ADC payloads are critical components of the ADC structure, and their selection and design are crucial for achieving optimal therapeutic efficacy and minimizing toxicity.

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A Comprehensive Guide to ADC Payload
Classes
Antibody-drug conjugates (ADCs) are a class of targeted cancer therapies that
combine the specificity of monoclonal antibodies (mAbs) with the cytotoxic
activity of chemotherapeutic drugs. ADCs use mAbs to selectively deliver a
cytotoxic payload to cancer cells, resulting in targeted killing of tumor cells
while sparing normal cells.
Figure 1. The general components of ADC. Source: reference [3]
In 2000, the first ADC drug, Mylotarg, was marketed, and no other ADC drugs
were marketed for the next decade. As ADC drug technology matured, the
FDA approved three ADC drugs in the next seven or eight years, and even
three ADC drugs were approved consecutively in one year in 2019. To
date, 15 drugs have been approved and hundreds of clinical trials are
underway to explore new targets and indications.

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Figure 2. Approved ADCs
These approved drug drugs drive a new round of growth of ADC, with the
market size gradually expanding. In 2021, the ADC market size is about $5.4
billion, and in 2022, according to incomplete statistics (some companies'
annual reports are not published), the market size has exceeded $7.6 billion.
ADC payloads are critical components of the ADC structure, and their
selection and design are crucial for achieving optimal therapeutic
efficacy and minimizing toxicity.
Payload Selection for ADC
Payloads are the components that exert the tumor-killing effect. After the ADC
drug enters the cell, the payload is the main agent that ultimately causes the
death of the target cell; therefore, the toxicity and physicochemical properties
of the payload directly affect the ability of the drug to kill the tumor and
consequently impact the efficacy. Payloads for conjugation must have a clear
mechanism of action, small molecular weight, high cytotoxicity, and retain
antitumor activity after chemical conjugation to antibodies.

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▶ Higher cytotoxicity. Considering the poor penetration and endocytosis
efficiency of the antibody, as well as the low antigen expression on the cell
surface, the amount of payload that can ultimately be delivered to the target
cells is limited. Assuming an efficiency of 50% for each step in the ADC
mechanism of action, only 1.56% of the toxin is able to enter the cell and exert
its effect, and the actual figure in humans is even lower. Therefore, to ensure
the drug effeciency, the toxin selected for ADC needs to have a high enough
toxic potency to effectively kill tumors.
▶ Smaller molecular weight. An increase in the overall molecular weight of
ADC may lead to the aggregation of ADC drugs, causing them to be cleared
faster. Therefore, the molecular weight of the toxin should be controlled in a
reasonable range. In addition, the smaller molecular weight allows the toxin to
diffuse through the cell membrane to neighboring cells, exerting a bystander
effect and further increasing the tumor elimination effect.
▶ Explicit mechanism of action. Since ADC drugs exert their effects by
being internalized by the target cells, the toxin is mainly released intracellularly,
which requires targeting intracellular targets and inducing cancer cell death
through apoptotic mechanisms, so the mechanism of action needs to be
clarified.
ADC Payload Classes
The payloads currently used for ADCs fall into the following three major
categories: tubulin inhibitor (e.g., maytansine analogs and auristatin
analogs), DNA damaging agent (e.g., calicheamicin and
pyrrolobenzodiazepine analogs) and transcription inhibitors.
Figure 3. Summary diagram of the different classes of cytotoxic molecules
used in ADC construction. Source: reference [1]

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Tubulin Inhibitors as Payloads of ADCs
Microtubules exist in all eukaryotic cells and are one of the critical components
that make up the cytoskeleton. Microtubules play a crucial role in supporting
cell structure maintenance, cell division, and intracellular transport. Disruption
of microtubules induces cell cycle arrest in the G2/M phase, which makes
microtubules an attractive target for drug discovery. Tubulin inhibitors can be
classified into two major categories according to their mechanisms of
action: agents promoting tubulin polymerization (e.g., paclitaxel,
epothilones, discodermolide and taccalonolides) or causing tubulin
depolymerization (such as maytansinoids, auristatins, vinblastine and
vincristine).
Figure 4. Structure, polymerization and depolymerization of microtubules.
Source: reference [3]
Auristatins
The auristatins originate from dolastatin-10, a natural compound found in the
sea hare Dolabella auricularia. In order to produce effective cytotoxic payloads
for antibody-drug conjugates (ADCs), monomethyl auristatin-E (MMAE) and
monomethyl auristatin-F (MMAF) were developed based on the structure of
auristatin. An example of an ADC utilizing MMAE is Brentuximab vedotin
(Adcetris®), which contains approximately 4 MMAE molecules conjugated
through cysteines of reduced interchain disulfide bonds via a

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protease-cleavable linker. Brentuximab vedotin was granted accelerated
approval in 2011 and full approval in 2015 for the treatment of classical
Hodgkin’s lymphoma, systemic anaplastic large cell lymphoma, and peripheral
T-cell lymphoma.
Figure 5. Structure of Brentuximab vedotin, Source: reference [2]
Maytansinoids
Maytansinoids are anti-mitotic tubulin inhibitors, which are derived from
maytansine. This benzoansamacrolide was initially isolated from an alcoholic
extract in the bark of African shrubs Maytenus serrata and Maytenus
buchananii in 1972. Maytansine and maytansinoids attach to the maytansine
site, leading to the suppression of microtubule dynamics and causing cell cycle
arrest in the G2/M phase. Through a semi-synthesis approach, a series of
maytansine analogs (DM1, DM3, and DM4) containing disulfide or thiol groups
that enable covalent linkage with monoclonal antibodies (mAbs) were created
in two steps. Ado-trastuzumab emtansine (Kadcyla®, T-DM1) is a conjugate of
approximately 3.5 maytansinoid DM1 molecules attached to the anti-HER2
antibody trastuzumab through surface-exposed lysines. Trastuzumab
emtansine received FDA approval in 2013 for the treatment of HER-2
positive metastatic breast cancer (mBC), with additional approved uses
including monotherapy and combination administration, as well as an adjuvant
treatment for early breast cancer.
Figure 6. Structure of trastuzumab emtansine, Source: reference [2]

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The most widely used tubulin inhibitors are the aforementioned auristatins and
maytansinoids, but other kinds of tubulin inhibitors have been tried in ADC,
including derivatives of paclitaxel (Taxol), vincristine, and colchicine. Recently,
Eribulin has also been used as a toxin molecule in the ADC drug MORAb-202,
and has entered the clinical phase.
DNA Damaging Agents as Payloads of ADCs
DNA damaging agents may be more effective than microtubule inhibitors,
which can kill target cells at any stage of their life cycle. There are at least four
mechanisms of action exerted by DNA-damaging agents, which are as follows:
(a) DNA double-strand breakage, (b) DNA alkylation, (c) DNA intercalation,
and (d) DNA cross-linking. The most used DNA-damaging payloads are
pyrrolobenzodiazepine, duocarmycins, doxorubicin, and calicheamicins
Gemtuzumab ozogamicin (Mylotarg®), with calicheamicin derivatives as
payload, was the first ADC approved by the FDA and was effective in treating
acute myeloid leukemia, but it was withdrawn from the market in 2010 due to
unstable linker toxicity. In 2017, Myelotarg was once again approved by the
FDA.
Among the DNA damaging agents, camptothecin (CPT) is of comparative
interest. Unlike other DNA damaging agents, CPT inhibits DNA replication and
transcription by acting on DNA topoisomerase Ӏ leading to tumor cell death.
CPT has strong in vitro antitumor activity, but poor water solubility and low
bioavailability have limited clinical application.
In contrast, 7-ethyl-10-hydroxycamptothecin (SN-38), the active metabolite of
CPT derivative irinotecan (CPT-11), is widely used in ADC drugs due to its
high bioavailability and antitumor activity, such as Sacituzumab govitecan and
Labetuzumab govitecan both use SN-38 as payload. More notably, Exatecan
methanesulfonate (DXd/Dx-8951f) has stronger topoisomerase Ӏ inhibition
activity and antitumor activity compared with other CPT-based derivatives, and
has been successfully used in the development of next-generation ADC drugs
such as T-DXd and Dato-DXd.

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Figure 7. Structure of Sacituzumab govitecan and Trastuzumab Deruxtecan,
source: Reference [2]
Transcription inhibitors as Payloads of ADCs
The final class of promising payloads are the transcription inhibitors targeting
RNA polymerase II. Example of these compounds are the amatoxins. To date,
α-amatoxins are the most potent and specific inhibitors of RNA polymerase II,
leading to apoptosis. The development of amatoxins as ADC drug "warheads"
has the following characteristics: first, high solubility in aqueous media to
facilitate conjugation reactions; second, less potential for aggregation of
amatoxin-based ADC drugs; and third, fewer side effects and rapid excretion
from urine. Based on these advantages, amatoxin is a very promising ADC
payload.
Conclusion
Payloads are an important part of ADCs, and payload diversification will play a
key role in the future development of ADCs.
A key issue in the development of these novel payloads is the mitigation of
their side effects. Currently approved ADCs have been shown to have
expected (myelosuppression, neurotoxicity) or unexpected (e.g., ocular or
pulmonary) toxicity. Therefore, obtaining satisfactory therapeutic indices will
be key to the future development of innovative ADC payloads.

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Huateng Pharma is dedicated to being your most reliable partner to provide
chemical synthesis and high-quality PEG linkers for ADC drugs. We are
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References:
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Conjugates: The New Frontier of Chemotherapy. Int J Mol Sci. 2020 Jul
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PMC7432430.
[2]Twomey JD, Zhang B. Targeting cancer with antibody-drug conjugates:
Promises and challenges [published correction appears in MAbs. 2021
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doi:10.1080/19420862.2021.1951427
[3] Chen H, Lin Z, Arnst KE, Miller DD, Li W. Tubulin Inhibitor-Based
Antibody-Drug Conjugates for Cancer Therapy. Molecules. 2017 Aug
1;22(8):1281. doi: 10.3390/molecules22081281. PMID: 28763044; PMCID:
PMC6152078.
[4] Fu Y, Ho M. DNA damaging agent-based antibody-drug conjugates for
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Epub 2018 Aug 30. PMID: 30294716; PMCID: PMC6161754.
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