An overview to the third generation of antibody-drug conjugates

Antibody-drug conjugates (ADCs) can be defined as prodrugs, which recognize targets that express tumor antigens and that are coupled to cytotoxins by “linkers” to form targeted delivery system for tumor cells. Under ideal conditions, the prodrug is not toxic when it is administered systemically, and when the antibody in the ADC drug binds to a target cell expressing a tumor antigen, and the entire ADC drug is endocytosed by the tumor cell, the small molecule cytotoxin component will be released in a sufficient amount and a highly active form to kill the tumor cells.

Therefore, an ideal ADC needs a perfect combination of four parts:

1. Selection of antibodies(targets): for tumor-specific (related) antigens, higher expression levels, good endocytic efficiency, good antigen affinity, no immunogenicity, drug accessibility;
2. Selection of linkers: stable in the blood circulation, rapid disintegration in the cells, and efficient release of poisons;
3. Coupling method: site-directed coupling, a uniform antibody conjugate;
4. Cytotoxic agents: generally, the toxicity of the agent is required to be strong enough to achieve IC50 <1 nmol for single use; sufficient water solubility and stability in serum; the target of the toxin is located in the cell.

The third-generation of antibody — drug conjugates (ADCs)

A large number of methods have been developed by researchers to conjugate cytotoxic drugs with defined sites and stoichiometric antibodies. Here we listed the main ways as follows:

1. Thiomab technology: Thiomab technology was first reported by Genentech’s Junutula et al., using genetic engineering techniques to insert cysteine ​​residues at specific positions in the light chain V110C and heavy chain A114C of trastuzumab, and then a site-specific antibody-drug conjugate was synthesized by coupling a thiol group on cysteine ​​with monomethyl auristatin E (MMAE), with a Drug-to-Antibody ratio (DAR) of 2 up to 92.1% (shows as the figure 1).

Figure 1. Thiomab technology

1. ThioBridge technology: the disulfide bond of the mAb is reduced, and the dibromo (or disulfonate) reagent is reacted with the reduced interchain disulfide to provide a re-bridged mAb, and the DAR is mainly 4.

Figure 2. ThioBridge technology

1. Introduction of the non-natural amino acid method: This method uses an evolved tyrosyl-t RNA/aminoacyl-t RNA synthetase that specifically recognizes unnatural amino acids, transfected by Chinese hamster ovary cells (CHO) instead of the 21st amino acid of the amber codon, the resulting cells can be used to synthesize various antibodies with a gene-decoding para-acetylphenylalanine residue, and then the hydroxylamine is deuterated. The main DAR is 2.

Figure 3. Introduction of the non-natural amino acid

1. Enzymatic catalysis: for example, sortase A (Srt A), an enzyme with membrane-bound thiol transpeptidase catalyzing function, recognizes the main sequence of protein LPETG, lyses the peptide bond of threonine and glycine, forms a stable intermediate, and attaches the thiol group in Srt A to the threonine carboxyl group via a thioester bond.

Figure 4. Enzymatic catalysis

In my opinion, ADCs are actually designed to improve chemotherapy drugs. Because traditional chemotherapeutic drugs do not have the ability to recognize tumor specifically, while the targeted delivery of cytotoxic drugs to cancer cells by antibodies increases the percentage of drug molecules that reach the tumor, thereby reducing the minimum effective dose of the chemotherapeutic drug and increasing the maximum tolerated dose. However, the first and second generation of ADCs still have off-target toxicity and heterogeneous antibody conjugates due to linkers and ligation methods. Nowadays, scientists have developed a variety of site-directed coupling methods, linkers, and cytotoxic agents. And the therapeutic applications of ADCs have been greatly improved.