Payload is the key component of ADC to exert cytotoxic effects, MMAE and MMAF are the most commonly used ADC payloads.

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Antibody–Drug Conjugate Payloads: MMAE &
MMAF
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.

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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.

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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
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
Polivy (polatuzumab vedotin), consists of three components: 1) a humanized
immunoglobulin G1 (IgG1) monoclonal antibody, specific for human CD79b; 2) the anti

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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
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

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Blenrep
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
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

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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
References:
[1] 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
[2] 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
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