For conjugated antibody LBAs, the anticytotoxic drug antibodies are used as capture or detection reagents paired with the
capture and detection reagents outlined above to measure the antibody which conjugates at least one drug (see Figure 2).
Figure 2: Example of conjugated antibody assays.
The merit of conjugated antibody assays is their ability to quantify the possible progressive loss of drug from the ADCs in
circulation. However, extra caution should be taken with the format using antidrug antibodies for detection because the drug
load of ADCs might change in vivo, compared with the ADC standard material used for the assays.
A successful design of an ADC is a combination of high drug-linker stability in circulation with efficient intratumoral cytotoxic
drug release. Significant achievements have been made in the past years to develop more stable linkers (4). However, the nonspecific
release of drug from the carrier antibody in circulation is still a crucial factor in determining the half-life of ADCs. To
measure the drug moieties that have been released from the carrier antibody (i.e., free drug), a competitive LBA format could
be used with antidrug antibodies coated for capture and a constant concentration of HRP-drug as the reporter (see Figure 3).
Figure 3: Competitive LBA for free drug.
Because the clearance of free drug released from the ADC is much faster than the clearance of the ADC itself, the free drug
might not be detectable. To solve this problem, one could measure the remaining drug that is bound to the ADC. This measurement
can be achieved through the use of cathepsin B digestion to release the drug previously bound to the carrier antibody, followed
by quantification of the free drug either via a competitive LBA assay or LC–MS.
Ideally, the assay methods for nonclinical PK bioanalysis should be developed during the early stages of ADC development and
characterization. Evaluating the assays could be done with the recovery of enriched or purified drug antibody ratio (DAR)
fractions compared with the average DAR standard to ensure that the different assay formats recover drug equally. If this
analysis is not possible, detailed information of the ADC's mechanism of action, targeted tumor antigen, type of linker, drug
antibody ratio, cytotoxic drug, and so forth, are necessary for the bioanalytical method design. In addition to LBA methods,
hydrophilic interaction liquid chromatography (HILIC), HPLC, and LC–MS are being used to quantify ADCs. These methods, however,
are beyond the scope of the current discussion and are reported elsewhere (1, 3, 4).
IMMUNOGENICITY OF ADCS
Although the same methods used for determining the immunogenicity of general therapeutic antibodies can be used to determine
the immunogenicity of ADCs, further characterization of anti-ADC antibodies for the targeting antibody, the linker, and the
drug components are required to address the specificity of positive samples. The complexity of the ADC structure raises additional
questions not previously encountered in the analysis of monoclonal antibody therapeutics or small molecule drugs. For instance,
does an antibody response against the linker or the cytotoxic drug affect ADC internalization? Alternatively, do only neutralizing
antibodies against the complementarity determining region (CDR) of the targeting antibody reduce the efficacy of the ADC?
Antibodies are commonly recognized as stable; therefore, most bioanalytical assays for therapeutical monoclonal antibodies
are established in serum. This blanket approach, however, is not appropriate for the bioanalysis of ADCs where the conjugation
of small molecule drug to the antibody via a linker creates a molecule whose overall stability depends upon the least stable
of the three components. To this end, plasma is suggested as being the preferred matrix used for PK and immunogenicity sample
analysis because the inhibition of the clotting cascade in plasma results in much less proteolysis than in serum. Moreover,
protease inhibitors could be added during sample collection to further stabilize the ADCs in plasma.