Most of the ADCs currently in clinical use rely on effective internalization and intracellular lysosome transport to achieve bio-cleavage of linkers to release drugs. And, the cleavaged drug must escape from the lysosome to achieve its therapeutic effect. However, not all tumor antigens can ensure the effective ADC treatment described above, especially in solid tumors. Besides, current internalized ADCs are also sensitive to acquired tumor resistance mechanisms.
Extracellular ADC cleavage in the tumor microenvironment (TME) can be a valuable alternative to conventional ADCs. In this case, the drug, once released, can be passively spread into the tumor mass, penetrating and killing adjacent antigen-negative tumor cells, thereby maximizing the bystander effect. Specific targeting of TMEs in solid tumors can be done by the use of weak or non-internalized antigens that exist in large quantities and selectively on cell membranes (e.g., CAIX, VEGFR, cadherin), components of the extracellular matrix (e.g., fibronectin and tenascin-C splice variants, fibrin, type IV collagen, αvβ3 integrin), or by proteins (e.g., VEGF) secreted by tumor cells in TME. If drugs can be selectively released from TMEs, all of these would be excellent targets for ADC therapy.
Significant therapeutic effects of disulfide- or peptide-linked biologically cleavable ADCs against non-internalized tumor targets have been found. Extracellular cleavage of linkers containing disulfide bonds is thought to be due to the release of reducing agents (such as glutathione) by dead cells, which leads to more cell death and thus the release of more reducing agents. In addition, levels of extracellular proteases (e.g. cathepsin, matrix metalloproteinases, and urokinase plasminogen) that are involved in tumor angiogenesis, invasion, and metastasis are elevated, and ADCs targeting non-endogenous antiproteinase-sensitive elements outside tumor cells in TMEs have also been shown to be effective in several mouse xenograft models. However, compared with intracellular bio-cleavage, extracellular bio-cleavage is not ubiquitous and is less efficient. Recently, therefore, researchers have explored ways in which ADC linkers can be chemically triggered. In this approach, the ADC binds to an extracellular tumor target, and after the unbound ADC is cleared from the bloodstream, an exogenous chemical probe (activator) is injected intravenously to release the drug in a selective and rapid reaction with the ADC connector, thus bypassing the dependence on tumor biology to release the drug. Due to the high antigen density of ADCs and the typically rapid pharmacokinetics of activators, the reagent concentration and reaction time in vivo is low, so this approach requires the use of fast and highly selective reactions, such as bioorthogonal reaction.
Fig 1. The mechanism of click chemistry ADC1
The first "click-to-release" ADC was based on a CC49 monoclonal antibody with TCO-Dox (DAR approximately 2) targeting the non-internalized tumor antigen TAG72. The ADC was very stable and showed PK characteristics similar to that of the parent CC49 antibody in tumor-bearing mice. However, the low click reaction binding of the activator limited its further application.
Fig 2. The first-generation click-to-release ADC based on an anti-TAG72 (CC49) antibody coupled with adriamycin2
In the second generation of click-to-release ADCs, the TAG72-targeting diabody with a short half-life was selected. This TCO-MMAE payload was attached to engineered Cys residues via PEG linkers, resulting in a diabody-based tc-ADC (containing the chemically cleavable TCO linker). It had high tumor uptake and very low levels in the blood and other non-target tissues. Pharmacokinetic studies were conducted in mice, where a two-day interval was selected between ADC and activator administration, as ADC was almost completely cleared from the blood. For the activator, a small molecule containing high-release 3,6-dialkyltetrazine motifs and a PEG11-DOTA regulating clearance was developed to fully react with tumor-bound TCO when the dosage was 0.33 mmol/kg.
Fig 3. Diabody-based ADCs tc-ADC3
Three ADCs, click-to-release ADC (tc-ADC), non-binding ADC (nb-ADC), and vc-MMAE ADC, were comparatively evaluated in colorectal cancer (LS174T) and ovarian cancer (OVCAR-3) models overexpressed in TAG72. Mice treated with the click chemistry-based tc-ADC were found to be well tolerated with no obvious signs of toxicity. And further results suggested that mice treated with four cycles of tc-ADC and activator over two weeks experienced significant and lasting tumor regression, whereas vc-ADC showed only limited therapeutic benefit.
Liu's team developed a bioorthogonal cleavage reaction derived from the organic deprotection reaction rather than the click chemistry for ligation. The team synthesized an aromatic linker containing an ortho-carbamoylmethylenesilyl-phenolic ether system and removed the silicon group using fluoride, followed by electron 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 phenylalanine trifuoroborate (Phe-BF3), while very little payload release was observed in the presence of hydrogen peroxide, glutathione, and cysteine. Phe-BF3 mimics natural phenylalanine and was actively taken up by tumor cells via LAT-1. The team, therefore, developed a linker containing tert-butyldimethyl silyl functionalized phenol (TBSO) and used it to attach trastuzumab to MMAE, resulting in a chemically cleavable internalized ADC. The release of MMAE was demonstrated in a proof-of-concept study of HER2-positive gastric cancer transplanted tumors (BGC823). A 4.5mpk ADC was administered, followed by 15 mg/ kg Ph-BF3 after 48 and 96 h. Mass spectrometry (MS) analysis confirmed the presence of a large number of free MMAE in the tumors of mice injected with ADC and activator compared to the control group.
Fig 4. A click chemistry ADC that the linker containing TBSO connects trastuzumab to MMAE4
Click chemistry has great application potential. Click chemistry ADCs can be activated independent of tumor biology, thus allowing the range to be extended to non-endocytic cancer targets and a stronger bystander effect by selecting the appropriate payload. In heterogeneous solid tumors, extracellular cleavage may provide a more even distribution of drugs, thereby increasing therapeutic effectiveness. However, the clinical application of click chemistry reaction has put forward high requirements for the safety and sufficient in vivo stability and reactivity of the reagent. 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.