Antibody drug conjugates (ADCs) are multi-domain molecules composed of monoclonal antibodies, linkers and small molecule cytotoxins. ADCs specifically bind target antigens highly expressed on the surface of tumor cells through monoclonal antibodies, enter cells through antigen-mediated internalization and release cytotoxins, which can improve efficacy and reduce the toxicity of cytotoxins to normal tissues and cells. Like conventional monoclonal antibodies, ADCs serve as protein drugs and are immunogenic. In addition, because conjugation introduces new epitopes, ADCs may induce more complex immune responses in the body, which may affect the effectiveness and safety of the drugs. Further characterization of anti-drug antibodies (ADA) induced by ADCs, especially neutralizing activity tests, will help analyze and predict the possible impact on efficacy and safety caused by the production of ADA. Due to the particularity of the structure and mechanism of ADCs, ADA produced for different domains may have neutralizing activity, which blocks the internalization of ADCs through a variety of mechanisms, or hinders the release of cytotoxic molecules, thereby reducing cell killing effect or leading to off-target toxicity. Therefore, the design and development of neutralizing antibody detection methods for ADCs are different from conventional antibody drugs and pose greater challenges.
Immunogenicity Risk Assessment and Evaluation Strategy of ADCs
- Risk Assessment of Immunogenicity of ADCs
The immunogenicity of macromolecule therapeutic drugs is closely related to the characteristics of the drug itself, as well as the subject’s or patient’s autoimmune status and disease-related factors. The unique structural characteristics of ADCs increase the complexity of their immunogenicity risks. Not only monoclonal antibodies can cause immune responses as antigens, but also linkers and linker-cytotoxins can act as haptens to cause immune responses. The conjugation of cytotoxins to monoclonal antibodies through linkers may lead to the generation of new immune epitopes and increase the risk of immune reactions to ADCs. Cytotoxins are hydrophobic, while monoclonal antibodies are hydrophilic. The coupling of the two increases the possibility of drug aggregation and also increases the risk of immune reactions caused by ADCs. Due to the existence of the “epitope spreading” effect, the immune response against a certain epitope may extend to other epitopes on the same molecule. The multiple immunogenic epitopes of ADCs may complicate the form of immune response or enhance the degree of immunity. The presence of linker-cytotoxins in ADCs may promote the cross-linking of immune complexes mediated by anti-linker-cytotoxin antibodies, and the macromolecular complexes formed affect the internalization of ADCs.
ADCs have no corresponding endogenous components, so immune responses directed against ADCs do not neutralize important endogenous proteins. In view of the limited clinical research experience and the specificity of ADCs, ADCs are identified as macromolecule drugs with moderate immunogenicity risk.
- Evaluation Strategies for Immunogenicity of ADCs
The immunogenicity evaluation of therapeutic protein drugs is mostly qualitative or semi-quantitative tests, usually using multi-level analysis strategies. Step 1 is a screening test to identify potentially positive samples. Step 2 is a confirmatory test to confirm the drug specificity of the antibody. Confirmatory tests are generally based on screening tests, adding an excess amount of drug to competitively bind ADA, and confirm the specificity of the antibody through the reduction of response signals. Step 3 is to determine the titer, neutralization activity, subtype, epitope and affinity of ADA by titer test, neutralization activity test, antibody typing/subtype test, epitope analysis (domain analysis) or affinity test, so as to better analyze the possible abnormal pharmacokinetics, efficacy reduction, allergy and other adverse events according to the characteristics of antibodies.
As special biological macromolecules, the immunogenicity evaluation of ADCs also follows the above strategy. In view of the structural characteristics of ADCs, it is recommended to add epitope analysis tests to evaluate the production and impact of ADA on different epitopes such as monoclonal antibodies, linkers, and linker-drugs. At present, bridging tests can basically detect most types of ADA, so bridging-based enzyme-linked immunoassays (ELISA) or electrochemiluminescence tests (ECL) are mostly used for the analysis of macromolecule drugs ADA. Anti-drug antibody epitope analysis of ADCs can use competition methods or direct detection methods. The principle of competitive epitope analysis is similar to that of the confirmatory test. It also uses an excess of unlabeled ADCs to pre-incubate with the sample before detection. The direct detection method uses ADCs as capture reagents and antibodies targeting monoclonal antibodies or cytotoxic molecules as detection reagents to directly detect the phenotype of ADA.
Analysis Method for Neutralizing Activity of Anti-Drug Antibodies of ADCs
The selection of the analysis mode for the neutralizing activity detection method needs to be considered based on a variety of factors, including but not limited to the mechanism of action of the drug itself, the correlation between the detection method and the real situation in the body, the selectivity of the method itself, the degree of interference from the biological matrix, sensitivity and robustness, etc. When selecting an analytical mode, it is recommended that the relevance of the assay method to the in vivo mechanism of action of the drug be a primary consideration. The test formats commonly used for neutralizing antibody analysis of antibody drugs are cell-based biological assays and non-cell competitive ligand-binding assays (CLBA). The selection of test mode should consider factors such as the mechanism of action of the drug, the sensitivity, selectivity, precision of the test, drug tolerance, and the impact of the produced NAb on the subjects.
- Cell-Based Bioactivity Assays
The effector cytotoxic molecules used by ADCs are divided into two categories: cytotoxins that inhibit mitosis and cytotoxins that promote DNA cleavage. Cytotoxicants such as maytansine analogs DM1 and DM4; auisatin analogs MMAE and MMAF inhibit cell mitosis by interfering with the production of tubulin in the cell cycle; calicheamicin analogs and pyrrolobenzodiazepine analogs induce DNA damage by binding to the minor groove of DNA, thereby directly killing cells or inducing apoptosis. Some ADCs can also work through antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Different domains such as monoclonal antibodies and cytotoxins of ADCs participate in the cell killing effect in a certain order. NAbs targeting monoclonal antibodies or cytotoxins may block the cell killing activity of ADCs. Therefore, cell proliferation and apoptosis are used alone. The functional test of other cells can better reflect the mechanism of action of ADCs and is used to evaluate the neutralizing activity of ADCs to produce ADA. In view of the mechanism of action of ADCs, regulatory agencies such as the FDA and EMA first recommend cell-based bioactivity tests for the analysis of neutralizing antibodies of ADCs.
a. Cellular assays based on cellular enzymatic reactions: During the process of cell proliferation, the activity of metabolic enzymes increases, but after the action of ADCs, cell death or apoptosis occurs, and the enzyme activity decreases. First, according to the action mechanism of ADCs, select appropriate cell lines and detection systems, and then establish a cell activity and toxicity detection system, that is, the activity curve of the drug. Then, based on the drug’s activity profile, appropriate drug concentrations of ADCs are determined at which NAbs present in the sample can reduce ADCs-mediated inhibition of cell viability or proliferation. Finally, the presence or absence of NAb is determined based on the changes in cell activity after the action of a certain concentration of ADCs.
b. Cell tests using cellular metabolic markers as detection indicators: The concentration changes of markers of cell metabolism, such as adenosine triphosphate (ATP) and lactate dehydrogenase (LDH), can be used as detection indicators to evaluate cell activity. The drug activity analysis method of ADCs, which uses cellular metabolites as detection indicators, detects the decrease in metabolic marker levels after drug administration. The degree of decrease is positively correlated with drug concentration. The combination of NAb and ADCs in the sample leads to a reduction in the drug activity of the ADCs, which in turn causes an increase in the levels of metabolic markers. The degree of increase is positively correlated with the activity or concentration of the NAb.
- Competitive Ligand Binding Assays (CLBAs)
The cell-based neutralizing antibody analysis test evaluates the neutralizing activity of anti-drug antibodies through the inhibition of the biological activity of ADCs, which can better reflect the in vivo effects of ADCs than the CLBA test. However, cell tests are greatly affected by matrix or drug interference, and the sensitivity of the test is low. Acid hydrolysis treatment of samples may affect the viability of cells, so in some cases, if fully justified, CLBA may also be considered to analyze the neutralizing activity of anti-drug antibodies of ADCs. CLBA is based on the competitive binding of NAb to the drug target protein. If the sample contains NAb, the binding of the drug to the target protein is inhibited to a certain extent. Research by Finco et al. shows that CLBA test systems generally have higher sensitivity and precision than cell-based test systems, have good drug tolerance, and can also tolerate strong matrix interference.
References
- Püsküllüoğlu, M. et al. Antibody-drug conjugates in HER-2 negative breast cancers with poor prognosis. Biochimica et Biophysica Acta (BBA) – Reviews on Cancer. 2023, 1878(6): 188991.