Antibodies with pH-dependent binding to both target antigens and neonatal Fc receptor (FcRn) provide an alternative tool to conventional neutralizing antibodies, particularly for therapies where reduction in antigen level is challenging due to high target burden. However, the requirements for optimal binding kinetic framework and extent of pH dependence for these antibodies to maximize target clearance from circulation are not well understood. We have identified a series of naturally-occurring high affinity antibodies with pH-dependent target binding properties. By in vivo studies in cynomolgus monkeys, we show that pH-dependent binding to the target alone is not sufficient for effective target removal from circulation, but requires Fc mutations that increase antibody binding to FcRn. Affinity-enhanced pH-dependent FcRn binding that is double-digit nM at pH 7.4 and single-digit nM at pH 6 achieved maximal target reduction when combined with similar target binding affinities in reverse pH directions. Sustained target clearance below the baseline level was achieved 3 weeks after single-dose administration at 1.5 mg/kg. Using the experimentally derived mechanistic model, we demonstrate the essential kinetic interplay between target turnover and antibody pH-dependent binding during the FcRn recycling, and identify the key components for achieving maximal target clearance. These results bridge the demand for improved patient dosing convenience with the “know-how” of therapeutic modality by design.
Aim: Volumetric absorptive microsampling (VAM) is being increasingly applied in nonclinical pharmacokinetic studies. Although there are published results for VAM use in small molecule pharmacokinetics (PK) studies, there is limited data on the utility of VAM for protein therapeutics. Results: We describe the use of Mitra® microsampler for blood sampling, ELISA quantitation and PK analysis of two marketed therapeutic monoclonal antibodies administered to rat. Results generated for these monoclonal antibodies using Mitra® were compared with both serum and whole blood sampling methods in the same study. Conclusion: The low relative standard deviation among the three sets of PK data suggest that Mitra® microsampler could be useful in early nonclinical PK studies for protein therapeutics where reduction and refinement of animal use is desirable.
Ang1 is a soluble ligand to receptor Tie2, and increasing the circulating Ang1 level may improve vascular stabilization under certain disease conditions. Here, we found that the circulating Ang1 level was significantly increased in cynomolgus monkeys treated with non-neutralizing anti-Ang1 antibodies. Improving the antibodies’ pharmacokinetic properties by IgG Fc mutations further increased the circulating Ang1 level. However, the mutations decreased the thermal stability of the molecules, which may limit their use as therapeutic antibodies. Nevertheless, we showed that non-neutralizing antibodies may have therapeutic potential by increasing the level of a target molecule in the circulation.
Prior to clinical studies, the pharmacokinetics (PK) of antibody-based therapeutics are characterized in preclinical species; however, those species can elicit immunogenic responses that can lead to an inaccurate estimation of PK parameters. Immunodeficient (SCID) transgenic hFcRn and C57BL/6 mice were used to characterize the PK of three antibodies that were previously shown to be immunogenic in mice and cynomolgus monkeys. Four mouse strains, Tg32 hFcRn SCID, Tg32 hFcRn, SCID and C57BL/6, were administered adalimumab (Humira Ò ), mAbX and mAbX-YTE at 1 mg/kg, and in SCID strains there was no incidence of immunogenicity. In non-SCID strains, drug-clearing ADAs appeared after 4-7 days, which affected the ability to accurately calculate PK parameters. Single species allometric scaling of PK data for Humira Ò in SCID and hFcRn SCID mice resulted in improved human PK predictions compared to C57BL/6 mice. Thus, the SCID mouse model was demonstrated to be a useful tool for assessing the preclinical PK of immunogenic therapeutics.
This improvement was essential to enable detection of GDF11 in biological samples, and without the technology the sensitivity achieved on the other platforms would not have been sufficient. Other factors such as ease of use, cost, assay time and automation capability can also be considered when developing custom immunoassays, based on the requirements of the bioanalyst.
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