Projecting human pharmacokinetics of monoclonal antibodies from nonclinical data: comparative evaluation of prediction approaches in early drug development.

Currently, more than 350 monoclonal antibodies (mAbs) and mAb derivatives are under development as therapeutics. The prediction of mAb pharmacokinetics (PK)/pharmacodynamics (PD) plays a key role in starting dose selection for first-in-human (FIH) studies. This article presents a brief overview of the biology and mechanisms of absorption, distribution, metabolism and excretion (ADME) for mAbs. In addition, a detailed review of mAb human PK/PD prediction from nonclinical data is provided, including allometry for mAbs with linear or nonlinear PK, species-invariant time method, physiologically based PK (PBPK) modeling and target-mediated drug disposition (TMDD) model, bioavailability projection and immunogenicity impact on PK prediction. Finally, from an industry perspective a decision tree of mAb human PK projection is proposed to facilitate drug development.

Comparative physiology of mice and rats: radiometric measurement of vascular parameters in rodent tissues.

A solid understanding of physiology is beneficial in optimizing drug delivery and in the development of clinically predictive models of drug disposition kinetics. Although an abundance of data exists in the literature, it is often confounded by the use of various experimental methods and a lack of consensus in values from different sources. To help address this deficiency, we sought to directly compare three important vascular parameters at the tissue level using the same experimental approach in both mice and rats. Interstitial volume, vascular volume, and blood flow were radiometrically measured in selected harvested tissues of both species by extracellular marker infusion, red blood cell labeling, and rubidium chloride bolus distribution, respectively. The latter two parameters were further compared by whole-body autoradiographic imaging. An overall good interspecies agreement was observed for interstitial volume and blood flow on a weight-normalized basis in most tissues. In contrast, the measured vascular volumes of most rat tissues were higher than for mouse. Mice and rats, the two most commonly utilized rodent species in translational drug development, should not be considered as interchangeable in terms of vascular volume per gram of tissue. This will be particularly critical in biodistribution studies of drugs, as the amount of drug in the residual blood of tissues is often not negligible, especially for biologic drugs (e.g., antibodies) having long circulation half-lives. Physiologically based models of drug pharmacokinetics and/or pharmacodynamics also rely on accurate knowledge of biological parameters in tissues. For tissue parameters with poor interspecies agreement, the significance and possible drivers are discussed.

Vascular physiology and protein disposition in a preclinical model of neurodegeneration.

The development of clinically relevant preclinical models that mimic the hallmarks of neurodegenerative disease is an ongoing pursuit in early drug development. In particular, robust physiological characterization of central nervous system (CNS) disease models is necessary to predict drug delivery to target tissues and to correctly interpret pharmacodynamic responses to disease-modifying therapeutic candidates. Efficient drug delivery across the blood-CNS barrier is a particularly daunting task, prompting our strategy to evaluate the biodistribution of five distinct molecular probes in a well-characterized mouse model of neurodegeneration. A transgenic mouse model of amyotrophic lateral sclerosis was selected based on a phenotype resembling clinical symptoms, including loss of motor neurons from the spinal cord and paralysis in one or more limbs, due to expression of a G93A mutant form of human superoxide dismutase (SOD1). The tissue distributions of two proteins, albumin and a representative immunoglobulin G antibody, as well as two blood flow markers, the lipophilic blood flow marker Ceretec (i.e., (99m)Tc-HMPAO) and the polar ionic tracer, rubidium-86 chloride ((86)RbCl), were measured following intravenous injection in SOD1(G93A) and age-matched control mice. The radiopharmaceutical TechneScan PYP was also used to measure the distribution of (99m)Tc-labeled red blood cells as a blood pool marker. Both the antibody and (86)Rb were able to cross the blood-spinal cord barrier in SOD1(G93A) mice to a greater extent than in control mice. Although the biodistribution patterns of antibody, albumin, and RBCs were largely similar, notable differences were detected in muscle and skin. Moreover, vastly different biodistribution patterns were observed for a lipophilic and polar perfusion agent, with SOD1(G93A) mutation resulting in reduced renal filtration rates for the former but not the latter. Overall, the multiprobe strategy provided an opportunity to efficiently collect an abundance of physiological information, including the degree and regional extent of blood-CNS barrier permeability, in a preclinical model of neurodegeneration.

Impact of drug conjugation on pharmacokinetics and tissue distribution of anti-STEAP1 antibody-drug conjugates in rats.

Antibody-drug conjugates (ADCs) are designed to combine the exquisite specificity of antibodies to target tumor antigens with the cytotoxic potency of chemotherapeutic drugs. In addition to the general chemical stability of the linker, a thorough understanding of the relationship between ADC composition and biological disposition is necessary to ensure that the therapeutic window is not compromised by altered pharmacokinetics (PK), tissue distribution, and/or potential organ toxicity. The six-transmembrane epithelial antigen of prostate 1 (STEAP1) is being pursued as a tumor antigen target. To assess the role of ADC composition in PK, we evaluated plasma and tissue PK profiles in rats, following a single dose, of a humanized anti-STEAP1 IgG1 antibody, a thio-anti-STEAP1 (ThioMab) variant, and two corresponding thioether-linked monomethylauristatin E (MMAE) drug conjugates modified through interchain disulfide cysteine residues (ADC) and engineered cysteines (TDC), respectively. Plasma PK of total antibody measured by enzyme-linked immunosorbent assay (ELISA) revealed ∼45% faster clearance for the ADC relative to the parent antibody, but no apparent difference in clearance between the TDC and unconjugated parent ThioMab. Total antibody clearances of the two unconjugated antibodies were similar, suggesting minimal effects on PK from cysteine mutation. An ELISA specific for MMAE-conjugated antibody indicated that the ADC cleared more rapidly than the TDC, but total antibody ELISA showed comparable clearance for the two drug conjugates. Furthermore, consistent with relative drug load, the ADC had a greater magnitude of drug deconjugation than the TDC in terms of free plasma MMAE levels. Antibody conjugation had a noticeable, albeit minor, impact on tissue distribution with a general trend toward increased hepatic uptake and reduced levels in other highly vascularized organs. Liver uptakes of ADC and TDC at 5 days postinjection were 2-fold and 1.3-fold higher, respectively, relative to the unmodified antibodies. Taken together, these results indicate that the degree of overall structural modification in anti-STEAP1-MMAE conjugates has a corresponding level of impact on both PK and tissue distribution.

Effects of charge on antibody tissue distribution and pharmacokinetics.

Antibody pharmacokinetics and pharmacodynamics are often governed by biological processes such as binding to antigens and other cognate receptors. Emphasis must also be placed, however, on fundamental physicochemical properties that define antibodies as complex macromolecules, including shape, size, hydrophobicity, and charge. Electrostatic interactions between anionic cell membranes and the predominantly positive surface charge of most antibodies can influence blood concentration and tissue disposition kinetics in a manner that is independent of antigen recognition. In this context, the deliberate modification of antibodies by chemical means has been exploited as a valuable preclinical research tool to investigate the relationship between net molecular charge and biological disposition. Findings from these exploratory investigations may be summarized as follows: (I) shifts in isoelectric point of approximately one pI unit or more can produce measurable changes in tissue distribution and kinetics, (II) increases in net positive charge generally result in increased tissue retention and increased blood clearance, and (III) decreases in net positive charge generally result in decreased tissue retention and increased whole body clearance. Understanding electrostatic interactions between antibodies and biological matrices holds relevance in biotechnology, especially with regard to the development of immunoconjugates. The guiding principles and knowledge gained from preclinical evaluation of chemically modified antibodies will be discussed and placed in the context of therapeutic antibodies that are currently marketed or under development, with a particular emphasis on pharmacokinetic and disposition properties.

Pharmacokinetics of humanized monoclonal anti-tumor necrosis factor-{alpha} antibody and its neonatal Fc receptor variants in mice and cynomolgus monkeys.

The neonatal Fc receptor (FcRn) plays a critical role in maintaining homeostasis of IgG antibodies. Recent studies have shown that the FcRn-IgG interaction can be modulated to alter the pharmacokinetics of the antibody. This has been achieved by altering amino acid residues in the FcRn-binding domain of the antibody, resulting in a change in the pH-dependent binding affinity of the antibody to FcRn. The purpose of this study was to examine the impact of the pH-dependent FcRn binding affinity on the pharmacokinetics of the antibody with changes in the Asn434 residue. Two anti-tumor necrosis factor-alpha monoclonal antibody (mAb) FcRn variants (N434A and N434H) were engineered, and pharmacokinetic studies of the two FcRn variants together with the wild type (WT) were conducted in mice and cynomolgus monkeys. N434A, which had binding properties to murine FcRn similar to those of the WT, had the same pharmacokinetic profile as the WT in mice. N434H, with the highest binding affinity to murine FcRn at pH 7.4, had a faster clearance (16.1 ml/day/kg) and a lower bioavailability (61.3%) compared with the WT (5.07 ml/day/kg, 73.2%) and N434A (5.90 ml/day/kg, 72.4%) in mice. N434A and N434H, which had higher binding affinity at pH 6.0 to monkey FcRn with comparable affinity at pH 7.4, had significantly higher areas under the serum concentration-time curve from time 0 to day 7 than the WT (749 +/- 71.9 and 819 +/- 81.5 versus 592 +/- 56.8 microg/ml . day) in monkeys. Thus, increasing the binding affinity of mAbs to FcRn at pH 6.0 while keeping a low binding affinity at pH 7.4 improves the pharmacokinetics of these molecules.

Effect of immune complex formation on the distribution of a novel antibody to the ovarian tumor antigen CA125.

3A5 is a novel antibody that binds repeated epitopes within CA125, an ovarian tumor antigen that is shed into the circulation. Binding to shed antigen may limit the effectiveness of therapeutic antibodies because of unproductive immune complex (IC) formation and/or altered antibody distribution. To evaluate this possibility, we characterized the impact of shed CA125 on the in vivo distribution of 3A5. In vitro, 3A5 and CA125 were found to form ICs in a concentration-dependent manner. This phenomenon was then evaluated in vivo using quantitative whole-body autoradiography to assess the tissue distribution of (125)I-3A5 in an orthotopic OVCAR-3 tumor mouse model at different stages of tumor burden. Low doses of 3A5 (75 µg/kg) and pathophysiological levels of shed CA125 led to the formation of ICs in vivo that were rapidly distributed to the liver. Under these conditions, increased clearance of 3A5 from normal tissues was observed in mice bearing CA125-expressing tumors. Of importance, despite IC formation, 3A5 uptake by tumors was sustained over time. At a therapeutically relevant dose of 3A5 (3.5 mg/kg), IC formation was undetectable and distribution to normal tissues followed that of blood. In contrast, increased levels of radioactivity were observed in the tumors. These data demonstrate that CA125 and 3A5 do form ICs in vivo and that the liver is involved in their uptake. However, at therapeutic doses of 3A5 and clinically relevant CA125 levels, IC formation consumes only a minor fraction of 3A5, and tumor targeting seems to be unaffected.

Development and evaluation of a novel method for preclinical measurement of tissue vascular volume.

Identification of clinically predictive models of disposition kinetics for antibody therapeutics is an ongoing pursuit in drug development. To encourage translation of drug candidates from early research to clinical trials, clinical diagnostic agents may be used to characterize antibody disposition in physiologically relevant preclinical models. TechneScan PYP was employed to measure tissue vascular volumes (V(v)) in healthy mice. Two methods of red blood cell (RBC) labeling were compared: a direct in vivo method that is analogous to a clinical blood pool imaging protocol, and an indirect method in which radiolabeled blood was transfused from donor mice into recipient mice. The indirect method gave higher precision in RBC labeling yields, lower V(v) values in most tissues, and lower (99m)Tc uptake in kidneys and bladder by single photon emission computed tomographic (SPECT) imaging relative to the direct method. Furthermore, the relative influence of each method on the calculated area under the first 7 days of the concentration-time curve (AUC(0-7)) of an IgG in nude mice was assessed using a physiologically based pharmacokinetic model. The model was sensitive to the source of V(v) values, whether obtained from the literature or measured by either method, when used to predict experimental AUC(0-7) values for radiolabeled trastuzumab in healthy murine tissues. In summary, a novel indirect method for preclinical determination of V(v) offered higher precision in RBC labeling efficiency and lower renal uptake of (99m)Tc than the direct method. In addition, these observations emphasize the importance of obtaining accurate physiological parameter values for modeling antibody uptake.

Modeling the efficacy of trastuzumab-DM1, an antibody drug conjugate, in mice.

Trastuzumab-DM1 (T-DM1) is a novel antibody-drug conjugate under investigation for the treatment of human epidermal growth factor receptor 2 (HER2)-positive metastatic breast cancer. One challenge in oncologic drug development is determining the optimal dose and treatment schedule. A novel dose regimen-finding strategy was developed for T-DM1 using experimental data and pharmacokinetic/pharmacodynamic modeling. To characterize the disposition of T-DM1, pharmacokinetic studies were conducted in athymic nude and beige nude mice. The pharmacokinetics of T-DM1 were described well by a two-compartment model. Tumor response data were obtained from single-dose, multiple-dose and time-dose-fractionation studies of T-DM1 in animal models of HER2-positive breast cancer, specifically engineered to be insensitive to trastuzumab. A sequential population-based pharmacokinetic/pharmacodynamic modeling approach was developed to describe the anti-tumor activity of T-DM1. A cell-cycle-phase nonspecific tumor cell kill model incorporating transit compartments captured well the features of tumor growth and the activity of T-DM1. Key findings of the model were that tumor cell growth rate played a significant role in the sensitivity of tumors to T-DM1; anti-tumor activity was schedule independent; and tumor response was linked to the ratio of exposure to a concentration required for tumor stasis.

Complex formation of anti-VEGF-C with VEGF-C released during blood coagulation resulted in an artifact in its serum pharmacokinetics.

A phage-derived human monoclonal antibody against VEGF-C was developed as a potential anti-tumor therapeutic and exhibited fast clearance in preclinical species, with notably faster clearance in serum than in plasma. The purpose of this work was to understand the factors contributing to its fast clearance. In vitro incubations in animal and human blood, plasma, and serum were conducted with radiolabeled anti-VEGF-C to determine potential protein and cell-based interactions with the antibody as well as any matrix-dependent recovery dependent upon the matrix. A tissue distribution study was conducted in mice with and without heparin infusion in order to identify a tissue sink and determine whether heparin could affect antibody recovery from serum and/or plasma. Incubation of radiolabeled anti-VEGF-C in human and animal blood, plasma, or serum revealed that the antibody formed a complex with an endogenous protein, likely VEGF-C. This complex was trapped within the blood clot during serum preparation from blood, but not within the blood cell pellet during plasma preparation. Low level heparin infusion in mice slowed down clot formation during serum preparation and allowed for better recovery of the radiolabeled antibody in serum. No tissue sink was found in mice. Thus, during this characterization, we determined that the blood sampling matrix greatly impacted the amount of antibody recovered in the samples, therefore, altering its derived pharmacokinetic parameters. Target biology should be considered when selecting appropriate sampling matrices for PK analysis.

Proteomic analysis of breast cancer molecular subtypes and biomarkers of response to targeted kinase inhibitors using reverse-phase protein microarrays.

Although breast cancer molecular subtypes have been extensively defined by means of gene expression profiling over the past decade, little is known, at the proteomic level, as to how signaling pathways are differentially activated and serve to control proliferation in different breast cancer subtypes. We used reverse-phase protein arrays to examine phosphorylation status of 100 proteins in a panel of 30 breast cancer cell lines and showed distinct pathway activation differences between different subtypes that are not obvious from previous gene expression studies. We also show that basal levels of phosphorylation of key signaling nodes may have diagnostic utility in predicting response to selective inhibitors of phosphatidylinositol 3-kinase and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase. Finally, we show that reverse-phase protein arrays allow the parallel analysis of multiple pharmacodynamic biomarkers of response to targeted kinase inhibitors and that inhibitors of epidermal growth factor receptor and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase result in compensatory up-regulation of the phosphatidylinositol 3-kinase/Akt signaling pathway.

Evidence for an asialoglycoprotein receptor on nonparenchymal cells for O-linked glycoproteins.

B cell-activating factor receptor 3 (BR3)-Fc is an IgG1-receptor dimeric fusion protein that has multiple O-linked glycosylation sites and sialylation levels that can vary in the manufacturing process. Increased sialic acid levels resulted from increased site occupancy with the O-linked N-acetylgalactosamine (GalNAc-Gal), but because the ratio of sialic acid per mole of oligosaccharide remained approximately 1, this led to increased asialo terminal GalNAc. Previous studies have demonstrated an effect of terminal asialo Gal or GalNAc on the clearance of glycoproteins due to uptake and degradation by lectin receptors in the liver. However, the previous studies examined N-linked oligosaccharides, and there are less data regarding O-linked oligosaccharides. The objective of these studies was to determine the effects on the pharmacokinetics and distribution of the asialo terminal GalNAc and varying amounts of sialic acid residues on BR3-Fc. The results of the data presented here suggest that exposed Gal on the desialylated BR3-Fc led to rapid clearance due to uptake and degradation in the liver that was associated with nonparenchymal cells. It is interesting to note that the data indicated a decreased clearance and increased exposure of BR3-Fc as the sialic acid levels increased, even though increased sialic acid was associated with increased asialo GalNAc. Therefore, the exposed GalNAc did not seem to play a role in the clearance of BR3-Fc; although the Gal linked to the hydroxyl group at position 3 may have prevented an interaction. Because we did not see uptake of desialylated BR3-Fc in hepatocytes where the asialoglycoprotein receptor is localized, this nonparenchymal cell lectin may have preference for O-linked glycoproteins.

Development and Translational Application of an Integrated, Mechanistic Model of Antibody-Drug Conjugate Pharmacokinetics.

Antibody drug conjugates (ADC), in which small molecule cytotoxic agents are non-specifically linked to antibodies, can enable targeted delivery of chemotherapeutics to tumor cells. ADCs are often produced and administered as a mixture of conjugated antibodies with different drug to antibody ratios (DAR) resulting in complex and heterogeneous disposition kinetics. We developed a mechanism-based platform model that can describe and predict the complex pharmacokinetic (PK) behavior of ADCs with protease-cleavable valine-citrulline (VC) linker linked to Monomethylmonomethyl auristatin F/E by incorporating known mechanisms of ADC disposition. The model includes explicit representation of all DAR species; DAR-dependent sequential deconjugation of the drug, resulting in the conversion of higher DAR to lower DAR species; and DAR-dependent antibody/ADC clearance. PK profiles of multiple analytes (total antibody, drug-conjugated antibody, and/or antibody-conjugated drug) for different ADC molecules and targets in rodents and cynomolgus monkeys were used for model development. The integrated cross-species model was successful in capturing the multi-analyte PK profiles after administration of purified ADCs with defined DAR species and ADCs with mixtures of DAR. Human PK predictions for DSTP3086S (anti-STEAP1-vc-MMAE) with the platform model agreed well with PK (total antibody and antibody-conjugated drug concentrations) measurements in the dose-ranging phase I clinical study. The integrated model is applicable to various other ADCs with different formats, conjugated drugs, and linkers, and provides a valuable tool for the exploration of mechanisms governing disposition of ADCs and enables translational predictions.

Mechanism-Based Competitive Binding Model to Investigate the Effect of Neonatal Fc Receptor Binding Affinity on the Pharmacokinetic of Humanized Anti-VEGF Monoclonal IgG1 Antibody in Cynomolgus Monkey.

The quantitative relationship between neonatal Fc receptor (FcRn) binding affinity at both acidic and physiological pH and the pharmacokinetics of protein engineered FcRn IgG1 variants has not yet been reported. Our objective was to develop a quantitatively mechanism-based competitive binding model to describe the effects of FcRn binding affinity at acidic and physiological pH on the pharmacokinetics of anti-VEGF IgG1 antibodies when both endogenous and exogenous antibodies are competing for the same FcRn. Pharmacokinetic (PK) and FcRn binding data from five Fc variants of humanized anti-VEGF IgG1 monoclonal antibodies with wide range of FcRn binding affinity were used for the analysis. Sixty-seven anti-VEGF IgG1 antibody-treated animals and 25 control animals with simulated endogenous IgG levels were used to develop the final model. A hybrid iterative two stages and Monte Carlo parametric expectation-maximization method was used to obtain the final model parameters estimates. The final model well described the observed PK data. Quantitative FcRn binding affinity-pharmacokinetics relationships was constructed to provide important biological insights in better understanding of the FcRn binding effect on pharmacokinetics of anti-VEGF IgG1 antibodies in cynomolgus monkeys and served as an important model-based drug discovery platform to guide the design and development of the future generation of anti-VEGF or other therapeutic IgG1 antibodies.

Efficacy of a long-acting growth hormone (GH) preparation in patients with adult GH deficiency.

CONTEXT: Treatment of adult GH deficiency (AGHD) with daily injections of GH results in decreased adipose mass, increased lean body mass (LBM), increased bone mineral density, and improved quality of life. OBJECTIVE: This study seeks to determine whether a depot preparation of GH given every 14 d would lead to comparable decreases in trunk adipose tissue as daily GH. DESIGN: This open-label, randomized study compares subjects receiving depot GH, daily GH, or no therapy. SETTING: The study was performed at 23 university or local referral endocrine centers. PATIENTS OR OTHER PARTICIPANTS: One hundred thirty-five adults with AGHD syndrome participated in the study. INTERVENTION: Subjects were randomized to receive depot GH (n = 51), daily GH (n = 53), or no treatment (n = 31) for 32 wk. The dose of GH was titrated so that IGF-I was less than or equal to +2 SD of the age-adjusted normal range. MAIN OUTCOME MEASURE: Trunk adipose tissue was the main outcome measure as measured by dual energy x-ray absorptiometry. RESULTS: The percentage of the trunk region that is fat increased by 0.4 in the no treatment group, but decreased by 3.2 (P = 0.001 vs. untreated) in the GH depot group and by 2.5 (P < 0.004 vs. untreated) in the daily GH group. Visceral adipose tissue area decreased by 9.1% in the GH depot group and by 6.8% in the daily GH group. LBM and high-density lipoprotein increased in both treatment groups. Side effect profiles were similar. Three subjects receiving GH experienced serious episodes of adrenal insufficiency. CONCLUSIONS: GH diminishes trunk and visceral adipose tissue and increases LBM in AGHD. A depot form of GH that is administered every 14 d is as safe and effective as daily GH injections.

Sustained delivery of human growth hormone from a novel gel system: SABER.

PURPOSE: The purpose of this study was to evaluate the release of recombinant human growth hormone (rhGH) from a novel non-polymeric sustained release system, SABER. METHODS: The SABER system consists of sucrose acetate isobutryate, a solvent and a polymeric release modifier. Spray dried formulations of zinc complexed rhGH in sodium bicarbonate containing sucrose and polysorbate 20 were homogenized with various SABER systems (10% w/v rhGH) and assessed in vitro and in vivo (rat studies). The effect of protein to sucrose ratio in the spray dried formulation and a release modifier, poly-D,L-lactic acid (PLA), in the SABER system, on the initial release was investigated along with the effect of dose volume. RESULTS: The in vitro release studies with rhGH SABER suspensions indicate that increasing the sucrose content from 2 to 5 mg/ml in the rhGH formulations increased the initial release (24 h) from 78.0% to 93.5%. When the protein formulation was held constant and 1.0% w/w PLA was added to the solvent phase, the initial release was reduced from 78.0% to less than 5.0%. The initial release in vivo after subcutaneous administration (SC) in rats (15 mg/kg rhGH) decreased with increasing PLA content (1.0% w/w PLA, Cmax = 342.8 ng/ml; 10% w/w PLA, Cmax = 35.4 ng/ml), while increased sucrose content increased both the initial release (AUC(0-2) days) and persistence (AUC(2-7) days) over the 7 days from 64.2 to 228.4 ng day/ml (total AUC). A linear dose response (rhGH serum levels) was observed after SC administration of different rhGH SABER volumes greater than 100 microl. Histological examination of the injection sites indicated a mild inflammatory response similar to that observed after injection of PLA microspheres. CONCLUSIONS: The addition of PLA reduced the initial release rate of protein release from SABER, while increasing the sucrose content of the protein formulation yielded increased rhGH persistence. These results demonstrate that the SABER delivery system allows weight-based dosing at volumes greater than 100 microl to achieve sustained release of intact rhGH in vivo for at least 7 days.

Modeling approach to investigate the effect of neonatal Fc receptor binding affinity and anti-therapeutic antibody on the pharmacokinetic of humanized monoclonal anti-tumor necrosis factor-α IgG antibody in cynomolgus monkey.

PURPOSE: Several neonatal Fc receptor (FcRn) variants of an anti-tumor necrosis factor (TNF)-α humanized monoclonal IgG antibodies (mAbs) were developed but the effect of their differential FcRn binding affinities on pharmacokinetic (PK) behavior were difficult to be definitively measured in vivo due to formation of anti-therapeutic antibody (ATA). A semi-mechanistic model was developed to investigate the quantitative relationship between the FcRn binding affinity and PK of mAbs in cynomolgus monkey with the presence of ATA. METHODS: PK and ATA data from cynomolgus monkeys which received a single intravenous dose of adalimumab, wild-type or two FcRn variant (N434H and N434A) anti-TNF-α mAbs were included in the analysis. Likelihood-based censored data handling method was used to include many PK observations with BQL values for model development. A fully integrated PK-ATA model was developed and used to fit simultaneously to the PK/ATA data. RESULTS AND CONCLUSIONS: The PK and ATA time-profiles and effect of FcRn-binding affinity on PK of mAbs were well described by the model and the parameters were estimated with good precision. The model was used successfully to construct quantitative relationships between FcRn binding affinity and PK of anti-TNF-α mAbs in the presence of the ATA-mediated elimination and interferences.

Quantitative cumulative biodistribution of antibodies in mice: effect of modulating binding affinity to the neonatal Fc receptor.

The neonatal Fc receptor (FcRn) plays an important and well-known role in antibody recycling in endothelial and hematopoietic cells and thus it influences the systemic pharmacokinetics (PK) of immunoglobulin G (IgG). However, considerably less is known about FcRn's role in the metabolism of IgG within individual tissues after intravenous administration. To elucidate the organ distribution and gain insight into the metabolism of humanized IgG1 antibodies with different binding affinities FcRn, comparative biodistribution studies in normal CD-1 mice were conducted. Here, we generated variants of herpes simplex virus glycoprotein D-specific antibody (humanized anti-gD) with increased and decreased FcRn binding affinity by genetic engineering without affecting antigen specificity. These antibodies were expressed in Chinese hamster ovary cell lines, purified and paired radiolabeled with iodine-125 and indium-111. Equal amounts of I-125-labeled and In-111-labeled antibodies were mixed and intravenously administered into mice at 5 mg/kg. This approach allowed us to measure both the real-time IgG uptake (I-125) and cumulative uptake of IgG and catabolites (In-111) in individual tissues up to 1 week post-injection. The PK and distribution of the wild-type IgG and the variant with enhanced binding for FcRn were largely similar to each other, but vastly different for the rapidly cleared low-FcRn-binding variant. Uptake in individual tissues varied across time, FcRn binding affinity, and radiolabeling method. The liver and spleen emerged as the most concentrated sites of IgG catabolism in the absence of FcRn protection. These data provide an increased understanding of FcRn's role in antibody PK and catabolism at the tissue level.

Enhanced tumor retention of a radiohalogen label for site-specific modification of antibodies.

A known limitation of iodine radionuclides for labeling and biological tracking of receptor targeted proteins is the tendency of iodotyrosine to rapidly diffuse from cells following endocytosis and lysosomal degradation. In contrast, radiometal-chelate complexes such as indium-111-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (In-111-DOTA) accumulate within target cells due to the residualizing properties of the polar, charged metal-chelate-amino acid adduct. Iodine radionuclides boast a diversity of nuclear properties and chemical means for incorporation, prompting efforts to covalently link radioiodine with residualizing molecules. Herein, we describe the Ugi-assisted synthesis of [I-125]HIP-DOTA, a 4-hydroxy-3-iodophenyl (HIP) derivative of DOTA, and demonstration of its residualizing properties in a murine xenograft model. Overall, this study displays the power of multicomponent synthesis to yield a versatile radioactive probe for antibodies across multiple therapeutic areas with potential applications in both preclinical biodistribution studies and clinical radioimmunotherapies.

Compartmental tissue distribution of antibody therapeutics: experimental approaches and interpretations.

Monoclonal antibodies have provided many validated and potential new therapeutic candidates for various diseases encompassing the realms of neurology, ophthalmology, immunology, and especially oncology. The mechanism of action for these biological molecules typically involves specific binding to a soluble ligand or cell surface protein in order to block or alter a molecular pathway, induce a desired cellular response, or deplete a target cell. Many antigens reside within the interstitial space, the fluid-filled compartment that lies between the outer endothelial vessel wall and the plasma membranes of cells. This mini-review examines the concepts relevant to the kinetics and behavior of antibodies within the interstitium with a special emphasis on radiometric measurement of quantitative pharmacology. Molecular probes are discussed to outline chemical techniques, selection criteria, data interpretation, and relevance to the study of antibody pharmacokinetics. The importance of studying the tissue uptake of antibodies at a compartmental level is highlighted, including a brief overview of receptor occupancy and its interpretation in radiotracer studies. Experimental methods for measuring the spatial composition of tissues are examined in terms of relative vascular, interstitial, and cellular volumes using solid tumors as a representative example. Experimental methods and physiologically based pharmacokinetic modeling are introduced as distinct approaches to distinguish between free and bound fractions of interstitial antibody. Overall, the review outlines the available methods for pharmacokinetic measurements of antibodies and physiological measurements of the compartments that they occupy, while emphasizing that such approaches may not fully capture the complexities of dynamic, heterogeneous tumors and other tissues.