Bioanalytical Assay Strategies for The Development of Antibody–Drug Conjugate Biotherapeutics

Abstract

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.

Immunogenicity of Antibody Drug Conjugates: Bioanalytical Methods and Monitoring Strategy for a Novel Therapeutic Modality

“…In contrast, an ADA incidence of 4.2% (eight out of 190) was reported in ado-trastuzumab emtansine (T-DM1)-dosed cynomolgus monkeys across eight preclinical studies, with no impact on pharmacokinetics (PK) (41). These results (among others (20)) for trastuzumab confirm that the particular linker and small-molecule drug may contribute unique immunogenicity properties. Hoofring et al also demonstrate that ADA in a rat preclinical study can impact PK (43).…”

“…Some of those differences may be due to the biophysical alterations that accompany conjugation of linker and smallmolecule drug to a mAb, resulting in altered antigen presentation. As a consequence, both the antigenicity and immunogenicity of any conjugated protein is influenced by site of conjugation and the chemical moiety itself (as demonstrated by the results discussed previously in the "PRECLINICAL MONITORING STRATEGIES" section (20,41)). Our expectation is that these evolving topics will be considered during a risk assessment that accompanies the development of a clinical immunogenicity strategy for each ADC.…”

“…The pharmacokinetics and pharmacology, toxicology, and bioanalytical strategies used for these ADCs have been reviewed (17)(18)(19)(20)(21)(22). Immunogenicity support strategies (and the resulting data) for these approved ADCs form the knowledge base on what is an expanding field (Table I illustrates the examples of published ADC immunogenicity information).…”

Preclinical pharmacokinetics and pharmacodynamics of DCLL9718A: An antibody-drug conjugate for the treatment of acute myeloid leukemia

“…11 Herein, we investigated the pharmacokinetics (PK) and pharmacodynamics (PD) of DCLL9718A in cynomolgus monkeys (binding species), and the PK in mouse and rat (non-binding species). Similar to previously described bioanalytical strategies for ADCs, 14 the characterization of DCLL9718A PK included the quantification of three key analytes: DCLL9718A total antibody (measurement of all drug-antibody ratios of the ADC, including fully conjugated, partially deconjugated, and fully deconjugated antibodies), antibody-conjugated PBD dimer (acPBD dimer, measurement of PBD dimer conjugated to the antibody), and unconjugated PDB dimer (measurement of PBD dimer that is not conjugated to the antibody through the linker). In addition, we also characterized the PK of the unconjugated antibody, MCLL0517A, in cynomolgus monkey and mouse to evaluate the effect of conjugation on the PK of DCLL9718A.…”

Utility of a Bayesian Mathematical Model to Predict the Impact of Immunogenicity on Pharmacokinetics of Therapeutic Proteins

“…In this study, we utilized specific and sensitive assays for the bioanalytical measurements of PK and ADA levels that have minimal interference from matrix associated factors using the approach of integrating the Bfree^and Btotal^TP detections (Thway et al (4,22,23)). Further accuracy of the ADA and TP measurements requires a comprehensive evaluation of interference due to circulating ligands (soluble form or shed form of membrane receptors), matrix associated factors like excess TP or ADA, biodegraded or transformed fragments as in fusion proteins and small peptides or toxins as in antibody drug conjugates (24,25). These analyses ensured the accuracy of the PK and ADA data in generation of the model.…”