Frank-Peter Theil

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.

Projecting human pharmacokinetics of therapeutic antibodies from nonclinical data: What have we learned?

The pharmacokinetics (PK) of therapeutic antibodies is determined by target and non-target mediated mechanisms. These antibody-specific factors need to be considered during prediction of human PK based upon preclinical information. Principles of allometric scaling established for small molecules using data from multiple animal species cannot be directly applied to antibodies. Here, different methods for projecting human clearance (CL) from animal PK data for 13 therapeutic monoclonal antibodies (mAbs) exhibiting linear PK over the tested dose ranges were examined: simple allometric scaling (CL versus body weight), allometric scaling with correction factors, allometric scaling based on rule of exponent and scaling from only cynomolgus monkey PK data. A better correlation was obtained between the observed human CL and the estimated human CL based on cynomolgus monkey PK data and an allometric scaling exponent of 0.85 for CL than other scaling approaches. Human concentration-time profiles were also reasonably predicted from the cynomolgus monkey data using species-invariant time method with a fixed exponent of 0.85 for CL and 1.0 for volume of distribution. In conclusion, we expanded our previous work and others and further confirmed that PK from cynomolgus monkey alone can be successfully scaled to project human PK profiles within linear range using simplify allometry and Dedrick plots with fixed exponent.

Pharmacokinetics and toxicology of therapeutic proteins: Advances and challenges

Significant progress has been made in understanding pharmacokinetics (PK), pharmacodynamics (PD), as well as toxicity profiles of therapeutic proteins in animals and humans, which have been in commercial development for more than three decades. However, in the PK arena, many fundamental questions remain to be resolved. Investigative and bioanalytical tools need to be established to improve the translation of PK data from animals to humans, and from in vitro assays to in vivo readouts, which would ultimately lead to a higher success rate in drug development. In toxicology, it is known, in general, what studies are needed to safely develop therapeutic proteins, and what studies do not provide relevant information. One of the major complicating factors in nonclinical and clinical programs for therapeutic proteins is the impact of immunogenicity. In this review, we will highlight the emerging science and technology, as well as the challenges around the pharmacokinetic- and safety-related issues in drug development of mAbs and other therapeutic proteins.

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.

Whole body physiologically-based pharmacokinetic models: their use in clinical drug development

Background: Whole-body physiologically-based pharmacokinetic (WB-PBPK) models mathematically describe an organism as a closed circulatory system consisting of compartments that represent the organs important for compound absorption, distribution, metabolism and elimination. Objectives: To review the current state of WB-PBPK model use in the clinical phases of drug development. Methods: A qualitative description of the WB-PBPK model structure is included along with a review of the varying methods available for input parameterisation. Current and potential WB-PBPK model application in clinical development is discussed. Conclusions: This modelling tool is at present used for small and large molecule drug development primarily as a means to scale pharmacokinetics from animals to humans based on physiology. The pharmaceutical industry is active in employing these models to clinical drug development although the applications in use now are narrow in comparison to the potential. Expanded integration of WB-PBPK models into the drug development process will only be achieved with staff training, managerial will, success stories and regulatory agency openness.

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.

Effects of Anti-VEGF on Pharmacokinetics, Biodistribution, and Tumor Penetration of Trastuzumab in a Preclinical Breast Cancer Model

Both human epidermal growth factor receptor 2 (HER-2/neu) and VEGF overexpression correlate with aggressive phenotypes and decreased survival among breast cancer patients. Concordantly, the combination of trastuzumab (anti-HER2) with bevacizumab (anti-VEGF) has shown promising results in preclinical xenograft studies and in clinical trials. However, despite the known antiangiogenic mechanism of anti-VEGF antibodies, relatively little is known about their effects on the pharmacokinetics and tissue distribution of other antibodies. This study aimed to measure the disposition properties, with a particular emphasis on tumor uptake, of trastuzumab in the presence or absence of anti-VEGF. Radiolabeled trastuzumab was administered alone or in combination with an anti-VEGF antibody to mice bearing HER2-expressing KPL-4 breast cancer xenografts. Biodistribution, autoradiography, and single-photon emission computed tomography–X-ray computed tomography imaging all showed that anti-VEGF administration reduced accumulation of trastuzumab in tumors despite comparable blood exposures and similar distributions in most other tissues. A similar trend was also observed for an isotype-matched IgG with no affinity for HER2, showing reduced vascular permeability to macromolecules. Reduced tumor blood flow (P < 0.05) was observed following anti-VEGF treatment, with no significant differences in the other physiologic parameters measured despite immunohistochemical evidence of reduced vascular density. In conclusion, anti-VEGF preadministration decreased tumor uptake of trastuzumab, and this phenomenon was mechanistically attributed to reduced vascular permeability and blood perfusion. These findings may ultimately help inform dosing strategies to achieve improved clinical outcomes. Mol Cancer Ther; 11(3); 752–62. ©2012 AACR.

Antitumor Activity of Targeted and Cytotoxic Agents in Murine Subcutaneous Tumor Models Correlates with Clinical Response

Purpose: Immunodeficient mice transplanted with subcutaneous tumors (xenograft or allograft) are widely used as a model of preclinical activity for the discovery and development of anticancer drug candidates. Despite their widespread use, there is a widely held view that these models provide minimal predictive value for discerning clinically active versus inactive agents. To improve the predictive nature of these models, we have carried out a retrospective population pharmacokinetic–pharmacodynamic (PK–PD) analysis of relevant xenograft/allograft efficacy data for eight agents (molecularly targeted and cytotoxic) with known clinical outcome. Experimental Design: PK–PD modeling was carried out to first characterize the relationship between drug concentration and antitumor activity for each agent in dose-ranging xenograft or allograft experiments. Next, simulations of tumor growth inhibition (TGI) in xenografts/allografts at clinically relevant doses and schedules were carried out by replacing the murine pharmacokinetics, which were used to build the PK–PD model with human pharmacokinetics obtained from literature to account for species differences in pharmacokinetics. Results: A significant correlation (r = 0.91, P = 0.0008) was observed between simulated xenograft/allograft TGI driven by human pharmacokinetics and clinical response but not when TGI observed at maximum tolerated doses in mice was correlated with clinical response (r = 0.36, P = 0.34). Conclusions: On the basis of these analyses, agents that led to greater than 60% TGI in preclinical models, at clinically relevant exposures, are more likely to lead to responses in the clinic. A proposed strategy for the use of murine subcutaneous models for compound selection in anticancer drug discovery is discussed. Clin Cancer Res; 18(14); 3846–55. ©2012 AACR.

Development of a novel method for predicting human volume of distribution at steady-state of basic drugs and comparative assessment with existing methods.

The parameters characterizing tissue distribution refer to the tissue/plasma partition coefficients (Kp), which can be used to derive volume of distribution at steady-state (V(ss)). The effort for predicting drug distribution in human has been further expanded to calculation methods using in vitro-based algorithms. The objective of the present study was to develop a novel prediction method to estimate human V(ss) for moderate-to-strong bases. The predictive performance of the novel method was compared with other well established in vitro-based methods available in the literature. Relevant information collected from previous prediction studies of V(ss) facilitated the development of the novel method. This was based on the calculation of V(ss) from data on Kp, which were estimated by correlating the unbound tissue/plasma ratio in vivo (Kpu) with the unbound red blood cells partitioning (RBCu) determined in vitro. The comparative assessment of the novel correlation method with existing prediction methods of human V(ss) was done using a literature dataset of 61 basic drugs (at least one pK(a) > or = 7). The five existing V(ss) prediction methods published in the literature are comprised of four versions of tissue composition-based models along with the model of Lombardo using the principle of Oie-Tozer. The statistical analysis of the prediction performance indicated that the novel method demonstrated a greater degree of accuracy compared to all other published methods. The maximum percentage of predicted values that fall within a twofold-error range is 77% for the basic drugs tested. Overall, the present study describes the development and the assessment of the predictive performance of the novel prediction method of human V(ss) based upon in vitro data, which appears to be superior based on the current dataset studied for basic drugs.

Minipig as a potential translatable model for monoclonal antibody pharmacokinetics after intravenous and subcutaneous administration

Subcutaneous (SC) delivery is a common route of administration for therapeutic monoclonal antibodies (mAbs) with pharmacokinetic (PK)/pharmacodynamic (PD) properties requiring long-term or frequent drug administration. An ideal in vivo preclinical model for predicting human PK following SC administration may be one in which the skin and overall physiological characteristics are similar to that of humans. In this study, the PK properties of a series of therapeutic mAbs following intravenous (IV) and SC administration in Göttingen minipigs were compared with data obtained previously from humans. The present studies demonstrated: (1) minipig is predictive of human linear clearance; (2) the SC bioavailabilities in minipigs are weakly correlated with those in human; (3) minipig mAb SC absorption rates are generally higher than those in human and (4) the SC bioavailability appears to correlate with systemic clearance in minipigs. Given the important role of the neonatal Fc-receptor (FcRn) in the PK of mAbs, the in vitro binding affinities of these IgGs against porcine, human and cynomolgus monkey FcRn were tested. The result showed comparable FcRn binding affinities across species. Further, mAbs with higher isoelectric point tended to have faster systemic clearance and lower SC bioavailability in both minipig and human. Taken together, these data lend increased support for the use of the minipig as an alternative predictive model for human IV and SC PK of mAbs.