Anti-PCSK9 antibody pharmacokinetics and low-density lipoprotein-cholesterol pharmacodynamics in nonhuman primates are antigen affinity-dependent and exhibit limited sensitivity to neonatal Fc receptor-binding enhancement.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) has emerged as an attractive therapeutic target for cardiovascular disease. Monoclonal antibodies (mAbs) that bind PCSK9 and prevent PCSK9:low-density lipoprotein receptor complex formation reduce serum low-density lipoprotein-cholesterol (LDL-C) in vivo. PCSK9-mediated lysosomal degradation of bound mAb, however, dramatically reduces mAb exposure and limits duration of effect. Administration of high-affinity mAb1:PCSK9 complex (1:2) to mice resulted in significantly lower mAb1 exposure compared with mAb1 dosed alone in normal mice or in PCSK9 knockout mice lacking antigen. To identify mAb-binding characteristics that minimize lysosomal disposition, the pharmacokinetic behavior of four mAbs representing a diverse range of PCSK9-binding affinities at neutral (serum) and acidic (endosomal) pH was evaluated in cynomolgus monkeys. Results revealed an inverse correlation between affinity and both mAb exposure and duration of LDL-C lowering. High-affinity mAb1 exhibited the lowest exposure and shortest duration of action (6 days), whereas mAb2 displayed prolonged exposure and LDL-C reduction (51 days) as a consequence of lower affinity and pH-sensitive PCSK9 binding. mAbs with shorter endosomal PCSK9:mAb complex dissociation half-lives (<20 seconds) produced optimal exposure-response profiles. Interestingly, incorporation of previously reported Fc-region amino acid substitutions or novel loop-insertion peptides that enhance in vitro neonatal Fc receptor binding, led to only modest pharmacokinetic improvements for mAbs with pH-dependent PCSK9 binding, with only limited augmentation of pharmacodynamic activity relative to native mAbs. A pivotal role for PCSK9 in mAb clearance was demonstrated, more broadly suggesting that therapeutic mAb-binding characteristics require optimization based on target pharmacology.

Measurement of in vivo therapeutic mAb concentrations: comparison of conventional serum/plasma collection and analysis to dried blood spot sampling.

BACKGROUND: Development of an alternative sampling method that uses small amounts of whole blood, such as dried blood spots (DBS), would be an advance in the quantitative assay field. Previously, we assessed the ability to quantitate therapeutic monoclonal antibodies present in DBS compared with a typical serum sample-based method, and concluded that measurements in DBS were reproducible and yielded methods that met requirements for precision, accuracy and sensitivity. The goal herein was to assess the measurement of therapeutic antibodies in DBS compared with serum and plasma in vivo. RESULTS: Comparison of DBS versus serum in Sprague-Dawley rats and DBS versus plasma in cynomolgus monkeys for measurement of antibody concentrations revealed a two- to three-fold difference in exposure between the samples. CONCLUSION: Overall, there was good correlation between DBS versus serum and DBS versus plasma, but there was a discrepancy in DBS exposures, presumably attributable to hematocrit and recovery effects.

Assessment of DBS technology for the detection of therapeutic antibodies.

BACKGROUND: Quantitating levels of therapeutic proteins in serum or plasma is critical for determining exposure levels and establishing pharmacokinetic parameters. Traditionally, whole blood is not used, mostly due to its inability to be frozen and potential issues with assay interference. To investigate the ability to reproducibly quantitate therapeutic antibodies present in dried blood spots (DBS) compared with a typical serum sample-based method, we used ELISA and electrochemiluminescence immunoassays to measure therapeutic drug levels present in DBS samples. RESULTS: Measurements of antibody therapeutics in DBS were reproducible, yielding methods that are precise, accurate and met expected sensitivity requirements compared with plasma-based methods. CONCLUSIONS: DBS are a viable alternative to the use of conventional serum or plasma samples for quantitative therapeutic antibody measures.