Abstract. The objectives of this investigation were as follows: (a) to validate a mechanism-based pharmacokinetic (PK) model of ADC for its ability to a priori predict tumor concentrations of ADC and released payload, using anti-5T4 ADC A1mcMMAF, and (b) to analyze the PK model to find out main pathways and parameters model outputs are most sensitive to. Experiential data containing biomeasures, and plasma and tumor concentrations of ADC and payload, following A1mcMMAF administration in two different xenografts, were used to build and validate the model. The model performed reasonably well in terms of a priori predicting tumor exposure of total antibody, ADC, and released payload, and the exposure of released payload in plasma. Model predictions were within two fold of the observed exposures. Pathway analysis and local sensitivity analysis were conducted to investigate main pathways and set of parameters the model outputs are most sensitive to. It was discovered that payload dissociation from ADC and tumor size were important determinants of plasma and tumor payload exposure. It was also found that the sensitivity of the model output to certain parameters is dose-dependent, suggesting caution before generalizing the results from the sensitivity analysis. Model analysis also revealed the importance of understanding and quantifying the processes responsible for ADC and payload disposition within tumor cell, as tumor concentrations were sensitive to these parameters. Proposed ADC PK model provides a useful tool for a priori predicting tumor payload concentrations of novel ADCs preclinically, and possibly translating them to the clinic.
Non-invasive imaging using radiolabels is a common technique used to study the biodistribution of biologics. Due to the limited shelf-life of radiolabels and the requirements of specialized labs, non-invasive optical imaging is an attractive alternative for preclinical studies. Previously, we demonstrated the utility of fluorescence molecular tomography (FMT) an optical imaging modality in evaluating the biodistribution of antibody-drug conjugates. As FMT is a relatively new technology, few fluorophores have been validated for in vivo imaging. The goal of this study was to characterize and determine the utility of near-infrared (NIR) fluorophores for biodistribution studies using interleukin-13 receptor subunit alpha-2 antibody (IL13Rα2-Ab). Eight fluorophores (ex/em: 630/800 nm) with an N-hydroxysuccinimide (NHS) linker were evaluated for Ab conjugation. The resulting antibody-fluorophore (Ab-F) conjugates were evaluated in vitro for degree of conjugation, stability and target-binding, followed by in vivo/ex vivo FMT imaging to determine biodistribution in a xenograft model. The Ab-F conjugates (except Ab-DyLight800) showed good in vitro stability and antigen binding. All Ab-F conjugates (except for Ab-BOD630) resulted in a quantifiable signal in vivo and had similar biodistribution profiles, with peak tumor accumulation between 6 and 24 h post-injection. In vivo/ex vivo FMT imaging showed 17-34% ID/g Ab uptake by the tumor at 96 h. Overall, this is the first study to characterize the biodistribution of an Ab using eight NIR fluorophores. Our results show that 3-dimensional optical imaging is a valuable technology to understand biodistribution and targeting, but a careful selection of the fluorophore for each Ab is warranted.
There are many sources of analytical variability in ligand binding assays (LBA). One strategy to reduce variability has been duplicate analyses. With recent advances in LBA technologies, it is conceivable that singlet analysis is possible. We retrospectively evaluated singlet analysis using Gyrolab data. Relative precision of duplicates compared to singlets was evaluated using 60 datasets from toxicokinetic (TK) or pharmacokinetic (PK) studies which contained over 23,000 replicate pairs composed of standards, quality control (QC), and animal samples measured with 23 different bioanalytical assays. The comparison was first done with standard curve and QCs followed by PK parameters (i.e., Cmax and AUC). Statistical analyses were performed on combined duplicate versus singlets using a concordance correlation coefficient (CCC), a measurement used to assess agreement. Variance component analyses were conducted on PK estimates to assess the relative analytical and biological variability. Overall, 97.5% of replicate pairs had a %CV of <11% and 50% of the results had a %CV of ≤1.38%. There was no observable bias in concentration comparing the first replicate with the second (CCC of 0.99746 and accuracy value of 1). The comparison of AUC and Cmax showed no observable difference between singlet and duplicate (CCC for AUC and Cmax >0.99999). Analysis of variance indicated an AUC inter-subject variability 35.3-fold greater than replicate variability and 8.5-fold greater for Cmax. Running replicates from the same sample will not significantly reduce variation or change PK parameters. These analyses indicated the majority of variance was inter-subject and supported the use of a singlet strategy.
The pharmacokinetics of an antibody (huA1)-drug (auristatin microtubule disrupting MMAF) conjugate, targeting 5T4-expressing cells, were characterized during the discovery and development phases in female nu/nu mice and cynomolgus monkeys after a single dose and in S-D rats and cynomolgus monkeys from multidose toxicity studies. Plasma/serum samples were analyzed using an ELISA-based method for antibody and conjugate (ADC) as well as for the released payload using an LC-MS/MS method. In addition, the distribution of the Ab, ADC, and released payload (cys-mcMMAF) was determined in a number of tissues (tumor, lung, liver, kidney, and heart) in two tumor mouse models (H1975 and MDA-MB-361-DYT2 models) using similar LBA and LC-MS/MS methods. Tissue distribution studies revealed preferential tumor distribution of cys-mcMMAF and its relative specificity to the 5T4 target containing tissue (tumor). Single dose studies suggests lower CL values at the higher doses in mice, although a linear relationship was seen in cynomolgus monkeys at doses from 0.3 to 10 mg/kg with no evidence of TMDD. Evaluation of DAR (drug-antibody ratio) in cynomolgus monkeys (at 3 mg/kg) indicated that at least half of the payload was still on the ADC 1 to 2 weeks after IV dosing. After multiple doses, the huA1 and conjugate data in rats and monkeys indicate that exposure (AUC) increases with increasing dose in a linear fashion. Systemic exposure (as assessed by Cmax and AUC) of the released payload increased with increasing dose, although exposure was very low and its pharmacokinetics appeared to be formation rate limited. The incidence of ADA was generally low in rats and monkeys. We will discuss cross species comparison, relationships between the Ab, ADC, and released payload exposure after multiple dosing, and insights into the distribution of this ADC with a focus on experimental design as a way to address or bypass apparent obstacles and its integration into predictive models.
Non-invasive imaging using radiolabels is a common technique used to study the biodistribution of biologics. Due to the limited shelf-life of radiolabels and the requirements of specialized labs, non-invasive optical imaging is an attractive alternative for preclinical studies. Previously, we demonstrated the utility of fluorescence molecular tomography (FMT) an optical imaging modality in evaluating the biodistribution of antibody-drug conjugates. As FMT is a relatively new technology, few fluorophores have been validated for in vivo imaging. The goal of this study was to characterize and determine the utility of near-infrared (NIR) fluorophores for biodistribution studies using interleukin-13 receptor subunit alpha-2 antibody (IL13Rα2-Ab). Eight fluorophores (ex/em: 630/800 nm) with an N-hydroxysuccinimide (NHS) linker were evaluated for Ab conjugation. The resulting antibody-fluorophore (Ab-F) conjugates were evaluated in vitro for degree of conjugation, stability and target-binding, followed by in vivo/ex vivo FMT imaging to determine biodistribution in a xenograft model. The Ab-F conjugates (except Ab-DyLight800) showed good in vitro stability and antigen binding. All Ab-F conjugates (except for Ab-BOD630) resulted in a quantifiable signal in vivo and had similar biodistribution profiles, with peak tumor accumulation between 6 and 24 h post-injection. In vivo/ex vivo FMT imaging showed 17–34% ID/g Ab uptake by the tumor at 96 h. Overall, this is the first study to characterize the biodistribution of an Ab using eight NIR fluorophores. Our results show that 3-dimensional optical imaging is a valuable technology to understand biodistribution and targeting, but a careful selection of the fluorophore for each Ab is warranted.
The pharmacokinetics of ezlopitant were determined in the rat, gerbil, guinea pig, ferret, dog and monkey after intravenous and oral administration. In general, ezlopitant is marked by high clearance values that approach or exceed hepatic blood flow values, moderate to high values for steady-state volume of distribution (3. 9-28 L/kg), and terminal phase half-life values ranging from 0.6 h in the guinea pig to 7.7 h in the rat. Oral bioavailability ranged from <0.2% (guinea pig) to 28% (dog). Data from portal vein cannulated dogs suggested that 37% of an oral dose of ezlopitant enters the portal vein as an unchanged drug in this species. Ezlopitant is metabolized to two pharmacologically active metabolites, an alkene (CJ-12 458) and a benzyl alcohol (CJ-12 764). After administration of the parent compound, CJ-12 764 was found in greater abundance than CJ-12 458 in all species examined. Ezlopitant and CJ-12 458 were highly protein bound in plasma (or serum), whereas the protein binding of CJ-12 764 was somewhat lower. Measurement of the kinetics of ezlopitant, CJ-12 458 and CJ-12 764 in the cerebrospinal fluid (CSF) of dogs demonstrated that all three compounds can partition into the CSF, and thereby, be capable of contributing to centrally mediated pharmacological effects. Thus, these data suggest that the pharmacological activity exhibited by ezlopitant in preclinical species in vivo is likely a result of the parent compound plus the active metabolites. Furthermore, the contributions of ezlopitant and the active metabolites to pharmacological activity probably varies with the identity of the model species, as well as the dose and route of ezlopitant administration.
2539 Background: CD40 is expressed on B-cells, monocytes, dendritic cells, other normal tissues and tumors. Previous studies showed that CD40 stimulation enhances antigen presentation, breaks tolerance, bypasses T-cell help, and induces apoptosis in CD40pos tumor cells. We report the in vitro activity and primate pharmacokinetics of a human anti-CD40 agonist antibody, CP-870,893, currently in clinical trials for the treatment of cancer. Methods: CP-870,893 was identified as a CD40 agonist antibody by screening lead molecules generated through the Abgenix Xenomouse® platform. Agonist activity was determined using upregulation of B-cell and monocytes derived dendritic cell surface markers, as well as dendritic cell IL-12 induction. BIAcore and equilibrium binding were utilized to determine affinity, and competition studies with CD40L were conducted on BIAcore. CP-870,893 was administered to cynomolgus monkeys i.v. at various doses, serum antibody levels were evaluated over time in an ELISA assay, and B-cell markers were monitored by FACS. Results: CP-870,893 (IgG2, kappa) binds CD40 with sub-nanomolar affinity, and does not block binding of CD40L. When human whole blood is incubated with CP-870,893, upregulation of key surface molecules involved in antigen presentation (MHC Class II, CD80, CD86, CD23 and ICAM-1) is observed with an EC50 of 5–50 ng/ml. Human monocytes derived dendritic cells, when stimulated with CP-870,893, upregulate activation markers (MHC Class II, CD80 and CD83) with an EC50 of 100–300 ng/ml, and secrete high levels of IL-12p40. In the presence of a second stimulus, such as LPS, human dendritic cells also secreted bioactive IL12-p70 when stimulated with CP-870,893 (EC50 ∼ 150 ng/ml). In addition, a CD40 positive human B-cell tumor line, when stimulated with CP-870,893, becomes susceptible to killing by human CTLs. In cynomolgus monkey studies, the clearance of CP-870,893 decreased with increasing dose. Circulating B-cell numbers decreased, and surface molecules were upregulated on B-cells. Conclusions: These data support the potential utility of CP-870,893 as an immune enhancing agent in cancer immunotherapy, by activating antigen presenting cells, and by enhancing the immunogenicity of CD40 positive tumor cells. [Table: see text]
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