title: Evaluation of ADC Physicochemical Characteristics and Their Impact on Pharmacokinetics in Transgenic huFcRn Mice
Creators: name: Kaempffe, Anna
Publisher: name: UNSPECIFIED
Year: 2022

description: Antibody-drug conjugates (ADCs) are a promising and fast-growing class of targeted anti-cancer therapeutics. They consist of highly potent chemotherapeutic drugs that are covalently linked to monoclonal antibodies (mAbs) which guide the drug to cancer cells. The concept of targeted delivery of a chemotherapeutic agent via an ADC aims at enhanced therapeutic efficacy and safety compared to the individual components of an ADC - the mAb and cytotoxic agent. Despite various successful ADC approvals, their development is complex and associated with a high risk of costly late-stage failures. It has been shown that the overall design of an ADC and every component can influence the critical physicochemical properties which in turn can lead to fast non-specific clearance of the ADCs. Elevated non-specific clearance can directly affect the efficacy and safety and limit the therapeutic window of ADCs. To address this problem, the aim of this study was to evaluate physicochemical property assays that indicate a risk for poor pharmacokinetics (PK) of ADCs. Physicochemical property assays were applied to an ADC series to evaluate whether ADC hydrophobicity (hydrophobic interaction chromatography (HIC)), thermal stability (nano differential scanning fluorimetry (nanoDSF)), and the binding behavior to the human neonatal Fc-receptor (huFcRn; huFcRn binding kinetics by Bio-Layer Interferometry (BLI)) could serve as an indicator of the PK of the ADCs that was analyzed in huFcRn transgenic Tg276 mice. For this, eight trastuzumab-based ADCs with a homogenous DAR of 2 (drug-to-antibody ratio of 2) were generated by conjugation to cysteines, genetically introduced at positions N325, L328, S239, D265, or S442, and by enzymatic conjugation via microbial transglutaminase (mTG) either to C-terminal light (LC) or heavy chain (HC) recognition motifs or to the endogenous position Q295 of the native antibody. Interestingly, pronounced differences in the ADC PK profiles were observed which allowed to confirm the recently described position L328 as favorable site for cysteine conjugation, comparable to the well-established position S239, and emphasizes the favorable position Q295 of native antibodies and the tagged LC antibody variant for enzymatic conjugations via mTG. Furthermore, to explore if conjugation site and method effects would apply to antibody scaffolds other than trastuzumab, two of the positions, L328 and the mTG LC variant, were evaluated in context of the clinically evaluated mAbs briakinumab and secukinumab. This showed that the influence of conjugation sites and methods on PK resulted in same ADC PK rankings also for briakinumab- and secukinumab-based variants. Physicochemical property assay data were obtained for all study ADCs and correlated with ADC clearance (CL) that serves as important PK parameter. Interestingly, similar huFcRn binding and differently pronounced hydrophobicity and thermal stabilities were observed which did not correlate with the ADC CL values. Consequently, additional physicochemical property assays were explored in this study that could serve as indicators for poor PK of ADCs and that were adapted from various reports where they allowed prediction of poor PK of mAbs. To enable a more robust correlation between PK and the in vitro assay outcome, 13 ADCs were used that varied not only in the conjugation site and method but also in the DAR and antibody scaffold and for which a broad range of CL values (1.18-8.38 mL/h/kg) were observed in hemizygous huFcRn Tg276 mice. These ADCs were assessed by seven physicochemical property assays that were implemented to analyze either the degree of self-association (clone self-interaction Bio-Layer Interferometry (CSI-BLI) and affinity-capture self-interaction nanoparticle spectroscopy (AC-SINS)), non-specific binding (polyspecificity reagent Bio-Layer Interferometry (PSR-BLI), baculovirus particle (BVP) and heparin enzyme-linked immunosorbent assay (ELISA)), hydrophobicity (bis-ANS), or huFcRn binding (huFcRn affinity chromatography). Assay data was used for the correlation to CL values. Overall, ADCs with lower CL showed lower assay results compared to ADCs with fast CL (about &gt;4 mL/h/kg) which showed elevated assay results. These results indicate for the first time that the selected in vitro assays could be a powerful tool for the early development not only for mAbs but also for ADCs allowing the selection of ADCs with favorable PK characteristics. Consequently, application of the in vitro assay panel could serve as screening paradigm for PK risk mitigation during early ADC development. However, further work is needed to expand the data set and to conduct robust statistical analyses which could serve as basis to define thresholds for each assay.