1. Antibody selection

Antibodies are the basis for targeted delivery of ADCs, so the selection of antibodies is one of the keys to ADCs. For the screening of antibodies, the following aspects are mainly considered:

(1) The antigen recognized by the antibody is a tumor-specific antigen, which is highly expressed on the surface of tumor cells and is not expressed or rarely expressed in normal cells, which is an important condition for specific recognition. (2) The antigen recognized by the antibody must be expressed on the surface of the tumor cell, not inside. (3) After antibody and antigen recognition, it can be rapidly endocytosized, and cannot endocytose is not conducive to the design of ADC drugs (except ADCs that target the extracellular environment of tumors). (4) After the antibody is modified by small molecules and linkers, it does not affect the recognition activity of the antibody

2. Connection sub-selection

One of the biggest challenges in ADC development is providing appropriate linkers for antibody and drug conjugation. Linkers are critical to the preclinical, clinical efficacy, and safety of ADC drugs. The ideal linker must be sufficiently stable in the circulatory system, as early release of small molecule toxins in the circulatory system can produce untargeted and unintended toxicity. After the ADC drug is internalized and transported to a specific intracellular body, the linker must be able to efficiently release cytotoxins in a highly active form in the cytoplasm of the target cell. There are a number of important considerations to consider when designing linkers, including the junction site and internalization of the antibody, the nature of the toxin, and the polarity of the linker. ADC linkers currently on the market and in clinical trials are divided into two main categories: non-cleavage linkers and cleavage linkers.

2.1 Non-cleavage linkers

Non-lythic linkers are highly stable in blood circulation, and the effective release of cytotoxins is mainly through the degradation of antibodies in lysosomes to release cytotoxins with charged amino acids to play a tumor killing effect. Therefore, when designing ADC molecules using non-cleavage linkers, it should be required that the metabolites of charged amino acids formed by cytotoxins are also highly toxic. For example, Kadcyla uses non-cleavage linkers, and its Lys-MCC-DM1 released after antibody degradation in lysosomes has strong antitumor activity.

However, ADCs with non-cleavage linkers also have some disadvantages, which tend to be more dependent on the biological characteristics of the target cell and require a good internalization process to be activated by degradation within the cell. In addition, because the complex composed of amino acid residues, linkers and small molecule cytotoxins has good hydrophilicity, the membrane permeability will be greatly reduced, and therefore the bystander effect will also be reduced.

2.2 Lyzable linkers

At present, most ADC drugs on the market and under development use lyptic linkers, which use certain characteristics of the tumor microenvironment that are different from normal cells to release cytotoxins to kill tumors, requiring high stability in blood circulation and rapid release of cytotoxins at the target site. Mainstream cleavage linkers include acid-labile hydrazone linkers, glutathione-sensitive linkers, and protease-hydrolyzed linkers.

2.2.1 Acid-labile hydrazon linkers

Acid-unstable hydrazone linkers were the first class of linkers to be used to build ADCs. This type of linker remains stable in the neutral pH environment of blood circulation (pH=7.3-7.5), and when the ADC is endocytosed by tumor cells into the acidic environment of endosomes (pH=5.0-6.5) and lysosomes (pH=4.5-5.0), hydrolysis reaction can occur immediately to release cytotoxins, and Mylotarg and Besponsa, which have been approved by the FDA for marketing, are ADCs of this type of linker. Although several clinical studies have shown that this pH-dependent hydrazon linker has the disadvantage of non-specific drug release and can produce obvious toxic side effects, this technique is still in use, and in addition, the stability and in vivo half-life of hydrazone linker can also be increased by chemical modification to minimize the damage of the drug in the circulatory system, and obtain a more effective therapeutic effect while reducing side effects.

2.2.2 Glutathione-sensitive linker

Disulfide bonds in disulfide linkers can be cleavaged by intracytosolic sulfhydryl cofactors such as glutathione (GSH) and cysteine (Cys). Generally, the concentration of reduced GSH in cells is high, between 0.5-10 mM, especially in tumor tissues, the hypoxic environment caused by its irregular vascular distribution can increase the activity of reductase, thereby further increasing the concentration of GSH. In contrast, in the human blood circulation, the concentration of GSH and Cys is very low, about 5uM. This thousand-fold concentration difference allows the ADC to achieve a selective release process, maintain a relatively stable presence in the human blood circulation, and release cytotoxins immediately after being internalized into the strong reducing environment of tumor cells. However, disulfide linkers are unstable in electrophilic and nucleophilic polar solvents, and increasing their steric hindrance can partially improve the stability in blood circulation, but such linkers are rarely used in ADC drugs and are gradually withdrawing from the stage of ADC drugs.

2.2.3 Protease hydrolysis of ligators

The more common enzymatic hydrolytic linkers include cathepsin-cleaved linkers and β-glucuronidase-based linkers, among which cathepsin-cleaved linkers have been widely used in many marketed ADC drugs.

Cathepsin B is a Cys protease that is typically highly expressed in lysosomes in mammalian cells, and at the same time, this protease has also been implicated in tumor progression and can be overexpressed in a variety of tumor cells. Cathepsin B can be digested by a specific sequence of dipeptides, providing an opportunity for dipeptide linkers to be selectively cleaved within the cell.

In the study of early linkers, it was found that if the dipeptide fragment was directly connected to the cytotoxin, the toxin drug form released after enzymatic cleavage often contained adducts of amino acids, which reduced the toxicity of some cytotoxins and could not exert the original killing effect. Therefore, a self-excisioning spacer such as PAB is introduced to separate the enzymatic cleavage site of the drug and the dipeptide linker to avoid the adverse effects of the above phenomenon. The amino terminal of the PAB can be linked to the peptide to form an amide bond, and the benzyl end can be attached to the amine in the toxin molecule via a p-aminobenzyloxycarbonyl group (PABC). Once the ADC is internalized into lysosomes and cleaved by cathepsin B, a 1,6-elimination reaction occurs immediately after the PABC, releasing the residual structure of unmodified cytotoxins, carbon dioxide, and linkers. The dipeptide linkers valine-citrullinated-PABC (Val-Cit-PABC) and valine-alanine-PABC (Val-Ala-PABC) developed using this principle, especially Val-Cit-PABC, are now widely used in the coupling of ADC drugs, and 14 of the 2022 ADC drugs (excluding Blenrep, which was delisted in 7), use this linker.

Schematic diagram of Rongchang bio's vedicitumab

β-Glucuronidase is an acid hydrolase of the lysosomal glycosidase class that catalyzes the decomposition of β-glucuronic acid residues in polysaccharides. The enzyme, like cathepsin, has very low activity outside the cell, and is abundant in lysosomes, and overexpressed in some tumor types, the β-glucuronic acid linker developed for the enzyme, its release method is similar to the dipeptide linker, after β-glucuroniderase digestion in lysosomes, the spacer PABC will immediately undergo 1,6-elimination to complete the release of small molecule cytotoxins.

3. Cytotoxin selection

At present, the more mature cytotoxins in marketing and clinical application include: (1) microtubule inhibitors, such as Auristadin cytotoxin MMAE and MMAF, and 6 ADC drugs such as Adcetris and Polivy that have been marketed use such cytotoxins; Medensulin cytotoxins DM1, DM4, etc., such as the marketed Kadcyla. (2) DNA alkylating agents, such as PBD dimer; (3) DNA damaging agents, such as kachimycin, have been successfully applied to the marketed drugs Mylotarg and Besponsa. (4) Topoisomerase inhibitors, such as camptothecin derivatives SN38, DXd, etc.

In addition to the above traditional cytotoxins, Akalux, which was launched in September 2020, uses the dye IR9 as a carrier, coupled to the antibody to form an antibody-IR700 as a complex, the complex binds to tumor cells in large quantities, under near-infrared irradiation, IR700 produces chemical changes, absorbs light energy and heats up, causing tumor cells to rapidly expand, destroy and necrosis, and the substances inside tumor cells are released outside the cell and are quickly eliminated by the neighboring immune system. In addition, immunostimulants (TLR700/7, STING), radiation agents (Thorium-8), etc. have also emerged in clinical applications, diversifying antitumor toxins.