Currently, there are numerous in vitro and in vivo non-clinical research methods, as well as the experience from the development of marketed drugs. However, predicting clinical efficacy and safety through optimized non-clinical studies remains challenging, and this article conducts an in-depth analysis on this issue.
The ideal ADCs should possess at least the following three characteristics:
1. Targeting binding ability: The ideal ADC target antigen is only expressed on the surface of tumors and at high levels, without easy shedding.
2. Transformation process: Before reaching the target cells, the cytotoxic molecule should ideally not shed from the ADC, meaning the entire ADC is internalized into the cells.
3. Release of the toxin small molecule: The small molecule should be rapidly and massively released within the tumor cells; the small molecule should possess high toxicity to efficiently kill tumor cells.
Pharmacological Evaluation Strategy for ADCs
Pharmacological action research is usually conducted during the drug discovery or preclinical evaluation phase. The main experimental projects that need to be carried out include target binding activity, antibody-mediated internalization, in vivo and in vitro tumor proliferation inhibition (for anti-tumor ADCs), antibody-dependent cellular cytotoxicity (ADCC), and complement-dependent cytotoxicity (CDC).
When examining the pharmacodynamics of ADCs in animals, the first consideration is species differences. If ADCs do not have binding activity or have low binding activity with antigens in mice, it is necessary to consider using transgenic animals for pharmacodynamic experiments, or conducting in vivo pharmacodynamic studies with animal-derived antibodies. Transgenic animals can avoid the use of alternative antibodies, and when the target antigen is expressed in the host's blood vessels or stroma, transgenic animals can not only evaluate the pharmacodynamic effects but also observe toxicity reactions to some extent. If the target antigen is only expressed in human tumor cells, there is no need for transgenic animals; the pharmacodynamic effects can be examined in nude mouse models implanted with human tumor cells. When conducting pharmacodynamic studies at the cellular level or animal model level, simultaneously conducting PK/PD studies, analyzing drug targeting distribution, receptor occupancy rate, and the relationship between drug exposure and effect can help in the design of clinical dosing regimens and the analysis of safety study results.
Toxicological Evaluation Strategy for ADCs
Animal species selection: To fully expose the toxicity characteristics of ADCs in non-clinical safety studies to support the clinical development of ADCs, assessing the relevance of animal test species and the translatability of non-clinical study results is crucial. This determination usually involves checking the binding affinity of ADCs to target antigens in non-clinical species compared to humans, as well as comparing the immunohistochemical staining spectrum in tissue cross-reactivity studies. The effector functions or immunogenicity of antibodies may also vary due to species differences.
Exposure parameters and metabolism: In addition, like other drugs and biological products, the extrapolation of non-clinical study results to humans should include a comparison of exposure parameters and metabolism. For example, BR-96 is an ADC formed by covalently linking doxorubicin (DOX) to the cysteine residues of a humanized BR96 monoclonal antibody through a 6-maleimidohexanoic acid hydrazide linker. Toxicology studies have shown that, unlike in rats and monkeys, dogs are more sensitive to the toxic effects of BR-96, with hemorrhagic enteritis as the dose-limiting toxicity. Surprisingly, BR96-DOX and the unmodified monoclonal antibody BR96 have the same dose-limiting toxicity, indicating that the toxicity is produced by the monoclonal antibody. Unlike unmodified DOX, ADCs did not induce cardiomyopathy in rat models. Therefore, the dose selection for the Phase I clinical trial of BR96-Dox in human subjects was based on the toxicology results in dogs.
Antibody target expression: Like therapeutic monoclonal antibodies, the non-clinical evaluation of ADCs may be hindered by the lack of antigen targets in animal test species. The ICH S6 (R1) guidelines on preclinical safety evaluation of biotechnology-derived pharmaceuticals (2011) state, "The species selection for antibody-drug/ toxin conjugates (ADCs) containing novel toxins/toxins should follow the same general principles as for the unmodified antibodies. The relevant animal species for testing monoclonal antibodies are those that express the epitope required and display a tissue cross-reactivity spectrum similar to that of human tissues. Therefore, species and disease specificity may determine or limit non-clinical evaluation."
Normal tissue expression of the target antigen is a major safety concern for ADC therapy. Ideally, the target antigen should be expressed at very low levels on normal cells; however, as mentioned in the above two examples, antibodies are not always specific in practice. Other potential toxicity examples related to a lack of specificity for ADC targets include NaPi2b, a sodium-dependent phosphate transporter, which is expressed in several tumor types but also detectably in normal tissues, where it plays a role in inorganic phosphate homeostasis. An ADC (anti-NaPi2b-vc-MMAE) was developed by conjugating a humanized IgG1 anti-NaPi2b monoclonal antibody to MMAE through a vc peptide linker, and it was confirmed to have specific binding affinity in normal human and crab monkey tissues. However, despite high-level expression in the normal lungs of monkeys, the safety of this cross-reactivity can be acceptable, with dose-limiting toxicities unrelated to normal tissue expression. The toxicological effects in normal rats, non-binding species, and monkeys are consistent with the pharmacology of MMAE.
General toxicology design principles: In general, there is no need to set separate groups for the monoclonal antibody/naked antibody, linker/Linker-toxin, as the monoclonal antibody molecule is one of the main factors affecting or determining systemic exposure. Dose design should consider the pharmacodynamics dose of ADCs, the characteristics of the antibody molecule, and the toxicity features of the small molecule toxin.
Focus on Payload-related toxicity: Although targeted release of small molecule compounds achieves drug enrichment in target tissues, improving the therapeutic window, the main toxicity characteristics of ADCs are still similar to those of the connected small molecule compounds. Most ADC toxicity reactions are less severe than direct administration of small molecule compounds, but some ADCs may increase tissue toxicity reactions associated with antibody target binding or off-target binding. Although ADCs increase targeted release of small molecule compounds, some small molecule compounds are still prematurely released, and some free small molecule compounds can rapidly diffuse or transfer to surrounding tissues and systemic circulation from target cells, or apoptosis and damage of target cells may lead to free small molecule compounds entering systemic circulation. The antibody part of ADCs may also bind to other non-target tissue organ cell surface receptors or epitopes, such as Fc receptor-mediated cellular phagocytosis, and the drug may exert toxic effects on corresponding tissue cells after metabolic breakdown. Therefore, the toxicity of ADCs is closely related to the characteristics of the antibody, small molecule compounds, and linkers, and changes in the toxicity characteristics of ADCs will occur with the variation of the antibody, small molecule compounds, and linkers. In non-clinical safety studies, attention should be paid to the impact of drug composition structure and pharmacokinetic characteristics on toxicity, and the results of the experiments should be comprehensively analyzed.
Genotoxicity: Biological macromolecules generally do not directly interact with DNA, and the ADC and its antibody part usually do not need to undergo genotoxicity tests. The potential genotoxicity of ADCs comes from the small molecule compounds. If the payload small molecule compound is a new compound, genotoxicity tests should be conducted; for new free small molecule compounds and/or new linkers produced in the body, the target test materials should be evaluated based on the physicochemical properties, cleavage characteristics, and structure of the cleavage products of ADCs; if sufficient data indicate that the target test material has genotoxicity, no genotoxicity studies are needed.
Reproductive toxicity: The small molecule compounds, linkers, and naked antibodies in ADCs may have adverse effects on reproductive organs, fertility, embryo-fetal development, and offspring development, so the reproductive toxicity risk of ADCs should be paid attention to. The research strategy, experimental design, implementation, and evaluation of ADC reproductive toxicity should refer to the ICH S5 guidelines, and combine the mechanism of action and target population of ADCs to adopt a specific problem-specific analysis strategy. ADCs intended for late-stage tumor patients can refer to the ICH S9 guidelines for reproductive toxicity research.
For biological products, reproductive toxicity is usually evaluated in pharmacologically relevant animal species. If both rodents and rabbits are target-binding related animal species, two animal species should be used to conduct embryo-fetal development toxicity tests, unless embryonic lethality or teratogenicity has been confirmed in one species. If the target-binding animal species are non-human primates or there are no relevant animal species, rats or rabbits are usually considered first for reproductive toxicity tests to investigate the reproductive toxicity of free small molecule compounds. If the study results confirm positivity, there is no need to investigate reproductive toxicity using target-binding related animal species; otherwise, non-human primates or transgenic animals or alternative molecules should be considered for testing to identify antigen-mediated reproductive toxicity. If the antibody or small molecule compound in ADCs has literature data clearly showing potential reproductive toxicity risks, reproductive toxicity studies are not needed and relevant risk control can be based on literature information.
Carcinogenicity: Based on the characteristics of ADCs, the necessity of carcinogenicity studies can be considered according to ICH S1, ICH S6, ICH S9, and other related guidelines.
Immunogenicity/Immunotoxicity: ADCs may cause immunogenicity after entering the body, producing anti-drug antibodies. The evaluation of immunogenicity of ADCs helps analyze the pharmacokinetics, efficacy, and safety results of the drug, so anti-drug antibody detection is usually accompanied in non-clinical safety studies of ADCs. Anti-drug antibodies may be produced from the antibody part and linker part of ADCs, and whether further examination of antigen epitopes is needed should be based on the risk of immunogenicity. The development, validation, and analytical detection strategy for immunogenicity analysis of ADCs should follow the "Technical Guidelines for Drug Immunogenicity Research". ADCs can refer to ICH S6, ICH S8, ICH S9, and other guidelines, and reasonably design immunotoxicity detection indicators based on factors such as the type and mechanism of action of ADCs. In addition to routine immunotoxicity studies, additional immunotoxicity studies should be considered if necessary.
Optical safety: Before Phase I clinical trials, the potential phototoxicity should be preliminarily evaluated based on the photochemical properties and pharmacological/chemical categories of the small molecule compounds (including linkers). If these data suggest potential risks, appropriate protective measures should be taken for clinical trial subjects. If non-clinical data or clinical experience cannot fully evaluate the risk of photo safety, photo safety assessment in line with the principles described in ICH S10 should be provided before large-sample clinical trials (Phase III).
Tissue cross-reactivity: Tissue cross-reactivity studies are in vitro tissue binding tests conducted using immunohistochemical techniques to determine the binding characteristics of antibodies and antigen epitopes in tissues. Tissue cross-reactivity studies can provide useful supplementary information for the understanding of target distribution and can also provide information on potential unexpected binding. Tissue cross-reactivity studies using human tissues are an integral part of the initial clinical dosing safety evaluation series for antibody drugs. When there are no target-binding related animal species, tissue cross-reactivity information is particularly important for predicting human toxicity risks. For ADCs intended for late-stage tumor indications, tissue cross-reactivity studies are not necessary if there are no special concerns after evaluating toxicological effects in relevant animal species.
Currently, ICH, FDA, NMPA, and other agencies have issued relevant technical guidelines for the non-clinical pharmacology and toxicology research of ADCs. Researchers can design scientifically reasonable non-clinical pharmacology and toxicology studies based on the characteristics of the drugs, maximizing the extrapolation of non-clinical animal-level pharmacodynamic effects and toxicity reactions to humans, predicting the efficacy and safety of ADCs in clinical applications, thereby improving the success rate of clinical trials and reducing development risks. Drug developers can communicate with regulatory agencies based on preliminary research results and existing issues to obtain scientifically standardized non-clinical research support information.
