A strategy for risk mitigation of antibodies with fast clearance

A majority of human therapeutic antibody candidates show pharmacokinetic properties suitable for clinical use, but an unexpectedly fast antibody clearance is sometimes observed that may limit the clinical utility. Pharmacokinetic data in cynomolgus monkeys collected for a panel of 52 antibodies showed broad distribution of target-independent clearance values (2.4–61.3 mL/day/kg), with 15 (29%) having clearance > 10 mL/day/kg. Alteration in the interaction with the recycling FcRn receptor did not account for the faster than expected clearance observed for the antibodies; off-target binding was presumed to account for the fast clearance. We developed an assay based on ELISA detection of non-specific binding to baculovirus particles that can identify antibodies having increased risk for fast clearance. This assay can be used during lead generation or optimization to identify antibodies with increased risk of having fast clearance in both humans and cynomolgus monkeys, and thus increase the likelihood of obtaining a suitable drug candidate.     

A strategy for risk mitigation of antibodies with fast clearance

A majority of human therapeutic antibody candidates show pharmacokinetic properties suitable for clinical use, but an unexpectedly fast antibody clearance is sometimes observed that may limit the clinical utility. Pharmacokinetic data in cynomolgus monkeys collected for a panel of 52 antibodies showed broad distribution of target-independent clearance values (2.4–61.3 mL/day/kg), with 15 (29%) having clearance > 10 mL/day/kg. Alteration in the interaction with the recycling FcRn receptor did not account for the faster than expected clearance observed for the antibodies; off-target binding was presumed to account for the fast clearance. We developed an assay based on ELISA detection of non-specific binding to baculovirus particles that can identify antibodies having increased risk for fast clearance. This assay can be used during lead generation or optimization to identify antibodies with increased risk of having fast clearance in both humans and cynomolgus monkeys, and thus increase the likelihood of obtaining a suitable drug candidate.     

Framework selection can influence pharmacokinetics of a humanized therapeutic antibody through differences in molecule charge

Pharmacokinetic (PK) testing of a humanized (κI, VH3 framework) and affinity matured anti-hepatitis C virus E2-glycoprotein (HCV-E2) antibody (hu5B3.κ1VH3.v3) in rats revealed unexpected fast clearance (34.9 mL/day/kg). This antibody binds to the rat recycling receptor FcRn as expected for a human IgG1 antibody and does not display non-specific binding to baculovirus particles in an assay that is correlated with fast clearance in cynomolgus monkey. The antigen is not expressed in rat so target-dependent clearance does not contribute to PK. Removal of the affinity maturation changes (hu5B3.κ1VH3.v1) did not restore normal clearance. The antibody was re-humanized on a κ4, VH1 framework and the non-affinity matured version (hu5B3.κ4VH1.v1) was shown to have normal clearance (8.5 mL/day/kg). Since the change in framework results in a lower pI, primarily due to more negative charge on the κ4 template, the effect of additional charge variation on antibody PK was tested by incorporating substitutions obtained through phage display affinity maturation of hu5B3.κ1VH3.v1. A variant having a pI of 8.61 gave very fast clearance (140 mL/day/kg) whereas a molecule with pI of 6.10 gave slow clearance (5.8 mL/kg/day). Both antibodies exhibited comparable binding to rat FcRn, but biodistribution experiments showed that the high pI variant was catabolized in liver and spleen. These results suggest antibody charge can have an effect on PK through alterations in antibody catabolism independent of FcRn-mediated recycling. Furthermore, introduction of affinity maturation changes into the lower pI framework yielded a candidate with PK and virus neutralization properties suitable for clinical development.     

Preclinical pharmacokinetics and pharmacodynamics of DCLL9718A: An antibody-drug conjugate for the treatment of acute myeloid leukemia

ABSTRACT

Few treatment options are available for acute myeloid leukemia (AML) patients. DCLL9718A is an antibody-drug conjugate that targets C-type lectin-like molecule-1 (CLL-1). This receptor is prevalent on monocytes, neutrophils, and AML blast cells, and unlike CD33, is not expressed on hematopoietic stem cells, thus providing possible hematopoietic recovery. DCLL9718A comprises an anti-CLL-1 IgG1 antibody (MCLL0517A) linked to a pyrrolobenzodiazepine (PBD) dimer payload, via a cleavable disulfide-labile linker. Here, we characterize the in vitro and in vivo stability, the pharmacokinetics (PK) and pharmacodynamics (PD) of DCLL9718A and MCLL0517A in rodents and cynomolgus monkeys. Three key PK analytes were measured in these studies: total antibody, antibody-conjugated PBD dimer and unconjugated PBD dimer. In vitro, DCLL9718A, was stable with most (> 80%) of the PBD dimer payload remaining conjugated to the antibody over 96 hours. This was recapitulated in vivo with antibody-conjugated PBD dimer clearance estimates similar to DCLL9718A total antibody clearance. Both DCLL9718A and MCLL0517A showed linear PK in the non-binding rodent species, and non-linear PK in cynomolgus monkeys, a binding species. The PK data indicated minimal impact of conjugation on the disposition of DCLL9718A total antibody. Finally, in cynomolgus monkey, MCLL0517A showed target engagement at all doses tested (0.5 and 20 mg/kg) as measured by receptor occupancy, and DCLL9718A (at doses of 0.05, 0.1 and 0.2 mg/kg) showed strong PD activity as evidenced by notable reduction in monocytes and neutrophils.     

Generation and characterization of a high affinity anti-human FcRn antibody,rozanolixizumab, and the effects of different molecular formats on the reduction of plasma IgG concentration

Rozanolixizumab (UCB7665), a humanized high-affinity anti-human neonatal Fc receptor (FcRn) monoclonal antibody (IgG4P), has been developed to reduce pathogenic IgG in autoimmune and alloimmune diseases. We document the antibody isolation and compare rozanolixizumab with the same variable region expressed in various mono-, bi- and trivalent formats. We report activity data for rozanolixizumab and the different molecular formats in human cells, FcRn-transgenic mice, and cynomolgus monkeys. Rozanolixizumab, considered the most effective molecular format, dose-dependently and selectively reduced plasma IgG concentrations in an FcRn-transgenic mouse model (no effect on albumin). Intravenous (IV) rozanolixizumab dosing in cynomolgus monkeys demonstrated non-linear pharmacokinetics indicative of target-mediated drug disposition; single IV rozanolixizumab doses (30 mg/kg) in cynomolgus monkeys reduced plasma IgG concentration by 69% by Day 7 post-administration. Daily IV administration of rozanolixizumab (initial 30 mg/kg loading dose; 5 mg/kg daily thereafter) reduced plasma IgG concentrations in all cynomolgus monkeys, with low concentrations maintained throughout the treatment period (42 days). In a 13-week toxicology study in cynomolgus monkeys, supra-pharmacological subcutaneous and IV doses of rozanolixizumab (≤ 150 mg/kg every 3 days) were well tolerated, inducing sustained (but reversible) reductions in IgG concentrations by up to 85%, with no adverse events observed. We have demonstrated accelerated natural catabolism of IgG through inhibition of IgG:FcRn interactions in mice and cynomolgus monkeys. Inhibition of FcRn with rozanolixizumab may provide a novel therapeutic approach to reduce pathogenic IgG in human autoimmune disease. Rozanolixizumab is being investigated in patients with immune thrombocytopenia (NCT02718716) and myasthenia gravis (NCT03052751).     

Integrated pharmacokinetic,pharmacodynamic and immunogenicity profiling of an anti-CCL21 monoclonal antibody in cynomolgus monkeys

QBP359 is an IgG1 human monoclonal antibody that binds with high affinity to human CCL21, a chemokine hypothesized to play a role in inflammatory disease conditions through activation of resident CCR7-expressing fibroblasts/myofibroblasts. The pharmacokinetics (PK) and pharmacodynamics (PD) of QBP359 in non-human primates were characterized through an integrated approach, combining PK, PD, immunogenicity, immunohistochemistry (IHC) and tissue profiling data from single- and multiple-dose experiments in cynomolgus monkeys. When compared with regular immunoglobulin typical kinetics, faster drug clearance was observed in serum following intravenous administration of 10 mg/kg and 50 mg/kg of QBP359. We have shown by means of PK/PD modeling that clearance of mAb-ligand complex is the most likely explanation for the rapid clearance of QBP359 in cynomolgus monkey. IHC and liquid chromatography mass spectrometry data suggested a high turnover and synthesis rate of CCL21 in tissues. Although lymphoid tissue was expected to accumulate drug due to the high levels of CCL21 present, bioavailability following subcutaneous administration in monkeys was 52%. In human disease states, where CCL21 expression is believed to be expressed at 10-fold higher concentrations compared with cynomolgus monkeys, the PK/PD model of QBP359 and its binding to CCL21 suggested that very large doses requiring frequent administration of mAb would be required to maintain suppression of CCL21 in the clinical setting. This highlights the difficulty in targeting soluble proteins with high synthesis rates.     

Linear pharmacokinetic parameters for monoclonal antibodies are similar within a species and across different pharmacological targets: A comparison between human,cynomolgus monkey and hFcRn Tg32 transgenic mouse using a population-modeling approach

The linear pharmacokinetics (PK) of therapeutic monoclonal antibodies (mAbs) can be considered a class property with values that are similar to endogenous IgG. Knowledge of these parameters across species could be used to avoid unnecessary in vivo PK studies and to enable early PK predictions and pharmacokinetic/pharmacodynamic (PK/PD) simulations. In this work, population-pharmacokinetic (popPK) modeling was used to determine a single set of ‘typical’ popPK parameters describing the linear PK of mAbs in human, cynomolgus monkey and transgenic mice expressing the human neonatal Fc receptor (hFcRn Tg32), using a rich dataset of 27 mAbs. Non-linear PK was excluded from the datasets and a 2-compartment model was applied to describe mAb disposition. Typical human popPK estimates compared well with data from comparator mAbs with linear PK in the clinic. Outliers with higher than typical clearance were found to have non-specific interactions in an affinity-capture self-interaction nanoparticle spectroscopy assay, offering a potential tool to screen out these mAbs at an early stage. Translational strategies were investigated for prediction of human linear PK of mAbs, including use of typical human popPK parameters and allometric exponents from cynomolgus monkey and Tg32 mouse. Each method gave good prediction of human PK with parameters predicted within 2-fold. These strategies offer alternative options to the use of cynomolgus monkeys for human PK predictions of linear mAbs, based on in silico methods (typical human popPK parameters) or using a rodent species (Tg32 mouse), and call into question the value of completing extensive in vivo preclinical PK to inform linear mAb PK.     

Framework selection can influence pharmacokinetics of a humanized therapeutic antibody through differences in molecule charge

Pharmacokinetic (PK) testing of a humanized (κI, VH3 framework) and affinity matured anti-hepatitis C virus E2-glycoprotein (HCV-E2) antibody (hu5B3.κ1VH3.v3) in rats revealed unexpected fast clearance (34.9 mL/day/kg). This antibody binds to the rat recycling receptor FcRn as expected for a human IgG1 antibody and does not display non-specific binding to baculovirus particles in an assay that is correlated with fast clearance in cynomolgus monkey. The antigen is not expressed in rat so target-dependent clearance does not contribute to PK. Removal of the affinity maturation changes (hu5B3.κ1VH3.v1) did not restore normal clearance. The antibody was re-humanized on a κ4, VH1 framework and the non-affinity matured version (hu5B3.κ4VH1.v1) was shown to have normal clearance (8.5 mL/day/kg). Since the change in framework results in a lower pI, primarily due to more negative charge on the κ4 template, the effect of additional charge variation on antibody PK was tested by incorporating substitutions obtained through phage display affinity maturation of hu5B3.κ1VH3.v1. A variant having a pI of 8.61 gave very fast clearance (140 mL/day/kg) whereas a molecule with pI of 6.10 gave slow clearance (5.8 mL/kg/day). Both antibodies exhibited comparable binding to rat FcRn, but biodistribution experiments showed that the high pI variant was catabolized in liver and spleen. These results suggest antibody charge can have an effect on PK through alterations in antibody catabolism independent of FcRn-mediated recycling. Furthermore, introduction of affinity maturation changes into the lower pI framework yielded a candidate with PK and virus neutralization properties suitable for clinical development.     

Correlations between pharmacokinetics of IgG antibodies in primates vs. FcRn-transgenic mice reveal a rodent model with predictive capabilities

Transgenic mice expressing human neonatal Fc receptor (FcRn) instead of mouse FcRn are available for IgG antibody pharmacokinetic (PK) studies. Given the interest in a rodent model that offers reliable predictions of antibody PK in monkeys and humans, we set out to test whether the PK of IgG antibodies in such mice correlated with the PK of the same antibodies in primates. We began by using a single research antibody to study the influence of: (1) different transgenic mouse lines that differ in FcRn transgene expression; (2) homozygous vs. hemizygous FcRn transgenic mice; (3) the presence vs. absence of coinjected high-dose human intravenous immunoglobulin (IVIG), and (4) the presence vs. absence of coinjected high-dose human serum albumin (HSA). Results of those studies suggested that use of hemizygous Tg32 mice (Tg32 hemi) not treated with IVIG or HSA offered potential as a predictive model for PK in humans. Mouse PK studies were then done under those conditions with a panel of test antibodies whose PK in mice and primates is not significantly affected by target binding, and for which monkey or human PK data were readily available. Results from the studies revealed significant correlations between terminal half-life or clearance values observed in the mice and the corresponding values reported in humans. A significant relationship in clearance values between mice and monkeys was also observed. These correlations suggest that the Tg32 hemi mouse model, which is both convenient and cost-effective, can offer value in predicting antibody half-life and clearance in primates.     

Utility of immunodeficient mouse models for characterizing the preclinical pharmacokinetics of immunogenic antibody therapeutics

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®), 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® 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.     

Pharmacokinetics and Biodistribution of a Human Monoclonal Antibody to Oxidized LDL in Cynomolgus Monkey Using PET Imaging

Purpose

Oxidized low-density lipoprotein (LDL) plays an essential role in the pathogenesis of atherosclerosis. The purpose of this study was to characterize the pharmacokinetics (PK) of a human recombinant IgG1 antibody to oxidized LDL (anti-oxLDL) in cynomolgus monkey. The tissue biodistribution of anti-oxLDL was also investigated using positron emission tomography (PET) imaging.

Methods

Anti-oxLDL was conjugated with the N-hydroxysuccinimide ester of DOTA (1,4,7,10-tetraazacyclododecane 1,4,7,10-tetraacetic acid) and radiolabeled by chelation of radioactive copper-64 (⁶⁴Cu) for detection by PET. Anti-oxLDL was administered as a single intravenous (IV) dose of 10 mg/kg (as a mixture of radiolabeled and non-labeled material) to two male and two female cynomolgus monkeys. Serum samples were collected over 29 days. Two ELISA methods were used to measure serum concentrations of anti-oxLDL; Assay A was a ligand binding assay that measured free anti-oxLDL (unbound and partially bound forms) and Assay B measured total anti-oxLDL. The biodistribution was observed over a 48-hour period following dose administration using PET imaging.

Results

Anti-oxLDL serum concentration-time profiles showed a biphasic elimination pattern that could be best described by a two-compartment elimination model. The serum concentrations obtained using the two ELISA methods were comparable. Clearance values ranged from 8 to 17 ml/day/kg, while beta half-life ranged from 8 to12 days. The initial volume of distribution and volume of distribution at steady state were approximately 55 mL/kg and 150 mL/kg, respectively. PET imaging showed distribution predominantly to the blood pool, visible as the heart and great vessels in the trunk and limbs, plus diffuse signals in the liver, kidney, spleen, and bone marrow.

Conclusions

The clearance of anti-oxLDL is slightly higher than typical IgG1 antibodies in cynomolgus monkeys. The biodistribution pattern appears to be consistent with an antibody that has no large, rapid antigen sink outside the blood space.     

Pharmacokinetics of an intracerebroventricularly administered antibody in rats

The pharmacokinetics (PK) of an antibody in the brain and the spinal cord is insufficiently understood, which is an obstacle to the discovery of antibody drugs that target diseases in the central nervous system. In this study, we focused on the elimination of IgG from cerebrospinal fluid (CSF) circulating in the brain and the spinal cord in rats, and, to evaluate the influence of CSF bulk flow on the clearance of IgG, also examined the PK of inulin in CSF. To monitor their concentrations in CSF, IgG and inulin were co-administered into the lateral ventricle via a catheter, and CSF was collected from the cisterna magna via another catheter time-sequentially. Blood was also obtained from the same individuals, and the concentrations of IgG and inulin in CSF and plasma were measured. The results revealed that PK parameters of IgG were similar to those of inulin; half-life and clearance of IgG were 47.0 ± 6.49 min and 29.0 ± 15.2 mL/day/kg, and those of inulin were 52.8 ± 25.4 min and 29.0 ± 13.3 mL/day/kg. Moreover, deconvolution analysis indicated that all of the IgG administered in the lateral ventricle was transferred to plasma from CSF within 24 hours. This study demonstrated that IgG in CSF was eliminated by bulk flow and transferred totally to blood circulation.     

Prediction of non-linear pharmacokinetics in humans of an antibody-drug conjugate (ADC) when evaluation of higher doses in animals is limited by tolerability: Case study with an anti-CD33 ADC

For antibody-drug conjugates (ADCs) that carry a cytotoxic drug, doses that can be administered in preclinical studies are typically limited by tolerability, leading to a narrow dose range that can be tested. For molecules with non-linear pharmacokinetics (PK), this limited dose range may be insufficient to fully characterize the PK of the ADC and limits translation to humans. Mathematical PK models are frequently used for molecule selection during preclinical drug development and for translational predictions to guide clinical study design. Here, we present a practical approach that uses limited PK and receptor occupancy (RO) data of the corresponding unconjugated antibody to predict ADC PK when conjugation does not alter the non-specific clearance or the antibody-target interaction. We used a 2-compartment model incorporating non-specific and specific (target mediated) clearances, where the latter is a function of RO, to describe the PK of anti-CD33 ADC with dose-limiting neutropenia in cynomolgus monkeys. We tested our model by comparing PK predictions based on the unconjugated antibody to observed ADC PK data that was not utilized for model development. Prospective prediction of human PK was performed by incorporating in vitro binding affinity differences between species for varying levels of CD33 target expression. Additionally, this approach was used to predict human PK of other previously tested anti-CD33 molecules with published clinical data. The findings showed that, for a cytotoxic ADC with non-linear PK and limited preclinical PK data, incorporating RO in the PK model and using data from the corresponding unconjugated antibody at higher doses allowed the identification of parameters to characterize monkey PK and enabled human PK predictions.     

Biomeasures and mechanistic modeling highlight PK/PD risks for a monoclonal antibody targeting Fn14 in kidney disease

Discovery of the upregulation of fibroblast growth factor-inducible-14 (Fn14) receptor following tissue injury has prompted investigation into biotherapeutic targeting of the Fn14 receptor for the treatment of conditions such as chronic kidney diseases. In the development of monoclonal antibody (mAb) therapeutics, there is an increasing trend to use biomeasures combined with mechanistic pharmacokinetic/pharmacodynamic (PK/PD) modeling to enable decision making in early discovery. With the aim of guiding preclinical efforts on designing an antibody with optimized properties, we developed a mechanistic site-of-action (SoA) PK/PD model for human application. This model incorporates experimental biomeasures, including concentration of soluble Fn14 (sFn14) in human plasma and membrane Fn14 (mFn14) in human kidney tissue, and turnover rate of human sFn14. Pulse-chase studies using stable isotope-labeled amino acids and mass spectrometry indicated the sFn14 half-life to be approximately 5 hours in healthy volunteers. The biomeasures (concentration, turnover) of sFn14 in plasma reveals a significant hurdle in designing an antibody against Fn14 with desired characteristics. The projected dose (>1 mg/kg/wk for 90% target coverage) derived from the human PK/PD model revealed potential high and frequent dosing requirements under certain conditions. The PK/PD model suggested a unique bell-shaped relationship between target coverage and antibody affinity for anti-Fn14 mAb, which could be applied to direct the antibody engineering towards an optimized affinity. This investigation highlighted potential applications, including assessment of PK/PD risks during early target validation, human dose prediction and drug candidate optimization.     

Preclinical pharmacokinetics,pharmacodynamics, tissue distribution,and tumor penetration of anti-PD-L1 monoclonal antibody,an immune checkpoint inhibitor

MPDL3280A is a human monoclonal antibody that targets programmed cell death-1 ligand 1 (PD-L1), and exerts anti-tumor activity mainly by blocking PD-L1 interaction with programmed cell death-1 (PD-1) and B7.1. It is being investigated as a potential therapy for locally advanced or metastatic malignancies. The purpose of the study reported here was to characterize the pharmacokinetics, pharmacodynamics, tissue distribution and tumor penetration of MPDL3280A and/or a chimeric anti-PD-L1 antibody PRO304397 to help further clinical development.

The pharmacokinetics of MPDL3280A in monkeys at 0.5, 5 and 20 mg·kg^?1 and the pharmacokinetics / pharmacodynamics of PRO304397 in mice at 1, 3 10 mg·kg^?1 were determined after a single intravenous dose. Tissue distribution and tumor penetration for radiolabeled PRO304397 in tumor-bearing mouse models were determined.

The pharmacokinetics of MPDL3280A and PRO304397 were nonlinear in monkeys and mice, respectively. Complete saturation of PD-L1 in blood in mice was achieved at serum concentrations of PRO304397 above ～0.5 µg·mL^?1. Tissue distribution and tumor penetration studies of PRO304397 in tumor-bearing mice indicated that the minimum tumor interstitial to plasma radioactivity ratio was ～0.3; saturation of target-mediated uptake in non–tumor tissues and desirable exposure in tumors were achieved at higher serum concentrations, and the distribution into tumors was dose-and time-dependent.

The biodistribution data indicated that the efficacious dose is mostly likely higher than that estimated based on simple pharmacokinetics/pharmacodynamics in blood. These data also allowed for estimation of the target clinical dose for further development of MPDL3280A.     

Conjugation of DM1 to anti-CD30 antibody has potential antitumor activity in CD30-positive hematological malignancies with lower systemic toxicity

ABSTRACT

An anti-CD30 antibody-drug conjugate incorporating the antimitotic agent DM1 and a stable SMCC linker, anti-CD30-MCC-DM1, was generated as a new antitumor drug candidate for CD30-positive hematological malignancies. Here, the in vitro and in vivo pharmacologic activities of anti-CD30-MCC-DM1 (also known as F0002-ADC) were evaluated and compared with ADCETRIS (brentuximab vedotin). Pharmacokinetics (PK) and the safety profiles in cynomolgus monkeys were assessed. Anti-CD30-MCC-DM1 was effective in in vitro cell death assays using CD30-positive lymphoma cell lines. We studied the properties of anti-CD30-MCC-DM1, including binding, internalization, drug release and actions. Unlike ADCETRIS, anti-CD30-MCC-DM1 did not cause a bystander effect in this study. In vivo, anti-CD30-MCC-DM1 was found to be capable of inducing tumor regression in subcutaneous inoculation of Karpas 299 (anaplastic large cell lymphoma), HH (cutaneous T-cell lymphoma) and L428 (Hodgkin’s disease) cell models. The half-lives of 4 mg/kg and 12 mg/kg anti-CD30-MCC-DM1 were about 5 days in cynomolgus monkeys, and the tolerated dose was 30 mg/kg in non-human primates, supporting the tolerance of anti-CD30-MCC-DM1 in humans. These results suggest that anti-CD30-MCC-DM1 presents efficacy, safety and PK profiles that support its use as a valuable treatment for CD30-positive hematological malignancies.     

Minimal physiologically-based pharmacokinetic modeling of DSTA4637A,A novel THIOMAB™ antibody antibiotic conjugate against Staphylococcus aureus,in a mouse model

DSTA4637A, a THIOMAB? antibody-antibiotic conjugate targeting Staphylococcus aureus, has shown promising bactericidal activity in a mouse model. DSTA4637A consists of a monoclonal anti-S. aureus antibody with an average of two rifalogue antibiotic molecules, dmDNA31, linked to its light chains. The goal of this study was to develop a minimal physiologically-based pharmacokinetic (mPBPK) model to characterize the pharmacokinetic (PK) properties of three analytes of DSTA4637A (i.e., total antibody, antibody-conjugated dmDNA31, and unconjugated dmDNA31) in mice, and to predict pharmacokinetics of DSTA4637A analytes in humans, as well as to provide an initial assessment for potential PK drug-drug interactions (DDI) in clinical trials via cross-species scaling of the mPBPK model. In the proposed model, selected organs, including heart, liver, and kidney, were connected anatomically with plasma and lymph flows. Mouse plasma and tissue concentrations of the three analytes of DSTA4637A were fitted simultaneously to estimate the PK parameters. Cross-species scaling of the model was performed by integrating allometric scaling and human physiological parameters. The final mPBPK model was able to successfully capture PK profiles of three DSTA4637A analytes in mouse plasma and in investigated organs. The model predicted a steady-state peak unbound dmDNA31 concentration lower than 5% of the IC₅₀ of dmDNA31 towards cytochrome P450 following 100 mg/kg weekly intravenous dose, which suggests a low risk of PK DDI in humans for DSTA4637A with co-administered cytochrome P450 substrates. The proposed mPBPK modeling and cross-species scaling approaches provide valuable tools that facilitate the understanding and translation of DSTA4637A disposition from preclinical species to humans.     

Dose dependent pharmacokinetics,tissue distribution,and anti-tumor efficacy of a humanized monoclonal antibody against DLL4 in mice

Delta-like-4 ligand (DLL4) plays an important role in vascular development and is widely expressed on the vasculature of normal and tumor tissues. Anti-DLL4 is a humanized IgG1 monoclonal antibody against DLL4. The purpose of these studies was to characterize the pharmacokinetics (PK), tissue distribution, and anti-tumor efficacy of anti-DLL4 in mice over a range of doses. PK and tissue distribution of anti-DLL4 were determined in athymic nude mice after administration of single intravenous (IV) doses. In the tissue distribution study, radiolabeled anti-DLL4 (mixture of ¹²⁵Iodide and ¹¹¹Indium) was administered in the presence of increasing amounts of unlabeled anti-DLL4. Dose ranging anti-DLL4 anti-tumor efficacy was evaluated in athymic nude mice bearing MV522 human lung tumor xenografts. Anti-DLL4 had nonlinear PK in mice with rapid serum clearance at low doses and slower clearance at higher doses suggesting the involvement of target mediated clearance. Consistent with the PK data, anti-DLL4 was shown to specifically distribute to several normal tissues known to express DLL4 including the lung and liver. Maximal efficacy in the xenograft model was seen at doses ≥ 10 mg/kg when tissue sinks were presumably saturated, consistent with the PK and tissue distribution profiles. These findings highlight the importance of mechanistic understanding of antibody disposition to enable dosing strategies for maximizing efficacy.     

Evaluating the Use of Antibody Variable Region (Fv) Charge as a Risk Assessment Tool for Predicting Typical Cynomolgus Monkey Pharmacokinetics

The pharmacokinetic (PK) behavior of monoclonal antibodies in cynomolgus monkeys (cynos) is generally translatable to that in humans. Unfortunately, about 39% of the antibodies evaluated for PKs in cynos have fast nonspecific (or non-target-mediated) clearance (in-house data). An empirical model relating variable region (Fv) charge and hydrophobicity to cyno nonspecific clearance was developed to gauge the risk an antibody would have for fast nonspecific clearance in the monkey. The purpose of this study was to evaluate the predictability of this empirical model on cyno nonspecific clearance with antibodies specifically engineered to have either high or low Fv charge. These amino acid changes were made in the Fv region of two test antibodies, humAb4D5-8 and anti-lymphotoxin α. The humAb4D5-8 has a typical nonspecific clearance in cynos, and by making it more positively charged, the antibody acquires fast nonspecific clearance, and making it less positively charged did not impact its clearance. Anti-lymphotoxin α has fast nonspecific clearance in cynos, and making it more positively charged caused it to clear even faster, whereas making it less positively charged caused it to clear slower and within the typical range. These trends in clearance were also observed in two other preclinical species, mice and rats. The effect of modifying Fv charge on subcutaneous bioavailability was also examined, and in general bioavailability was inversely related to the direction of the Fv charge change. Thus, modifying Fv charge appears to impact antibody PKs, and the changes tended to correlate with those predicted by the empirical model.     

Preclinical and translational pharmacokinetics of a novel THIOMAB™ antibody-antibiotic conjugate against Staphylococcus aureus

ABSTRACT

DSTA4637S, a novel THIOMAB? antibody-antibiotic conjugate (TAC) against Staphylococcus aureus (S. aureus), is currently being investigated as a potential therapy for complicated S. aureus bloodstream infections. DSTA4637S is composed of a monoclonal THIOMAB^TM IgG1 recognizing S. aureus linked to a rifamycin-class antibiotic (dmDNA31) via a protease-cleavable linker. The pharmacokinetics (PK) of DSTA4637A (a liquid formulation of DSTA4637S) and its unconjugated antibody MSTA3852A were characterized in rats and monkeys. Systemic concentrations of three analytes, total antibody (TAb), antibody-conjugated dmDNA31 (ac-dmDNA31), and unconjugated dmDNA31, were measured to describe complex TAC PK in nonclinical studies. In rats and monkeys, following intravenous administration of a single dose of DSTA4637A, systemic concentration-time profiles of both TAb and ac-dmDNA31 were bi-exponential, characterized by a short distribution phase and a long elimination phase as expected for a monoclonal antibody-based therapeutic. Systemic exposures of both TAb and ac-dmDNA31 were dose proportional over the dose range tested, and ac-dmDNA31 cleared 2–3 times faster than TAb. Unconjugated dmDNA31 plasma concentrations were low (<4 ng/mL) in every study regardless of dose. In this report, an integrated semi-mechanistic PK model for two analytes (TAb and ac-dmDNA31) was successfully developed and was able to well describe the complicated DSTA4637A PK in mice, rats and monkeys. DSTA4637S human PK was predicted reasonably well using this model with allometric scaling of PK parameters from monkey data. This work provides insights into PK behaviors of DSTA4637A in preclinical species and informs clinical translatability of these observed results and further clinical development.

Abbreviations: ADC: Antibody-drug conjugate; AUC_inf: time curve extrapolated to infinity; ac-dmDNA31: antibody-conjugated dmDNA31; C_max: maximum concentration observed; DAR: drug-to-antibody ratio; CL: clearance; CL_D: distribution clearance; CL₁: systemic clearance of all DAR species; k_DC: deconjugation rate constant; PK: Pharmacokinetics; IV: Intravenous; IgG: Immunoglobulin G; mAb: monoclonal antibody; S. aureus: Staphylococcus aureus; TAC: THIOMAB^TM antibody-antibiotic conjugate; TDC: THIOMAB^TM antibody-drug conjugate; TAb: total antibody; t_{1/2, λz}: terminal half-life; vc linker: valine-citrulline linker; V_ss: volume of distribution at steady state; V_c: volume of distribution for the central compartment; V_p: the volume of distribution for the peripheral compartment.