Antibody–drug conjugates nonclinical support Bioanalytical Challenge
Detection reagent
(Biotin-antihuman mAb or pAb, Biotin-anti-ld/anti-CDR mAb, Biotin-antigen)
ADC (DAR ≥ 0) Capture reagent
(antigen, anti-ld/anti-CDR mAb, antihuman mAb or pAb)
Figure 1. Ligand-binding assay for antibody–drug conjugate total antibody analyte. ADC: Antibody–drug conjugate; CDR: Complementarity determining region; DAR: Drug–antibody ratio; Id: Idiotype; mAb: Monoclonal antibody; pAb: Polyclonal antibody.
and generic reagents against human IgG or (Fab’)2 region or Fc region, or light chain (LC), or heavy plus
light (H+L) chain regions could be used as critical reagents for total antibody assay. All of these reagents offer their own advantages and limitations. Theoreti- cally, the assay formats utilizing specific reagents owing to their higher binding affinity and specificity should provide better
specificity and selectivity, extended
dynamic range, higher assay sensitivity and should be relatively easily transferred from nonclinical to clinical matrices. But the availability of specific reagents, par- ticularly targeted tumor antigen may be limited. Even if the antigen protein is available, the application of anti- gen protein as the critical reagent may render assay sus- ceptible to the target interference. Similarly, though the use of anti-Id or anti-CDR antibodies may offer high assay specificity and sensitivity over generic reagents, their binding to antibody component of ADC may be impacted by the conjugation site on the ADC [16]. Thus, the critical reagent employed for total antibody analyte quantitation depends on the availability of the reagents as well as on the requirement of DAR-sensitive or DAR-insensitive PK profile and PK parameters to evaluate safety, efficacy and in vivo stability of the drug candidate. It has been reported that while for some ADCs, only generic reagents could yield DAR-insen- sitive total antibody assays, for other ADCs both the generic or specific reagents could yield DAR-insensitive assays [15,17]. The physicochemical characteristics of the ADC such as conjugation chemistry, linker type and small molecule drug type and conjugation site govern whether the generic or specific or both types of reagents would yield the DAR-insensitive assay. For instance, if a DAR-insensitive assay is needed for an ADC that has conjugation sites largely located on a certain region of the antibody moiety, the usage of a critical reagent that binds to this region may not be the right choice, particularly for higher DAR species.
future science group An example of the impact of change in critical
reagent on the DAR sensitivity of a total antibody assay is illustrated in Table 4. For an ADC with conventional lysine-based conjugation chemistry, when the assay format involving target protein as capture reagent and anti-human Fc as detection reagent was employed for total antibody quantitation, it yielded an overestima- tion in concentrations for samples prepared with Ab relative to the average DAR standard. The observed over-recovery of samples prepared with Ab suggested that the binding of either the capture or the detection reagent to the antibody component of ADC had been compromised by the small molecule drug conjugation. It has been reported that conventional
lysine-based
conjugation occurs primarily on regions of the heavy and light chains that offer areas of large solvent acces- sibility and structural flexibility, and no conjugation sites were observed in CDRs of the antibody moi- ety [30]. Based on this information, when the detection reagent was changed from the generic antihuman Fc antibody to the LC specific polyclonal antibody in the total antibody assay format, a DAR-insensitive assay was generated. This new assay format yielded accept- able recovery for both Ab and average DAR standard as shown in Table 4. Alternative assay formats involving coincubation of
samples and critical reagents have also been reported to offer better assay performance against various DAR species [9,16,17]. However, since not all ADCs are same, there is no single DAR-insensitive assay format for the total antibody analyte that could fit all ADCs inde- pendent of their physicochemical characteristics. The assay strategy for total antibody quantitation would thus need to be adapted on a case-by-case basis. Total antibody assay performance was evaluated in
rodent serum for an ADC containing lysine-based con- ventional conjugation chemistry. The assay format on an ELISA platform involved capture of the total (uncon-
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