half-life of 95-99 h (3·9-4·1 days) at the 10 mg/kg and 20 mg/kg doses. Target occupancy was greater than 90% at doses of 3 mg/kg and 10 mg/kg. Antidrug antibodies were detected in two (4%) of 53 patients. No substantial differences were found in absolute lymphocyte count or multiple immune cell subsets, including those expressing PD-L1, after treatment with avelumab. 31 (58%) of 53 patients in the overall safety population died; no deaths were related to treatment on study. INTERPRETATION: Avelumab has an acceptable toxicity profile up to 20 mg/kg and the maximum tolerated dose was not reached. Based on pharmacokinetics, target occupancy, and immunological analysis, we chose 10 mg/kg every 2 weeks as the dose for further development and phase 3 trials are ongoing. FUNDING: National Cancer Institute and Merck KGaA.

Avelumab internalization by human circulating immune cells is mediated by both Fc gamma receptor and PD-L1 binding.

Avelumab is an IgG1 anti-programmed death ligand 1 (anti-PD-L1) monoclonal antibody that has been approved as a monotherapy for metastatic Merkel cell carcinoma and advanced urothelial carcinoma, and in combination with axitinib for advanced renal cell carcinoma. Avelumab is cleared faster and has a shorter half-life than other anti-PD-L1 antibodies, such as atezolizumab and durvalumab, but the mechanisms underlying these differences are unknown. IgG antibodies can be cleared through receptor-mediated endocytosis after binding of the antibody Fab region to target proteins, or via Fcγ receptor (FcγR)-mediated endocytosis. Unlike other approved anti-PD-L1 antibodies, avelumab has a native Fc region that retains FcγR binding capability. We hypothesized that the rapid clearance of avelumab might be due to the synergistic effect of both FcγR-mediated and PD-L1 target-mediated internalization. To investigate this, we performed in vitro and in vivo studies that compared engineered variants of avelumab and atezolizumab to determine mechanisms of cellular internalization. We found that both FcγR and PD-L1 binding contribute to avelumab internalization. While FcγR binding was the dominant mechanism of avelumab internalization in vitro, with CD64 acting as the most important FcγR, studies in mice and cynomolgus monkeys showed that both FcγR and PD-L1 contribute to avelumab elimination, with PD-L1 binding playing a greater role. These studies suggest that the rapid internalization of avelumab might be due to simultaneous binding of both PD-L1 and FcγR in trans. Our findings also provide a basis to alter the clearance and half-life of monoclonal antibodies in therapeutic development.

Changing Body Weight-Based Dosing to a Flat Dose for Avelumab in Metastatic Merkel Cell and Advanced Urothelial Carcinoma.

Avelumab, an anti-programmed death-ligand 1 monoclonal antibody approved for the treatment of metastatic Merkel cell carcinoma and platinum-treated urothelial carcinoma, was initially approved with a 10 mg/kg weight-based dose. We report pharmacokinetic (PK)/pharmacodynamic analyses for avelumab comparing weight-based dosing and a flat 800 mg dose, developed using data from 1,827 patients enrolled in 3 clinical trials (NCT01772004, NCT01943461, and NCT02155647). PK metrics were simulated for weight-based and flat-dosing regimens and summarized by quartiles of weight. Derived exposure metrics were used in simulations of exposure-safety (various tumors) and exposure-efficacy (objective responses; Merkel cell or urothelial carcinoma). Flat dosing was predicted to provide similar exposure to weight-based dosing, with slightly lower variability. Exposure-safety and exposure-efficacy simulations suggested similar benefit:risk profiles for the two dosing regimens. These pharmacometric analyses provided the basis for the US Food and Drug Administration approval of a flat dose of avelumab 800 mg every 2 weeks in approved indications.

Radiation sensitization of glioblastoma by cilengitide has unanticipated schedule-dependency.

We investigated whether cilengitide could amplify the antitumor effects of radiotherapy in an orthotopic rat glioma xenograft model. Cilengitide is a specific inhibitor of alphav series integrins, and acts as an antiangiogenic. U251 human glioma cells express alphavbeta3 and alphavbeta5 integrins. We used in vitro assays of adhesion and growth of tumor and endothelial cells to evaluate cytotoxicity and the potential for cilengitide to enhance radiation toxicity. Treatment was then evaluated in an orthotopic model to evaluate synergy with therapeutic radiation in vivo. In vitro, cilengitide blocked cell adhesion, but did not influence the effects of radiation on U251 cells; cilengitide strongly amplified radiation effects on endothelial cell survival. In vivo, radiotherapy prolonged the survival of U251 tumor-bearing rats from 50 to over 110 days. Cotreatment with cilengitide and radiation dramatically amplified the effects of radiation, producing survival over 200 days and triggering an enhanced apoptotic response and suppression of tumor growth by histology at necropsy. Signaling pathways activated in the tumor included NFkappab, a documented mediator of cellular response to radiation. Because cilengitide has a short plasma half-life (t((1/2)) approximately 20 min), antiangiogenic scheduling typically uses daily injections. We found that a single dose of cilengitide (4 mg/kg) given between 4 and 12 hr prior to radiation was sufficient to produce the same effect. Our results demonstrate that blockade of alphav integrins mediates an unanticipated rapid potentiation of radiation, and suggests possible clinical translation for glioma therapy.

Time-Varying Clearance and Impact of Disease State on the Pharmacokinetics of Avelumab in Merkel Cell Carcinoma and Urothelial Carcinoma.

Avelumab, a human anti-programmed death ligand 1 immunoglobulin G1 antibody, has shown efficacy and manageable safety in multiple tumors. A two-compartment population pharmacokinetic model for avelumab incorporating intrinsic and extrinsic covariates and time-varying clearance (CL) was identified based on data from 1,827 patients across three clinical studies. Of 14 tumor types, a decrease in CL over time was more notable in metastatic Merkel cell carcinoma and squamous cell carcinoma of the head and neck, which had maximum decreases of 32.1% and 24.7%, respectively. The magnitude of reduction in CL was higher in responders than in nonresponders. Significant covariate effects of baseline weight, baseline albumin, and sex were identified on both CL and central distribution volume. Significant covariate effects of black/African American race, C-reactive protein, and immunogenicity were found on CL. None of the covariate or time-dependent effects were clinically important or warranted dose adjustment.

A generic approach to engineer antibody pH-switches using combinatorial histidine scanning libraries and yeast display.

There is growing interest in the fast and robust engineering of protein pH-sensitivity that aims to reduce binding at acidic pH, compared to neutral pH. Here, we describe a novel strategy for the incorporation of pH-sensitive antigen binding functions into antibody variable domains using combinatorial histidine scanning libraries and yeast surface display. The strategy allows simultaneous screening for both, high affinity binding at pH 7.4 and pH-sensitivity, and excludes conventional negative selection steps. As proof of concept, we applied this strategy to incorporate pH-dependent antigen binding into the complementary-determining regions of adalimumab. After 3 consecutive rounds of separate heavy and light chain library screening, pH-sensitive variants could be isolated. Heavy and light chain mutations were combined, resulting in 3 full-length antibody variants that revealed sharp, reversible pH-dependent binding profiles. Dissociation rate constants at pH 6.0 increased 230- to 780-fold, while high affinity binding at pH 7.4 in the sub-nanomolar range was retained. Furthermore, binding to huFcRn and thermal stability were not affected by histidine substitutions. Overall, this study emphasizes a generalizable strategy for engineering pH-switch functions potentially applicable to a variety of antibodies and further proteins-based therapeutics.

The immunocytokine NHS-IL12 as a potential cancer therapeutic.

Targeted delivery of IL-12 might turn this cytokine into a safer, more effective cancer therapeutic. Here we describe a novel immunocytokine, NHS-IL12, consisting of two molecules of IL-12 fused to a tumor necrosis-targeting human IgG1 (NHS76). The addition of the human IgG1 moiety resulted in a longer plasma half-life of NHS-IL12 than recombinant IL-12, and a selective targeting to murine tumors in vivo. Data from both in vitro assays using human PBMCs and in vivo primate studies showed that IFN-gamma production by immune cells is attenuated following treatment with the immunocytokine, suggesting an improved toxicity profile than seen with recombinant IL-12 alone. NHS-IL12 was superior to recombinant IL-12 when evaluated as an anti-tumor agent in three murine tumor models. Mechanistic studies utilizing immune cell subset-depleting antibodies, flow cytometric methods, and in vitro cytotoxicity and ELISA assays all indicated that the anti-tumor effects of NHS-IL12 were primarily CD8+ T cell-dependent and likely IL-12-mediated. Combining NHS-IL12 treatment with a cancer vaccine, radiation, or chemotherapy resulted in greater anti-tumor effects than each individual therapy alone. These preclinical findings provide a rationale for the clinical testing of this immunocytokine, both as a single agent and in combination with vaccines, radiation and chemotherapy.

Therapeutic assessment of SEED: a new engineered antibody platform designed to generate mono- and bispecific antibodies.

The strand-exchange engineered domain (SEED) platform was designed to generate asymmetric and bispecific antibody-like molecules, a capability that expands therapeutic applications of natural antibodies. This new protein engineered platform is based on exchanging structurally related sequences of immunoglobulin within the conserved CH3 domains. Alternating sequences from human IgA and IgG in the SEED CH3 domains generate two asymmetric but complementary domains, designated AG and GA. The SEED design allows efficient generation of AG/GA heterodimers, while disfavoring homodimerization of AG and GA SEED CH3 domains. Using a clinically validated antibody (C225), we tested whether Fab derivatives constructed on the SEED platform retain desirable therapeutic antibody features such as in vitro and in vivo stability, favorable pharmacokinetics, ligand binding and effector functions including antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. In addition, we tested SEED with combinations of binder domains (scFv, VHH, Fab). Mono- and bivalent Fab-SEED fusions retain full binding affinity, have excellent biochemical and biophysical stability, and retain desirable antibody-like characteristics conferred by Fc domains. Furthermore, SEED is compatible with different combinations of Fab, scFv and VHH domains. Our assessment shows that the new SEED platform expands therapeutic applications of natural antibodies by generating heterodimeric Fc-analog proteins.

Interferon beta-1a overdose in a multiple sclerosis patient.

OBJECTIVE: To report on a patient with multiple sclerosis (MS) who attempted suicide by taking an overdose of interferon (IFN) beta-1a. CASE SUMMARY: A 38-year-old man with MS and depressive symptoms self-administered approximately 6 or 7 prefilled syringes containing 44 mug (12 MIU) of subcutaneous IFN beta-1a in a suicide attempt. Clinical examination in the emergency department revealed a modest rise in body temperature and diffuse redness of the skin of the limbs and truncal region. The patient's signs and symptoms resolved over the following 24 hours. IFN beta-1a was temporarily withdrawn and treatment with citalopram, which the patient had spontaneously discontinued before the suicide attempt, was resumed. Laboratory assessment showed no modifications in biochemical and hematologic parameters. The IFN beta-1a concentration in the serum sample taken at 48 hours after the suicide attempt confirmed that the patient had taken a very high dose of IFN. DISCUSSION: Depression, a common condition in MS patients, was precipitated in this patient by the spontaneous discontinuation of citalopram and not influenced by IFN beta-1a therapy, which the patient resumed at 44 mug 3 times per week. CONCLUSIONS: This case demonstrates the risks of depression in MS patients and the danger of discontinuing treatment with antidepressants. This report also shows that approximately 264-308 mug of IFN beta-1a, the highest single dose of IFN beta-1a reported taken by a human as of this writing, caused only a transient and self-limiting malaise.

Pharmacokinetics and pharmacodynamics of PEGylated IFN-beta 1a following subcutaneous administration in monkeys.

PURPOSE: To characterize the pharmacokinetic/pharmacodynamic (PK/PD) properties of a new polyethylene glycol (PEG) conjugate formulation of interferon (IFN)-beta la following subcutaneous (SC) administration in monkeys. METHODS: Single SC injections of 0.3, 1, and 3 million international units (MIU)/kg of PEG-IFN-beta 1a were administered to 3 groups of cynomolgus monkeys (n = 4 each). Plasma concentrations of drug and neopterin, a classic biomarker for IFN-beta PD, were measured at various time-points after dosing. PK/PD profiles were described by noncompartmental methods and pooled data by an integrated mathematical model, where fixed and delayed concentration-time profiles were used as driving functions in an indirect stimulatory response model. RESULTS: PEG-IFN-beta 1a was rapidly absorbed, with peak concentrations observed at about 4-5 h. Compared to previous identical SC doses of IFN-beta la, administration of 1 and 3 MIU/kg of pegylated drug resulted in 27- and 16-fold increases in area under the concentration-time curves. Neopterin concentrations followed a typical dose-dependent biphasic pattern. Pooled PD profiles were well-described by the PK/PD model, and the neopterin elimination rate (0.0190 h(-1)) is consistent with previous estimates. CONCLUSIONS: The PEG-modification of IFN-beta la provides enhanced drug exposure and similar pharmacodynamics of neopterin compared to the unmodified formulation.

Receptor-mediated pharmacokinetics and pharmacodynamics of interferon-beta1a in monkeys.

A pharmacokinetic/pharmacodynamic (PK/PD) model was developed to simultaneously characterize interferon after i.v. and s.c. dosing at various dose levels. A sequential study in monkeys (n = 18) was conducted, where single doses of 1, 3, and 10 MIU/kg of recombinant-human interferon-beta (IFN-beta) 1a were given i.v. and then s.c. Plasma concentrations of IFN-beta were determined and biphasic neopterin concentrations were used as the pharmacodynamic (PD) endpoint. Multiple dosing also was evaluated by giving 1 MIU/kg s.c. doses once daily for 7 days (n = 3). The integrated model uses target-mediated drug disposition to describe drug elimination by receptor binding and internalization, and well characterizes the observed nonlinear pharmacokinetic (PK) profiles. The s.c. doses exhibited an absorption phase (Tmax = 3 h) and incomplete bioavailability (F = 0.3-0.7). An indirect response model for stimulation of neopterin triphosphate production by activated receptor complex followed by conversion to neopterin was used to jointly model the formation and loss of neopterin with a capacity factor Smax = 23.8. Greater relative neopterin response after s.c. dosing was accounted for by prolonged receptor activation relative to the SC50 value. Repeated daily s.c. dosing produced modestly elevated IFN-beta1a concentrations and neopterin concentrations that were lower than simulated from single-dose modeling. Although several mechanisms could be involved, these phenomena were simply remodeled as down-regulation of Smax and receptors. The PK/PD model for IFN-beta1a depicts receptor binding as a key feature controlling nonlinear elimination, nonstationary kinetics, and neopterin induction in a manner consistent with known processes controlling its disposition and pharmacological effects.