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After the antibody-drug conjugates (ADCs) drug enters the blood circulation and binds to the target antigen receptor on the surface of tumor cells, the newly formed ADC-antigen complex is internalized and degraded by lysosomes, releasing the payload and inducing tumor cell death. Therefore, the payload is an important part of the ADC design. The activity and physicochemical properties of the payload directly affect the anti-tumor efficacy of ADC drugs. The mechanism of action of the payload is an important factor in determining the performance of the ADC. In addition, other properties of the ADC payload such as cytotoxicity, immunogenicity, stability during preparation and circulation, water solubility, and modifiability are also important.
Fig. 1. The evolution of ADC payloads (Acta Pharmaceutica Sinica B. 2023, 13(10): 4025-4059).
An ideal ADC payload should have the following characteristics:
1. First, it should have sufficiently high cytotoxicity and a limited number of tumor surface antigens, especially in solid tumors. Furthermore, due to the low permeability and poor internalization activity of monoclonal antibodies, the amount of ADC payload that can be endocytosed into tumor cells via antibody-antigen binding is very low.
2. The ADC payload should have sufficiently low immunogenicity. Antibody-related drugs run the risk of inducing immunogenicity, which may negatively impact ADC efficacy and even lead to patient death. Currently, although ADCs use humanized monoclonal antibodies and small molecule payloads, they may still have an increased risk of immunogenicity compared with therapeutic monoclonal antibodies. To solve this problem, scientists extract highly toxic payloads from plants, animals, or microorganisms to ensure that the immunogenicity of the human payload is negligibly small. Using smaller molecular payloads is also a way to reduce the risk of immunogenicity.
3. The ADC payload should have high stability. Due to the long half-life of antibodies in circulation, ADCs should remain stable in the blood circulation to avoid release or breakdown. Under low pH conditions, the payload remains stable in the cytoplasm and lysosomes without significant degradation.
4. ADC payloads should have functional groups that can be modified without significantly affecting their effectiveness. The payload must have a modifiable functional group or a site that can bind to the monoclonal antibody. The site of modification must be chosen carefully to maintain drug effectiveness. When using non-cleavable linkers, the payload must maintain its potency even after the antibody is degraded.
5. The ADC payload should have a bystander killing effect. Some ADC drugs are internalized and release small, uncharged, membrane-permeable hydrophobic molecules that diffuse through the cell membrane and kill adjacent tumor cells negative for antigen expression. This process is called the "bystander killing effect" and is of great significance to tumor cells with uneven antigen expression. In general, payloads that promote bystander killing in tumor cells are more suitable for cancers with low or heterogeneous expression of target antigens. In order to construct ADC drugs with bystander effects, the structure of the ADC must meet conditions where the linker can be cleaved and the payload can penetrate into the cell membrane. Hydrophobic molecules with good cell membrane penetration have a strong bystander killing effect, but this may also make the drug easily absorbed by healthy tissues, leading to severe systemic toxicity. Therefore, the balance between the two is very critical for the toxic-effect effect of ADC.
6. The ADC payload should have appropriate water solubility to facilitate conjugation with the antibody and ensure sufficient solubility of the conjugate under physiological conditions. When excess hydrophobic payload binds to the antibody, the resulting ADC tends to aggregate and become unstable. In addition, the hydrophilicity of the payload can affect the cell membrane permeability of the ADC or its metabolites, thereby affecting its bystander killing activity.
7. The target of the payload should be intracellular, as most ADCs need to enter tumor cells to release their payload. Many membrane-targeting payloads from microorganisms, plants, and animals, such as those on neurons that act primarily by blocking ion channels or disrupting blood coagulation, are not suitable as ADC payloads.
As the main component of the cytoskeleton, tubulin not only supports the structural integrity of cells, but also plays a crucial role in the mitosis stage of cell proliferation. By inhibiting the production and aggregation of tubulin (polymerization), it can not only kill tumor cells, but also inhibit the rapid proliferation of tumor cells. Tubulin inhibitors commonly used in ADC construction include maytansines, Auristatins, and Tubulysins.
DNA damaging agents are divided into three categories due to different mechanisms of action: DNA double-strand destroying agents, DNA inserting agents, and DNA alkylating agents. DNA is crucial in the growth and proliferation process of cells. Its destruction can effectively kill tumor cells and inhibit their rapid proliferation.
Fig. 2. DNA Damage and DNA repair (Cancer Genetics. 2021, (252-253): 6-24).
DNA double-strand destroying agents: The chemical structure of DNA double-strand destroying agents contains an enedyne fragment. After entering the nucleus, this fragment undergoes a Bergman cyclization reaction to form a benzene ring diradical transition state, inducing DNA double bond cleavage. The representative of this type of toxin small molecule is calicheamicin γ 1.
DNA inserting agents: During the DNA replication and transcription stages, DNA topoisomerase I plays an important role. It forms a cleavable complex with DNA in the form of a covalent bond, thereby creating a single-stranded nick. The other undamaged single strand rotates back from the gap, relaxing the supercoiled DNA to facilitate replication and transcription. When unwinding is complete, topoisomerase I dissociates and promotes DNA chain recovery. Therefore, if a molecule can be introduced at the stage when topoisomerase I forms a cleavable complex with DNA, which can block the rotation of undamaged single strands, it can prevent DNA replication and transcription and induce tumor cell death. Camptothecin is one such type of topoisomerase I inhibitor compound. Currently, two commercial ADC drugs, Enhertu® and Trodelvy®, use this type of compound as a toxin small molecule. The cell-killing activity of camptothecin compounds is 1 to 2 orders of magnitude weaker than that of tubulin inhibitors, which can expand the therapeutic window of ADC drugs and increase the dosage.
DNA alkylating agents: DNA alkylating agents generally bind to the minor groove of DNA, and then use functional groups in the molecule that are easily reactive with amino groups, such as carbon-nitrogen double bonds or cyclopropane structures, to undergo nucleophilic reactions with guanine or adenine to form chains in DNA. This causes DNA to form inter- or intra-strand cross-links, preventing its replication and transcription, leading to tumor cell death. The cell killing activity of this type of compound is at the picomolar level, which is more active than tubulin inhibitors. Therefore, the dosage of ADC can be significantly reduced, but the therapeutic window will also be reduced accordingly. Commonly used DNA alkylating reagents are pyrrolobenzodiazepine dimer (PBD dimer) and Duocarmycin.
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