Application of blood microsampling in cynomolgus monkey and demonstration of equivalent monoclonal antibody PK parameters compared to conventional sampling.

PURPOSE: The purpose of this study was to evaluate the suitability of whole blood microsampling procedures in non-human primate (NHP) to support toxicokinetic assessments of biotherapeutics in non-human primates. METHOD: A one-month single dose intravenous pharmacokinetic (PK) study was performed in male cynomolgus monkeys with a human IgG1 control monoclonal antibody (mAb) as a surrogate monoclonal antibody biotherapeutic. In this study, both serum samples (conventional sample collection) and microsampling samples were collected. Microsampling samples were collected from two sites on cynomolgus monkey, with each site using two different devices for the whole blood collection. The drug concentrations from all sample types were determined using a quantitative ligand binding assay (LBA). The PK parameters obtained from microsampling samples and serum samples were examined using a standard PK analysis method. The comparability of key PK parameters from both sample types were analyzed statistically. RESULTS: Similar profiles of drug concentrations versus timepoints from all sampling procedures were observed. The correlations of PK concentration data obtained from serum and microsampling samples were ≥ 0.97 using Brand Alman Plot analysis. The key PK parameters obtained from microsampling samples were comparable to those obtained from serum samples (the % differences of mean PK parameters obtained from both sample types were within ±25%). CONCLUSION: This study confirmed that PK parameters obtained from samples using microsampling were comparable to that of serum samples in cynomolgus monkeys. Therefore, the microsampling procedure described can be used as a substitute for conventional sampling procedure to support PK/TK studies of biotherapeutics in non-clinical product developments.

Translating slow-binding inhibition kinetics into cellular and in vivo effects.

Many drug candidates fail in clinical trials owing to a lack of efficacy from limited target engagement or an insufficient therapeutic index. Minimizing off-target effects while retaining the desired pharmacodynamic (PD) response can be achieved by reduced exposure for drugs that display kinetic selectivity in which the drug-target complex has a longer half-life than off-target-drug complexes. However, though slow-binding inhibition kinetics are a key feature of many marketed drugs, prospective tools that integrate drug-target residence time into predictions of drug efficacy are lacking, hindering the integration of drug-target kinetics into the drug discovery cascade. Here we describe a mechanistic PD model that includes drug-target kinetic parameters, including the on- and off-rates for the formation and breakdown of the drug-target complex. We demonstrate the utility of this model by using it to predict dose response curves for inhibitors of the LpxC enzyme from Pseudomonas aeruginosa in an animal model of infection.

A Phase I trial of the kinesin spindle protein (Eg5) inhibitor AZD4877 in patients with solid and lymphoid malignancies.

BACKGROUND: This Phase I study assessed the safety and maximum tolerated dose (MTD) of the kinesin spindle protein inhibitor AZD4877 in patients with relapsed/refractory solid tumors and lymphoma. METHODS: In this multicenter study, a standard 3 + 3 dose-escalation design was used. AZD4877 was given as an intravenous infusion on days 1, 4, 8 and 11 of each 21-day cycle. Responses were assessed with CT scans +/- PET after 6 and 12 weeks, then every 12 weeks while on therapy. An additional four patients with lymphoma were enrolled at the MTD. RESULTS: 29 patients were enrolled and 22 patients received at least one dose of AZD4877 and were evaluable for safety. The MTD was 11 mg. Dose-limiting toxicity was neutropenia (n = 2 patients, 15 mg cohort). The most common adverse events were grade 1/2 fatigue, nausea, neutropenia and dyspnea. AZD4877 exposure generally increased with dose, with mean elimination half-life approximately 16 h at the MTD. Pharmacodynamic analyses demonstrated moderate correlation between plasma drug concentrations at 6 or 24 h and monoaster formation in peripheral blood mononuclear cells (PBMCs). CONCLUSIONS: AZD4877 is generally well-tolerated with pharmacodynamic evidence of target inhibition in circulating PBMCs.

Determination of Anti-drug Antibody Affinity in Clinical Study Samples Provides a Tool for Evaluation of Immune Response Maturation.

Characterization of clinical anti-drug antibody (ADA) responses to biotherapeutics can be important to understanding the consequences of immunogenicity. ADA are expected to be polyclonal, with composition and affinities that evolve over time. Measuring ADA binding affinity can be complicated by the polyclonal nature of response, residual drug in sample, and low ADA levels. We developed a novel workflow to determine the apparent ADA affinity (KD) against a monoclonal antibody biotherapeutic, PF-06480605. An affinity capture elution pre-treatment step was used to isolate ADA and remove residual drug interference from samples. Solution-phase equilibrium incubation was performed using drug and sample ADA as variable and fixed binding interactants, respectively. Unbound ADA concentration was measured using a Singulex Erenna ligand-binding assay (LBA) method. Apparent ADA KD values were calculated using a custom R Shiny algorithm. KD values determined for ADA positive samples showed good correlation with other immunogenicity parameters, including titers and neutralizing antibody (NAb) activity with a general increase in affinity over time, indicative of a maturing immune response. Time of onset of high affinity responses (KD < 100 pM) varied between patients, ranging from 16 to 24 weeks. Antibody responses appeared monophasic at earlier time points, trending towards a biphasic response with a variable transition time and general increase in proportion of high affinity ADA over time. Herein, we provide a novel, sensitive bioanalytical method to determine the KD of ADA in clinical samples. The observed decrease in ADA KD is consistent with evidence of a maturing immune response.

Feasibility of singlicate-based analysis in bridging ADA assay on Meso-Scale Discovery platform: comparison with duplicate analysis.

Aim: To investigate the feasibility of singlicate analysis in anti-drug antibody (ADA) assay by comparing performance characteristics for assays qualified in duplicate and singlicate formats. Materials & methods: We employed modeling to assess and quantify the impact of singlicate to cut point factor (CPF) in scenarios with the duplicate precision from 1-20% and the proportion of well-to-well variance to overall assay variance from 0.01-0.90. The impact to CPF by singlicate is marginal if the well-to-well coefficient of variation is <10% and represents <25% of the overall variability. Results & conclusion: The assay parameters including sensitivity, precision, selectivity, drug and target tolerance were comparable between singlicate and duplicate based assays. Our results suggested the minimal impact of singlicate analysis on ADA assay with good duplicate precision. The study provided additional supportive evidence that the singlicate-based analysis is feasible in ADA ligand binding assays.

Evaluation of pKa estimation methods on 211 druglike compounds.

The pK(a) values of 211 discovery (druglike) compounds were determined experimentally using capillary electrophoresis coupled with ultraviolet spectroscopy and a novel fitting algorithm. These values were compared to those predicted by five different commercially available pK(a) estimation packages: ACDLabs/pK(a), Marvin (ChemAxon), MoKa (Molecular Discovery), Epik (Schrodinger), and Pipeline Pilot (Accelrys). Even though the topological method MoKa was noticeably faster than ACD, the accuracy of those two methods and Marvin was statistically indistinguishable, with a root-mean-squared error of about 1 pK(a) unit compared to experiment. Pipeline Pilot and EpiK both produced pK(a) estimates in significantly worse agreement with the experiment. Interestingly, on a number of compounds, the predictions due to ACD v12 were in poorer agreement with the experiment than ACD v10. Microscopic and "apparent" pK(a) predictions were also compared using ACD v10. Microscopic pK(a)s gave significantly worse agreement with the experiment than the "apparent" values. In all cases, the errors appeared to be randomly distributed across chemical series.

Fit-for-Purpose Validation and Establishment of Assay Acceptance and Reporting Criteria of Dendritic Cell Activation Assay Contributing to the Assessment of Immunogenicity Risk.

Validation of key analytical and functional performance characteristics of in vitro immunogenicity risk assessment assays increases our confidence in utilizing them for screening biotherapeutics. Herein, we present a fit-for-purpose (FFP) validation of a dendritic cell (DC) activation assay designed to assess the immunogenicity liability of protein biotherapeutics. Characterization of key assay parameters was achieved using monocyte-derived DCs (MoDCs) treated with cell culture medium only (i.e., background control (BC)), keyhole limpet hemocyanin (KLH) as system positive control (SPC), and 2 therapeutic monoclonal antibodies (mAbs) with known clinical immunogenicity profiles (bococizumab and TAM163) as therapeutic controls (TCs). In the absence of established validation guidelines for primary cell-based assays, the present DC activation assay was validated using a novel FFP approach which allows more flexibility in selection of validation parameters and designing of experiments based on the intended use of the assay. The present FFP validation allowed us to understand the impact of experimental variables on assay precision, develop a clear concise readout for DC activation results, establish a reliable response threshold to define a result as a positive DC activation response, and define in-study donor acceptance criteria and cohort size. FFP validation of this DC activation assay indicated that the assay is sufficient to support its context of use, a preclinical immunogenicity risk management tool.

Criteria to Reevaluate Anti-drug Antibody Assay Cut Point Suitability in the Target Population.

After tier 1 and 2 cut points for anti-drug antibody (ADA) assays are derived during pre-study assay validation in a population, there is a need to verify the continued appropriateness of the previously derived cut points during sample analysis in the same or different populations, per FDA guidance (US HHS, FDA, CDER, CBER, 2019). Proper sample size-dependent criteria with statistical underpinning were derived and presented in this technical note to aid in assessing the appropriateness of tier 1 and tier 2 cut points, respectively.