Antibody-drug conjugate (ADC) consists of antibodies, linkers and small molecule toxins. While having the advantages of high targeting and high cytotoxicity, the diversity and complexity of ADC structure and the low content of small molecule toxins released in the circulatory system have brought many challenges to its pharmacokinetic research. In ADC molecules, the selection of targets and antibodies is the starting point of ADC drug design and the decisive factor of drug indications. The selected target antigens should usually be expressed at a high level associated with tumors or diseases. The linker should be stable enough during the circulation of ADC in vivo, and ADC drugs should be able to rapidly release small molecule toxins in an efficient and active form after entering the target cells. Small molecule toxins should have an effective killing effect on tumor cells. Targets, antibodies, linkers and other factors will affect the efficacy and safety of ADC drugs, which will be discussed below.

Target Antigens and Antibodies

The selection of targets and antibodies is the starting point of ADC drug design and the decisive factor of drug indications. The selected target antigens should usually be expressed at a high level associated with tumors or diseases. Ideally, the selected antigen should be highly homogeneously expressed on the surface of the target cell, but not or rarely expressed on the surface of normal tissues or cells. Antigen should be non-secretory. The secreted antigens can bind to ADC drugs or naked antibodies in the circulatory system of the body, resulting in less binding of ADC drugs to tumor cells, affecting the efficacy and safety of drugs. After the ADC drug binds to the antigen, it is necessary to have a suitable endocytosis pathway and a certain endocytosis rate to release small molecule toxins through enzymatic degradation in the cell.

Fig. 1. Antibody-drug conjugate (ADC) assembly and interaction with target cells (Clin Pharmacokinet. 2018, 57(6): 687-703).

At present, the lack of efficacy and off-target toxicity are the main challenges for ADC drugs. One of the important reasons is the low expression level and limited internalization rate of target antigen. Researchers are currently developing methods to solve the problems of low antigen expression and low internalization rate, such as using anti-tumor angiogenesis antibodies or bispecific antibodies to design non-internalized ADC drugs:

• Anti-angiogenic antibodies are used to avoid the internalization process, but off-target effects may occur and affect normal angiogenesis. It is necessary to carefully select the target antigen and corresponding antibody;
• The bispecific antibody is used to target two non-overlapping epitopes of the antigen to enhance the affinity between the antibody and the antigen.

In the ADC drugs designed by ROSSIN et al., double antibodies lacking the Fc region are used to target the antigen, and additional chemical activators are used to lyse the junctions outside the tumor cells, thereby releasing free small molecule drugs and penetrating into the tumor cells. This method avoids the lack of internalization caused by tumor cell interstitial pressure and epithelial barrier, thereby improving anti-tumor activity. Bispecific antibodies can also selectively bind to two different antigens on tumor cells, thereby reducing off-target toxicity.

Studies have shown that some ADC drugs can use the physical and chemical properties of the adaptor and the tumor microenvironment to release free small molecule toxins to kill adjacent antigen-negative tumor cells. This process is called bystander killing effect. After internalization, some ADC drugs can metabolize and release uncharged cytotoxic metabolites that can penetrate cell membranes and kill adjacent antigen-negative cancer cells. The bystander killing effect is important for tumor cells with heterogeneous antigen expression.

It should be noted that even for the same target, different tumor types can affect the therapeutic effect of ADC. That is to say, the same ADC drug may show different PK characteristics and effective safety in patients with different indications. For example, Besponsa is an antibody-drug conjugate that targets CD22-targeted Inotuzumab and enediyne toxin Ozogamicin. It was approved by the FDA in 2017 for the treatment of adult relapsed refractory B-cell acute lymphoblastic leukemia. However, Besponsa is used in relapsed and refractory non-Hodgkin 's patients. Phase III clinical trial of lymphoma was terminated due to poor efficacy.

Small Molecule Drugs

Factors such as limited tumor penetration ability of antibodies, low antigen expression, and limited endocytosis efficiency can cause low concentrations of small molecule toxins in cells, so small molecule toxins need to have high cytotoxicity.

In general, the target of small molecule toxins of ADC drugs is located in the cell. If ADC drugs cannot be transported to the cell, it will affect the effectiveness and safety of the drug, and may be toxic to normal cells outside the cell or after dissociation. In addition, it is necessary to consider the influence of small molecules on the overall properties of ADC drugs, such as the endocytosis efficiency of ADC drugs, the polarity and immunogenicity of ADC drugs. At the same time, small molecule toxins usually need to have appropriate solubility in aqueous buffer solution to facilitate coupling with antibodies, and the coupled small molecule toxins should have certain stability. At present, the main small molecule toxins used are maytansine, auristatin, anthracyclines and camptothecin analogs.

Our Featured ADC Toxins

BOC Sciences is a leading provider of ADC cytotoxins. We are committed to providing a wide range of high-purity cytotoxic payloads such as maytansinoid alkaloids, auristatins, duocarmycins, calicheamicins and pyrrolobenzodiazepines. These cytotoxic agents are conjugated to monoclonal antibodies via specialized linkers to form ADCs that specifically target cancer cells expressing specific antigens.

ADC Linker

The selected linker needs to be stable in plasma to avoid the early release of small molecule toxins to damage normal tissues or cells. When ADC drugs are endocytosed into target cells, the selected linker needs to be able to quickly release effective active ingredients. In addition, the influence of the molecular weight and polarity of the selected linkers on the overall properties of ADC drugs should also be considered.

The linker can be divided into cleavable and non-cleavable types. The cleavable linkers can use the difference between the tumor microenvironment and the normal physiological environment to release small molecule toxins that may penetrate the membrane and produce bystander effects. The non-cleavage type of linker usually breaks the connection between the antibody and the linker after the antigen-antibody complex enters the intracellular lysosome. The two types of linkers have their own advantages and disadvantages:

• The non-cleavage linker is more stable than the cleavable linker, which can reduce off-target toxicity and improve multi-drug resistance (MDR);
• The passive diffusion of metabolites produced by cleavable linker is more likely to enter the cell to produce bystander killing effect, which is of great significance for tumors with heterogeneous target antigen expression, but it is more likely to be off-target than non-cleavage linkers.

Compared with the cleavable linker, the non-cleavable linker is more stringent in antigen selection. Kadcyla is a non-cleavage thioether linker connected to the meydenine derivative. Because ADC produces ionized metabolites in intracellular metabolism, poor permeability, and less impact on surrounding normal cells, Kadcyla shows acceptable safety. The properties of the linker will have a great influence on the metabolic pathway of the drug in the body, and have an important influence on the design of ADC drugs.

Our Featured ADC Linkers

BOC Sciences offers a variety of linker technologies, including cleavable linkers (such as hydrazone linkers, disulfide linkers, and peptide linkers) and non-cleavable linkers (such as maleimides and thioethers), to meet different therapeutic needs. One of the key advantages of our ADC linker products is their high quality and purity, ensuring the safety and efficacy of ADC formulations. Our products undergo strict quality control measures to meet the highest industry standards and regulatory requirements. BOC Sciences also offers custom synthesis services to develop novel ADC components suitable for specific research or clinical applications.

Attachment Sites and Drug-to-Antibody Ratios (DAR)

The efficacy of ADC drugs mainly depends on the concentration of small molecule toxins in tumor cells, so the drug-to-antibody ratio (DAR) is an important factor affecting the efficacy of ADC drugs. At present, many studies are devoted to improving the DAR of ADC drugs in order to increase the concentration of drugs in tumor cells. However, studies have found that the higher the DAR, the better the efficacy, which may be related to factors such as the polarity of small molecule toxins. From a safety point of view, the higher the DAR, the greater the toxicity to normal tissues. In the study of Zhang et al., the activity of ADC drugs did not increase further when DAR increased to a certain extent. Choosing the appropriate DAR is of great significance for the effective concentration in tumor cells.

The connection site is related to the uniformity of the ADC drug and is also one of the important considerations in the design of DAR molecules. The cysteine (8) and lysine (80) residues on the antibody are easier to undergo chemical reactions and be modified, so they are often used as binding sites for effector molecules. In early ADC development studies, lysine on the antibody was usually selected as the binding site, because there can be up to 80 lysine residues on each antibody, resulting in great heterogeneity. There are only 8 free cysteines on each antibody that can be connected to the linker through disulfide bonds. Using cysteine as a connection site helps reduce the heterogeneity of ADC. Junutula et al. reported a new type of Thiomab-drug conjugates (TDC), which use engineered site-specific cysteine to have a clearer DAR and less heterogeneity.