Currently clinically used ADCs rely in most cases on efficient internalization and intracellular lysosomal transport for bio-cleavage of the linker and thus release of the drug. In addition, the cleaved drug must escape from the lysosome to achieve its therapeutic effect. However, not all tumor antigens ensure efficient ADC treatment as described above, especially in solid tumors. Furthermore, currently internalized ADCs are also sensitive to acquired tumor resistance mechanisms.

Research Status of ADC Therapies

Extracellular ADC cleavage in the tumor microenvironment (TME) could serve as a valuable alternative to traditional ADCs. In this case, once released, the drug can passively diffuse into the tumor mass, penetrate and kill adjacent antigen-negative tumor cells, thereby maximizing the bystander effect. Specific targeting of the TME in solid tumors can be achieved through the use of weak or non-internalized antigens (e.g. CAIX, VEGFR, cadherins), components of the extracellular matrix (e.g. splice variants of fibronectin and Tenascin-C, fibrin, collagen type IV, αvβ3 integrin) or proteins secreted by tumor cells in the TME (e.g. VEGF). All of these would be excellent targets for ADC therapy if the drug could be selectively released from the TME.

Mechanism of ADC Click Chemistry

Currently, significant therapeutic efficacy of bio-cleavable ADCs based on disulfide linkers or peptide linkers against non-internalizing tumor targets has been found. Extracellular cleavage of linkers containing disulfide bonds is thought to be due to the release of reducing agents (such as glutathione) by dying cells leading to more cell death and thus release of more reducing agents. However, extracellular bio-cleavage is less ubiquitous and less efficient than intracellular bio-cleavage. Researchers have thus recently explored ways in which ADC linkers can be chemically triggered.

In this approach, the ADC binds to the extracellular tumor target, and the unbound ADC is cleared from the blood. Then, an intravenously injected exogenous chemical probe (activator) selectively and rapidly reacts with the ADC linker to release the drug, thereby bypassing the dependence on tumor biology for drug release. Due to the high antigen density of ADCs and the fast pharmacokinetics typical of activators, in vivo reagent concentrations and reaction times are low, this approach requires the use of fast and highly selective reactions such as bio-orthogonal.

Classical Click-Release Reaction-IEDDA Pyridazine Elimination Orthogonal Cleavage Reaction

The fastest bio-orthogonal chemical reaction is the inverse electron demand Diels-Alder (IEDDA) reaction between trans-cyclooctene (TCO) and tetrazine, which is the basis for the orthogonal cleavage reaction of pyridazine elimination. It introduces a carbamate-linked payload at the TCO allylic position. In the first step (click), TCO reacts rapidly and selectively with a 1,2,4,5-tetrazine derivative (activator) to generate several DHP tautomers. The 1,4-DHP isomer then rapidly releases the amine-containing payload and carbon dioxide in a second electron-cascade elimination step.

Tetrazine-triggered pyridazine elimination is a robust and broadly applicable chemical reaction with good TCO linker and tetrazine activator stability coupled with rapid and high-yield release in vitro and in vivo. One disadvantage of using TCOs embedded in the cleavable linker of ADCs compared to typical TCOs for bioconjugation is the reduced reactivity towards tetrazine due to steric hindrance from the allyl substituent. To further enhance click responsiveness and potentially reduce in vivo dose levels, the researchers developed a novel click-release strategy. This strategy is still based on the robust pyridazine elimination reaction between TCO and tetrazine, but uses TCO as the activator and embeds the tetrazine into the linker (Fig. 3). In such a system, derivatives of sTCO can be used to release payloads with three orders of magnitude greater click reactivity relative to tetrazine-triggered pyridazine elimination.

Application of Click Chemistry ADC

The first click-cleavable ADC was based on the CC49 mAb with TCO-Dox (DAR ~2), targeting the non-internalized tumor antigen TAG72 (Fig. 4). The ADC is very stable, showing similar PK properties to the parental CC49 antibody in tumor-bearing mice. However, the low click-binding response of activators limits their further applications.

The second-generation click-cleavable ADC selected TAG72-targeted Diabody with a shorter half-life. This Diabody-TCO-linked MMAE payload was attached to an engineered Cys residue via a PEG linker, resulting in a click-cleavable ADC (tc-ADC) (Fig. 5). It has high tumor uptake and very low levels in blood and other non-target tissues. Pharmacokinetic studies in mice showed that ADC was almost completely cleared from blood when there was a two-day interval between ADC and activator administration. The activators are small molecules containing a high-release 3,6-dialkyltetrazine motif and a clearance-modulating PEG11-DOTA. In addition, studies have shown that when the dosage is 0.33 mmol/kg, it can completely react with tumor-bound TCO.

In addition to pyridazine elimination reactions, some other bio-orthogonal cleavage reactions were explored for ADC applications. Recently, Chen's group set out to establish a metal-based bio-orthogonal cleavage reaction into a linker cleavage reaction. The speed of these cleavage reactions can be as fast as pyridazine elimination reactions, or even faster. To this end, the group conducted a systematic survey of 24 different species containing copper, palladium, ruthenium, nickel, cobalt, and iron. Among all the compounds tested, the copper(I) complexes had a positive effect on the presence of propargyloxyacetyl or propargyl functional linkers were shown to cleave efficiently and rapidly and release amine- or phenol-containing payloads.

Liu's group developed a bio-orthogonal cleavage reaction derived from an organic deprotection reaction rather than a click ligation reaction. The group synthesized an aromatic linker comprising an ortho-carbamoylmethylene silyl-phenol ether system, and removed the silicon group using fluoride or a fluoride transfer agent, followed by electronic rearrangement, resulting in an amino-containing payload and release of carbon dioxide. In PBS, 90% of the payload was released within 24 h in the presence of trifluoroboron phenylalanine (Phe-BF3). In the presence of hydrogen peroxide, glutathione, and cysteine, only little payload release was observed. Mechanistically, Phe-BF3 mimics natural phenylalanine and is actively taken up by tumor cells through LAT-1.

Based on this, the research group developed a linker containing tert-butyldimethylsilyl-functionalized phenol (TBSO). This linker links trastuzumab to MMAE, resulting in a chemically cleavable internalized ADC (Fig. 7). In a proof-of-concept study in HER2-positive gastric cancer xenografts (BGC823), significant free MMAE was present in the tumors of mice injected with ADC and activator, demonstrating efficient release of MMAE.

Click chemistry has great application potential in ADCs. Click-chemistry ADCs can activate them independent of tumor biology, thus allowing expansion to non-endocytic cancer targets. In addition, click chemistry ADCs can achieve stronger bystander effects by selecting appropriate payloads. In heterogeneous solid tumors, extracellular cleavage may provide more uniform drug distribution, thereby increasing therapeutic efficacy. However, the clinical application of click chemistry puts high demands on the safety and sufficient in vivo stability and reactivity of the reagents. So far, only the pyridazine elimination reaction developed by Tagworks has been shown to have clinical potential. However, it is believed that with the accumulation and maturity of the technology, the new generation of click chemistry ADC is expected to be more widely used in the patient population.

Source: https://adc.bocsci.com/resource/application-of-click-chemistry-in-the-next-generation-of-novel-antibody-drug-conjugation-adcs.html