Ben-Quan Shen

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.

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.

Highly specific off-target binding identified and eliminated during the humanization of an antibody against FGF receptor 4

Off-target binding can significantly affect the pharmacokinetics (PK), tissue distribution, efficacy and toxicity of a therapeutic antibody. Herein we describe the development of a humanized anti- fibroblast growth factor receptor 4 (FGFR4) antibody as a potential therapeutic for hepatocellular carcinoma (HCC). A chimeric anti FGFR4 monoclonal antibody (chLD1) was previously shown to block ligand binding and to inhibit FGFR4 mediated signaling as well as tumor growth in vivo. A humanized version of chLD1, hLD1.vB, had similar binding affinity and in vitro blocking activity, but it exhibited rapid clearance, poor target tissue biodistribution and limited efficacy when compared to chLD1 in a HUH7 human HCC xenograft mouse model. These problems were traced to instability of the molecule in rodent serum. Size exclusion high performance liquid chromatography, immunoprecipitation and mass spectral sequencing identified a specific interaction between hLD1.vB and mouse complement component 3 (C3). A PK study in C3 knock-out mice further confirmed this specific interaction. Subsequently, an affinity-matured variant derived from hLD1.vB (hLD1.v22), specifically selected for its lack of binding to mouse C3 was demonstrated to have a PK profile and in vivo efficacy similar to that of chLD1 in mice. Although reports of non-specific off-target binding have been observed for other antibodies, this represents the first report identifying a specific off-target interaction that affected disposition and biological activity. Screens developed to identify general non-specific interactions are likely to miss the rare and highly specific cross-reactivity identified in this study, thus highlighting the importance of animal models as a proxy for avoiding unexpected clinical outcomes.

Site-Specific Trastuzumab Maytansinoid Antibody–Drug Conjugates with Improved Therapeutic Activity through Linker and Antibody Engineering

Antibody-drug conjugates (ADCs) have a significant impact toward the treatment of cancer, as evidenced by the clinical activity of the recently approved ADCs, brentuximab vedotin for Hodgkin lymphoma and ado-trastuzumab emtansine (trastuzumab-MCC-DM1) for metastatic HER2+ breast cancer. DM1 is an analog of the natural product maytansine, a microtubule inhibitor that by itself has limited clinical activity and high systemic toxicity. However, by conjugation of DM1 to trastuzumab, the safety was improved and clinical activity was demonstrated. Here, we report that through chemical modification of the linker-drug and antibody engineering, the therapeutic activity of trastuzumab maytansinoid ADCs can be further improved. These improvements include eliminating DM1 release in the plasma and increasing the drug load by engineering four cysteine residues into the antibody. The chemical synthesis of highly stable linker-drugs and the modification of cysteine residues of engineered site-specific antibodies resulted in a homogeneous ADC with increased therapeutic activity compared to the clinically approved ADC, trastuzumab-MCC-DM1.

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.

Maximizing tumour exposure to anti-neuropilin-1 antibody requires saturation of non-tumour tissue antigenic sinks in mice

BACKGROUND AND PURPOSE Neuropilin‐1 (NRP1) is a VEGF receptor that is widely expressed in normal tissues and is involved in tumour angiogenesis. MNRP1685A is a rodent and primate cross‐binding human monoclonal antibody against NRP1 that exhibits inhibition of tumour growth in NPR1‐expressing preclinical models. However, widespread NRP1 expression in normal tissues may affect MNRP1685A tumour uptake. The objective of this study was to assess MNRP1685A biodistribution in tumour‐bearing mice to understand the relationships between dose, non‐tumour tissue uptake and tumour uptake.

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.

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 125I-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.

Physiologically Based Pharmacokinetic Modeling as a Tool to Predict Drug Interactions for Antibody-Drug Conjugates

Background and ObjectivesMonomethyl auristatin E (MMAE, a cytotoxic agent), upon releasing from valine-citrulline-MMAE (vc-MMAE) antibody-drug conjugates (ADCs), is expected to behave like small molecules. Therefore, evaluating the drug–drug interaction (DDI) potential associated with MMAE is important in the clinical development of ADCs. The objective of this work was to build a physiologically based pharmacokinetic (PBPK) model to assess MMAE–drug interactions for vc-MMAE ADCs.MethodsA PBPK model linking antibody-conjugated MMAE (acMMAE) to its catabolite unconjugated MMAE associated with vc-MMAE ADCs was developed using a mixed ‘bottom-up’ and ‘top-down’ approach. The model was developed using in silico and in vitro data and in vivo pharmacokinetic data from anti-CD22-vc-MMAE ADC. Subsequently, the model was validated using clinical pharmacokinetic data from another vc-MMAE ADC, brentuximab vedotin. Finally, the verified model was used to simulate the results of clinical DDI studies between brentuximab vedotin and midazolam, ketoconazole, and rifampicin.ResultsThe pharmacokinetic profile of acMMAE and unconjugated MMAE following administration of anti-CD22-vc-MMAE was well described by simulations using the developed PBPK model. The model’s performance in predicting unconjugated MMAE pharmacokinetics was verified by successful simulation of the pharmacokinetic profile following brentuximab vedotin administration. The model simulated DDIs, expressed as area under the concentration-time curve (AUC) and maximum concentration (Cmax) ratios, were well within the two-fold of the observed data from clinical DDI studies.ConclusionsThis work is the first demonstration of the use of PBPK modelling to predict MMAE-based DDI potential. The described model can be extended to assess the DDI potential of other vc-MMAE ADCs.