Click chemistry is an efficient, selective and modular chemical reaction that is widely used in the field of biomedicine, and has shown great potential in the development of antibody-drug conjugates (ADCs). ADCs are an innovative class of drugs that combine the targeting ability of antibodies with the cytotoxicity of small molecule drugs and are widely used in cancer treatment. However, the development of ADCs faces many challenges, one of which is how to establish a stable and effective connection between antibodies and drugs. This is where click chemistry comes into play.

Antibody-Drug Conjugate

ADCs are a new class of targeted therapeutics consisting of a monoclonal antibody (mAb) backbone covalently linked to a cytotoxic payload via a chemical linker. Each component of an ADC plays a critical role in its stability, efficacy, and toxic side effects. ADC platforms are designed to improve the therapeutic index and minimize off-target toxicity by releasing their cytotoxic payload upon interaction with cognate antigens. Early clinical development of ADCs was hampered by their significant immunogenicity, suboptimal target selectivity, and narrow therapeutic window.

Fig. 1. Click Chemistry in ADC (Nat Commun. 2018, 9(1): 1484).

Most recent ADCs are built on IgG1 antibodies, which have higher solubility, longer half-life, and minimized nonspecific immunogenicity. The cognate antigens of these antibodies are selected by their high and uniform expression on the cell surface of tumor cells or other components of the tumor microenvironment (TME) to achieve targeted binding and drug internalization. Chemical linkers attach the cytotoxic payload to the mAb and prevent premature systemic drug release while allowing the payload to be effectively delivered into tumor cells. Currently available linkers are divided into cleavable linkers and non-cleavable linkers, which are degraded by the extracellular acidic environment, proteolysis or elevated glutathione levels or by intracellular lysosomes, respectively. While non-cleavable linkers are more stable in the circulation, cleavable linkers tend to increase the bystander effect, that is, neighboring cells that spread from the targeted tumor cells are killed by cytotoxic drug molecules. ADCs are designed to carry highly effective chemotherapy and are not suitable for systemic infusion in free form due to unacceptable toxicity.

Click Chemistry

Click chemistry is a new technology developed by K. Barry Sharpless, a 2001 Nobel Prize winner in Chemistry and a researcher at the Institute of Chemical Biology at the Skaggs Institute. Its high efficiency and high controllability have set off a storm in the field of chemical synthesis and become an attractive development direction in the current international pharmaceutical field. The basic idea of click chemistry is to use carbon-heteroatom bonding reactions to quickly achieve molecular diversity. Generally, azide and alkyne form covalent bonds, which have the advantages of high efficiency, stability, and high specificity. The reaction is not affected by pH and can be carried out in water at room temperature, and even in living cells. Click chemistry is widely used in bioconjugation because it can accurately control the modification sites of biomolecules.

* Click chemistry linkers in ADCs:

In recent years, as studies have confirmed the benefits of homogeneity and site specificity of drugs on antibodies, there are more and more demonstrations of using this chemical method to construct chemically defined ADCs. In the synthesis of ADCs, click chemistry provides an efficient, rapid and highly selective connection method. The most typical click chemistry reaction is the 1,3-dipolar cycloaddition reaction, also known as the copper-catalyzed cycloaddition of azide and alkyne (CuAAC). This reaction can be carried out under mild conditions and is highly compatible with biological macromolecule systems.

Click Reaction Chemistry

Through click chemistry, the linker arm can be precisely designed between the antibody and the drug to ensure that the drug can be effectively released after binding to the target cell. For example, in some studies, by introducing an azide or alkyne group on the antibody and the corresponding reactive group on the drug, the two can be effectively connected by a simple click reaction after the immunolabeling step. This method not only improves the coupling efficiency of the ADC, but also ensures the location and number of connections, thereby optimizing the drug properties of the ADC. Another important application of click chemistry is to improve the stability and uniformity of the ADC. Traditional ADC preparation methods often produce heterogeneous products, which will affect the efficacy and safety of the drug. With the help of click chemistry, precise control of the connection site can be achieved to ensure the uniformity and stability of the ADC, thereby improving the therapeutic effect of the drug and reducing side effects. In addition, click chemistry also provides flexibility and diversity for the improvement of ADC. By designing different click reactions, a variety of functional molecules (such as fluorescent dyes, diagnostic probes, etc.) can be coupled to antibodies to develop multifunctional ADCs. This multifunctional ADC not only has the ability to kill cancer cells, but can also be used for cancer diagnosis and disease course monitoring, achieving the effect of integrated diagnosis and treatment. At present, the three common types of click chemistry reactions include:

• CUAAC Click Chemistry

Following the introduction of the concept of click chemistry, the copper-catalyzed azide-alkyne cycloaddition reaction (CuAAC) was independently reported by Sharpless and Medal groups in 2002. This reaction can be regarded as the first classic in click chemistry. Azide and terminal alkyne remain stable under most chemical conditions, but can be efficiently and specifically converted into 1,3-substituted triazoles (Formula 1) under copper-catalyzed conditions. Linking groups with the same structure have not yet been found in nature, but the characteristics of mild conditions, high yield, high chemical selectivity and no interference from water and oxygen have become the outstanding advantages of this reaction.

• SPAAC Click Chemistry

The strain-promoted azide-alkyne cycloaddition (SPAAC) reaction was developed by Bertozzi et al. in 2004. It does not require the use of metal catalysts, reducing agents, or stabilizing ligands. Instead, the reaction utilizes the enthalpy released by ring strain into cyclooctynes (such as OCT, BCN, DBCO, DIBO, and DIFO) to form stable triazoles (Formula 2). Although the reaction kinetics of SPAAC are slower than CuAAC, its biocompatibility in living cells is unquestionable. To date, the reaction has been widely used in the fields of hybrid and block polymer formation, metabolic engineering, nanoparticle functionalization, and oligonucleotide labeling.

• Diels-Alder Reaction

Trans-cyclooctenes (TCO) react in the Inverse Electron Demand Diels-Alder (IEDDA) reaction, which is characterized by the absence of catalysts, fast reaction rates, and good biocompatibility under physiological conditions. TCOs are widely used in biological and material science research, especially pre-targeting methods and related kits for targeted medical imaging or therapy. Tetrazines are a class of click chemistry labeling reagents containing a reactive tetrazine group, a six-membered heterocyclic compound containing four nitrogen atoms, and have three isomers: 1,2,3,4-tetrazine, 1,2,4,5-tetrazine, and 1,2,3,5-tetrazine. Tetrazine reagents are highly reactive with TCO in the Inverse Electron Demand Diels-Alder reaction and the Inverse Diels-Alder reaction to eliminate nitrogen. This is a very fast reaction for bioconjugation at low concentrations in labeling living cells, molecular imaging, and other bioconjugation applications.

ADC Click Chemistry

The first click-cleavable ADC was based on CC49 mAb with TCO-Dox (DAR of approximately 2), targeting the non-internalized tumor antigen TAG72. This ADC was very stable and showed similar PK properties to the parent CC49 antibody in tumor-bearing mice. However, the low click binding response of the activator limited its further application.

Fig. 2. First generation click-cleavable ADCs (Bioconjug Chem. 2016, 27(7): 1697-706).

The second generation of clickable ADCs selected a TAG72-targeted Diabody with a shorter half-life. This Diabody was linked to a TCO-linked MMAE payload and connected to an engineered Cys residue via a PEG linker, resulting in a click-cleaved ADC (tc-ADC). It has high tumor uptake and very low levels in the blood and other non-target tissues. Through pharmacokinetic studies in mice, a two-day interval was selected between ADC and activator administration because the ADC was almost completely cleared from the blood at this time. For the activator, a small molecule containing a high-release 3,6-dialkyltetrazine motif and a clearance-regulated PEG11-DOTA was developed, which completely reacted with the tumor-bound TCO when used at a dose of 0.33 mmol/kg.

Fig. 3. Second generation click-cleavable ADCs (Nat Commun. 2018, 9(1): 1484).

This new click-cleavable ADC (tc-ADC) was compared with non-binding ADC (nb-ADC) and vc-ADC prepared by vc-linked MMAE in colorectal cancer (LS174T) and ovarian cancer (OVCAR-3) models overexpressing TAG72. The study found that mice treated with click chemistry tc-ADC were well tolerated with no obvious signs of toxicity. After mice received four cycles of tc-ADC and activator treatment over two weeks, tumors regressed significantly and durably, while vc-ADC showed only limited therapeutic effects.

* ADC related products:

In addition to the pyridazine elimination reaction, several other bioorthogonal cleavage reactions have been explored for use in ADCs. Recently, the Chen group set out to establish the conversion of metal-based bioorthogonal cleavage reactions to linker cleavage reactions. These cleavage reactions can be as fast or even faster than the pyridazine elimination reaction. To this end, the group systematically surveyed 24 different species containing copper, palladium, ruthenium, nickel, cobalt, and iron. Among all the compounds tested, copper(I) complexes showed efficient and rapid cleavage of linkers containing propargyloxyacetyl or propargyl functional groups, and released amine- or phenol-containing payloads.

The Liu group developed a bioorthogonal cleavage reaction that was derived from an organic deprotection reaction rather than a click ligation reaction. The group synthesized an aromatic linker containing an ortho-carbamoylsilyl-phenol ether system and used fluoride or fluoride transfer agents to remove the silicon group, followed by electronic rearrangement, resulting in the release of an amino-containing payload and carbon dioxide. In PBS, 90% of the payload was released within 24 h in the presence of trifluoroboron phenylalanine (Phe-BF3), whereas only minimal payload release was observed in the presence of hydrogen peroxide, glutathione, and cysteine. Phe-BF3 mimics natural phenylalanine and is actively taken up by tumor cells via LAT-1. The research team therefore developed a linker containing tert-butyldimethylsilyl-functionalized phenol (TBSO) and used it to link trastuzumab to MMAE, resulting in a chemically cleavable internalizing ADC. MMAE release was demonstrated in a proof-of-concept study of HER2-positive gastric cancer xenografts (BGC823). 4.5 mpk of ADC was administered first, followed by 15 mg/kg of Phe-BF3 48 and 96 h later. Mass spectrometry (MS) analysis confirmed the presence of significant amounts of free MMAE in tumors of mice injected with the ADC and activator compared to controls.

Conclusion

Click chemistry has great potential for application. Click chemistry ADCs can be activated independently of tumor biology, thus allowing the scope to be expanded to non-endocytic cancer targets and a stronger bystander effect to be obtained by selecting appropriate payloads. In heterogeneous solid tumors, extracellular cleavage may provide a more uniform drug distribution, thereby increasing the therapeutic effect. However, the clinical application of click chemistry reactions places high demands on the safety of reagents and sufficient in vivo stability and reactivity. So far, only the pyridazine elimination reaction developed by Tagworks has been shown to have clinical potential. But it is believed that with the accumulation and maturity of technology, the new generation of click chemistry ADCs is expected to be more widely used in patient populations.