Inquiry Basket
Antibody-drug conjugates (ADCs) are a new class of biotherapeutics that typically consist of a cytotoxic payload covalently bound to an antibody via a linker. ADC undergoes continuous decoupling and biotransformation processes in the body, resulting in the formation of complex and structurally heterogeneous mixtures. In addition to decoupled free antibodies and free payloads, there are also ADC molecules conjugated to small molecule drugs to varying degrees. Therefore, it poses great challenges to the development of bioanalytical methods. In order to understand the pharmacokinetic and pharmacodynamic characteristics of ADC drugs in non-clinical and clinical research and development stages, as well as the immunogenicity and safety, different molecular forms of entities such as drug-coupled antibodies, naked antibodies and total antibodies in the body after administration need to be analyzed using different methods. Among them, the biological analysis of total antibodies, conjugated antibodies, free loads and related metabolites runs through the entire process of early discovery/screening, preclinical and clinical of conjugated drugs.
Fig. 1. LBA assay format for TAb and ADC (Molecules. 2022, 27(19): 6299).
ADC has macromolecule parts and small molecule parts. Different components have different requirements for analysis methods. It is difficult to use a biological analysis method to describe the process, status, and whereabouts of ADC in the body. Coupling linkers in ADCs are usually divided into cleavable linkers and non-cleavable linkers, which behave completely differently in biological systems and release toxins in different forms in the body. Specifically, cleavable linkers (e.g., valine-citrulline dipeptides, hydrazine linker, and disulfide bond linker) are specifically sensitive to intracellular properties of cancer cells, such as the expression of certain proteases, pH and glutathione, in order to achieve selective release of toxins. In contrast, non-cleavable linkers do not contain a specific release mechanism and rely on intracellular proteolysis following ADC internalization. From an analytical perspective, while ADCs with cleavable linkers release free toxins in biological systems, ADCs with non-cleavable linkers typically release the active toxin-linker-amino acid moiety as a product of complete degradation of the ADC. Therefore, different bioanalytical methods should be implemented for different analytes.
In vivo biotransformations of ADCs (such as toxin shedding, protein mass gain/loss, and payload metabolism) frequently occur due to the instability and complexity of the internal environment, coupling sites, and linkers. This biotransformation may reduce efficacy and increase off-target toxicity. Although some new engineering technologies (such as cystine conjugation, unnatural amino acid conjugation, enzyme-mediated conjugation, peptide tag conjugation, novel heterobifunctional reagents) use site-specific conjugation methods to produce more uniform and stable ADC, but in vivo biotransformation may still persist. If the detection site on the ADC undergoes biotransformation, it will interfere with the accuracy of the bioanalytical method.
In clinical practice, it is impossible to measure the concentration of drugs in tumor tissues. We can only measure the concentration of ADC in the blood circulation. How to reflect the final drug effect through limited data is a challenge for clinical PK models. The drug-to-antibody ratio (DAR) is an important parameter describing the amount of payload conjugated to an antibody, but DAR species can change dynamically in vivo, which poses additional challenges to bioanalysis, adding ADME (absorption, distribution, metabolism and excretion) research complexity.
ADC goes through many steps before entering the body and finally acting at the target site. When only about 50% of the drug can proceed at each step, the amount of drug that can reach the target site is very small. In the end, less than 2% of the drug is effective, so the toxicity requirements of the toxin are quite high. When the toxin toxicity is so high, the therapeutic window of ADC is very narrow, and the toxin of ADC must not be shed in the circulation system. Therefore, the sensitivity requirements for the free drug determination method are very high.
A 2013 American Society of Pharmaceutical Scientists document recommends that the following components should be measured for evaluation in the early stages of ADC drug development: total antibody, ADC, and free toxin (Table 1). In addition to these three basic evaluation components, based on the structure of the ADC molecule and the characteristics of the toxin, bound toxins, in vivo DAR values, immunogenicity, etc. can also be additionally measured to better understand the pharmacokinetics and ADME. LC-MS/MS is typically used for small molecule free toxin analysis; ligand binding assay (LBA) or LC-MS methods are used to quantify total antibodies and ADC.
| ADC | ADC Detection Method | Total Antibody Detection Method | Free Toxin Determination Method |
| Tisotumab vedotin-tftv | LBA | LBA | LC-MS/MS |
| Loncastuximab tesirine-lpyl | LBA | LBA | LC-MS/MS |
| Sacituzumab govitecan-hziy | Derivedb | LBA | LC-MS/MS |
| Belantamab mafodotin-blmf | LBA | LBA | LC-MS/MS |
| Fam-trastuzumab deruxtecan-nxki | LBA | LBA | LC-MS/MS |
| Polatuzumab vedotin-piiq | Hybrid LBA LC-MS/MSc | N/A | LC-MS/MS |
| Enfortumab vedotin-ejfv | LBA | LBA | LC-MS/MS |
| Inotuzumab ozogamicin | LC-MS/MSc | N/A | LC-MS/MS |
| Trastuzumab emtansine | LBA | LBA | LC-MS/MS |
Table 1. FDA-approved bioanalytical method for regulatory filing of ADC.
Detected by the antibody moiety against the conjugated drug. The determination of total antibodies includes steps such as capture and enzyme digestion. Reagent selection, reaction time and dosage need to be carefully explored to achieve better recovery rates.
Total antibody analysis methods: ELISA, electrochemiluminescence immunoassay ECLA.
In non-clinical PK and TK studies of ADC drugs, the detection antibody categories selected are:
In clinical trials of ADC drugs, antibodies that bind recombinant antigens, anti-idiotypic or anti-complementary defined regions can be used to reduce non-specific binding and minimize serum protein background. For total antibody testing of ADCs used in preclinical and clinical development, ensure that all expected DARs in vivo are accurately quantified as total antibody concentrations.
Conjugated antibody ADC analysis: ELISA, electrochemiluminescence immunoassay ECLA.
For PK analysis of conjugated antibodies, anti-small molecule antibodies are used as coating reagents, and the detection antibodies are consistent with the total antibody analysis. ELISA is usually used to measure antibody conjugates in serum.
Through common pre-treatment methods for small molecule bioanalysis such as protein precipitation, liquid-liquid extraction or solid-phase extraction, cytotoxins are extracted and directly detected by LC-MS/MS. The analysis of free toxins should pay attention to sensitivity and stability. The plasma stability of the linker also needs to be investigated.
Small molecule cytotoxin analysis: LC-MS/MS.
Before using LC-MS/MS to quantitatively analyze free small molecules, protein precipitation and/or solid-phase extraction are required to remove plasma proteins. If drug-related metabolites need to be quantified, the metabolites need to be identified first and their standards prepared. The structure of catabolites is determined, stable isotope-labeled internal standards or simulated molecules are synthesized, and small molecule LC-MS/MS analysis methods can be used for research related to small molecule catabolites.
Anti-drug antibody (ADA) is the main method for evaluating the immunogenicity of antibody drugs. ADC may cause immunogenicity in the human body and produce corresponding ADA, affecting PK, PD and safety.
1. Treat antibody drugs as foreign proteins, similar to vaccination, to generate an immune response;
2. The body's tolerance mechanism for self-proteins is destroyed, similar to the mechanism of autoimmune diseases and the production of autoantibodies.
1. Screening: Preliminary screening of biological samples. Samples that are higher than the threshold are potentially positive, and samples that are lower than the threshold are negative samples.
2. Confirmation: Perform immune confirmation on the screened positive samples to confirm the specificity between the antibody and the drug; determine whether it is a positive sample based on the inhibition rate before and after adding the drug.
3. Titer: Dilute the positive sample differently so that the diluted sample is the dilution factor at the screening threshold, which is the antibody titer of ADA.
4. Neutralization: Neutralizing antibodies are a special type of ADA. NAb interferes with the in vivo activity of the drug by blocking the product from reaching its target or interfering with the binding of the receptor/ligand.
The biological activity measurement of ADC is also crucial. Biological activity measurement, that is, the determination of the active ingredient content and drug potency of antibody drugs, is an important quality control indicator to ensure the effectiveness of antibody drugs, so a complete system needs to be established for characterization.
Binding activity can directly reflect the binding ability of the antibody to the antigen, as well as the binding ability to effector molecules/receptors, such as FcγRIII, C1q, etc. On this basis, through the determination of cytological biological activity, such as ADCC (antibody-dependent cell-mediated cytotoxicity), CDC (complement dependent cytotoxicity), cell proliferation inhibition, cytotoxicity, cytokine secretion, internalization, etc., a comprehensive characterization system for detecting antibody activity in vitro is formed, which is the basis for antibody drugs to enter in vivo experiments and the guarantee for the success of clinical experiments. Different antibody drugs exert different effects through different mechanisms, and different biological activity measurement methods need to be established. Biological activities include: internalization, inhibition of cell proliferation, cytotoxicity, cytokine secretion ability, ADCC, CDC, etc.
BOC Sciences is a leading provider of ADC analysis services, offering comprehensive solutions to support the development and analysis of ADCs. One of the key strengths of our bioanalytical services is our expertise in method development and validation. Our team of experienced scientists has a deep understanding of the unique challenges associated with analyzing ADCs, such as their complex structure, heterogeneity, and low concentrations in biological samples. We utilize state-of-the-art instrumentation and cutting-edge technologies to develop custom bioanalytical methods that meet the specific needs of each project.
Compared with traditional macromolecule or small molecule drugs, bioanalysis of ADCs is complex and challenging, requiring innovative bioanalytical technologies to overcome challenges in biotransformation, ADME, and tissue sample analysis. A well-established bioanalytical strategy is critical to the clinical success of ADCs. With the development of technology and instruments, bioanalytical methods such as LBA or hybrid LBA-LC-MS continue to be powerful and reliable tools, while some new methods such as HRMS are also slowly taking shape. These tools are accompanied by research from the early stages of drug development to laboratory toxicology and clinical research, which helps to better analyze the pharmacokinetics (PK), metabolism and biological distribution of ADC, master the exposure, in vivo catabolism and biotransformation of ADC, and grasp the efficacy and toxicity of ADC.
Reference
