Next-Generation of Novel Antibody-Drug Conjugates: Click Chemistry ADCs

Currently, ADCs used in clinical settings mostly rely on effective internalization and transport to intracellular lysosomes to achieve biocleavage of the linker and subsequently release the drug. Moreover, the cleaved drug must escape from lysosomes to exert its therapeutic effect. However, not all tumor antigens can ensure the effective processing of ADCs, especially in solid tumors, and current internalized ADCs are also sensitive to acquired tumor resistance mechanisms.

Extracellular ADC cleavage in the tumor microenvironment (TME) can serve as a valuable alternative to traditional ADCs. In this case, once the drug is released, it can passively diffuse into tumor masses, penetrate, and kill neighboring antigen-negative tumor cells, thereby maximizing the “bystander effect”. Specific targeting of TME in solid tumors can be achieved by using weak or non-internalizing antigens (such as CAIX, VEGFR, calreticulin) that are abundant and selectively present on cell membranes, extracellular matrix components (such as splice variants of fibronectin and Tenascin-C, fibrinogen, collagen IV, αvβ3 integrin), or proteins secreted by tumor cells in TME (such as VEGF). If drugs can be selectively released from TME, all of these will be excellent targets for ADC therapy.

Significant therapeutic effects of biocleavable ADCs targeting non-internalizing tumor targets have been found based on disulfides or peptide-based linkers. Extracellular cleavage of connectors containing disulfide bonds is believed to be due to the release of reducing agents (such as glutathione) from dead cells, leading to more cell death and thus releasing more reducing agents. Moreover, the levels of proteases involved in tumor angiogenesis, invasion, and metastasis outside tumor cells (such as tissue proteases, matrix metalloproteinases, and urokinase-type plasminogen activator) are elevated, and ADCs targeting extracellular non-internalizing antigen protease-sensitive proteins in TME have also been shown to be effective in several mouse xenograft models. However, compared with intracellular biocleavage, extracellular biocleavage is not universally present and is less efficient. Therefore, researchers have recently explored chemically triggered methods for ADC connectors (Figure 1). In this approach, ADC binds to extracellular tumor targets, and after unbound ADCs are cleared from the blood, an exogenous chemical probe (activator) is intravenously injected, which selectively and rapidly reacts with the ADC connector to release the drug, thus bypassing the dependence on tumor biology to release the drug. Due to the high antigen density of ADCs and the typical rapid pharmacokinetics of activators, both in vivo reagent concentrations and reaction times are low, so this method requires fast and highly selective reactions, such as bioorthogonal reactions. (You maybe interested in reading: In Vitro vs In Vivo Studies of Polyethylene Glycol)

Figure 1. Schematic Diagram of Click Chemistry ADC Mechanism
Figure 1. Schematic Diagram of Click Chemistry ADC Mechanism

Classic Click Release Reaction: IEDDA Quinone Elimination Orthogonal Cleavage Reaction

The inverse electron-demand Diels-Alder (IEDDA) reaction between trans-cyclooctene (TCO) and tetrazine is the fastest bioorthogonal chemical reaction, forming the basis of the quinone elimination orthogonal cleavage reaction. It introduces an aminoformate-linked payload at the TCO alkene position. In the first step (click), TCO reacts rapidly and selectively with 1,2,4,5-tetrazine derivative (activator), generating several DHP isomers. Then, the 1,4-DHP isomers undergo rapid electron cascade elimination in the second step, releasing the amine-containing payload and carbon dioxide (Figure 2).

 Schematic Diagram of IEDDA-Mediated Quencher Elimination Mechanism
Figure 2. Schematic Diagram of IEDDA-Mediated Quencher Elimination Mechanism

IEDDA related products: Tetrazine-NHS ester, Tetrazine-PEG3-Azide, Methyltetrazine-PEG5-Alkyne, Methyltetrazine-NHS ester, VdU (5-Vinyl-2′-deoxyuridine).

The quencher elimination triggered by tetrazine (Tz) is robust and widely applicable, featuring good stability of the TCO linker and tetrazine activator, as well as rapid and high-yield release both in vitro and in vivo. One drawback of using a TCO embedded in a cleavable linker within an ADC is the reduced reactivity towards tetrazine compared to typical TCOs used for bioconjugation, due to steric hindrance from the vinyl group substituent. To further enhance the click reactivity and potentially lower in vivo dosage levels, a novel click-release strategy has been developed, still based on the potent quencher elimination reaction between TCO and tetrazine, but utilizing TCO as the activator and embedding tetrazine within the linker (Figure 3). In such a system, derivatives of sTCO can be employed to release the payload, exhibiting a three-order-of-magnitude increase in click reactivity relative to tetrazine-triggered quencher elimination.

Figure 3. Transition from Tetrazine-Triggered Drug Release to TCO-Triggered Drug Release for Enhanced Click Reactivity

TCO related products: TCO-NHS Acetate, TCO-acid, TCO-PEG6-acid, TCO-triethoxysilane, TCO-PEG8-amine, TCO-tBu ester, TCO-trimethoxysilane, TCO-PEG12-DBCO, TCO-PEG3-alcohol, etc.

Applications of Click Chemistry ADCs

The first clickable cleavable ADC was based on the CC49 monoclonal antibody with TCO-Dox (DAR approximately 2), targeting the non-internalizing tumor antigen TAG72 (Figure 4). This ADC demonstrated high stability and exhibited PK characteristics similar to the parental CC49 antibody in tumor-bearing mice. However, the low click binding reaction of the activator restricted its further applications.
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Figure 4. First Generation Clickable Cleavable ADC Based on Anti-TAG72 (CC49) Antibody Conjugated with Doxorubicin

The second-generation clickable cleavable ADC opted for a TAG72-targeting Diabody with a shorter half-life. This Diabody was conjugated with TCO-linked MMAE payload via a PEG linker to engineered Cys residues, resulting in a clickable cleavable ADC (tc-ADC) (Figure 5). It exhibited high tumor uptake with minimal levels in blood and other non-target tissues. Pharmacokinetic studies in mice were conducted with a two-day interval between ADC and activator administration, as the ADC was nearly completely cleared from the bloodstream by this time. For the activator, a small molecule was developed containing a highly releasable 3,6-dialkyltetrazine motif and a PEG11-DOTA for clearance modulation, which achieved complete reaction with the tumor-binding TCO at a dose of 0.33 mmol/kg.

Figure 5. Second Generation Clickable Cleavable ADC (tc-ADC)

This novel clickable cleavable ADC (tc-ADC) was evaluated in comparison with non-binding ADC (nb-ADC) and vc-linked MMAE-prepared vc-ADC in overexpressing TAG72 colorectal cancer (LS174T) and ovarian cancer (OVCAR-3) models. The study found that mice treated with the click chemistry tc-ADC exhibited good tolerance with no apparent signs of toxicity. Mice treated with four cycles of tc-ADC and activator therapy over two weeks showed significant and sustained tumor regression, whereas vc-ADC demonstrated only limited therapeutic efficacy (Figure 6).

Figure 6. Therapeutic Efficacy of tc-ADC in Ovarian Cancer (OVCAR-3) Mouse Model

MMAE related products: Amino-PEG4-MMAE, MC-Val-Cit-PAB-MMAE, MMAE, Amino-PEG4-Val-Cit-PAB-MMAE, Azido-PEG4-MMAE, DBCO-PEG4-Val-Cit-PAB-MMAE, DBCO-Val-Cit-PAB-MMAE, Gly3-Val-Cit-PAB-MMAE, NHS ester-PEG4-Val-Cit-PAB-MMAE, etc.

In addition to quencher elimination reactions, other bioorthogonal cleavage reactions have been explored for their application in ADCs. Recently, the Chen research group embarked on establishing metal-based bioorthogonal cleavage reactions for linker cleavage. These cleavage reactions exhibit speeds comparable to, or even faster than, quencher elimination reactions. To this end, the group systematically investigated 24 different species containing copper, palladium, ruthenium, nickel, cobalt, and iron. Among all tested compounds, copper(I) complexes showed effective and rapid cleavage of linkers containing propargyloxyethyl acyl or propargyl functional groups, releasing payloads containing amine or phenol.

The newly developed propargyl-containing ADC linkers were used to conjugate Doxorubicin or etoposide to anti-HER2 antibodies (Figure 7). These ADCs were demonstrated to release drugs in the presence of copper(I)-BTTAA as a chemical activator and kill HER2 overexpressing cells in vitro. However, potential toxicity of copper(I) complexes in vivo and instability over time in biological environments pose certain safety risks for metal-based bioorthogonal clickable ADCs.

Figure 7. Click Chemistry ADCs with Propargyl-containing Linkers Conjugating Doxorubicin or Etoposide to HER2 Antibodies

The Liu research group developed a bioorthogonal cleavage reaction derived from an organic deprotection reaction rather than a click conjugation reaction. The group synthesized an aromatic linker system containing ortho-aminomethylphenylsilyl-phenyl ether moiety and used fluoride or fluoride transfer agents to remove the silane, followed by electron rearrangement, resulting in the release of amino-containing payloads and carbon dioxide. In PBS, 90% of the payload was released within 24 hours in the presence of Phe-BF3, while 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. Therefore, the research group developed a linker containing tert-butyldimethylsilyl-functionalized phenol (TBSO) and used it to link trastuzumab to MMAE, obtaining a chemically cleavable internalizing ADC (Figure 8). Concept validation studies in HER2-positive gastric cancer xenografts (BGC823) confirmed the release of MMAE. Initially, mice were dosed with 4.5 mpk ADC, followed by administration of 15 mg/kg Phe-BF3 at 48 and 96 hours. Mass spectrometry (MS) analysis confirmed significant free MMAE in the tumors of mice injected with both ADC and activator compared to the control group. (LCMS Analysis, LCMSMS Analysis Services, GCMS Analysis, Q-TOF-LC-MS and TQ-LC-MS analysis of Oligonucleotides)

Figure 8. Click Chemistry ADC with TBSO-containing Linker Conjugating Trastuzumab to MMAE

Click chemistry reactions hold significant potential applications. Click chemistry ADCs can be activated independent of tumor biology, allowing for expansion to non-internalizing cancer targets and achieving stronger bystander effects by selecting appropriate payloads. Extracellular cleavage in heterogeneous solid tumors may provide more uniform drug distribution, thereby enhancing therapeutic efficacy. However, clinical application of click chemistry reactions requires high demands on reagent safety, sufficient in vivo stability, and reactivity. To date, only tetrazine elimination reactions developed by Tagworks have been shown to have clinical potential. Nevertheless, with accumulating and maturing technologies, the next generation of click chemistry ADCs is expected to have broader applications within patient populations.

1. Chemical Linkers in Antibody–Drug Conjugates (ADCs).
2. Triggered drug release from an antibody–drug conjugate using fast “click-to-release” chemistry in mice.
3. Chemically triggered drug release from an antibody-drug conjugate leads to potent antitumour activity in mice.
4. A bioorthogonal system reveals antitumour immune function of pyroptosis.

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