Pharmacokinetics

Assessments of Antibody Biodistribution

The Journal of Clinical Pharmacology 2015 55(S3) S29–S38 © 2015, The American College of Clinical Pharmacology DOI: 10.1002/jcph.365

Patrick M. Glassman, BS1, Lubna Abuqayyas, PhD2, and Joseph P. Balthasar, PhD1

Abstract Monoclonal antibody (mAb) therapeutics are in use for several disease conditions, and have generally shown excellent clinical benefit, in large part due to their high specificity and affinity for target proteins. As this therapeutic class continues to grow in size, improved understanding of the mechanisms controlling mAb biodistribution and protein binding may be expected to allow better prediction of safety and efficacy. Due to the large size and polarity of antibodies, rates of mAb distribution and elimination are typically much slower than those reported for small molecule drugs. Additionally, high affinity interaction with target proteins will often influence mAb pharmacokinetics, leading to complex, nonlinear tissue distribution and elimination. In this report, we summarize key determinants of mAb disposition, methods for assessing antibody exposure and protein binding, and model-based approaches that may be utilized to predict mAb pharmacokinetics.

Keywords biologics, biotechnology (BTN), pharmaceutics (PCT), pharmacodynamics (PDY), pharmacokinetics and metabolism

The origination of hybridoma technology by Kohler and Milstein,1 followed by the advent of modern biotechnology techniques, has led to a significant interest in the development of monoclonal antibodies (mAbs) for the treatment of many diseases. There are currently 30 mAb products approved by the FDA for a wide variety of indications, with an additional
300 mAbs in various stages of clinical development. Safety, efficacy, and dosepotency of therapeutic mAbs is influenced by pharmacokinetic rate processes associated with mAb absorption, distribution, and elimination.2 In this review, we will discuss key determinants of mAb biodistribution, analytical methods for assessing mAb concentrations in plasma and in tissues, and model-based approaches to describe and predict mAb pharmacokinetics (PK).

Determinants of Antibody Biodistribution Tissue distribution of monoclonal antibodies is governed by a myriad of factors related to the antibody molecular structure, vascular endothelium, fluid flow rates, interaction with target, and interaction with Fc receptors. Understanding the quantitative influence of these factors on the kinetics of mAb disposition is necessary for accurate predictions of antibody exposure in plasma and in tissues, and facilitates the prediction of mAb efficacy, toxicity, and dose-potency. In this section, we outline several key determinants of antibody biodistribution, and discuss their influence on the observed in vivo disposition of monoclonal antibody drugs. Distribution Kinetics Antibody extravasation may occur by paracellular or by transcellular transport.2 Due to the relatively large

molecular weight and polarity of IgG antibodies, mAbs show slow rates of diffusion through vascular endothelial cell membranes and, thus, convective transport through paracellular pores is considered to be the primary mechanism of antibody extravasation in tissues. The rate and efficiency of convective transport of mAb into tissue interstitial fluids is dependent on the net fluid flow rate and on the nature of the paracellular pores within the vascular endothelium. The larger the pore size, the lower the restriction for convective transport. Hence, higher antibody concentrations are often observed within tissues associated with fenestrated or discontinuous capillary endothelia (spleen, liver). The distribution of mAb to the brain is limited due to the tight junctions in the brain vascular endothelium, and plasma:brain concentration ratios are typically in the range of 500:1.3 On the other hand, the vasculature in most tumors is quite porous, facilitating the penetration and accumulation of macromolecules within tumors.4 Receptor-mediated endocytosis of IgG may also contribute to the movement of IgG from blood to tissues (discussed below).

1

Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, University at Buffalo, The State University of New York, Buffalo, NY 14214, USA 2 Amgen, Thousand Oaks, CA, USA Submitted for publication 30 April 2014; accepted 14 July 2014. Corresponding Author: Joseph P. Balthasar, Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, University at Buffalo, 452 Kapoor Hall, Buffalo, NY 14214, USA Email: [email protected]

S30 Antibody–Antigen Binding Following entry of mAb into tissue interstitial fluids, antibody spreads from the point of extravasation via convection and diffusion. Again, owing to the relatively large size of macromolecular proteins, diffusion proceeds slowly. For example, the effective diffusivity coefficients for bovine albumin (67 kDa) and IgM (900 kDa) in LS174T tumor xenografts have been estimated by Brown et al5 to be 1.19  107 and 7.68  108 cm2/s. Similarly, Jain and Baxter6 estimated the effective diffusion coefficient for IgG to be 1.3  108 cm2/s. Antibody distribution within tissues may be hindered by binding to tissue proteins. Several groups have reported that high affinity mAb binding in tissues creates a barrier to mAb distribution following extravasation. This phenomenon, known as the “binding site barrier,” is considered to be partly responsible for the heterogeneous distribution of high affinity mAbs within tumors.7 The binding site barrier may be overcome by increasing the dose (saturation of target) and/or by lowering antibody affinity to its target antigen.7–10 The influence of mAb– target binding affinity on mAb distribution in tumors was nicely demonstrated, with use of a series of anti-HER2 mAb, in an elegant study by Rudnick et al.11 The authors measured the average distance of mAb distribution from tumor capillaries, and found an inverse relationship between mAb affinity and intra-tumoral distribution, where moderate affinity antibodies were found to distribute more extensively within tumors when compared to high affinity mAb. Although reducing affinity to target antigens may allow more homogenous mAb distribution within tissue, this benefit may be offset by decreased tissue selectivity. The binding site barrier may also be overcome by simply administering saturating doses of mAb. Of note, the relationship between dose and the degree of target saturation will be determined by several factors, including antibody-target affinity, target “body-load” or binding capacity, target accessibility, and rates of target turnover. Saturation of target binding in tissue may not only promote mAb distribution by minimizing the influence of the binding site barrier, but target saturation may also lead to a substantial decrease in the efficiency of target-mediated mAb elimination in tissue, which may lead to increased tissue:plasma exposure ratios (eg, in conditions where target-mediated elimination acts as a “sink” within tissue).12 Interactions With Fc Receptors FcRn. FcRn is a heterodimeric protein comprised of b2-microglobulin (b2m) and a 50 kDa a-chain.13 FcRn is widely expressed in many tissues, including the vascular endothelium, which is considered to be a primary site of IgG catabolism.14 The binding of IgG to FcRn is strikingly pH dependent, with no interaction occurring at physiological pH (7.4), and high affinity binding at the

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mildly acidic pH associated with early endosomes (6.0– 6.5).15 The receptor functions to rescue IgG antibodies from intracellular catabolism, “recycling” antibody to extracellular fluid following fluid-phase endocytosis. Saturation of FcRn, or knock-out of FcRn, leads to substantial increases in the rate of IgG elimination.16–20 For example, Hansen and Balthasar showed that the clearance of 7E3, a murine IgG1 mAb, was increased by
240% in rats after administration of high doses of pooled IgG to saturate FcRn.21 The IgG–FcRn interaction has been hypothesized to facilitate IgG transport from blood to tissues, including transport across the gastro-epithelium,22 placenta,23 and pulmonary epithelium.24 However, the quantitative significance of FcRn-mediated transport as a determinant of IgG distribution kinetics has not been convincingly demonstrated to date. Preclinical experiments have shown that FcRn-knockout leads to 10–15-fold reductions in plasma and tissue exposure of mAb (relative to data collected from wild-type, control mice); however, no substantial changes in plasma:tissue mAb exposure ratios were found.25 These data are consistent with a significant influence of FcRn on mAb elimination, but with an absence of substantial effects of FcRn on the kinetics of mAb distribution. The role of FcRn in IgG brain distribution has been the focus of several investigations, and conflicting data have been reported. Based on investigations of the kinetics of IgG efflux from the brain, and following their demonstration of FcRn expression in brain capillaries, Pardridge and coworkers first hypothesized that FcRn mediates “reverse transcytosis” of IgG from brain to blood, thus minimizing the extent of IgG exposure in the brain.26 This hypothesis has been supported by some in vitro and in vivo investigations.27,28 However, studies in FcRn a-chain/  and in b2m/ mice,3,29 with intravenous administration of mAb and with extensive sampling of brain tissue, have not shown significant differences in plasma:brain or blood:brain area under the curve (AUC) ratios. Given the differing reports in the literature, the role of FcRn in IgG distribution remains somewhat uncertain. FcgR. FcgR are expressed on several cell types, including macrophages, monocytes, neutrophils, eosinophils, beta cells, T-lymphocytes, mast cells and in professional antigen-presenting cells in blood and in tissues (eg, bone marrow, thymus, lung, liver, and spleen).30–34 FcgR differ substantially with respect to IgG affinity and function. The class includes low to intermediate affinity activating receptors (FcgRIII and IV, Kd ¼ 106 to 107 M), high affinity activating receptors (FcgRI, KD ¼ 108 to 109 M), and low affinity inhibitory receptors (FcgRII, Kd
106).35–38 The influence of FcgR on mAb effects is well accepted; however, despite substantial discussion,39–44 there is a no clear consensus of opinion regarding the role

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of FcgR on the tissue distribution and elimination of monoclonal antibodies. Cartron and coworkers have provided clear evidence of the significance of FcgR polymorphism on mAb (rituximab) efficacy in the treatment of non-Hodgkin lymphoma. In their study, they reported significantly improved survival for patients who express an FcgRIIIA polymorph with high affinity for IgG, relative to survival data found for patients who express a low affinity FcgRIIIA polymorph.45 Subsequent investigations demonstrated that rituximab pharmacokinetics were not significantly different in groups of patients expressing high, intermediate, or low affinity FcgRIIIA polymorphs.46 On the other hand, different elimination rates were observed for engineered IgG variants with a range of affinities to FcgR in other studies.43,44 Recently, Abuqayyas and Balthasar investigated the role of FcgR in the disposition of mAbs via the use of FcgRI/RIII/ and FcgRIIb/ mice and have found that knockout of these receptors did not lead to altered plasma and tissue disposition of non-specific29,47 or targeted48 mAbs. This work is consistent with findings from earlier cell culture experiments49 and with recent in vivo investigations in monkeys.50 Although FcgR may be a determinant of the PK of selected mAbs (such as those that form large immune complexes in vivo, or mAbs that have been engineered for high-affinity FcgR binding), FcgR do not appear to provide a substantial “general” effect on mAb PK (in contrast to the well-described protective function of FcRn). Tissue Clearance The rate of antibody clearance from tissue is thought to be primarily dependent on the rate of convective elimination of antibody from tissue and the rate of antibody catabolism.2,51 Fluid is continuously drained from the tissue interstitial space via the lymphatic system. Lymphatic capillaries are much larger in diameter, and more fenestrated, than blood capillaries; as such, there is little restriction to the convective clearance of IgG antibodies from tissues. More efficient convective clearance from tissue, relative to the efficiency of convective uptake into tissue, largely explains why unbound antibody concentrations are lower in tissues relative to the concentrations found in plasma. Antibody catabolism within tissues may occur via specific interaction with target proteins (target-mediated elimination) or by non-specific processes (eg, fluid phase endocytosis and intracellular catabolism). The significance of target-mediated elimination is a function of antibody dose, target accessibility, target expression level, target turnover kinetics, antibody–antigen binding rates, and rates of internalization of the mAb–target complex. Target-mediated disposition, which is commonly observed for therapeutic mAbs,52 may significantly affect the distribution of antibodies in tissue.

S31 Additional Factors Glycosylation may affect the rate and extent of mAb elimination and distribution by masking or exposing immunogenic sites. Additionally, it is postulated that disposition kinetics may be significantly influenced by mAb surface charge, as mAb with a net negative charge at physiological pH may be repelled from the surface of cell membranes, which also show a net negative charge due to the presence of charged membrane components (eg, sialic acids, glycosaminoglycans).53 This charge repulsion has been proposed to limit rates of extravasation and fluid phase endocytosis of negatively charged mAb, decreasing mAb distribution and clearance. Increases in rates of distribution and clearance are expected for mAb bearing a net positive charge. However, there are conflicting reports regarding the effects of charge on mAb pharmacokinetics,53–56 and more thorough investigations are needed to assess the effect of charge, and the effect of the surface distribution of charge, on mAb disposition. The amount of antibody in tissues and at the biophase is highly dependent on antibody plasma concentration. Factors that may affect antibody concentrations in plasma will eventually affect antibody exposure in tissues. Antidrug antibodies (ADA), if present within the subject, may bind to mAb, forming large immune complexes, with rapid elimination of the ADA–drug complexes by phagocytosis. On the other hand, ADA–drug complexes may, in certain cases, demonstrate slow elimination from the systemic circulation, leading to increased exposure, localization, or accumulation of the mAb in plasma and in tissues.57 Concomitant administration of an antibody with other antibodies or with small molecule drugs may also affect the disposition of antibody in tissues. Drugs that affect any of the aforementioned determinants of antibody disposition may lead to alterations in mAb distribution in tissues. For example, we found that treatment of xenograft bearing mice with an anti-VEGF mAb decreased the tumor distribution of an anti-CEA antibody by
50%, presumably by anti-VEGF mediated reductions in tumor vascularization, blood flow, and vascular permeability.58 Additionally, co-administration of trastuzumab and paclitaxel resulted in 1.5-fold increase in trastuzumab plasma concentrations, which may relate to effects of paclitaxel on HER2 expression and trastuzumab TMD.59

Methods to Quantify Antibody Concentrations in Tissues Thorough examination of mAb distribution requires the development and validation of bioanalytical methods capable of assessing antibody concentrations in tissues. Various techniques have been described in the literature, utilizing both labeled and unlabeled mAbs. In this section, we will discuss four assay types often used to assess mAb

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S32 tissue distribution: enzyme-linked immunosorbent assay (ELISA), radioisotope quantification, imaging, and liquid chromatography–mass spectrometry (LC–MS). For each, we will describe the general considerations required for use of the technique, as well as strengths and limitations (summarized in Table 1). Additionally, we will discuss the application of methods to assess the interaction of mAbs with their target proteins. ELISA ELISA assays are often used as an indirect measurement of mAb concentrations in biological fluids, utilizing a secondary antibody linked to an enzyme (eg, alkaline phosphatase, horseradish peroxidase) to report the presence of an unlabeled antibody of interest. These assays will typically have high sensitivity, although matrix effects may be significant, particularly when using ELISAs to assess mAb concentrations in tissue samples. Additionally, validation of ELISAs for quantification of mAb in tissue is often complicated by uncertainties in the efficiency of recovery. In most cases, recovery from tissue is evaluated by use of quality control samples where mAb

has been added (spiked in) to prepared homongenates. This type of assessment may overestimate recovery, in part due to the possible loss of mAb during the homogenization process. Antigen-capture ELISAs utilize a specific reagent (eg, purified antigen/peptide or anti-idiotypic antibody) to bind the mAb of interest in biological fluids. Assay performance may be highly dependent on the selectivity and affinity of the capture reagent. For example, Blasco et al60 compared the plasma PK of rituximab using peptide-based and anti-idiotypic antibody-based antigencapture ELISAs, and found dramatic differences in assayed mAb concentrations, possibly due to differences in the detection of rituximab bound to soluble target. Species-specific antibody capture ELISAs generally require little development time, as the capture reagent (eg, anti-human IgG) does not need to be specific for the mAb of interest. Although species-specific assays may be quickly and easily applied for preclinical investigations, it is not possible to carry these assays forward for clinical use (as endogenous human IgG would compete with mAb for binding to the capture antibody). Additionally, this

Table 1. Comparison of Assay Formats for Assessment of Monoclonal Antibody Biodistribution Assay format Enzyme-linked immunosorbent assay (ELISA) Antigen-capture ELISA Species-specific ELISA

Radioisotope quantification

Autoradiography

Microautoradiography

Whole body autoradiography

Positron emission tomography

Fluorescence imaging

Liquid chromatography–mass spectrometry

Strengths High sensitivity Does not require labeling of mAb Distinguishes free/total mAb Minimal time for development

Long half-life of label High sensitivity Minimal sample manipulation Long half-life of label

Assessment of distribution at cellular level High resolution and sensitivity Maintenance of tissue structure Can be combined with other methods Quantitative assessment of distribution Can visualize all tissues simultaneously Freezing allows snapshot of distribution Relatively high spatial resolution Non-invasive technique Can be used clinically High sensitivity May be coupled with other imaging techniques Non-invasive technique Enhanced signal-to-noise ratio Does not require radioisotopes Can detect unlabeled antibody Can be used clinically Allows detection of chemical stability

Weaknesses Significant matrix effects Nonlinear standard curves Relatively poor precision Cannot be used clinically Interference by catabolites Potential for cross-reactivity Does not distinguish free/total mAb Label may alter disposition Not suitable for chronic dosing Cannot be used clinically Label may alter disposition Relatively low-throughput Cannot be used clinically Generally considered tedious More technically challenging

Artifacts of freezing

Low resolution Short half-life of label Label may alter disposition Label may alter disposition Short path length of free light Challenging validation Potentially low-throughput

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type of ELISA is not likely to distinguish between total and free mAb concentrations, and the use of polyclonal capture reagents may lead to the detection of IgG catabolites in samples. Radiometric Tissue Analysis A common method for assessment of mAb tissue distribution in preclinical studies is via quantification of radiolabeled antibody. 125I is often employed in preclinical work, as it has a relatively long half-life (60 days), it is easy to detect using a gamma counter (gamma emitter), and there are several well-established methods for protein iodination (eg, using oxidizing agents such as Chloramine-T25). Of course, the analysis is via measurement of the radiolabel, and it is necessary to assure that the labeling method does not alter mAb disposition, and that mAb quantification is not skewed by the presence of labeled mAb catabolites. A second radiolabel that is often used to assess mAb distribution is 111In, which has a halflife of approximately 3 days and is conjugated to antibody using chelating agents.61 A common observation for indium-labeled mAbs is the accumulation of label within cells expressing internalizing target,62 due to the inability of the label to escape cells following antibody degradation within the lysosome. Because of this property of the label, while plasma PK may appear similar in a comparison of 125 I and 111In labeled mAbs, accumulation within targetexpressing organs may appear different. It is advisable to assess the utility of radiolabeled mAb for PK investigations by comparison of results with use of a second assay method (eg, ELISA). Additionally, it is best to limit the use of this method to short-term, single-dose PK studies, as it becomes increasingly difficult to rule out the confounding influence of radiolabeled catabolites with increasing time after initiation of dosing. Imaging Several imaging approaches have been described in the literature for the assessment of mAb tissue distribution, each with their own strengths and weaknesses. Autoradiography, which detects radioactivity using photographic film, imaging plates, or nuclear emulsions, may be applied at macro- and microscopic levels. Macroscopic, whole body autoradiography (WBA) was first described for use in preclinical biodistribution studies by Ullberg.63 WBA allows for assessment of distribution in all tissues simultaneously, and it often provides excellent resolution. WBA is well suited for use with long-lived radioisotopes (eg, 125I, 14C), which permits studies to be carried out over a longer duration at lower doses. In one recent example, WBA was employed to allow rapid, qualitative assessment of the disposition of mAb in control and FcgRknockout mice.47 Micro-autoradiography allows for investigation of intra-tissue drug distribution, via imaging of tissue sections of 5–10 mm thickness. This method has

S33 been applied to assess mAb distribution within tissues such as the eye64 and tumor.65 Compared to WBA, this method allows much greater resolution; however, sample processing and method validation is often quite challenging. There is growing interest in the use of non-invasive imaging techniques to allow assessment of mAb biodistribution without requirement for the collection of blood or tissue samples (enabling use in living subjects, including human patients). Positron emission tomography (PET), which detects the annihilation of positrons with electrons via coincidence events (two-photon emission), affords excellent sensitivity. Unfortunately, most positron-emitting radionuclides have very short half-lives relative to the pharmacokinetic half-life of mAbs (often on the order of 3–4 weeks). However, several PET tracers are often used for detection of mAbs, such as 124I, which has a physical half-life of 4.2 days.66,67 Additionally, 89Zr (physical half-life ¼ 3.27 days68) has become of interest as a PET tracer for mAb biodistribution studies.69 To improve the anatomical specificity of the distribution profile, PET imaging is commonly combined with other imaging techniques such as MRI or CT. Antibody distribution may also be assessed via optical methods, such as fluorescence imaging, which detects a signal emitted from a fluorophore which has been conjugated to an antibody. In part due to the short path length of fluorescent-emitted light in tissues, this approach does not offer sufficient resolution for assessing intra-tissue distribution patterns from living subjects (eg, assessing the heterogeneity of mAb distribution in tumors); however, fluorescence imaging may allow a rapid, quantitative assessment of tissue-specific drug disposition (eg, for assuring mAb distribution to a tissue of interest, and/or for diagnostic applications). As with any method requiring labeling of antibody, the fluorophore-to-antibody ratio must be optimized so as not to alter the disposition characteristics of the mAb. Multiple fluorophores have been used in this method to assess antibody distribution, including fluorescein isothiocyanate (488 and 515 nm)70 and far red and near infrared dyes (
700–900 nm) such as cyanine.71,72 Indocyanin-coupled antibodies have been successfully used to image tumor xenografts by multiple groups.73,74 In addition to in vivo imaging applications, fluorescence imaging may be applied ex vivo, with collection of tissue samples at specific time points (eg, as often possible in preclinical biodistribution studies). Tissue sections may then be analyzed using fluorescent microscopy to evaluate the localization of the compound within a tissue. Of note, mAb concentrations within the vascular space in tissues are often far greater than mAb concentrations within other tissue spaces (eg, within the interstitial space or cellular space). For many of the experimental methods discussed above, it is feasible to remove residual blood

S34 from tissues, prior to their collection, through the use of perfusion techniques (eg, perfusion of animals with large volumes of saline solution). This type of treatment is, of course, not possible for applications of non-invasive imaging of tissues in living subjects. Due to the relatively low spatial resolution of most imaging technologies, it is typically impossible to isolate and quantify mAb in different tissue regions of interest and, consequently, measured concentrations often represent tissue-averaged values. As such, application of imaging techniques for meaningful assessment of mAb concentrations in extravascular regions of tissues requires measurement (or estimation) of the volume of residual blood (ie, blood residing within the tissue of interest) and consideration of the concentration of mAb in blood. LC–MS Over the past decade, there has been a growing interest in the development of LC–MS assays for the quantification of mAb in both plasma and tissues. LC–MS analyses typically include steps for sample clean-up or mAb enrichment, followed by a controlled digest to create peptide fragments, with LC separation and MS quantification via detection of unique “signature” peptides. A key advantage of this type of assay is its ability to detect unlabeled antibody, which eliminates potential changes in disposition that may be introduced by addition of a label. Additionally, LC–MS can be used to investigate in vivo chemical stability, which is difficult or impossible to perform with other approaches. For example, Huang and colleagues have published an LC–MS assay that allowed assessment of site-specific mAb deamidation kinetics with samples collected from a pharmacokinetic study conducted in monkeys.75 Application of LC–MS to the analysis of mAb in tissue samples has been demonstrated by Duan et al, who developed a sensitive method for quantification of antibody disposition in brain, heart, liver, kidney, spleen, and lung.76 The use of “signature peptides” derived from mAb hypervariable domains has been shown to allow simultaneous quantification of several mAbs (eg, after cassette dosing).77 The main weakness of LC–MS assays is that considerable expertise may be required for assay development and validation, and assay throughput may lag behind that of competing methods.

Characterization and Prediction of Antibody Biodistribution Because of the interplay between factors controlling mAb disposition, the application of mathematical modeling may provide great value in describing the pharmacokinetics of mAbs and the relative importance of various physiological and cellular factors in controlling the observed behavior. In this section, we will describe, in

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order of increasing complexity, various approaches which are commonly used to characterize mAb biodistribution, both during pre-clinical studies and clinically. Non-Compartmental Analyses The application of non-compartmental analysis (NCA) is often one of the first steps taken when characterizing plasma data. These analyses allow the determination of summary parameters for drug exposure and distribution. However, a central assumption of NCA is that all drug is cleared from the blood space (or a space in rapid equilibrium with blood), which is generally appropriate for small molecule drugs (as they are primarily cleared by highly perfused organs such as the liver and kidney). In the case of mAbs, elimination often takes place in tissue spaces that are not in rapid equilibrium with blood. As a consequence of the violation of this key assumption of NCA, Vss may be significantly underestimated. For example, simulation-based approaches have shown that as the fraction of mAb eliminated in tissue increases, NCA approaches will trend towards significant (up to 5.51-fold) under-predictions of the true Vss.78 Mammillary Models The most common compartmental model structure applied to describe the disposition of mAbs is a 2-compartment mammillary model with either linear or nonlinear elimination.79,80 Similar to NCA approaches, the application of mammillary models is generally limited by the assumption that mAb elimination occurs only from the central compartment (often approximated as blood volume for mAbs). The application of simple mammillary models is most useful for mAbs that display minimal tissue binding, with an absence of target-mediated tissue elimination. As discussed for NCA, the application of a mammillary model for mAb exhibiting significant peripheral elimination will generally lead to significant underestimations of the volume of distribution. Nonetheless, mammillary models may be useful and appropriate for many mAbs, particularly those that display linear PK. Target-Mediated Disposition Models Due to the high-affinity binding of mAbs with their targets, particularly cell-surface proteins, antibodies are often susceptible to target-mediated elimination. As a result, target mediated disposition (TMD) models based on the general model of TMD first proposed by Mager and Jusko81 are often used to characterize mAb PK. This model structure places target within the central compartment of mAb distribution, and is useful for characterizing mAb disposition using plasma data for readily accessible targets. However, in cases where there target is located in a site that not in rapid equilibrium with plasma (eg, within the interstitial space of a solid tumor), application of the general model of TMD may significantly underestimate the

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affinity of mAb–target binding (eg, overestimate the equilibrium dissociation constant, KD). For such targets, use of a peripheral model of TMD is more appropriate, as described by Lammerts van Bueren et al.82 When comparing the general and peripheral models of TMD, drastic differences in predicted receptor occupancy and effective doses may be obtained. As such, application of the appropriate model structure, based on knowledge of target biology, becomes extremely important for subsequent, model-based predictions of mAb distribution and efficacy.

spaces and mAb distribution, and allow consideration of target-elimination within the tissue(s) of interest. These models may be applied to estimate tissue concentrations and receptor binding to predict efficacy and toxicity of mAb. However, development of PBPK models requires significant expertise, access to model parameters (which may require estimation or extraction from diverse reports in the literature), and access to tissue data (for model evaluation and validation).

Physiologically Based Pharmacokinetic Models Physiologically based pharmacokinetic (PBPK) modeling provides the opportunity to assess the interplay of many processes controlling mAb disposition, including tissue uptake and elimination, target interaction in physiologically relevant space(s), and FcRn-mediated protection. Additionally, PBPK models allow for relatively straightforward scaling from preclinical species (eg, mouse, rat, or monkey) to man, as key parameter values (eg, blood flows and organ volumes) are known across species. Covell et al83 published the first PBPK model for IgG (and fragments), and divided organs into plasma, interstitial, and cell-associated spaces, with uptake into tissue being through a combination of diffusion and convection. This initial model was modified and expanded by Baxter et al84 to consider specific mAb–target binding in tissues, and to introduce the concept of a “two-pore formalism” for transcapillary convective transport of mAb in tissues. During the past decade, groups have begun to incorporate the effects of FcRn-mediated IgG recycling in PBPK models for mAbs. This concept was first published in the 2005 model developed by Ferl et al; however, the effects of FcRn were only considered in the skin and muscle tissues.85 A model published by Garg and Balthasar,25 which was extended to incorporate TMD by Urva et al,12 incorporated FcRn in all tissues, assuming equilibrium binding between IgG and FcRn within the endosomal space. The Urva model structure was later applied successfully by Abuqayyas and Balthasar86 to predict the disposition of an anti-CEA mAb in control and tumor-bearing mice with different levels of target expression. Further expansion of this model allowed for pH-dependent association and dissociation of IgG with FcRn within the time course of endosomal transit, which has been proposed to allow better predictions of the pharmacokinetics of mAb engineered for altered FcRn binding.87 Shah and Betts88 have developed a “platform” PBPK model of mAb disposition, characterizing mAb PK in mouse, rat, monkey, and man simultaneously, and they have applied this model to infer tissue concentrations of mAb from plasma data alone.89 Overall, PBPK models may be considered as the “gold standard” for describing the biodistribution of mAbs, as they utilize physiologically relevant parameters for tissue

Future Prospectus In this review, we have summarized key determinants of monoclonal antibody biodistribution, as well as methods for assessment and prediction of antibody disposition. With the rapid growth of this class of drugs, it is likely that methods for assessment of antibody concentrations in plasma and tissue will improve in parallel. It is anticipated that improved analytical methods will help to facilitate the closure of key gaps of knowledge regarding mAb disposition, including the role played by mAb charge, FcgR binding, effects of disease and drug-drug interactions, and inter-individual variability in FcR expression. Improved mechanistic information may be expected to allow development of improved mechanistic mathematical models increasing the accuracy and precision of a priori predictions of mAb pharmacokinetics in plasma and in tissues, potentially allowing increased safety and efficiency of mAb development. References 1. Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 1975;256(5517):495– 497. 2. Lobo ED, Hansen RJ, Balthasar JP. Antibody pharmacokinetics and pharmacodynamics. J Pharm Sci. 2004;93(11):2645–2668. 3. Garg A, Balthasar JP. Investigation of the influence of FcRn on the distribution of IgG to the brain. AAPS J. 2009;11(3):553–557. 4. Hobbs SK, Monsky WL, Yuan F, et al. Regulation of transport pathways in tumor vessels: Role of tumor type and microenvironment. Proc Natl Acad Sci USA. 1998;95(8):4607–4612. 5. Brown EB, Boucher Y, Nasser S, Jain RK. Measurement of macromolecular diffusion coefficients in human tumors. Microvasc Res. 2004;67(3):231–236. 6. Jain RK, Baxter LT. Mechanisms of heterogeneous distribution of monoclonal antibodies and other macromolecules in tumors: Significance of elevated interstitial pressure. Cancer Res. 1988; 48(24 Pt 1):7022–7032. 7. Juweid M, Neumann R, Paik C, et al. Micropharmacology of monoclonal antibodies in solid tumors: Direct experimental evidence for a binding site barrier. Cancer Res. 1992;52(19): 5144–5153. 8. Blumenthal RD, Fand I, Sharkey RM, Boerman OC, Kashi R, Goldenberg DM. The effect of antibody protein dose on the uniformity of tumor distribution of radioantibodies: An autoradiographic study. Cancer Immunol Immunother. 1991;33(6):351– 358. 9. Langmuir VK, Mendonca HL, Woo DV. Comparisons between two monoclonal antibodies that bind to the same antigen but have

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