Click chemistry involves reactions that form carbon-heteroatom bonds with high selectivity and yield. It can be performed in living systems without interfering with biological processes. Click chemistry is being used in developing targeted drugs like antibody-drug conjugates (ADCs). Some companies are applying click chemistry to develop ADCs that target non-internalizing receptors by using a linker that can only be cleaved through a click reaction activated by an external agent, allowing drug release in the tumor microenvironment. This overcomes limitations of traditional ADCs that require internalization. Clinical trials show click chemistry-activated ADCs and prodrugs can significantly improve efficacy with minimal toxicity.

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"Click Chemistry" Holds Promise For
Non-internalized ADC Applications
What is "Click Chemistry"?
The term "click chemistry" was first coined by Professor K. B. Sharpless of the
Scripps Research Institute (2001 Nobel Laureate in Chemistry). It is a class of
reactions involving the formation of carbon-heteroatom bonds, which are
characterized by high yield and selectivity.
Bioorthogonal click chemistry can be performed in living systems without
interfering with normal life activities. Over the past two decades, bioinorganic
and medicinal chemists have increasingly used this chemical approach,
especially in the development of targeted drugs, such as antibody-drug
conjugates (ADCs).
What are the characteristics of "click chemistry"?
1. The chemical yield of the target product is very high.
2. It is a combination of simple and easily available structural fragments.
3. Almost no by-products are generated.
4. Product separation process is simple, no need to use chromatography for
separation.
5. The reaction can be carried out in water and organic solvents.
Examples of early applications of "click chemistry"
Click chemistry was first used for tracing, because some in vitro methods of
labelling reinjection are not fully effective and may also cause rejection.
In the figure below, if we want to study the distribution and changes of SiaNAz
(a sugar on the surface of cells) in vivo, we need to trace it. The operation of
click chemistry is as follows: Ac4ManNAz can be converted into SiaNAz in
vivo. The researchers added an N3 group (azide group) to the former, and
then injected AC4MannaZ-N3 into mice. A few days later, the mice were
injected with a FLAG-labeled molecule, which carried a group that could react
with N3 group (alkynyl group). When the molecule met SiaNAz in the process
of metabolism in vivo, it will "click" like the buckle of a safety belt, and the
FLAG will be carried to our sugar molecules by chemical reactions, thus
achieving the effect of tracing (and because N3 is so small, it won't affect the
bioconversion process of sugar molecules).

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Copper-free click chemistry in living animals, PNAS, 2010, 5, 1821-1826.
Source: Carolyn R. Bertozzi et al.
Application of click chemistry in ADC field
The application of click chemistry in the field of ADC is still in the clinical
stage, and there are not many research and development companies. It
belongs to an emerging field, but according to the current efficacy, it is very
worth exploring.
Biotech companies that use bioorthogonal click chemistry to prepare ADCs
Source: Nat Biotechnol. 2019 Aug;37(8):835-837.
1. Cleavage of non-internalized ADCs by click chemistry

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Tagworks Pharmaceuticals is developing a bioorthogonal click chemistry
strategy to overcome some of the shortcomings of ADCs in targeting solid
tumors. Traditional antibody-drug conjugates (ADCs) target internalized
receptors on cancer cells, resulting in drug release by intracellular cleavage of
linkers. However, only a subset of patients with solid tumors have adequate
expression of specific internalizing receptors.
Based on this, Tagworks Pharmaceuticals has developed
a Click-to-Release approach to make non-internalized targets applicable to
ADC therapeutics: ADC targets non-internalized receptors, which contain a
connector that can only be destroyed by bioorthogonal chemical click reaction.
When the ADC is enriched around tumor cells, systemic administration of
an activator reacts with the ADC linker outside the tumor cell, resulting in drug
release in the tumor microenvironment and subsequent uptake into
surrounding cancer cells and tumor-supporting stromal cells for action.
ADCs cleaved by click chemistry have expanded their use, enabling
pervasive and direct temporal control of drug release in the tumor
microenvironment. This would allow for more uniform drug delivery, potentially
improving the therapeutic effect of heterogeneous or poorly penetrating
tumors.
Tagworks Pharmaceuticals uses click chemistry to cleave non-internalized
ADCs
2. Tagworks: A Chemically-cleavable ADC for TAG72 Targets
Tumor-associated glycoprotein 72 (TAG72) is a non-internalized target that
is widely expressed in a range of human adenocarcinomas of epithelial origin,
such as breast, colorectal, gastric, lung, pancreatic, prostate, and ovarian
cancers. Tagworks is developing click-cleavable anti-TAG72 ADCs based on
antibody fragment types, conjugated to the anti-mitotic agent MMAE.

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The activator is administered 1-3 days later, thereby rapidly releasing MMAE in
the tumor microenvironment and spreading into surrounding tumor cells,
maximizing the bystander effect.
A dual-antibody-based anti-TAG72 ADC showed potent antitumor efficacy in
mice with colorectal and ovarian cancer xenografts without detectable toxicity.
In contrast, similar ADC controls with valine-citrulline linker were less effective
in these models.
"Chemically-cleavable ADC + activator" has significant efficacy and long
survival
3. Shasqi Bio: CAPAC™ Platform--SQ3370 (Click Activated Protodrugs
Against Cancer)
Shasqi, from San Francisco, California, is also working on bioorthogonal
chemical click reactions in patients with the Click Activated Protodrugs
Against Cancer (CAPAC™) platform, a way to activate cancer drugs at the
tumor site and reduce systemic toxicity. Shasqi is clinically validating the
efficacy of its platform by using SQ3370, which also marks the first time that
click chemistry has been performed in a patient.
The researchers locally injected an alginate hydrogel modified with a chemical
click handle (tetrazine) at the tumor site, followed by intravenous injection of a
prodrug with a complementary handle (for example,
trans-cyclooctene-modified doxorubicin). The prodrug is stable in vivo and has
little activity until it is metabolically contacted with the hydrogel, at which point
a chemical click reaction occurs, thereby releasing the drug. This reduces
off-target exposure and enables very high local doses of the drug.

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Click Activated Protodrugs Against Cancer (CAPAC™) platform
Source: ACS Cent Sci. 2016 Jul 27;2(7):476-82.
After treatment monitoring in the xenograft model over 100 days, the median
tumor size of the doxorubicin prodrug cohort remained unchanged and both
were undetectable (P = 0.021), while the control group rebounded rapidly at
day 50. At the same time, the body weight of the doxorubicin prodrug group
has been at a stable level, while the body weight of the control group mice has
been declining with the treatment time.
Therapeutic effect of doxorubicin prodrug in soft tissue sarcoma xenograft
model

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Conclusion
“Click Chemistry" has many different forms of application in the field of
ADCs, all of which are highly effective and are at an early stage of research,
and it relies on reactions that can operate in the organism without disrupting
biochemical processes, and has a wide application potential.
Huateng Pharma is a leading CDMO for PEG derivatives and pharmaceutical
intermediates which can meet customers’ needs from lab research to pilot
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reagents forclick chemistryreaction.
References:
[1] Click chemistry targets antibody-drug conjugates for the clinic
[2] Copper-free click chemistry in living animals
[3].In Vivo Bioorthogonal Chemistry Enables Local Hydrogel and Systemic
Pro-Drug To Treat Soft Tissue Sarcoma
[4] In vivo bioorthogonal chemistry enables local hydrogel and systemic
pro-drug to treat soft tissue sarcoma
Related articles:
[1] Antibody-Drug Conjugates for Cancer Therapy - Prospects and Challenges
[2] ADC Linker - Development and Challenges