A strategy for risk mitigation of antibodies with fast clearance.

A majority of human therapeutic antibody candidates show pharmacokinetic properties suitable for clinical use, but an unexpectedly fast antibody clearance is sometimes observed that may limit the clinical utility. Pharmacokinetic data in cynomolgus monkeys collected for a panel of 52 antibodies showed broad distribution of target-independent clearance values (2.4-61.3 mL/day/kg), with 15 (29%) having clearance > 10 mL/day/kg. Alteration in the interaction with the recycling FcRn receptor did not account for the faster than expected clearance observed for the antibodies; off-target binding was presumed to account for the fast clearance. We developed an assay based on ELISA detection of non-specific binding to baculovirus particles that can identify antibodies having increased risk for fast clearance. This assay can be used during lead generation or optimization to identify antibodies with increased risk of having fast clearance in both humans and cynomolgus monkeys, and thus increase the likelihood of obtaining a suitable drug candidate.

Noncompartmental kinetic analysis of DCE-MRI data from malignant tumors: Application to glioblastoma treated with bevacizumab.

Dynamic contrast enhanced MRI contrast agent kinetics in malignant tumors are typically complex, requiring multicompartment tumor models for adequate description. For consistent comparisons among tumors or among successive studies of the same tumor, we propose to estimate the total contrast agent-accessible volume fraction of tumor, including blood plasma, v(pe), and an average transfer rate constant across all tumor compartments, K(trans.av), by fitting a three-compartment tumor model and then calculating the area under the tumor impulse-response function (= v(pe)) and the ratio area under the tumor impulse response function over mean residence time in tumor (= K(trans.av)). If the duration of dynamic contrast enhanced MRI was too short to extrapolate the tumor impulse-response function to infinity with any confidence, then conditional parameters v(pe)(*) and K(trans.av*) should be calculated from the available incomplete impulse response function. Median decreases of 33% were found for both v(pe)(*) and K(trans.av*) in glioblastoma patients (n = 16) 24 hours after the administration of bevacizumab (P < 0.001). Median total contrast-enhancing tumor volume was reduced by 18% (P < 0.0001). The combined changes of tumor volume, v(pe)(*), and K(trans.av*) suggest a reduction of true v(pe), possibly accompanied by a reduction of true K(trans.av). The proposed method provides estimates of a scale and a shape parameter to describe contrast agent kinetics of varying complexity in a uniform way.

An automated method for nonparametric kinetic analysis of clinical DCE-MRI data: application to glioblastoma treated with bevacizumab.

Here, we describe an automated nonparametric method for evaluating gadolinium-diethylene triamine pentaacetic acid (Gd-DTPA) kinetics, based on dynamic contrast-enhanced-MRI scans of glioblastoma patients taken before and after treatment with bevacizumab; no specific model or equation structure is assumed or used. Tumor and venous blood concentration-time profiles are smoothed, using a robust algorithm that removes artifacts due to patient motion, and then deconvolved, yielding an impulse response function. In addition to smoothing, robustness of the deconvolution operation is assured by excluding data that occur prior to the plasma peak; an exhaustive analysis was performed to demonstrate that exclusion of the prepeak plasma data does not significantly affect results. All analysis steps are executed by a single R script that requires blood and tumor curves as the sole input. Statistical moment analysis of the Impulse response function yields the area under the curve (AUC) and mean residence time (MRT). Comparison of deconvolution results to fitted Tofts model parameters suggests that AUCMRT and AUC of the Impulse response function closely approximate fractional clearance from plasma to tissue (K(trans)) and fractional interstitial volume (v(e)). Intervisit variability is shown to be comparable when using the deconvolution method (11% [AUCMRT] and 13%[AUC]) compared to the Tofts model (14%[K(trans)] and 24%[v(e)]). AUC and AUCMRT both exhibit a statistically significant decrease (P < 0.005) 1 day after administration of bevacizumab.

A novel, Q-PCR based approach to measuring endogenous retroviral clearance by capture protein A chromatography.

Quantification of virus removal by the purification process during production is required for clinical use of biopharmaceuticals. The current validation approach for virus removal by chromatography steps typically involves time-consuming spiking experiments with expensive model viruses at bench scale. Here we propose a novel, alternative approach that can be applied in at least one instance: evaluating retroviral clearance by protein A chromatography. Our strategy uses a quantitative PCR (Q-PCR) assay that quantifies the endogenous type C retrovirus-like particle genomes directly in production Chinese Hamster Ovary (CHO) cell culture harvests and protein A pools. This eliminates the need to perform spiking with model viruses, and measures the real virus from the process. Using this new approach, clearance values were obtained that was comparable to those from the old model-virus spike/removal approach. We tested the concept of design space for CHO retrovirus removal using samples from a protein A characterization study, where a wide range of chromatographic operating conditions were challenged, including load density, flow rate, wash, pooling, temperature, and resin life cycles. Little impact of these variables on CHO retrovirus clearance was found, arguing for implementation of the design space approach for viral clearance to support operational ranges and manufacturing excursions. The viral clearance results from Q-PCR were confirmed by an orthogonal quantitative product-enhanced reverse transcriptase (Q-PERT) assay that quantifies CHO retrovirus by their reverse transcriptase (RT) enzyme activity. Overall, our results demonstrate that protein A chromatography is a robust retrovirus removal step and CHO retrovirus removal can be directly measured at large scale using Q-PCR assays.

Reactive hyperemia and BOLD MRI demonstrate that VEGF inhibition, age, and atherosclerosis adversely affect functional recovery in a murine model of peripheral artery disease.

PURPOSE: To develop magnetic resonace imaging (MRI) methods for functional assessment of arteriogenesis in a murine model of peripheral artery disease to quantify the influences of vascular endothelial growth factor (VEGF), age, and atherosclerosis. MATERIALS AND METHODS: Reactive hyperemia (RH), which was induced using a device designed for remote and transient occlusion of the aorta and vena cava, was measured by blood-oxygen-level-dependent MRI. Twenty-eight days after femoral artery ligation, peak height (PH) and time to peak (TTP) of the RH response was compared with sham-operated animals in 10-week-old C57Bl6, 9-month-old C57Bl6, and 9-month-old Ldlr(-/-)Apobec(-/-) mice. The contribution of VEGF to functional recovery was assessed in young mice. Angiogenesis was quantified using an anti-PECAM1 radioimmunoassay. RESULTS: In young animals, angiogenesis was maximal 7 days after ligation, whereas functional recovery took 28 days. Inhibition of VEGF eliminated the angiogenesis seen at 7 days and reduced RH (PH, P < 0.05). At day 28, RH was altered in old (TTP, P < 0.05) and atherosclerotic (PH, P < 0.05; TTP, P < 0.05) animals. RH was different in young, old, and atherosclerotic sham animals. Old and atherosclerotic mice showed reduced angiogenesis. CONCLUSION: The method presented herein can provide a sensitive assay for the functional assessment of arteriogenesis and highlights the contribution of VEGF, age, and atherosclerosis to this process.