Optimizing Early Clinical Investigations in Cancer Immunotherapy: The Translational Journey of RG6292, a Novel, Selective Treg-Depleting Antibody.

The multifaceted IL-2/IL-2R biology and its modulation by promising therapeutic agents are highly relevant topics in the cancer immunotherapy field. A novel CD25-Treg-depleting antibody (Vopikitug, RG6292) has been engineered to preserve IL-2 signaling on effector T cells to enhance effector activation and antitumor immunity, and is currently being evaluated in the clinic. The Entry into Human-enabling framework described here investigated the characteristics of RG6292, from in vitro quantification of CD25 and RG6292 pharmacology using human tissues to in vivo assessment of PK/PD/safety relationships in cynomolgus monkeys as non-human primate species (NHP). Fundamental knowledge on CD25 and Treg biology in healthy and diseased tissues across NHP and human highlighted the commonalities between these species in regard to the target biology and demonstrated the conservation of RG6292 properties between NHP and human. The integration of in vitro and in vivo PK/PD/safety data from these species enabled the identification of human relevant safety risks, the selection of the most appropriate safe starting dose and the projection of the pharmacologically-relevant dose range. The first clinical data obtained for RG6292 in patients verified the appropriateness of the described approaches as well as validated the full clinical relevance of the projected safety, PK, and PD profiles from animal to man. This work shows how the integration of mechanistic non-clinical data increases the predictive value for human, allowing efficient transition of drug candidates and optimizations of early clinical investigations.

Investigating brain uptake of a non-targeting monoclonal antibody after intravenous and intracerebroventricular administration.

Monoclonal antibodies play an important role in the treatment of various diseases. However, the development of these drugs against neurological disorders where the drug target is located in the brain is challenging and requires a good understanding of the local drug concentration in the brain. In this original research, we investigated the systemic and local pharmacokinetics in the brain of healthy rats after either intravenous (IV) or intracerebroventricular (ICV) administration of EGFRvIII-T-Cell bispecific (TCB), a bispecific monoclonal antibody. We established an experimental protocol that allows serial sampling in serum, cerebrospinal fluid (CSF) and interstitial fluid (ISF) of the prefrontal cortex in freely moving rats. For detection of drug concentration in ISF, a push-pull microdialysis technique with large pore membranes was applied. Brain uptake into CSF and ISF was characterized and quantified with a reduced brain physiologically-based pharmacokinetic model. The model allowed us to interpret the pharmacokinetic processes of brain uptake after different routes of administration. The proposed model capturing the pharmacokinetics in serum, CSF and ISF of the prefrontal cortex suggests a barrier function between the CSF and ISF that impedes free antibody transfer. This finding suggests that ICV administration may not be better suited to reach higher local drug exposure as compared to IV administration. The model enabled us to quantify the relative contribution of the blood-brain barrier (BBB) and Blood-CSF-Barrier to the uptake into the interstitial fluid of the brain. In addition, we compared the brain uptake of three monoclonal antibodies after IV dosing. In summary, the presented approach can be applied to profile compounds based on their relative uptake in the brain and provides quantitative insights into which pathways are contributing to the net exposure in the brain.

Assay concept for detecting anti-drug IgM in human serum samples by using a novel recombinant human IgM positive control.

Aim: Development and qualification of an easy-to-use ELISA for detection of IgM anti-drug antibodies (ADA) and its use in a clinical Phase I trial. Results & methodology: During the assay development two positive control (PC) approaches, the preparation of a chemically conjugated and a recombinant PC, were pursued. With both PCs, the assay was developed and successfully qualified considering the regulatory guidelines. For a case study, the IgM ADA isotyping assay with the recombinant PC was selected. Different courses and intensities of immune response regarding IgM signals were demonstrated. Conclusion: The easy-to-use ELISA allowed IgM-ADA detection in clinical samples. Conjugated and recombinant IgM PCs were comparable regarding assay sensitivity, precision and suitability.

Indatuximab ravtansine plus dexamethasone with lenalidomide or pomalidomide in relapsed or refractory multiple myeloma: a multicentre, phase 1/2a study.

BACKGROUND: Indatuximab ravtansine (BT062) is an antibody-drug conjugate that binds to CD138 and synergistically enhances the antitumor activity of lenalidomide in preclinical models of multiple myeloma. This phase 1/2a study was done to determine the safety, activity, and pharmacokinetics of indatuximab ravtansine in combination with immunomodulatory drugs in patients with relapsed or refractory multiple myeloma. METHODS: This open-label, phase 1/2a study took place at nine hospital sites in the USA. Eligible patients were aged 18 years or older, had relapsed or refractory multiple myeloma, and ECOG performance status or Zubrod score of 2 or below. Patients who received indatuximab ravtansine with lenalidomide and dexamethasone (indatuximab ravtansine plus lenalidomide) had failure of at least one previous therapy. Patients treated with indatuximab ravtansine with pomalidomide and dexamethasone (indatuximab ravtansine plus pomalidomide) had failure of at least two previous therapies (including lenalidomide and bortezomib) and had progressive disease on or within 60 days of completion of their last treatment. In phase 1, patients received indatuximab ravtansine intravenously on days 1, 8, and 15 of each 28-day cycle in escalating dose levels of 80 mg/m2, 100 mg/m2, and 120 mg/m2, with lenalidomide (25 mg; days 1 to 21 every 28 days orally) and dexamethasone (20-40 mg; days 1, 8, 15, and 22 every 28 days). In phase 2, the recommended phase 2 dose of indatuximab ravtansine was given to an expanded cohort of patients in combination with lenalidomide and dexamethasone. The protocol was amended to allow additional patients to be treated with indatuximab ravtansine plus pomalidomide (4 mg; days 1 to 21 every 28 days orally) and dexamethasone, in a more heavily pretreated patient population than in the indatuximab ravtansine plus lenalidomide group. The phase 1 primary endpoint was to determine the dose-limiting toxicities and the maximum tolerated dose (recommended phase 2 dose) of indatuximab ravtansine, and the phase 2 primary endpoint was to describe the objective response rate (ORR; partial response or better) and clinical benefit response (ORR plus minor response). All patients were analysed for safety and all patients with post-treatment response assessments were analysed for activity. This study is registered with ClinicalTrials.gov, number NCT01638936, and is complete. FINDINGS: 64 (86%) of 74 screened patients were enrolled between July 3, 2012, and June 30, 2015. 47 (73%) patients received indatuximab ravtansine plus lenalidomide (median follow-up 24·2 months [IQR 19·9-45·4]) and 17 (27%) received indatuximab ravtansine plus pomalidomide (24·1 months [17·7-36·7]). The maximum tolerated dose of indatuximab ravtansine plus lenalidomide was 100 mg/m2, and defined as the recommended phase 2 dose for indatuximab ravtansine plus pomalidomide. An objective response for indatuximab ravtansine plus lenalidomide was observed in 33 (71·7%) of 46 patients and in 12 (70·6%) of 17 patients in the indatuximab ravtansine plus pomalidomide group. The clinical benefit response for indatuximab ravtansine plus lenalidomide was 85% (39 of 46 patients) and for indatuximab ravtansine plus pomalidomide it was 88% (15 of 17 patients). The most common grade 3-4 adverse events in both groups were neutropenia (14 [22%] of 64 patients), anaemia (10 [16%]), and thrombocytopenia (seven [11%]). Treatment-emergent adverse events (TEAEs) that led to discontinuation occurred in 35 (55%) of the 64 patients. Five (8%) patients with a TEAE had a fatal outcome; none was reported as related to indatuximab ravtansine. INTERPRETATION: Indatuximab ravtansine in combination with immunomodulatory drugs shows preliminary antitumor activity, is tolerated, and could be further evaluated in patients with relapsed or refractory multiple myeloma. FUNDING: Biotest AG.

Immunogenicity testing of therapeutic antibodies in ocular fluids after intravitreal injection.

AIM: High drug concentrations in ocular fluids after intravitreal administration preclude the use of drug-sensitive immunoassays. A drug-tolerant immunoassay is therefore desirable for immunogenicity testing in ophthalmology. EXPERIMENTAL: Immune complex (IC) antidrug antibody (ADA) assays were established for two species. The assays were compared with the bridging assay in ocular and plasma samples from two preclinical studies. RESULTS: The IC assays showed high drug tolerance, which enabled a reliable ADA detection in ocular fluids after intravitreal administration. The IC assays were superior to the bridging assay in the analysis of ocular fluids with high drug concentrations. CONCLUSION: The IC assay allows a reliable ADA detection in matrices with high drug concentrations, such as ocular fluids.

Method establishment for discerned immunogenicity assessment of a recombinant glycoprotein containing nonhuman sialic acid Neu5Gc residues.

AIM: Recombinant glycoprotein produced in nonhuman mammalian cell lines can be modified with the immunogenic nonhuman sialic N-glycolylneuraminic acid (Neu5Gc). We describe here a validated method for detection of antidrug antibodies against both protein and Neu5Gc-containing glycan epitopes. RESULTS: An electrochemiluminescent method was established with drug conjugates as capture and detection reagents. Rabbit antidrug polyclonal antibodies were used as the positive control for protein moiety-specific antibodies, while chicken anti-Neu5Gc polyclonal antibodies were used as the positive control for antibodies against Neu5Gc glycan epitope. Specificity to Neu5Gc was verified by signal inhibition with bovine γ-globulin that contains Neu5Gc. CONCLUSION: The assay illustrated here discerns the immunogenicity of the protein backbone and the sialic acid Neu5Gc glycan moiety of a recombinant protein containing Neu5Gc.

Differential sensitivity of lipegfilgrastim and pegfilgrastim to neutrophil elastase correlates with differences in clinical pharmacokinetic profile.

To assess the basis of the different half-lives of long-acting human granulocyte colony-stimulating factor (G-CSF) drugs, the effect of neutrophil elastase on lipegfilgrastim and pegfilgrastim was investigated. Sensitivity to human neutrophil elastase (HNE) was evaluated by incubating the drugs with HNE followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Drugs were also incubated with isolated human neutrophils followed by Western blot analysis. Lipegfilgrastim was more resistant to degradation with HNE or neutrophils than pegfilgrastim and appeared more intact on SDS-PAGE gels and Western blots. Lipegfilgrastim retained more functional activity than pegfilgrastim after incubation with HNE (67% vs ∼ 9%, respectively) or neutrophils (80% vs ∼ 4%, respectively) as assessed in an NFS-60 cell-based [(3) H]-thymidine incorporation assay. The binding and affinity of untreated lipegfilgrastim and pegfilgrastim for G-CSF receptors were evaluated using an NFS-60 competitive G-CSF receptor-binding assay and surface plasmon resonance. Untreated drugs were also evaluated in the functional NFS-60 thymidine incorporation assay. G-CSF receptor binding, receptor affinity, and functional activity were comparable between untreated drugs. The results showed a greater resistance to neutrophil elastase degradation and concomitant retention of functional activity of lipegfilgrastim compared with pegfilgrastim, which potentially explains the clinical observations of a longer half-life of lipegfilgrastim versus pegfilgrastim.

Lipegfilgrastim: pharmacodynamics and pharmacokinetics for body-weight-adjusted and 6 mg fixed doses in two randomized studies in healthy volunteers.

OBJECTIVE: Two phase I, single-blind (subject blinded to treatment), randomized studies were conducted to assess the pharmacodynamics, pharmacokinetics, safety, and tolerability of lipegfilgrastim compared with pegfilgrastim in healthy adult volunteers. METHODS: Study 1 consisted of a pilot safety phase (N = 8) during which subjects received a single body-weight-adjusted subcutaneous dose of lipegfilgrastim 25 µg/kg and a dose escalation phase (N = 45) wherein subjects received lipegfilgrastim 50 or 100 µg/kg or pegfilgrastim 100 µg/kg. Study 2 was a single-blind, fixed-dose study (N = 36) comparing subcutaneous lipegfilgrastim 6 mg and pegfilgrastim 6 mg. RESULTS: Cumulative exposure (AUC0-t last and AUC 0-∞) and peak exposure (Cmax) were higher for lipegfilgrastim than pegfilgrastim after both weight-adjusted and fixed dosing. In both studies, the terminal elimination half-life of lipegfilgrastim was 5-10 hours longer than the terminal elimination half-life for pegfilgrastim at the maximum dose, and the time to maximum serum concentration (tmax) was observed later for lipegfilgrastim than for pegfilgrastim. The area over the baseline effect curve (AOBEC) for absolute neutrophil count (ANC) was approximately 30% greater after lipegfilgrastim dosing compared with the same dose of pegfilgrastim at the maximum dose. Both drugs were well tolerated, with a similar occurrence of adverse events between treatment groups. Key limitations of these studies include the small numbers of subjects and differences in dosage regimens between the two studies. CONCLUSIONS: In these studies, lipegfilgrastim provided a longer-lasting increase in ANC compared with pegfilgrastim at an equivalent dose, without increasing the peak ANC values. This may reflect the higher cumulative exposure and slower clearance (therefore longer body residence) of lipegfilgrastim. These data support the use of single-dose lipegfilgrastim 6 mg in subsequent phase III trials as prophylactic treatment for patients receiving myelosuppressive chemotherapy.

Assessing heterogeneity of peroxisomes: isolation of two subpopulations from rat liver.

Peroxisomes exhibit a heterogeneous morphological appearance in rat liver tissue. In this respect, the isolation and subsequent biochemical characterization of peroxisome species from different subcellular prefractions should help to solve the question of whether peroxisomes indeed diverge into functionally specialized subgroups in one tissue. As a means to address this question, we provide a detailed separation protocol for the isolation of peroxisomes from both the light (LM-Po) and the heavy (HM-Po) mitochondrial prefraction for their subsequent comparative analysis. Both isolation strategies rely on centrifugation in individually adapted Optiprep gradients. In case of the heavy mitochondrial fraction, free flow electrophoresis is appended as an additional separation step to yield peroxisomes of sufficient purity. In view of their morphology, peroxisomes isolated from both fractions are surrounded by a continuous single membrane and contain a gray-opaque inner matrix. However, beyond this overall similar appearance, HM-Po exhibit a smaller average diameter, float at lower density, and show a more negative average membrane charge when compared to LM-Po.

Convenient and versatile subcellular extraction procedure, that facilitates classical protein expression profiling and functional protein analysis.

Standardized sample preparation to reduce proteome complexity facilitates subsequent proteome analysis. Here we describe a robust sequential extraction method that enables simple fractionation of proteins in their native state according to their subcellular localization, yielding four subproteomes enriched in (a) cytosolic; (b) membrane and membrane organelle-localized; (c) soluble and DNA-associated nuclear and (d) cytoskeletal proteins. Efficiency and selectivity is demonstrated by morphological-, two-dimensional electrophoresis image-, immunological- as well as enzymatic-analysis. In pilot studies, subcellular redistribution of regulatory proteins was successfully measured.