Antibody-drug conjugate (ADC) research has been conducted for more than a century since Paul Ehrlich first introduced the concept of the "magic bullet" in 1913 for the use of antibodies to target diseases.
ADC consists of three main components: an antibody responsible for selectively recognizing cancer cell surface antigens capable of internalizing ADC, a drug payload responsible for killing cancer cells upon release within it, and a linker that connects the antibody and payload components. ADC drugs fill the gap between antibody drugs and traditional chemotherapeutic drugs, which improve drug specificity and therapeutic window, enabling targeted release with more controllable safety.
Figure 1. ADC Structure, source: Signal Transduct Target Ther. 2022, 7(1): 93-117
Payload is the key component of ADC to exert cytotoxic effects, which has to meet the following principal requirements: (1) highly cytotoxic, usually with the IC50 value at low nanomolar or picomolar level, (2) well-defined target and action mechanism, (3) (potential) chemical attachment site, (4) well solubility, (5) high stability during circulation, uptake, and release to the target.
There are two main groups of cytotoxic drugs (payload) employed in the ADC field. The first class contains microtubule inhibitors that disrupt microtubule assembly and affect mitosis, such as Auristatin (MMAE, MMA)、maytansinoid derivatives (DM2, DM4), Tubulysins. The second class consists of DNA-damaging drugs, such as PDB、Duocarmycins, Camptothecin.
Table 1. ADC Payload classification
ADCs Based on the Auristatins
In 1987, Pettit et al. first isolated dolastatin 10 from sea hare Dolabella auricularia featured by its ability to inhibit tubulin polymerization, binding at the vinca alkaloid sites in a noncompetitive manner. Monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF) are identified as the best analogs of dolastatin 10, which bind to microtubules and prevent cell proliferation by inhibiting mitosis. Their molecules are highly stable and show no signs of degradation in plasma, liver lysosomal extracts or proteases such as cathepsin B. In some clinical trials in lymphoma, leukemia and solid tumors (e.g. lung, gastric, prostate, and breast cancer), MMAE & MMAF has also shown potent activity.
Figure 2. MMAE & MMAF structure
Both MMAE and MMAF are peptide analogs composed of five amino acids. MMAF differs from MMAE in that it has a charged (carboxyl) phenylalanine at the C-terminus, while MMAE is uncharged. The negatively charged carboxylic acid group hinders the diffusion of MMAF in the cell, making MMAF slightly less toxic than MMAE. The conformational relationships of dolastatin 10 analogs have been extensively studied, mainly focusing on the terminal subunits: P1 (carbon terminus) and P5 (nitrogen terminus), with the most common approach being the introduction of carbamate on P1.
Among the 14 marketed ADC drugs, there are six ADC drugs with payloads of MMAE or MMAF, including Adcetris, Polivy, Padcev, Blenrep, Tivdak, and Aidixi (RC48). Notably, at the beginning of 2022, the FDA granted breakthrough therapy designation to the novel ADC telisotuzumab vedotin based on the excellent data from its phase 2 LUMINOSITY study.
Table 2. Summary of Auristatin-Based ADCs
Adcetris (Brentuximab vedotin), consists of three components: 1) a chimeric IgG1 antibody, cAC10, specific for human CD30; 2) the microtubule inhibitor MMAE; and 3) a protease-cleavable linker that covalently attaches MMAE to cAC10.
Brentuximab vedotin has a approximate molecular weight of 153 kDa, with approximately four MMAE molecules attached to each antibody molecule. Brentuximab vedotin is made by chemical conjugation of an antibody, produced by mammalian (Chinese hamster ovary) cells, and a small molecule component, produced by chemical synthesis.
Figure 3. Structure of Adcetris
Polivy (polatuzumab vedotin), consists of three components: 1) a humanized immunoglobulin G1 (IgG1) monoclonal antibody, specific for human CD79b; 2) the anti mitogenic agent MMAE; and 3) the protease-cleavable linker mc-vc-PAB, which covalently attaches MMAE to the (IgG1) monoclonal antibody.
The molecular weight of polatuzumab vedotin is approximately 150 kDa, with an average of 3.5 MMAE molecules attached to each antibody molecule. It is produced by chemical conjugation of antibodies, which are produced from mammalian (Chinese hamster ovary) cells, and small molecule components, which are produced by chemical synthesis.
Figure 4. Structure of Polivy
Padcev (Enfortumab vedotin) is comprised of a fully human anti-Nectin-4 immunoglobulin G1 kappa monoclonal antibody conjugated to the small molecule microtubule-disrupting agent monomethyl auristatin E (MMAE), via a protease-cleavable maleimidocaproyl valine-citrulline (vc) linker. The AGS-22C3 antibody (mAb) intermediate is a fully human IgG1, kappa subclass monoclonal antibody selectively binding to the nectin-4 extracellular domain on the surface of target cells.
Enfortumab vedotin has a approximate molecular weight of 152 kDa, with an average of four MMAE molecules attached to each antibody molecule.
Figure 5. Structure of Padcev
Blenrep (Belantamab mafodotin) consists of 3 components: 1) glycosylated humanized immunoglobulin G1 monoclonal antibody covalently linked to 2) microtubule inhibitor MMAF via 3) an anti-protease maleimide propyl linker. The antibody was produced in mammals (Chinese hamster ovaries) using recombinant DNA technology, and the microtubule inhibitor and linker were chemically synthesized. Approximately 4 mafodotin molecules were attached to each antibody molecule.
Figure 6. Structure of Blenrep
Disitamab vedotin (RC48) consists of three components: 1) anti-human epidermal growth factor receptor 2 extracellular region (HER2 ECD) antibody; 2) linker (MC-Val-Cit-PAB,Linker ); and 3) cytotoxic monomethyl Auristatin E (MMAE). The antibody ratio (DAR) value is about 4, i.e. about 4 MMAE molecules are attached to each antibody molecule.
Figure 7. Structure of Disitamab vedotin
Tivdak (Tisotumab vedotin) consists of 3 components: 1) human anti-TFIgG1-kappa antibody conjugated via 2) protease-cleavable vc (valine-citrulline) linker to 3) microtubule disruptor MMAE.
Monoclonal antibodies were produced in mammalian (Chinese hamster ovaries). Each monoclonal antibody molecule carries an average of 4 MMAE molecules.
Figure 8. Structure of Tivdak
 Cheng-Sánchez, I.; Moya-Utrera, F.; Porras-Alcalá, C.; López-Romero, J.M.; Sarabia, F. Antibody-Drug Conjugates Containing Payloads from Marine Origin. Mar. Drugs 2022, 20, 494. https://doi.org/10.3390/ md20080494
 Akaiwa M, Dugal-Tessier J, Mendelsohn BA. Antibody-Drug Conjugate Payloads; Study of Auristatin Derivatives. Chem Pharm Bull (Tokyo). 2020;68(3):201-211. doi:10.1248/cpb.c19-00853