Ola Saad

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.

Bioanalytical assay strategies for the development of antibody–drug conjugate biotherapeutics

Antibody-drug conjugates (ADCs) are monoclonal antibodies with covalently bound cytotoxic drugs. They are designed to target tumor antigens selectively and offer the hope of cancer treatment without the debilitating side-effects of conventional therapies. The concept of ADCs is not new; however, development of these therapeutics is challenging and only recently are promising clinical data emerging. These challenges include ADC bioanalysis, such as quantifying in serum/plasma for PK studies and strategies for assessing immunogenicity. ADCs have complex molecular structures incorporating large- and small-molecule characteristics and require diverse analytical methods, including ligand-binding assays and MS-based methods. ADCs are typically mixtures with a range of drug-to-antibody ratios. Biotransformations in vivo can lead to additional changes in drug-to-antibody ratios resulting in dynamically changing mixtures. Thus, a standard calibration curve consisting of the reference standard may not be appropriate for quantification of analytes in vivo and represents a unique challenge. This paper will share our perspective on why ADC bioanalysis is so complex and describe the strategies and rationale that we have used for ADCs, with highlights of original data from a variety of nonclinical and clinical case studies. Our strategy has involved novel protein structural characterization tools to help understand ADC biotransformations in vivo and use of the analyte knowledge gained to guide the development of quantitative bioanalytical assays.

Characterization of intact antibody–drug conjugates from plasma/serum in vivo by affinity capture capillary liquid chromatography–mass spectrometry

Antibody-drug conjugates (ADCs) are designed to facilitate the targeted delivery of cytotoxic drugs to improve their tumor fighting effects and minimize systemic toxicity. However, efficacy and safety can potentially be compromised due to the release of conjugated drugs from the ADC with time while in circulation, resulting in changes in the drug-to-antibody ratio (DAR). Current understanding of this process is limited because existing methods such as immunoassays fail to distinguish ADCs with different DARs. Here we demonstrate a novel method with bead-based affinity capture and capillary liquid chromatography-mass spectrometry to allow direct measurement of drug release by quantifying DAR distributions of the ADC in plasma/serum. This method successfully identified individual intact conjugated antibody species produced due to drug loss from ADCs (e.g., an engineered site-specific anti-MUC16 THIOMAB-drug conjugate) and measured the corresponding DAR distributions in vitro and in vivo. Information obtained can provide insights into the mechanisms involved in drug loss and help to optimize ADC therapeutics. Other potential applications of the method may include characterization of posttranslational modifications, protein adducts, and immunogenicity.

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.

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.

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.

Anti-CD22-MCC-DM1 and MC-MMAF Conjugates: Impact of Assay Format on Pharmacokinetic Parameters Determination

CD22 represents a promising target for antibody-drug conjugate therapy in the context of B cell malignancies since it rapidly internalizes, importing specifically bound antibodies with it. To determine the pharmacokinetic parameters of anti-CD22-MCC-DM1 and MC-MMAF conjugates, various approaches to quantifying total and conjugated antibody were investigated. Although the total antibody assay formats gave similar results for both conjugates, the mouse pharmacokinetic profile for the anti-CD22-MCC-DM1 and MC-MMAF appeared significantly different depending on the conjugated antibody assay format. Since these differences significantly impacted the PK parameters determination, we investigated the effect of the drug/antibody ratio on the total and conjugated antibody quantification using multiple assay formats. Our investigations revealed the limitations of some assay formats to quantify anti-CD22-MCC-DM1 and MC-MMAF with different drug load and in the context of a heterogeneous ADC population highlight the need to carefully plan the assay strategy for the total and conjugated antibody quantification in order to accurately determine the ADC PK parameters.

Potential Mechanisms for Thrombocytopenia Development with Trastuzumab Emtansine (T-DM1)

Purpose: Trastuzumab-emtansine (T-DM1) is an antibody–drug conjugate (ADC) comprising the cytotoxic agent DM1 conjugated to trastuzumab with a stable linker. Thrombocytopenia was the dose-limiting toxicity in the phase I study, and grade ≥3 thrombocytopenia occurred in up to 13% of patients receiving T-DM1 in phase III studies. We investigated the mechanism of T-DM1–induced thrombocytopenia. Experimental Design: The effect of T-DM1 on platelet function was measured by aggregometry, and by flow cytometry to detect the markers of activation. The effect of T-DM1 on differentiation and maturation of megakaryocytes (MK) from human hematopoietic stem cells was assessed by flow cytometry and microscopy. Binding, uptake, and catabolism of T-DM1 in MKs, were assessed by various techniques including fluorescence microscopy, scintigraphy to detect T-[H3]-DM1 and 125I-T-DM1, and mass spectrometry. The role of FcγRIIa was assessed using blocking antibodies and mutant constructs of trastuzumab that do not bind FcγR. Results: T-DM1 had no direct effect on platelet activation and aggregation, but it did markedly inhibit MK differentiation via a cytotoxic effect. Inhibition occurred with DM1-containing ADCs but not with trastuzumab demonstrating a role for DM1. MKs internalized these ADCs in a HER2-independent, FcγRIIa-dependent manner, resulting in intracellular release of DM1. Binding and internalization of T-DM1 diminished as MKs matured; however, prolonged exposure of mature MKs to T-DM1 resulted in a disrupted cytoskeletal structure. Conclusions: These data support the hypothesis that T-DM1–induced thrombocytopenia is mediated in large part by DM1-induced impairment of MK differentiation, with a less pronounced effect on mature MKs. Clin Cancer Res; 21(1); 123–33. ©2014 AACR.

PK assays for antibody–drug conjugates: case study with ado-trastuzumab emtansine

BACKGROUND Antibody-drug conjugates (ADCs) combine the characteristics of large-molecule biologics and small-molecule drugs and are heterogeneous mixtures that can biotransform in vivo, resulting in additional complexity. ADC bioanalytical strategies require novel analytical methods, as well as existing large- and small-molecule methods. Because ADCs in late-stage clinical development are relatively new, regulatory guidelines and standard industry best practices for developing strategies for bioanalytical PK assays are still being established. RESULTS A PK assay strategy was developed that included comprehensive novel reagent and assay characterization approaches for the ADC ado-trastuzumab emtansine (T-DM1). CONCLUSION The bioanalytical strategy was successfully applied to the drug development of T-DM1 and ensured that key analytes were accurately measured in support of nonclinical and clinical development.

Bioanalytical approaches for characterizing catabolism of antibody–drug conjugates

The in vivo stability and catabolism of antibody-drug conjugates (ADCs) directly impact their PK, efficacy and safety, and metabolites of the cytotoxic or small molecule drug component of an ADC can further complicate these factors. This perspective highlights the importance of understanding ADC catabolism and the associated bioanalytical challenges. We evaluated different bioanalytical approaches to qualitatively and quantitatively characterize ADC catabolites. Here we review and discuss the rationale and experimental strategies used to design bioanalytical assays for characterization of ADC catabolism and supporting ADME studies during ADC clinical development. This review covers both large and small molecule approaches, and uses examples from Kadcyla® (T-DM1) and a THIOMAB™ antibody-drug conjugate to illustrate the process.