The emergence of antibody-drug conjugates (ADCs) as a potential therapeutic avenue in cancer treatment has garnered significant attention. By combining the selective specificity of monoclonal antibodies with the cytotoxicity of drug molecules, ADCs aim to increase the therapeutic index, selectively targeting cancer cells while minimizing systemic toxicity. However, the manufacture of ADCs faces several challenges. These include identifying suitable target antigens, enhancing antibodies, suitable conjugation strategies, linkers, and payloads, and its complex in vivo metabolic processes pose difficulties for ADME. This review focuses on the optimization strategies used during ADC development and the technical approach used for ADME analysis.
Target and Antibody
ADCs have a broad range of targets, and while most can internalize following endocytosis mediated by receptors, some targets are found in the tumor vascular system or the cell surface. The optimal target should have high expression in tumor cells and low or non-existent expression in regular tissues and organs. The majority of the targets selected for current ADCs are antibody targets that have been successfully commercialized or developed based on pre-existing marketed monoclonal antibodies.
Antibody modifications can be structural or functional. Although early ADCs were primarily synthesized as heterogeneous mixtures, they were found to have subpar pharmacokinetics, stability, and efficacy. Current efforts are geared towards creating homogeneous constructs with accurate drug loading and predetermined, regulated attachment sites. One major advancement in antibody modification is the engineering of antibodies with specific modification sites. Structural modifications are also a method to optimize antibodies, such as utilizing smaller antibodies to achieve high tumor penetration.
The process of developing antibodies requires in vitro screening for efficacy evaluation. This includes primary screening of the antibody’s internalization ability and activity screening using the factor that antibodies can stimulate target degradation and directly kill tumor cells. Affinity of the antibody for the target antigen is also crucial. The ideal ADC drug’s KD value should not be lower than that of its bare antibody against the antigen. In recent years, ground-breaking ADCs have emerged based on platforms like PROTAC and LYTAC.
The process for developing antibodies requires in vitro screening for efficacy evaluation. This includes primary screening for the antibody’s internalisation ability, using a fluorescein-labeled antibody, or screening for activity based on the fact that antibodies can stimulate target degradation and directly eliminate tumor cells. Frequently used tools range from laser confocal microscopy for co-localisation analysis and FACS assays for internalisation, to WB assays for target degradation and mechanism studies. Other activity assays include colony formation or CCK-8, complemented by high-throughput screening using high content live cell imaging systems. It’s crucial not to overlook the affinity of the antibody of a qualified ADC drug for the target antigen, as it’s vitally important. At present, the common tests are Elisa, SPR technology, and the KD value of an ideal ADC drug should not be lower than the KD value of its unaccompanied antibody against the antigen.
Linker and Payload
The potency of the ADC is directly determined by the payload. The most advanced ones include microtubule disrupting agents (MMAE, MMAF, DM1, DM4) and DNA-damaging agents. Non-cleavable alkyl linkers such as N-maleimidomethylcyclohexane-1-carboxylate (MCC, used in Kadcyla) are the most frequently used linkers, along with enzymatically cleavable linkers like the self-immolative para-aminobenzyl (PAB) group attached to a cathepsin-labile valine-citrulline dipeptide (vc, used in ADCETRIS), and acid-labile hydrazone linkers (AcBut, used in Mylotarg). Most approved ADCs use peptide-based linkers which are sensitive to lysosomal proteases. In contrast to the common chemical and enzymatic environment in vivo, non-cleavable linkers are inert, offering the advantage of reduced off-target toxicity, but compromising the bystander effect of the payload.
The ideal linker prevents the payload’s premature release in plasma and also circumvents stability changes that lead to ADCs aggregation in vivo. Size exclusion chromatography (SEC-HPLC) is typically utilized to analyze the aggregation of antibodies and ADCs. Current strategies for linker improvement are as follows: Enhancing the hydrophilicity and chargeability of peptide-based linkers, creating branching linkers that enable ADC cleavage by tumour-specific/enhanced proteases, and developing branching linkers that can perform site-specific conjugation with different DARs.
See ADC linker Products:
The conjugation and DAR
The number of drug molecules that can be attached to a single antibody depends on the conjugation chemistry. This quantity is characterized by the Drug to Antibody Ratio (DAR), which represents the average number of drug molecules per antibody molecule. In the case of Antibody-Drug Conjugates (ADCs), the DAR and its changes in vivo serve as critical parameters. They can influence the ADC’s physicochemical properties, effectiveness, safety, and pharmacokinetics. ADCs with high DARs are prone to aggregation and might exhibit an accelerated clearance rate or potentially pose a higher toxicity risk as compared to non-conjugated monoclonal antibodies (mAb) or antibodies with lower loads. Consequently, rigorous in vivo testing is essential to find the optimal DARs for specific targets and antibodies, in order to attain the most beneficial therapeutic window.
As for determining the average DAR, the simplest method is to measure the concentration of both the drug and antibody using ultraviolet-visible (UV/VIS) spectroscopic measurements or calculating it using a Matrix-assisted Laser Desorption/Ionization Mass Spectrometer (MALDI-MS) or Electrospray Ionization Mass Spectrometer (ESI-MS). Several liquid-phase methods, like Hydrophobic Interaction Chromatography (HIC), Reversed-Phase Liquid Chromatography (RPLC), and Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS), are also useful for analyzing the DAR in homogeneous and non-homogeneous ADCs. However, the suitability of these methods in different sample types should be taken into consideration. This should be supplemented with in vivo and in vitro experiments to monitor the changes in half-life and thermal stability of ADCs.
ADME
The establishment of an ADME evaluation system is critical to the ADC development process. As previously mentioned, this is due to the variable physicochemical properties of ADCs, resulting from differences in linker, payload, conjugation strategy, and DARs. For a comprehensive assessment of a novel ADC’s ADME, it is necessary to characterize the disposition of both the intact molecule and its various components. This includes the target-mediated and catabolic clearance of the mAb, as well as the release, traditional small molecule distribution, metabolism, and excretion of the released drug. In ADC drug bioanalysis, the ligand binding assay (LBA) is the most commonly used bioanalytical method. It necessitates the use of platforms designed for different antibodies and load types. ELISA and ECLA, for instance, can be used to detect total antibodies and conjugated antibodies. Meanwhile, LC-HRMS serves as a more general detection method, and LC-MS/MS is mainly used to analyze free payload and metabolites for quantitative analysis.
Conclusions
The process of module optimization and ADME characterization for an ADC is complex, considering the need to account for both the mAb and small molecule components. The present review elucidates the experimental systems and strategies in current use, offering guidance to help researchers successfully develop novel ADCs with desirable ADME characteristics. As ADC technology is still under development, a continuous re-evaluation is necessary as the field matures in the coming years.
Reference:
1. Beaumont, Kevin, et al. “ADME and DMPK Considerations for the Discovery and Development of Antibody Drug Conjugates (ADCs).” Xenobiotica, vol. 52, no. 8, Aug. 2022, pp. 770–85.