Antibody-Drug Conjugates (ADCs) are new therapies that combine the high targeting of monoclonal antibodies with the strong cytotoxicity of small molecule cytotoxins. Due to its precise and powerful mechanism of action, ADC has become a new hot spot and important trend in the development and research of anti-tumor antibody drugs. ADC drugs can effectively kill cancer cells without harming normal tissues, thereby improving the therapeutic index of anti-cancer treatment. They are known as “precision-guided magic bullets.” ADC consists of three parts (Fig. 1), including the targeting antibody part, the small molecule payload that plays the main function, and the ADC linker connecting the two.
After the monoclonal antibodies in the ADC bind to the target antigen overexpressed on the tumor cells, the ADC is internalized into the tumor cells. Due to the presence of lysosomal proteases, the cytotoxic payload is partially separated from the antibody, and the cytotoxic payload initiates cell damage or apoptosis to kill cancer cells. For example, the payload can diffuse into the extracellular matrix and kill neighboring cancer cells, known as the bystander effect, which further enhances the efficacy of the ADC. A comprehensive understanding of the pharmacokinetic characteristics of ADC drugs is crucial to the design and development of ADC drugs. This article will focus on the bioanalytical strategy for pharmacokinetic evaluation of ADC drugs and the quantitative bioanalytical platform for ADC drugs, providing ideas for drug developers.
Preclinical DMPK Research Strategy for ADC Drugs
Compared with traditional antibody drugs or small molecule drugs, ADC drugs have complex structures. Various factors can affect the efficacy of ADC drugs. At the same time, the dynamic processes of ADC drugs in vivo are diverse. Evaluating the exposure-efficacy and exposure-safety relationships of ADC drugs from distribution, uncoupling, metabolism and elimination is crucial for drug design and subsequent development. The absorption, distribution, metabolism and excretion (ADME) of ADC drugs in various tissues directly affect the efficacy and safety of ADC, which is more complex and challenging than other drugs. ADC drugs not only need to analyze macromolecules (total antibodies and conjugated antibodies) but also small molecules (free load and conjugated load). The DMPK research strategy for ADC drugs includes in vitro stability and payload release experiments, in vivo PK/PD studies, etc.
Quantitative Bioanalysis of ADC Drugs
Quantitative bioanalysis to evaluate total antibodies, conjugated antibodies, free payload and related metabolites throughout all stages of ADC drug early discovery/screening, preclinical and clinical. In order to correctly evaluate the ADME properties of ADC drugs, comprehensive bioanalytical methods are required.
| Analyte | Definition | Platform or Method | Precautions |
| Total Antibody | DAR≥0 | *LBA *LC-MS/MS (Surrogate Peptide) *LC-HRMS | *The LBA platform requires ADC naked resistor bridging experiments *Surrogate peptide requires antibody sequence |
| Conjugated Antibody | DAR≥1 | *LBA *LC-MS/MS (Surrogate Peptide) | *If there are anti-payload antibodies, it is recommended to analyze on the LBA platform |
| Free Payload and Payload Related Species | Released toxic small molecules and their related small molecules | *LC-MS/MS | *Routine small molecule analysis *High sensitivity requirements |
In ADC drug bioanalysis, Ligand Binding Assay (LBA) is the most commonly used bioanalytical method. As the “gold standard” in the field of macromolecule quantitative research, LBA has the advantages of high sensitivity and high throughput, and is often used to detect total antibodies and conjugated antibodies. The difficulty in analyzing LBA lies in the need to optimize key reagents for different species and matrix types. Therefore, the development cycle of the LBA method is long, and it is unable to provide structural information of ADC and related biotransformation information. With the continuous development of mass spectrometry technology, liquid chromatography-high resolution mass spectrometry (LC-HRMS) technology is increasingly used for the characterization and quantification of macromolecules. As an alternative method, LC-HRMS is mainly used for the determination of total antibodies and conjugated antibodies, and has the advantages of short time cycle and good versatility.
- Free Payload: LC-MS/MS Quantitative Analysis
A major factor affecting ADC toxicity is free payload, because free payload may mediate unintended off-target toxicity. Therefore, it is necessary to monitor free loads, which has also become a very important part of ADC research. The concentration of free payload in plasma is very low, which poses a greater challenge to sensitivity. LC-MS/MS quantitative analysis of free payloads is often used to determine whether free payload is released during ADC drug storage, the stability of the linker plasma of antibodies and toxins, the stability of the toxin itself in the biological matrix, small molecule PK and tissue distribution.
- Total Antibody and Conjugation Site Analysis
Total antibodies are typically analyzed using the LBA platform. LBA is divided into specific method or generic method. At least two sets of key reagents are usually prepared to ensure the smooth completion of method development. At the same time, during method development, it is necessary to examine whether the signal response of the method to intact ADC drugs and naked antibodies conforms to the general pharmacokinetic laws. The consistency of the naked antibody and ADC signal should also be checked through bridging experiments. In addition, total antibody can also be determined using the surrogate peptide method. In order to reduce interference from the matrix, ADC was enriched using immunoaffinity magnetic beads, and then digested with trypsin to generate characteristic peptides, which were quantitatively analyzed using LC-MS/MS. This method has high sensitivity and strong specificity.
- Conjugated ADC Analysis
Conjugated antibodies are usually analyzed using the LBA platform and captured using anti payload antibody. If anti payload antibodies are not available, alternative methods can be used for conjugated antibody analysis. The LC-HRMS method captures targets from biological matrices with the help of antibodies or antigens that specifically bind to the antibody portion of the ADC. The LC-HRMS method analyzes ADC drugs at the intact or subunit level, and calculates the value of the conjugated antibody.
- DAR Value Analysis
Drug-antibody-ratio (DAR) represents the amount of conjugated load of the drug-antibody part of each ADC. The DAR value continuously changes with drug metabolism and is an important parameter to describe the safety and effectiveness of ADC drugs. Hydrophobic interaction chromatography (HIC) is considered the gold standard for DAR value analysis of cysteine-coupled ADC drugs, but this method requires a large amount of ADC (20-50 μg) and requires a high ADC concentration in the sample. In recent years, LC-HRMS has provided a feasible method to analyze ADC drug DAR values at the intact and subunit levels with its unique advantages. Compared with the HIC-UV method, the LC-HRMS method requires fewer test samples and can achieve the characterization of ADCs with different DAR values.
Since ADC drugs have complex structures and diverse processes in the body, the stability, safety, and effectiveness of ADC drugs require the assistance of multiple biological analysis platforms to observe the unique biotransformation of ADC and obtain rich and accurate data. As more innovative conjugation strategies, antibody scaffolds, and novel warheads are used in next-generation ADCs, future developments in bioanalytical technology will need to address these increasingly complex issues.
References
- Yu, J. et al. Novel ADCs and combination therapy in urothelial carcinoma: latest updates from the 2023 ASCO-GU Cancers Symposium. Journal of Hematology & Oncology. 2023, 16(1).
- Fu, Z. et al. Antibody drug conjugate: the “biological missile” for targeted cancer therapy. Sig Transduct Target Ther. 2022, 004: 007.