Tracey Clark

The Application of Target Information and Preclinical Pharmacokinetic/Pharmacodynamic Modeling in Predicting Clinical Doses of a Dickkopf-1 Antibody for Osteoporosis

PF-04840082 is a humanized prototype anti-Dickkopf-1 (Dkk-1) immunoglobulin isotype G2 (IgG2) antibody for the treatment of osteoporosis. In vitro, PF-04840082 binds to human, monkey, rat, and mouse Dkk-1 with high affinity. After administration of PF-04840082 to rat and monkey, free Dkk-1 concentrations decreased rapidly and returned to baseline in a dose-dependent manner. In rat and monkey, PF-04840082 exhibited nonlinear pharmacokinetics (PK) and a target-mediated drug disposition (TMDD) model was used to characterize PF-04840082 versus Dkk-1 concentration response relationship. PK/pharmacodynamic (PK/PD) modeling enabled estimation of antibody non-target-mediated elimination, Dkk-1 turnover, complex formation, and complex elimination. The TMDD model was translated to human to predict efficacious dose and minimum anticipated biological effect level (MABEL) by incorporating information on typical IgG2 human PK, antibody-target association/dissociation rates, Dkk-1 expression, and turnover rates. The PK/PD approach to MABEL was compared with the standard “no adverse effect level” (NOAEL) approach to calculating clinical starting doses and a pharmacological equilibrium method. The NOAEL method gave estimates of dose that were too high to ensure safety of clinical trials. The pharmacological equilibrium approach calculated receptor occupancy (RO) based on equilibrium dissociation constant alone and did not take into account rate of turnover of the target or antibody–target complex kinetics and, as a result, it likely produced a substantial overprediction of RO at a given dose. It was concluded that the calculation of MABEL according to the TMDD model was the most appropriate means for ensuring safety and efficacy in clinical studies.

Design of selective, ATP-competitive inhibitors of Akt.

This paper describes the design and synthesis of novel, ATP-competitive Akt inhibitors from an elaborated 3-aminopyrrolidine scaffold. Key findings include the discovery of an initial lead that was modestly selective and medicinal chemistry optimization of that lead to provide more selective analogues. Analysis of the data suggested that highly lipophilic analogues would likely suffer from poor overall properties. Central to the discussion is the concept of optimization of lipophilic efficiency and the ability to balance overall druglike propeties with the careful control of lipophilicity in the lead series. Discovery of the nonracemic amide series and subsequent modification produced an advanced analogue that performed well in advanced preclinical assays, including xenograft tumor growth inhibition studies, and this analogue was nominated for clinical development.

Synthesis and structure based optimization of novel Akt inhibitors

Based on a high throughput screening hit, pyrrolopyrimidine inhibitors of the Akt kinase are explored. X-ray co-crystal structures of two lead series results in the understanding of key binding interactions, the design of new lead series, and enhanced potency. The syntheses of these series and their biological activities are described. Spiroindoline 13j is found to have an Akt1 kinase IC(50) of 2.4+/-0.6 nM, Akt cell potency of 50+/-19 nM, and provides 68% inhibition of tumor growth in a mouse xenograft model (50 mg/kg, qd, po).

Plasma/serum protein binding determinations.

The binding of a drug to serum or plasma proteins enables the transport of drugs via the blood to sites of action throughout the body. For expediency we will use serum proteins throughout this discussion with the understanding that one can substitute the term plasma proteins in all experimental instances. Only the fraction of drug unbound from serum proteins is available to diffuse from the vascular system and accumulate in tissues thereby enabling interaction with therapeutic targets and accessibility to xenobiotic clearance pathways. Therefore, the extent of drug binding to serum proteins can have a significant impact on pharmacokinetic (PK) parameters such as clearance rates and volume of distribution. In addition, because only the unbound drug is available to interact with therapeutic targets, the extent of serum binding can have significant effects on the pharmacodynamic properties of a compound as well [1, 2] Determining the fraction of drug bound to serum proteins is a standard parameter evaluated in the process of drug discovery. Although the clinical importance of changes in serum protein binding has been questioned [3-8] the need for serum protein binding studies in the discovery and preclinical development stages is essential for the pharmacokinetic modeling of drugs [1, 3, 9]. The extent of serum protein binding is an important parameter used in many in vivo modeling calculations to estimate the volume of distribution, organ clearance, and for scale-up of pharmacokinetic and pharmacodynamic parameters from animal models to humans [3, 10, 11]. The convergence of several trends in the pharmaceutical industry including high speed chemical synthesis technologies, the increasing use of in silico ADME modeling together with early in vivo evaluations of lead compounds has increased the demand for serum protein binding determinations [12].

Neonatal Fc Receptor and its Role in the Absorption, Distribution, Metabolism and Excretion of Immunoglobulin G-Based Biotherapeutics

The neonatal Fc receptor (FcRn) is a heterodimeric membrane associated protein expressed in a variety of endothelial, epithelial and hematopoietic cells. FcRn regulates pH dependent intracellular trafficking of immunoglobulin G (IgG) and albumin, resulting in enhanced serum persistence and transcellular permeability of these proteins compared to other proteins of similar size. FcRn confers passive immunity during the early stages of life by facilitating maternal transmission of antibodies during gestation, and in some species during the neonatal period. The receptor continues to contribute to immunity beyond the perinatal period and throughout life by providing immunosurveillance in intestinal, pulmonary and genitourinary mucosa. In this capacity, FcRn facilitates bidirectional transport of IgG across mucosa and intracellular trafficking of antigen-antibody complexes in antigen presenting cells. Based on the functional roles of FcRn in regulating serum persistence and transcellular permeability, protein engineers have sought to exploit this receptor as a means of enhancing the absorption, distribution, metabolism and excretion (ADME) of IgG-based therapeutics. In this review, the current state of knowledge regarding the structural, mechanistic and functional properties of FcRn, as they relate to the ADME of IgG-based therapeutics, are discussed.

Optimization of Tubulysin Antibody–Drug Conjugates: A Case Study in Addressing ADC Metabolism

As part of our efforts to develop new classes of tubulin inhibitor payloads for antibody–drug conjugate (ADC) programs, we developed a tubulysin ADC that demonstrated excellent in vitro activity but suffered from rapid metabolism of a critical acetate ester. A two-pronged strategy was employed to address this metabolism. First, the hydrolytically labile ester was replaced by a carbamate functional group resulting in a more stable ADC that retained potency in cellular assays. Second, site-specific conjugation was employed in order to design ADCs with reduced metabolic liabilities. Using the later approach, we were able to identify a conjugate at the 334C position of the heavy chain that resulted in an ADC with considerably reduced metabolism and improved efficacy. The examples discussed herein provide one of the clearest demonstrations to-date that site of conjugation can play a critical role in addressing metabolic and PK liabilities of an ADC. Moreover, a clear correlation was identified between the hydrophobicity of an ADC and its susceptibility to metabolic enzymes. Importantly, this study demonstrates that traditional medicinal chemistry strategies can be effectively applied to ADC programs.

Where Did the Linker-Payload Go? A Quantitative Investigation on the Destination of the Released Linker-Payload from an Antibody-Drug Conjugate with a Maleimide Linker in Plasma

The reactive thiol of cysteine is often used for coupling maleimide-containing linker-payloads to antibodies resulting in the generation of antibody drug conjugates (ADCs). Currently, a numbers of ADCs in drug development are made by coupling a linker-payload to native or engineered cysteine residues on the antibody. An ADC conjugated via hinge-cysteines to an auristatin payload was used as a model in this study to understand the impact of the maleimide linkers on ADC stability. The payload was conjugated to trastuzumab by a protease-cleavable linker, maleimido-caproyl-valine-citruline-p-amino-benzyloxy carbonyl (mcVC-PABC). In plasma stability assays, when the ADC (Trastuzumab-mcVC-PABC-Auristatin-0101) was incubated with plasma over a 144-h time-course, a discrepancy was observed between the measured released free payload concentration and the measured loss of drug-to-antibody ratio (DAR), as measured by liquid chromatography-mass spectrometry (LC-MS). We found that an enzymatic release of payload from ADC-depleted human plasma at 144 h was able to account for almost 100% of the DAR loss. Intact protein mass analysis showed that at the 144 h time point, the mass of the major protein in ADC-depleted human plasma had an additional 1347 Da over the native albumin extracted from human plasma, exactly matching the mass of the linker-payload. In addition, protein gel electrophoresis showed that there was only one enriched protein in the 144 h ADC-depleted and antipayload immunoprecipitated plasma sample, as compared to the 0 h plasma immunoprecipitated sample, and the mass of this enriched protein was slightly heavier than the mass of serum albumin. Furthermore, the albumin adduct was also identified in 96 h and 168 h postdose in vivo cynomolgus monkey plasma. These results strongly suggest that the majority of the deconjugated mc-VC-PABC-auristatin ultimately is transferred to serum albumin, forming a long-lived albumin-linker-payload adduct. To our knowledge, this is the first report quantitatively characterizing the extent of linker-payload transfer to serum albumin and the first clear example of in vivo formation of an albumin-linker-payload adduct.

Antibody–drug conjugates nonclinical support: from early to late nonclinical bioanalysis using ligand-binding assays

The objective of antibody-drug conjugate (ADC) bioanalysis at different stages of drug development may vary and so are the associated bioanalytical challenges. While at early drug discovery stage involving candidate selection, optimization and preliminary nonclinical assessments, the goal of ADC bioanalysis is to provide PK, toxicity and efficacy data that assists in the design and selection of potential drug candidates, the late nonclinical and clinical drug development stage typically involves regulated ADC bioanalysis that delivers TK data to define and understand pharmacological and toxicological properties of the lead ADC candidate. Bioanalytical strategies and considerations involved in developing successful ligand binding assays for ADC characterization from early discovery to late nonclinical stages of drug development are presented here.

Insights into antibody–drug conjugates: bioanalysis and biomeasures in discovery

Author for correspondence: Department of Pharmacokinetics, Dynamics & Metabolism, Pfizer Global Research & Development, Eastern Point Rd, Groton, CT 06340, USA Tel.: +1 860 715 0641 Fax: +1 860 715 9501 E-mail: tracey.clark@pfizer.com Xiaogang Han Department of Pharmacokinetics, Dynamics & Metabolism, Pfizer Global Research & Development, CT, USA Lindsay King Department of Pharmacokinetics, Dynamics & Metabolism, Pfizer Global Research & Development, CT, USA Frank Barletta Department of Pharmacokinetics, Dynamics & Metabolism, Pfizer, NY, USA Antibody–drug conjugate (ADC) therapeutics utilize the specificity of monoclonal antibodies (mAbs) and potency of highly toxic small molecules. ADCs are typically composed of a mAb with a cytotoxin conjugated to it, resulting in a heterogeneous mixture of mAb with various numbers of toxins. Due to this heterogeneity, characterized by the therapeutic drug-to-antibody ratio (DAR), the selection of bioanalytical tools, biomeasure assays and analytes used to understand and develop ADCs can be challenging [1]. Since the therapeutic has both largeand small-molecule components, one can use bioanalytical tools in both spaces. Questions for ADC programs are typically: what should be measured, when, and by which method? Since all assays have limitations [1], a combination of bioanalytical tools is typically used to understand the ADC in vitro/in vivo, understand payload delivery to the site of action and to establish an exposure–response relationship.

Site Selection: a Case Study in the Identification of Optimal Cysteine Engineered Antibody Drug Conjugates

As the antibody drug conjugate (ADC) community continues to shift towards site-specific conjugation technology, there is a growing need to understand how the site of conjugation impacts the biophysical and biological properties of an ADC. In order to address this need, we prepared a carefully selected series of engineered cysteine ADCs and proceeded to systematically evaluate their potency, stability, and PK exposure. The site of conjugation did not have a significant influence on the thermal stability and in vitro cytotoxicity of the ADCs. However, we demonstrate that the rate of cathepsin-mediated linker cleavage is heavily dependent upon site and is closely correlated with ADC hydrophobicity, thus confirming other recent reports of this phenomenon. Interestingly, conjugates with high rates of cathepsin-mediated linker cleavage did not exhibit decreased plasma stability. In fact, the major source of plasma instability was shown to be retro-Michael mediated deconjugation. This process is known to be impeded by succinimide hydrolysis, and thus, we undertook a series of mutational experiments demonstrating that basic residues located nearby the site of conjugation can be a significant driver of succinimide ring opening. Finally, we show that total antibody PK exposure in rat was loosely correlated with ADC hydrophobicity. It is our hope that these observations will help the ADC community to build “design rules” that will enable more efficient prosecution of next-generation ADC discovery programs.