Illustration of D0 competing with ADC (D2 to D8) at similar binding sites of target cell to affect ADC action of Antibody Dependent Cell mediated Cytotoxicity (ADCC). (Image adapted from Axion Biosystems)
Antibody drug conjugates (ADC) are typically composed of an antibody chemically attached with small molecule drugs as payload through linkers¹. The drugs attach to the antibody in different proportions, which defines the drug-to-antibody ratio (DAR or abbreviated further as D). Different ratios indicate different degree of drug(s) appended to the antibody, affecting ADC potency and cytotoxicity to different extents, and are a critical quality attribute of ADCs²,³.
D0 refers to an antibody conjugated with no drug (0 payload or naked antibody⁴), D1 conjugated with 1 drug, D2 with 2 drugs and so on. So far, the maximum number of drugs conjugated to an antibody is limited to 8 (D8)² with D4 being the most common⁵.
Drug conjugation is a chemical process often yielding a distribution, with existence of minute levels of unconjugated (D0) species. ADC stability during purification process and storage may further generate D0.
Further, ADC in serum or plasma, as observed in animal or human PK time-course studies, the cleavable linkers of ADCs may get hydrolyzed at an appreciable rate, leading to appreciable D0 and payload. In such instances, the naked antibody (due to the premature release of payload) loses targeted cell killing (thus reducing efficacy), and the released payload in extra-tumoral compartments⁶ can cause off-targeted cytotoxicity (safety concern).
Both D0 and ADC recognize the same epitope on the tumor / antigen surface, thus competing for the target binding affecting ADC potency, as illustrated in Figure. Additionally, effector function in ADC (with multiple payload) could be diminished, possibly due to masking Fc moiety of ADC by conjugated payload¹.
Overall, in case of ADC, the effector cell mediated cytotoxicity of target molecule may be decreased due to
(a) competition with D0 for the same epitope,
(b) masking of Fc moiety by conjugated payload.
Binding studies, using ELISA and/or SPR, are generally used to characterize the affinity and specificity of a therapeutic antibody to a specific purified antigen. The Fab portion (CDR) of a therapeutic antibody and its ADC counterpart remain the same, and therefore, there may not be significant difference in the performance of these molecules in binding to the desired antigen.
Hence, such binding assays may not be sufficient for differentiating D0 in ADC.
These assays can still be used for product consistency in lot release due to their robustness and fast turnaround time. On the other hand, effector function assay can be achieved to differentiate D0 (without payload, not masking Fc moiety) from ADC (with multiple payloads, likely masking Fc moiety), as well as for cell-based assay with direct drug-induced cytotoxicity.
These effector function studies, carried out mainly by:
These form the backbone of potency experiments (Figure) and are likely to be performed early in development stages for evaluating efficacy and safety concerns.
Hydrophobic Interaction Chromatography (HIC), capable of separating the D0, D1, D2, etc., along with SEC-MS and UV spectroscopy, are often used for DAR distribution, identification, and quantitation, accompanied by Reverse Phase High Performance Liquid Chromatography (RP-HPLC) which provides DAR distribution in light-and-heavy-chain separately.
Fractions collected from RP-HPLC or HIC can be analyzed by Liquid Chromatography-Mass Spectroscopy (LC-MS) for peak identity⁷. These techniques can be built concurrently to build a robust analytic technique for characterization and release testing of an ADC.
A variety of immunological assays can also be employed to evaluate the ADC’s capacity to engage the immune system to trigger effector functions. This approach, if incorporated early-on in drug development, can avoid surprises later⁸.
Crystal Bio Solutions, a Contract Research Organization (CRO), specializes in providing comprehensive method development services for sterility and endotoxin assessment assays. The organization validates rapid 5–7-day sterility assessments against the traditional 14-day compendial testing for drug products (DP), alongside designing and executing suitable methods for endotoxin assessment. Following method development, Crystal Bio ensures a seamless technology transfer to GMP suites. This approach eliminates the need for an Analytical Method Transfer department at the prospective client or Intellectual Property (IP) holder's site.
This strategy not only reduces the costs associated with training personnel but also significantly saves time - a critical factor, particularly during the release phase of a DP. Such efficiencies are crucial for accelerating the time-to-market for new therapies, enhancing the overall productivity of the drug development and release process.