Monoclonal antibodies with identical Fc sequences can bind to FcRn differentially with pharmacokinetic consequences.

The neonatal Fc receptor (FcRn) is a key determinant of IgG homeostasis. It binds to the Fc domain of IgG in a strictly pH-dependent manner and protects IgG from lysosomal degradation. The impact of FcRn salvage pathway on IgG monoclonal antibody (mAb) pharmacokinetics (PK) has been well established. In this report, a set of mAbs with wild-type human Fc sequences but different Fab domains were used to examine the potential impact of Fab domain on in vitro FcRn binding and in vivo PK. We were surprised to find that mAbs with the same wild-type human Fc sequences but different Fab domains were shown to bind FcRn with considerable differences in both the binding at acidic pH and the dissociation at neutral pH, suggesting that the Fab domain may also have an impact on FcRn interaction. For these mAbs, no relationship between the FcRn binding affinity at acidic pH and in vivo PK was found. Instead, an apparent correlation between the in vitro FcRn dissociation at neutral pH and the in vivo PK in human FcRn mice, nonhuman primates and humans was observed. Our results suggested that the Fab domain of mAbs can affect their interaction with FcRn and thus their pharmacokinetic properties and that in vitro FcRn binding/dissociation assays can be a useful screening tool for pharmacokinetic assessment of mAbs with wild-type Fc sequences.

Pharmacokinetics of IgG1 monoclonal antibodies produced in humanized Pichia pastoris with specific glycoforms: a comparative study with CHO produced materials.

A glycoengineered Pichia pastoris host was used to produce an IgG1 with either afucosylated N-glycosylation (afucosylated biantennary complex) or without N-glycosylation (N297A) while a wild type P. pastoris host was used to produce an IgG1 containing fungal-type N- and O-linked glycosylation. The PK properties of these antibodies were compared to a commercial IgG1 produced in CHO cells following intravenous administration in wild type C57B6, FcγR-/- or hFcRn transgenic mice. MAbs produced in glycoengineered yeast exhibited similar PK properties in wild type mice or FcγR-/- mice with respect to clearance (CL), volume of distribution at steady-state (Vss) and half-life (t(1/2)) to that produced in mammalian (CHO) cells, while the mAb produced in wild type yeast exhibited ∼2-3-fold faster CL, which might be due to the high mannose content interacting with mannose receptors. Furthermore, in vitro binding affinity to human FcRn or mouse FcRn was similar between the reference mAb and mAbs produced in humanized yeast, and the glycovariants produced in humanized yeast exhibited similar PK patterns in human FcRn transgenic mice and in wild type mice. These results suggest the potential application of P. pastoris as a production platform for clinically viable mAbs.

Impact of methionine oxidation in human IgG1 Fc on serum half-life of monoclonal antibodies.

IgG monoclonal antibodies (mAbs) consist of two Fab fragments and one Fc fragment. The Fab fragments contain the variable regions and are responsible for drug specificity (via antigen binding); the Fc fragment contains constant regions and is responsible for effector functions (via interactions with Fcγ receptors) and extended serum half-life (via interaction with the neonatal Fc receptor, FcRn). There are two conserved methionine (Met) residues located in the FcRn binding site of the Fc fragment. It has been shown previously that oxidation of these two Met residues decreases the binding affinity to FcRn. We have further evaluated the impact of Met oxidation on serum half-lives of two humanized IgG1 mAbs in transgenic mice with human FcRn. Variable oxidation levels were obtained by several procedures: exposure to an oxidizing agent, accumulation during extended refrigerated storage, or chromatographic separation. Our results show that Met oxidation can result in a significant reduction of the serum circulation half-life and the magnitude of the change correlates well with the extent of Met oxidation and changes in FcRn binding affinities. The relatively low levels of Met oxidation accumulated during 3 years of refrigerated storage had minimal impact on FcRn binding and no detectable impact on the serum half-life.

Effects of the prototype serotonin 5-HT(1B/1D) receptor agonist sumatriptan and the calcitonin gene-related peptide (CGRP) receptor antagonist CGRP(8-37) on myocardial reactive hyperemic response in conscious dogs.

The triptans, serotonin 5-HT(1B/1D) receptor agonists exemplified by sumatriptan, are a mainstay migraine therapy but have class labeling contraindicating their use in patients with coronary artery disease. Triptans constrict human coronary artery in vitro, and there are case reports of myocardial infarction in patients using sumatriptan. However, preclinical studies with sumatriptan in normal dogs have failed to demonstrate effects on resting coronary flow. Calcitonin gene-related peptide (CGRP) receptor antagonism, exemplified by the prototype CGRP receptor antagonist peptide CGRP(8-37), is a new antimigraine mechanism which also has been reported to have no effect on coronary flow in normal, non-stressed animals. The goal of the present studies was to compare the effects of sumatriptan (10microg/kg/min i.v.) and CGRP(8-37) (30microg/kg/min i.v.) on systemic and coronary hemodynamics in conscious dogs under resting conditions and during myocardial reactive hyperemia following a brief 15s of coronary artery occlusion. Neither CGRP(8-37) nor sumatriptan affected resting coronary flow. However, whereas CGRP(8-37) had no effect on myocardial reactive hyperemic response, sumatriptan reduced peak reactive hyperemic coronary artery blood flow (baseline vs treatment: 75.4+/-12.7 vs 60.0+/-10.3ml/min, P<0.05), reactive hyperemic flow (16.7+/-5.2 vs 11.6+/-3.3ml, P<0.05) and the repayment of coronary blood flow debt following coronary artery occlusion (484+/-76 vs 369+/-57%, P<0.05), indicating an impairment in coronary blood flow reserve. The positive control nitric oxide synthase inhibitor L-NNA (30mg/kg/30min i.v.) likewise significantly attenuated myocardial reactive hyperemic response. These findings provide evidence for a differentiation between CGRP receptor antagonism and triptan effects on coronary vascular function.