Current strategies in the non-clinical safety assessment of biologics: New targets, new molecules, new challenges.

Nonclinical safety testing of biopharmaceuticals can present significant challenges to human risk assessment with these innovative and often complex drugs. Emerging topics in this field were discussed recently at the 2016 Annual US BioSafe General Membership meeting. The presentations and subsequent discussions from the main sessions are summarized. The topics covered included: (i) specialty biologics (oncolytic virus, gene therapy, and gene editing-based technologies), (ii) the value of non-human primates (NHPs) for safety assessment, (iii) challenges in the safety assessment of immuno-oncology drugs (T cell-dependent bispecifics, checkpoint inhibitors, and costimulatory agonists), (iv) emerging therapeutic approaches and modalities focused on microbiome, oligonucleotide, messenger ribonucleic acid (mRNA) therapeutics, (v) first in human (FIH) dose selection and the minimum anticipated biological effect level (MABEL), (vi) an update on current regulatory guidelines, International Council for Harmonization (ICH) S1, S3a, S5, S9 and S11 and (vii) breakout sessions that focused on bioanalytical and PK/PD challenges with bispecific antibodies, cytokine release in nonclinical studies, determining adversity and NOAEL for biologics, the value of second species for toxicology assessment and what to do if there is no relevant toxicology species.

Biotherapeutics in non-clinical development: Strengthening the interface between safety, pharmacokinetics-pharmacodynamics and manufacturing.

Biological drugs comprise a wide field of different modalities with respect to structure, pharmacokinetics and pharmacological function. Considerable non-clinical experience in the development of proteins (e.g. insulin) and antibodies has been accumulated over the past thirty years. In order to improve the efficacy and the safety of these biotherapeutics, Fc modifications (e.g. Fc silent antibody versions), combinations (antibody-drug conjugates, protein-nanoparticle combinations), and new constructs (darpins, fynomers) have been introduced. In the last decade, advanced therapy medicinal products (ATMPs) in research and development have become a considerable and strongly growing part of the biotherapeutic portfolio. ATMPs consisting of gene and cell therapy modalities or even combinations of them, further expand the level of complexity, which already exists in non-clinical development strategies for biological drugs and has thereby led to a further diversification of expertise in safety and PKPD assessment of biological drugs. It is the fundamental rationale of the BioSafe meetings, held yearly in the EU and in the US, to convene experts on a regular basis and foster knowledge exchange and mutual understanding in this fast growing area. In order to reflect at least partially the variety of the biotherapeutics field, the 2016 EU BioSafe meeting addressed the following topics in six sessions: (i) In vitro Meets in vivo to Leverage Biologics Development (ii) New developments and regulatory considerations in the cell and gene therapy field (iii) CMC Challenges with Biologics development (iv) Minipigs in non-clinical safety assessment (v) Opportunities of PKPD Assessment in Less Common Administration Routes In the breakout sessions the following questions were discussed: (i) Cynomolgus monkey as a reprotoxicology Species: Impact of Immunomodulators on Early Pregnancy Maintenance (ii) Safety Risk of Inflammation and Autoimmunity Induced by Immunomodulators (iii) Experience with non-GMP Material in Pivotal Non-clinical Safety Studies to Support First in Man (FiM) Trials (iv) Safety Assessment of Combination Products for Non-oncology.

Development of a Translational Physiologically Based Pharmacokinetic Model for Antibody-Drug Conjugates: a Case Study with T-DM1.

Systems pharmacokinetic (PK) models that can characterize and predict whole body disposition of antibody-drug conjugates (ADCs) are needed to support (i) development of reliable exposure-response relationships for ADCs and (ii) selection of ADC targets with optimal tumor and tissue expression profiles. Towards this goal, we have developed a translational physiologically based PK (PBPK) model for ADCs, using T-DM1 as a tool compound. The preclinical PBPK model was developed using rat data. Biodistribution of DM1 in rats was used to develop the small molecule PBPK model, and the PK of conjugated trastuzumab (i.e., T-DM1) in rats was characterized using platform PBPK model for antibody. Both the PBPK models were combined via degradation and deconjugation processes. The degradation of conjugated antibody was assumed to be similar to a normal antibody, and the deconjugation of DM1 from T-DM1 in rats was estimated using plasma PK data. The rat PBPK model was translated to humans to predict clinical PK of T-DM1. The translation involved the use of human antibody PBPK model to characterize the PK of conjugated trastuzumab, use of allometric scaling to predict human clearance of DM1 catabolites, and use of monkey PK data to predict deconjugation of DM1 in the clinic. PBPK model-predicted clinical PK profiles were compared with clinically observed data. The PK of total trastuzumab and T-DM1 were predicted reasonably well, and slight systemic deviations were observed for the PK of DM1-containing catabolites. The ADC PBPK model presented here can serve as a platform to develop models for other ADCs.

Key factors influencing ADME properties of therapeutic proteins: A need for ADME characterization in drug discovery and development.

Protein therapeutics represent a diverse array of biologics including antibodies, fusion proteins, and therapeutic replacement enzymes. Since their inception, they have revolutionized the treatment of a wide range of diseases including respiratory, vascular, autoimmune, inflammatory, infectious, and neurodegenerative diseases, as well as cancer. While in vivo pharmacokinetic, pharmacodynamic, and efficacy studies are routinely carried out for protein therapeutics, studies that identify key factors governing their absorption, distribution, metabolism, and excretion (ADME) properties have not been fully investigated. Thorough characterization and in-depth study of their ADME properties are critical in order to support drug discovery and development processes for the production of safer and more effective biotherapeutics. In this review, we discuss the main factors affecting the ADME characteristics of these large macromolecular therapies. We also give an overview of the current tools, technologies, and approaches available to investigate key factors that influence the ADME of recombinant biotherapeutic drugs, and demonstrate how ADME studies will facilitate their future development.

Non-Clinical Disposition and Metabolism of DM1, a Component of Trastuzumab Emtansine (T-DM1), in Sprague Dawley Rats.

DM1, a derivative of maytansine, is the cytotoxic component of the antibody-drug conjugate trastuzumab emtansine (T-DM1). Understanding the disposition and metabolism of DM1 would help to assess (1) any tissue-specific distribution and risk for potential drug-drug interactions and (2) the need for special patient population studies. To this end, the current study determined the disposition and metabolism of DM1 following single intravenous administration of [(3)H]-DM1 in Sprague Dawley rats. Blood, tissues, urine, bile, and feces were collected up to 5 days after dose administration and analyzed for total radioactivity and metabolites. Results showed that radioactivity cleared rapidly from the blood and quickly distributed to the lungs, liver, kidneys, spleen, heart, gastrointestinal tract, adrenal glands, and other tissues without significant accumulation or persistence. The majority of dosed radioactivity was recovered in feces (~100% of the injected dose over 5 days) with biliary elimination being the predominant route (~46% of the injected dose over 3 days). Excretion in urine was minimal (~5% of the injected dose over 5 days). Mass balance was achieved over 5 days. An analysis of bile samples revealed a small fraction of intact DM1 and a predominance of DM1 metabolites formed through oxidation, hydrolysis, S-methylation, and glutathione and its related conjugates. Collectively, these data demonstrate that DM1 is extensively distributed and quickly cleared from blood, and undergoes extensive metabolism to form multiple metabolites, which are mainly eliminated through the hepatic-biliary route, suggesting that hepatic function (but not renal function) plays an important role in DM1 elimination.

Mechanistic pharmacokinetic/pharmacodynamic modeling of in vivo tumor uptake, catabolism, and tumor response of trastuzumab maytansinoid conjugates.

PURPOSE: Trastuzumab emtansine (T-DM1), an antibody-drug conjugate (ADC) comprised of trastuzumab linked to the antimitotic agent DM1, has shown promising results in patients with human epidermal growth factor receptor 2-positive metastatic breast cancer. Investigations of the mechanisms of the action of ADCs, including T-DM1, have been primarily descriptive or semiquantitative. However, quantitative pharmacokinetic/pharmacodynamic (PK/PD) analysis may provide insights into their complex behavior. The analyses described herein applied PK/PD modeling to nonclinical studies of maytansinoid conjugates. METHODS: The maytansinoid conjugates T-DM1 and T-SPP-DM1, with thioether and disulfide linkers, respectively, were tested in mouse efficacy, PK, and tumor uptake studies. (3)[H]DM1-bearing ADCs were used to facilitate the quantitation of the ADCs in plasma, as well as ADC and ADC catabolites in tumors. Three mechanistic PK/PD models were used to characterize plasma ADC, tumor ADC, and tumor catabolite concentrations. Tumor catabolite concentrations were used to fit tumor response. Model parameters were estimated using R software and nonlinear least squares regression. RESULTS: Plasma ADC-associated DM1 concentrations of T-DM1 decreased more slowly than those of T-SPP-DM1, likely due to slower DM1 release. A comparison of the mechanistic models found that the best model allowed catabolism and catabolite exit rates to differ between ADCs, that T-DM1 exhibited both faster tumor catabolism and catabolite exit rate from tumors than T-SPP-DM1; findings inconsistent with expected behavior based on the physicochemical nature of the respective catabolites. Tumor catabolite concentrations adequately described tumor response with both ADCs showing similar potency. CONCLUSION: Mechanistic PK/PD studies described herein provided results that confirmed and challenged current hypotheses, and suggested new areas of investigation.

A mechanistic pharmacokinetic model elucidating the disposition of trastuzumab emtansine (T-DM1), an antibody-drug conjugate (ADC) for treatment of metastatic breast cancer.

Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate (ADC) therapeutic for treatment of human epidermal growth factor receptor 2 (HER2)-positive cancers. The T-DM1 dose product contains a mixture of drug-to-antibody ratio (DAR) moieties whereby the small molecule DM1 is chemically conjugated to trastuzumab antibody. The pharmacokinetics (PK) underlying this system and other ADCs are complex and have not been elucidated. Accordingly, we have developed two PK modeling approaches from preclinical data to conceptualize and understand T-DM1 PK, to quantify rates of DM1 deconjugation, and to elucidate the link between trastuzumab, T-DM1, and DAR measurements. Preclinical data included PK studies in rats (n = 34) and cynomolgus monkeys (n = 18) at doses ranging from 0.3 to 30 mg/kg and in vitro plasma stability. T-DM1 and total trastuzumab (TT) plasma concentrations were measured by enzyme-linked immunosorbent assay. Individual DAR moieties were measured by affinity capture liquid chromatography-mass spectrophotometry. Two PK modeling approaches were developed for T-DM1 using NONMEM 7.2 software: a mechanistic model fit simultaneously to TT and DAR concentrations and a reduced model fit simultaneously to TT and T-DM1 concentrations. DAR moieties were well described with a three-compartmental model and DM1 deconjugation in the central compartment. DM1 deconjugated fastest from the more highly loaded trastuzumab molecules (i.e., DAR moieties that are ≥3 DM1 per trastuzumab). T-DM1 clearance (CL) was 2-fold faster than TT CL due to deconjugation. The two modeling approaches provide flexibility based on available analytical measurements for T-DM1 and a framework for designing ADC studies and PK-pharmacodynamic modeling of ADC efficacy- and toxicity-related endpoints.

Optimized nonclinical safety assessment strategies supporting clinical development of therapeutic monoclonal antibodies targeting inflammatory diseases.

An increasing number of immunomodulatory monoclonal antibodies (mAbs) and IgG Fc fusion proteins are either approved or in early-to-late stage clinical trials for the treatment of chronic inflammatory conditions, autoimmune diseases and organ transplant rejection. The exquisite specificity of mAbs, in combination with their multi-functional properties, high potency, long half-life (permitting intermittent dosing and prolonged pharamcological effects), and general lack of off-target toxicity makes them ideal therapeutics. Dosing with mAbs for these severe and debilitating but often non life-threatening diseases is usually prolonged, for several months or years, and not only affects adults, including sensitive populations such as woman of child-bearing potential (WoCBP) and the elderly, but also children. Immunosuppression is usually a therapeutic goal of these mAbs and when administered to patients whose treatment program often involves other immunosuppressive therapies, there is an inherent risk for frank immunosuppression and reduced host defence which when prolonged increases the risk of infection and cancer. In addition when mAbs interact with the immune system they can induce other adverse immune-mediated drug reactions such as infusion reactions, cytokine release syndrome, anaphylaxis, immune-complex-mediated pathology and autoimmunity. An overview of the nonclinical safety assessment and risk mitigation strategies utilized to characterize these immunomodulatory mAbs and Fc fusion proteins to support first-in human (FIH) studies and futher clinical development in inflammatory disease indications is provided. Specific emphasis is placed on the design of studies to qualify animal species for toxicology studies, early studies to investigate safety and define PK/PD relationships, FIH-enabling and chronic toxicology studies, immunotoxicity, developmental, reproductive and juvenile toxicity studies and studies to determine the potential for immunosuppression and reduced host defence against infection and cancer. Nonclinical strategies to facilitate clinical and market entry in the most efficient timeframe are presented.

Preclinical safety profile of trastuzumab emtansine (T-DM1): mechanism of action of its cytotoxic component retained with improved tolerability.

Trastuzumab emtansine (T-DM1) is the first antibody-drug conjugate (ADC) approved for patients with human epidermal growth factor receptor 2 (HER2)-positive metastatic breast cancer. The therapeutic premise of ADCs is based on the hypothesis that targeted delivery of potent cytotoxic drugs to tumors will provide better tolerability and efficacy compared with non-targeted delivery, where poor tolerability can limit efficacious doses. Here, we present results from preclinical studies characterizing the toxicity profile of T-DM1, including limited assessment of unconjugated DM1. T-DM1 binds primate ErbB2 and human HER2 but not the rodent homolog c-neu. Therefore, antigen-dependent and non-antigen-dependent toxicity was evaluated in monkeys and rats, respectively, in both single- and repeat-dose studies; toxicity of DM1 was assessed in rats only. T-DM1 was well tolerated at doses up to 40 mg/kg (~4400 µg DM1/m(2)) and 30 mg/kg (~ 6000 µg DM1/m(2)) in rats and monkeys, respectively. In contrast, DM1 was only tolerated up to 0.2mg/kg (1600 µg DM1/m(2)). This suggests that at least two-fold higher doses of the cytotoxic agent are tolerated in T-DM1, supporting the premise of ADCs to improve the therapeutic index. In addition, T-DM1 and DM1 safety profiles were similar and consistent with the mechanism of action of DM1 (i.e., microtubule disruption). Findings included hepatic, bone marrow/hematologic (primarily platelet), lymphoid organ, and neuronal toxicities, and increased numbers of cells of epithelial and phagocytic origin in metaphase arrest. These adverse effects did not worsen with chronic dosing in monkeys and are consistent with those reported in T-DM1-treated patients to date.

American Association of Pharmaceutical Scientists National Biotechnology Conference Short Course: Translational Challenges in Developing Antibody-Drug Conjugates: May 24, 2012, San Diego, CA.

The American Association of Pharmaceutical Scientists (AAPS) National Biotechnology Conference Short Course "Translational Challenges in Developing Antibody-Drug Conjugates (ADCs)," held May 24, 2012 in San Diego, CA, was organized by members of the Pharmacokinetics, Pharmacodynamics and Drug Metabolism section of AAPS. Representatives from the pharmaceutical industry, regulatory authorities, and academia in the US and Europe attended this short course to discuss the translational challenges in ADC development and the importance of characterizing these molecules early in development to achieve therapeutic utility in patients. Other areas of discussion included selection of target antigens; characterization of absorption, distribution, metabolism, and excretion; assay development and hot topics like regulatory perspectives and the role of pharmacometrics in ADC development. MUC16-targeted ADCs were discussed to illustrate challenges in preclinical development; experiences with trastuzumab emtansine (T-DM1; Genentech) and the recently approved brentuximab vedotin (Adcetris; Seattle Genetics) were presented in depth to demonstrate considerations in clinical development. The views expressed in this report are those of the participants and do not necessarily represent those of their affiliations.

Pharmacokinetic considerations for antibody drug conjugates.

Antibody drug conjugates (ADCs) are a class of therapeutics that combine the target specificity of an antibody with the potency of a chemotherapeutic. This therapeutic strategy can significantly expand the therapeutic index of a chemotherapeutic by minimizing the systemic exposure and associated toxicity of the chemotherapeutic agent, while simultaneously maximizing the delivery of the chemotherapeutic to the target. The abundance of antibody targets, coupled with advances in antibody engineering, conjugation chemistry, and examples of early clinical success, have stimulated interest in developing ADCs. However, developing and optimizing the highly complex components of ADCs remain challenging. Understanding the pharmacokinetics (PK) and consequently the pharmacokinetic-pharmacodynamic (PKPD) properties of ADCs is critical for their successful development. This review discusses the PK properties of ADCs, with a focus on ADC-specific characteristics, including molecular heterogeneity, in vivo processing, and the implications of multiple analytes. The disposition of ADCs and the utility of PKPD modeling are discussed in the context of providing guidance to assist in the successful development of these complex molecules.

High-content live-cell imaging assay used to establish mechanism of trastuzumab emtansine (T-DM1)--mediated inhibition of platelet production.

Proplatelet production represents a terminal stage of megakaryocyte development during which long, branching processes composed of platelet-sized swellings are extended and released into the surrounding culture. Whereas the cytoskeletal mechanics driving these transformations have been the focus of many studies, significant limitations in our ability to quantify the rate and extent of proplatelet production have restricted the field to qualitative analyses of a limited number of cells over short intervals. A novel high-content, quantitative, live-cell imaging assay using the IncuCyte system (Essen BioScience) was therefore developed to measure the rate and extent of megakaryocyte maturation and proplatelet production under live culture conditions for extended periods of time. As proof of concept, we used this system in the present study to establish a mechanism by which trastuzumab emtansine (T-DM1), an Ab-drug conjugate currently in clinical development for cancer, affects platelet production. High-content analysis of primary cell cultures revealed that T-DM1 is taken up by mouse megakaryocytes, inhibits megakaryocyte differentiation, and disrupts proplatelet formation by inducing abnormal tubulin organization and suppressing microtubule dynamic instability. Defining the pathways by which therapeutics such as T-DM1 affect megakaryocyte differentiation and proplatelet production may yield strategies to manage drug-induced thrombocytopenias.

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.

Catabolic fate and pharmacokinetic characterization of trastuzumab emtansine (T-DM1): an emphasis on preclinical and clinical catabolism.

Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate in clinical development for the treatment of human epidermal growth factor receptor 2 (HER2)-positive cancers. Herein, we describe a series of studies to assess T-DM1 absorption, distribution, metabolism, and excretion (ADME) in rats as well as to assess human exposure to T-DM1 catabolites. Following administration of unlabeled and radiolabeled T-DM1 in female Sprague Dawley rats as a single dose, plasma, urine, bile and feces were assessed for mass balance, profiling and identification of catabolites. In rats, the major circulating species in plasma was T-DM1, while DM1 concentrations were low (1.08 to 15.6 ng/mL). The major catabolites found circulating in rat plasma were DM1, [N-maleimidomethyl] cyclohexane-1- carboxylate-DM1 (MCC-DM1), and Lysine-MCC-DM1. These catabolites identified in rats were also detected in plasma samples from patients with HER2-positive metastatic breast cancer who received single-agent T-DM1 (3.6 mg/kg every 3 weeks) in a phase 2 clinical study. There was no evidence of tissue accumulation in rats or catabolite accumulation in human plasma following multiple dosing. In rats, T-DM1 was distributed nonspecifically to the organs without accumulation. The major pathway of DM1-containing catabolite elimination in rats was the fecal/biliary route, with up to 80% of radioactivity recovered in the feces and 50% in the bile. The rat T-DM1 ADME profile is likely similar to the human profile, although there may be differences since trastuzumab does not bind the rat HER2- like receptor. Further research is necessary to more fully understand the T-DM1 ADME profile in humans.

The effect of different linkers on target cell catabolism and pharmacokinetics/pharmacodynamics of trastuzumab maytansinoid conjugates.

Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate consisting of the anti-HER2 antibody trastuzumab linked via a nonreducible thioether linker to the maytansinoid antitubulin agent DM1. T-DM1 has shown favorable safety and efficacy in patients with HER2-positive metastatic breast cancer. In previous animal studies, T-DM1 exhibited better pharmacokinetics (PK) and slightly more efficacy than several disulfide-linked versions. The efficacy findings are unique, as other disulfide-linked antibody-drug conjugates (ADC) have shown greater efficacy than thioether-linked designs. To explore this further, the in vitro and in vivo activity, PK, and target cell activation of T-DM1 and the disulfide-linked T-SPP-DM1 were examined. Both ADCs showed high in vitro potency, with T-DM1 displaying greater potency in two of four breast cancer cell lines. In vitro target cell processing of T-DM1 and T-SPP-DM1 produced lysine-N(ε)-MCC-DM1, and lysine-N(ε)-SPP-DM1 and DM1, respectively; in vivo studies confirmed these results. The in vitro processing rates for the two conjugate to their respective catabolites were similar. In vivo, the potencies of the conjugates were similar, and T-SPP-DM1 had a faster plasma clearance than T-DM1. Slower T-DM1 clearance translated to higher overall tumor concentrations (conjugate plus catabolites), but unexpectedly, similar levels of tumor catabolite. These results indicate that, although the ADC linker can have clear impact on the PK and the chemical nature of the catabolites formed, both linkers seem to offer the same payload delivery to the tumor.

Conjugation site modulates the in vivo stability and therapeutic activity of antibody-drug conjugates.

The reactive thiol in cysteine is used for coupling maleimide linkers in the generation of antibody conjugates. To assess the impact of the conjugation site, we engineered cysteines into a therapeutic HER2/neu antibody at three sites differing in solvent accessibility and local charge. The highly solvent-accessible site rapidly lost conjugated thiol-reactive linkers in plasma owing to maleimide exchange with reactive thiols in albumin, free cysteine or glutathione. In contrast, a partially accessible site with a positively charged environment promoted hydrolysis of the succinimide ring in the linker, thereby preventing this exchange reaction. The site with partial solvent-accessibility and neutral charge displayed both properties. In a mouse mammary tumor model, the stability and therapeutic activity of the antibody conjugate were affected positively by succinimide ring hydrolysis and negatively by maleimide exchange with thiol-reactive constituents in plasma. Thus, the chemical and structural dynamics of the conjugation site can influence antibody conjugate performance by modulating the stability of the antibody-linker interface.

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.

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.

Trastuzumab emtansine (T-DM1): a novel agent for targeting HER2+ breast cancer.

Increased understanding of the molecular mechanisms of tumorigenesis has led to the development of novel agents that target tumor cells with minimal effects on normal cells. The success of this approach is exemplified by the development of monoclonal antibodies directed toward antigens expressed selectively by tumor cells. The conjugation of these monoclonal antibodies with potent cytotoxic drugs has the potential to further improve efficacy while retaining a favorable safety profile. Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate (ADC) currently in clinical development. It combines the humanized antibody trastuzumab, which targets the human epidermal growth factor receptor 2 (HER2) receptor on cancer cells, and the potent antimicrotubule agent DM1 using a unique highly stable linker. When T-DM1 binds to HER2, a proportion of the receptors are thought to be internalized by the process of receptor endocytosis, followed by the intracellular release of an active form of DM1, which in turn kills the tumor cell. This review presents the rationale for the development of T-DM1 and summarizes the preclinical and clinical data for this novel agent for the treatment of breast cancer.

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.