Chemotherapy is a mainstay of a standardized treatment regimen for cancer. However, the nonspecific targeting of healthy cells
as well as tumor cells by cytotoxic small-molecule drugs often results in intolerable side effects. These side effects compromise
the efficacy of the treatment regimen and dramatically decrease the quality of life for cancer patients.
Antibody-drug conjugates (ADCs) are a new class of chemotherapeutics which comprise monoclonal antibodies (mAbs) that selectively
bind to tumor-associated antigens associated with a cytotoxic small-molecule payload (1). The payload is attached to the antibody
using enzyme-cleavable linkers. Much effort has been made to identify the highly tumor-specific mAbs, anticancer drugs with
the maximum efficacy, and linkers that are stable in circulation but allow for rapid cleavage to release the cell-killing
drugs following intracellular uptake of the ADCs. More than 30 targets have been investigated and more than 20 ADCs are now
in various phases of clinical development, including Trastuzumab-DM1 (Roche), SGN-35 (Seattle Genetics), HuN901-DM1 (ImmunoGen),
CR011-vcMMAE (Celidex Therapeutics), SAR3419 (Sanofi-Aventis), CMC-544 (Pfizer), and BIIB015 (Biogen Idec). Given the high
complexity of ADCs resulting from the addition of the drug payload to already complex antibodies, the development and validation
of analytical methods for ADC characterization, formulation analysis, and bioanalysis present significant challenges. A comprehensive
review of bioanalytical assays for ADCs was published by Stephan et al. (2). In the current discussion, attention is focused
on the development of bioanalytical assays for ADCs from a preclinical perspective.
Roger N. Hayes
ASSAY FORMATS FOR PHARMACOKINETIC METHODS
Figure 1: Example of total antibody assays.
The ADC is a heterogeneous mixture containing a cocktail of monoclonal antibodies with different drug payloads. Because of
this heterogeneous nature, ligand binding assays (LBAs) are generally used for ADC bioanalysis. A variety of platforms are
used, including colorimetric enzyme-linked immunosorbent assay, compact-disk, formatted Gyrolab, and electrochemiluminescence-based
Meso Scale Discovery. Total antibody assays can be used to quantify total antibody with or without the cytotoxic drug conjugated
to it. Targeted tumor antigens, anti-idiotype mAbs, anti-human immunoglobulin (IgG) (Fab')2, and anti-human IgG (Fc) antibodies can be used as capture reagents. Anti-human IgG (Fab')2-horseradish peroxidase (HRP)
or anti-human IgG (Fc)-HRP, or biotinylated anti-human antibodies with streptavidin-HRP, are then used for detection (see
To eliminate nonspecific binding, monkey adsorbed anti-human IgG antibodies are commonly used for nonhuman primate studies.
Depending on the type of linkers, the position of drug conjugation may be located on (Fab')2, or the hinge region of the carrier
antibodies. Increasing stoichiometry of drug conjugation may affect the binding of the ADCs to capture or detection reagents,
and significantly affect the assay performance. Moreover, some assay formats are sensitive to the drug load even though the
binding sites are not directly blocked. It has been reported that different assay formats yield different pharmacokinetic
(PK) profiles and markedly affect the calculation of critical PK parameters such as clearance and drug exposure (3). It appears
that most of the assay formats are drug-load sensitive, but the generic human IgG assay using anti-human IgG (Fc) for capture
and detection is an exception to this observation.