Pharmacokinetic Properties of Humanized IgG1 and IgG4 Antibodies in Preclinical Species: Translational Evaluation.

Assessment of the factors that regulate antibody exposure-response relationships in the relevant animal models is critical for the design of successful translational strategies from discovery to the clinic. Depending on the specific clinical indication, preclinical development paradigms may require that the efficacy or dosing-related attributes for the existing antibody be assessed in various species when cross-reactivity of the lead antibody to the intended species is justified. Additionally, with the success of monoclonal antibodies for management of various human conditions, a parallel interest in therapeutic use of these novel modalities in various veterinary species has followed. The protective role of neonatal Fc receptor (FcRn) in regulation of IgG homeostasis and clearance is now well recognized and the "nonspecific clearance" of antibodies through bone marrow-derived phagocytic and vascular endothelial cells (via lysosomal processes) is modulated by interactions with FcRn receptors. In this study, we have attempted to examine the PK properties of human IgG antibodies in dog and monkey. These studies establish a translational framework for evaluation of IgG antibody PK properties across species.

Generic automated method for liquid chromatography-multiple reaction monitoring mass spectrometry based monoclonal antibody quantitation for preclinical pharmacokinetic studies.

Quantitation of therapeutic monoclonal antibodies (mAb) using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for pharmacokinetic (PK) studies is becoming an essential complement to traditional antibody-based ligand binding assays (LBA). Here we show an automated method to perform LC-MS/MS-based quantitation, with IgG1 conserved peptides, a heavy isotope labeled mAb internal standard, and anti-human Fc enrichment. All reagents in the method are commercially available with no requirement to develop novel assay-specific reagents. The method met traditional quantitative LC-MS/MS assay analytical characteristics in terms of precision, accuracy, and specificity. The method was applied to the pharmacokinetic study of a mAb dosed in cynomolgus monkey, and the results were compared with the immunoassay data. This methodology has the potential to benefit and accelerate the early biopharmaceutical development process, particularly by enabling PK analysis across species and candidate molecules with minimal method development.

Effects of PEGylation and immune complex formation on the pharmacokinetics and biodistribution of recombinant interleukin 10 in mice.

Interleukin 10 (IL-10) is a potent cytokine homodimer with multiple immunoregulatory functions. Here, we have characterized the effects of PEGylation and formation of human IL-10 (hIL-10)/humanized anti-human IL-10 (hαhIL-10) immune complexes in the pharmacokinetics, biodistribution, and biotransformation of IL-10 in mice. To assess the fate of native, PEGylated, and antibody-bound IL-10; we implemented an analytical set of fluorescence emission-linked assays. Plasma size exclusion chromatography analysis indicated that fluoro-labeled native and PEGylated murine IL-10 (PEG-mIL-10) are stable in the circulation. PEGylation of IL-10 resulted in a 21-fold increased exposure, 2.7-fold increase in half-life, and 20-fold reduction in clearance. Kidney is the major organ of disposition for both native and PEGylated mIL-10 with renal uptake directly related to systemic clearance. The fluorescence signal in the kidneys reached tissue/blood ratios up to 150 and 20 for native and PEG-mIL-10, respectively. hIL-10/hαhIL-10 immune complexes are detectable in the circulation without evidence of unbound or degraded hIL-10. The exposure of hIL-10 present in immune complexes versus that of hIL-10 alone increased from 0.53 to 11.28 µg · day/ml, with a half-life of 1.16 days and a 23-fold reduction in clearance. Unlike hIL-10 alone, antibody-bound hIL-10 was targeted mainly to the liver with minimal renal distribution. In addition, we found an 11-fold reduction (from 9.9 to 113 nM) in binding to the neonatal Fc receptor (FcRn) when the hαhIL10 antibody is conjugated to hIL-10. The potential changes in FcRn binding in vivo and increased liver uptake may explain the unique pharmacokinetic properties of hIL-10/hαhIL-10 immune complexes.

IL-33, an interleukin-1-like cytokine that signals via the IL-1 receptor-related protein ST2 and induces T helper type 2-associated cytokines.

Cytokines of the interleukin-1 (IL-1) family, such as IL-1 alpha/beta and IL-18, have important functions in host defense, immune regulation, and inflammation. Insight into their biological functions has led to novel therapeutic approaches to treat human inflammatory diseases. Within the IL-1 family, IL-1 alpha/beta, IL-1Ra, and IL-18 have been matched to their respective receptor complexes and have been shown to have distinct biological functions. The most prominent orphan IL-1 receptor is ST 2. This receptor has been described as a negative regulator of Toll-like receptor-IL-1 receptor signaling, but it also functions as an important effector molecule of T helper type 2 responses. We report a member of the IL-1 family, IL-33, which mediates its biological effects via IL-1 receptor ST 2, activates NF-kappaB and MAP kinases, and drives production of T(H)2-associated cytokines from in vitro polarized T(H)2 cells. In vivo, IL-33 induces the expression of IL-4, IL-5, and IL-13 and leads to severe pathological changes in mucosal organs.

The CD200 receptor is a novel and potent regulator of murine and human mast cell function.

CD200R is a member of the Ig supergene family that is primarily expressed on myeloid cells. Recent in vivo studies have suggested that CD200R is an inhibitory receptor capable of regulating the activation threshold of inflammatory immune responses. Here we provide definitive evidence that CD200R is expressed on mouse and human mast cells and that engagement of CD200R by agonist Abs or ligand results in a potent inhibition of mast cell degranulation and cytokine secretion responses. CD200R-mediated inhibition of FcepsilonRI activation was observed both in vitro and in vivo and did not require the coligation of CD200R to FcepsilonRI. Unlike the majority of myeloid inhibitory receptors, CD200R does not contain a phosphatase recruiting inhibitory motif (ITIM); therefore, we conclude that CD200R represents a novel and potent inhibitory receptor that can be targeted in vivo to regulate mast cell-dependent pathologies.

Characterization of the CD200 receptor family in mice and humans and their interactions with CD200.

CD200 (OX2) is a broadly distributed cell surface glycoprotein that interacts with a structurally related receptor (CD200R) expressed on rodent myeloid cells and is involved in regulation of macrophage function. We report the first characterization of human CD200R (hCD200R) and define its binding characteristics to hCD200. We also report the identification of a closely related gene to hCD200R, designated hCD200RLa, and four mouse CD200R-related genes (termed mCD200RLa-d). CD200, CD200R, and CD200R-related genes were closely linked in humans and mice, suggesting that these genes arose by gene duplication. The distributions of the receptor genes were determined by quantitative RT-PCR, and protein expression was confirmed by a set of novel mAbs. The distribution of mouse and human CD200R was similar, with strongest labeling of macrophages and neutrophils, but also other leukocytes, including monocytes, mast cells, and T lymphocytes. Two mCD200 receptor-like family members, designated mCD200RLa and mCD200RLb, were shown to pair with the activatory adaptor protein, DAP12, suggesting that these receptors would transmit strong activating signals in contrast to the apparent inhibitory signal delivered by triggering the CD200R. Despite substantial sequence homology with mCD200R, mCD200RLa and mCD200RLb did not bind mCD200, and presently have unknown ligands. The CD200 receptor gene family resembles the signal regulatory proteins and killer Ig-related receptors in having receptor family members with potential activatory and inhibitory functions that may play important roles in immune regulation and balance. Because manipulation of the CD200-CD200R interaction affects the outcome of rodent disease models, targeting of this pathway may have therapeutic utility.