Antibody-drug Conjugate (ADC) is a chemotherapeutic drug with strong cytotoxicity, which has both the powerful lethality of small molecule drugs and the high targeting of pure monoclonal antibodies, so it has become a hot issue in the research and development of tumor targeted therapy.
Mechanistically, an ADC acts by binding to the target antigen on the cell surface, followed by its internalization via antigen-mediated endocytosis, trafficking into the lysosome, and the release of the payload through the proteolytic degradation of the antibody moiety and/or cleavage of the linker.
The rationale for developing an ADC is that linking a cytotoxic agent to a tumor-targeting antibody will enable its selective targeting to cancer cells, leading to their eradication while sparing cells in normal tissues.
ADCs are relatively complicated products which require careful assembly. In some cases, preparation of antibody and drug--linker and the final conjugation are done at different sites by specialist companies. ADC manufacturing is a multistep process that can be divided into three distinct stages: cGMP production of the antibody, cGMP synthesis of the drug-linker complex, and conjugation to form the ADC.
DAR is defined as the number of small drug molecules attached to an antibody, which can be obtained by testing methods such as HPLC-MS. A clear DAR value is essential for the later stages of ADC drug development. ADC drugs are taken up by tumor cells in limited quantities during circulation in vivo, so a higher DAR is generally beneficial for increased potency.
Antibody drug conjugates (ADCs) are a form of targeted immunotherapy. They are composed of three components: a monoclonal antibody (mAb) and a cytotoxic payload made from a chemotherapy agent, which are connected together using a chemical linker.
The key of next generation ADC drugs is site-specific binding that can ensure a clear DAR. Through the specific binding of small molecule drugs and monoclonal antibodies, the stability and pharmacokinetics of the drugs are significantly improved, and relative drug activity and binding activity to cells at lower antigen levels are enhanced as well. In addition, antibody optimization, linkers, and small molecule drugs can significantly improve the therapeutic effect of ADC drugs.
After decades of research and troubleshooting , appreciable technological advances and an improved mechanistic understanding of ADC activity has culminated in the FDA approval of 14 ADCs, each providing demonstrable therapeutic benefit to cancer patients.
To design a good ADC drug, choosing the right combinations of antibody backbone, linker chemistry (conjugation technology), and payloads is crucial. Each of these components must be adjusted and optimized to obtain the correct balance between effectiveness and safety. For example, the ideal affinity of an antibody depends on the density of the antigen on the target; bystander activity may or may not be required; the optimal DAR depends on the effectiveness of the payload.
The cytotoxic payload or warhead is an important part of ADC. It is activated after being released from ADC in the cytoplasm of tumor cells and can destroy tumor cells even at low doses. Mechanistically, after the entrance of ADC to cells, cytotoxins are responsible for inducing target cell death. Therefore, the toxicity and physicochemical properties of cytotoxins directly affect the efficacy of ADC in tumor elimination.
Generally, the toxicity of the cytotoxin is required to be strong enough, and the IC50 value is 0.01-0.1nM. In addition, the target of cytotoxin needs to be intracellular and have sufficient water solubility and stability in serum.
The auristatins, such as monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF), are analogs of dolastatin 10, a microtubule-impacting agent. To prepare auristatin conjugates, native disulfide bonds within the antibody are reduced to generate cysteine residues that are subsequently linked to a maleimido derivative of MMAE or MMAF, via a dipeptide linker.
Calicheamicins (CLMs) is a small molecule fat soluble compound, which has relatively high stability. Calicheamicins can induce DNA double-strand breaks. In ADC development, N-acetyl γ-calicheamicin can be linked to lysine residues of antibodies through an acid-labile hydrazone bond or a non-cleavable linker.
Camptothecins (CPTs) are a quinoline alkaloid isolated from camptothecaacuminata decne. As a natural inhibitor of topoisomerase I, Camptothecins have good therapeutic effects on malignant tumors (eg, liver cancer, breast cancer, lung cancer and ovarian cancer), viruses and skin diseases due to the cytotoxic activities.
Doxorubicin is a DNA interchelator that inhibits topoisomerase II thereby inhibiting cancer cell growth. It is a potent anthracycline antibiotic first discovered from the actinobacteria Streptomyces peucetius in the 1960s. It is routinely used in the clinic as a chemotherapeutic agent for the treatment for various cancers including both solid and hematological malignancies.
First isolated from Streptomyces bacteria in the 1970s, the duocarmycins are a class of minor groove-binding, DNA-alkylating natural products that form covalent bonds with the N3 positions of adenine bases and induce apoptosis. Byondis’ trastuzumab duocarmazine, a HER2-targeting ADC currently in phase 3 clinical trials, uses one such derivative, seco-DUBA, as its payload.
As the chemical derivatives of Maytansine, Maytansinoids are tubulin inhibitors with the strongest known activities. It has strong inhibitory activities against various tumor cells and solid tumor systems. Since the greater toxic side effects when directly used in clinical treatment, Maytansinoids are commonly used as potent cytotoxic payloads in antibody-drug conjugates (ADCs).
The piericidin family of microbial metabolites features a 4-pyridinol core linked with a methylated polyketide side chain. Piericidins are exclusively produced by actinomycetes, especially members of the genus Streptomyces. The close structural similarity with coenzyme Q renders the piericidins important NADH-ubiquinone oxidoreductase (complex I) inhibitors in the mitochondrial electron transport chain.
Pyrrolobenzodiazepines (PBDs) are well known naturally occurring DNA interactive antitumour antibiotics like anthramycin, chicamycin and DC-81, which are produced from various Streptomyces bacteria. This natural product was shown to be significantly cytotoxic toward tumour cells in vitro. Meanwhile, PBDs-dimers are DNA alkylators that exhibit extremely potent cytotoxic activities against multiple cancer cell lines. These molecules function by covalently binding to the 2-amino groups of guanine in the minor grooves of cellular DNA, thereby forming DNA adducts that disrupt normal cell functions.
The drug-linker complex is composed of two parts, an active cytotoxin and a specific linker. The cytotoxin is a highly toxic drug that can interact with intracellular targets, and the linker is mainly responsible for the toxicity, stability and specificity of the ADC drug. Drug-linker complexes can be conjugated to ready-to-use antibodies to generate cancer cell targeting agents with high selectivity and cytotoxicity.
Linker is not only the molecular moiety making the covalent connection between the antibody and the small molecule payload, but also a key element in targeted drug therapy having designed properties. Its incorporation should not induce aggregations, ensure acceptable PK properties of the construct while limiting premature release of the payload in plasma (stability) and enabling efficient release of the active molecule at the targeted site of action.
The linker is very important for the stability, toxicity, PK characteristics, and pharmacodynamics of ADC. Each linker has its advantages and disadvantages. When choosing linkers, many factors must be considered, including the existing groups in the monoclonal antibodies and cytotoxic drugs, the reactive groups and derived functional groups. In the end, it is necessary to determine how to optimize the selection of appropriate linkers, targets, and toxic molecules through case by case analysis to balance the effectiveness and toxicity of ADC drugs.
The linker of cleavable linker may be cleavable. Here, a chemical bond (or multiple chemical bonds) between the payload and the antibody attachment site (usually an amino acid) will be cleaved intracellularly. The cleavable linker can be degraded under different pH values or the action of intracellular enzymes to achieve the separation of chemical drugs from antibodies.
The non-cleavable linker maintains the coupling integrity of the antibody and the chemical drug throughout the entire drug action process. The final active metabolite, released by ADC in the lysosome after complete hydrolysis by the protease, contains the payloads and all the constituent elements of the linker still connected to the amino acid residues of the antibody, which is usually lysine or cysteine residue.
The chemically labile linkers, which include acid-cleavable linkers and disulfide linkers, are extensively applied to ADCs due to their ability to undergo fracture, increasing the acidity of the endosomal-lysosomal pathway, as well as the concentration of glutathione inside cells.
Enzyme-cleavable linkers have emerged as a particularly effective ADC linker type due to their ability to selectively release payloads in the lysosomes of target cells. The advantage of using enzyme-cleavable linkers refers to their ability to selectively induce drug release at target cells rather than in circulation.
Peptide linkers are the most widely used enzymatically cleavable linkers. The advantage of peptide linkers is that they not only maintain the ADCs' drug stability in blood circulation but also enable the rapid release of cytotoxins in the tumor site. Moreover, the release of drugs in an active structure without any additional modification maintains drug molecules' physicochemical properties and activity.
Acid-cleavable linkers are stable to alkaline environments but highly sensitive to acidic environments such as hydrazone. Acid-cleavable linkers take advantage of the low pH in the endosome and the lysosome to trigger the hydrolysis of the acid-labile hydrazone linker and subsequently release the payload.
Enzyme cleavable linkers take advantage of the abundance of hydrolytic enzymes with the specificity to recognize the sequences of peptides patterns of carbohydrate in order to degrade peptides and carbohydrates. The different contents of these enzymes between the blood and lysosomal compartment ensure a well-designed ADC undergoes cleavage only in the lysosomal environment. Currently, the commonly used enzymatic linkers for ADC development mainly include the following categories: