Due to improvements in payloads, linkers, and conjugation methods, antibody-drug conjugates (ADCs) have seen significant progress over the past decade.
In particular, linker design plays a critical role in regulating the stability of ADCs in systemic circulation and the efficiency of payload release in tumors, thus influencing the pharmacokinetics (PK), efficacy, and toxicity profiles of ADCs.
Several key linker parameters, such as conjugation chemistry, linker length, and linker steric hindrance, can impact the PK and effectiveness of ADC drugs.
The ideal linker should remain stable in the circulatory system and release the cytotoxic payload specifically within the tumor. However, existing linkers often release the payload linker non-specifically, inevitably leading to off-target toxicity.
Therefore, in the design of ADC drugs, correctly adjusting these crucial linker parameters to achieve a balance between ADC stability and payload release efficiency is essential to attain the desired therapeutic effects.
Over the past few years, numerous new linkers have been developed, including cathepsin-cleavable linkers, acid-cleavable linkers, GSH-cleavable linkers, Fe(II)-cleavable linkers, novel enzyme-cleavable linkers, light-responsive cleavable linkers, and bioorthogonal cleavable linkers.
Among these, cathepsin-cleavable, GSH-cleavable, and acid-cleavable linkers have been well-studied and applied in approved ADCs. Significant progress in linker design has been made, which will help guide the future direction of linker development.
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Cathepsin-Cleavable Linkers
In 2017, Caculitan et al. discovered that the valine-citrulline (Val-Cit) linker exhibits broad sensitivity to multiple cathepsins, including cathepsin B, cathepsin K, and cathepsin L.
To improve selectivity, Wei et al. designed a linker using the cyclobutane-1,1-dicarboxamide (cBu) structure, which primarily relies on cathepsin B.
Intracellularly, cathepsin B inhibitors can effectively inhibit (over 75%) the drug release from linkers containing cBu-Cit, whereas cathepsin K inhibitors show no significant effect.
In contrast, traditional Val-Cit linkers appear resistant to all single cathepsin inhibitors (inhibitors of cathepsin B, L, and K all show less than 15% inhibition).
Additionally, ADCs containing cBu-Cit linkers demonstrated greater tumor inhibitory effects in vitro compared to those with Val-Cit linkers.
Moreover, the optimization of peptide linkers is not limited to developing new structures; peptide linkers can be optimized with minimal structural changes, such as variations in amino acid type and structural chemistry.
Some studies have compared the payload conjugation of MMAE using Val-Cit and Val-Ala dipeptide structures. In the case of non-internalizing antibodies, Val-Cit and Val-Ala linkers conjugated to engineered cysteines exhibited similar characteristics and performed better than Val-Lys and Val-Arg analogs. When using anti-Her2 ADCs with randomly conjugated cysteines, Val-Ala demonstrated less aggregation in high DAR structures compared to Val-Cit. Conversely, both linkers showed similar buffer stability, cathepsin B release efficiency, cellular activity, and tissue pathology profiles.
Get to know more about payload conjugation of MMAE products:
Gly3-Val-Cit-PAB-MMAE | CAS:2684216-48-4
Val-Cit-PAB-MMAE | CAS:644981-35-1
The tetrapeptide Gly-Gly-Phe-Gly exhibits all the characteristics of a stable and effective cleavable linker, and the approved ADC drug Enhertu uses this type of linker.
Enhertu by Daiichi Sankyo is a plasma-stable ADC with a DAR of 7.7 that undergoes proteolytic degradation in lysosomes to release DX-8951f, an effective topoisomerase I inhibitor derived from exatecan. Achieving such a high DAR is notable because it contradicts the well-established principle that high DAR conjugates tend to have poor pharmacokinetic profiles. The self-immolation spacer used here is a simple and compact semicarbazide, unlike the PABC spacer used in Val-Cit linkers.
Acid-Cleavable Linkers
Acid-cleavable linkers utilize the pH difference between tumor tissue (pH 4.0–5.0) and plasma (pH ∼7.4) to selectively release payloads to the tumor tissue.
This strategy achieved early clinical success with Mylotarg and was later applied in Besponsa. However, the limited stability of acid-cleavable linkers has significantly restricted their application in ADCs. Benzophenone-derived hydrazone linkers hydrolyze in human and mouse plasma with a half-life of 2 days, and the serum stability of sacituzumab govitecan (Trodelvy) is also unsatisfactory, with a half-life of 36 hours.
Therefore, acid-labile ADCs require more stable linkers or must use moderately cytotoxic payloads.
In 2019, a novel silyl ether-based acid-labile ADC was developed, carrying the highly cytotoxic monomethyl auristatin E (MMAE). This design greatly enhanced the stability of acid-labile linkers. Additionally, the ADC using this novel silyl ether linker had a half-life of over 7 days in human plasma and showed good therapeutic effects in mouse xenograft models.
GSH-Cleavable Linkers
GSH-cleavable linkers rely on the higher levels of glutathione (1–10 mmol/L) in the cytoplasm compared to plasma (∼5 μmol/L). Disulfide bonds are most commonly used in these linkers. However, current disulfide bond structures cannot perfectly combine high circulation stability with efficient intracellular release.
In 2017, Thomas et al. attempted to address this issue by directly linking small molecule drugs to engineered cysteines in the antibody. Directly conjugating the antibody increases circulation stability due to the spatial protection provided by the antibody.
In vivo stability studies reveal that when DM1 is conjugated to the monoclonal antibody via a disulfide bond at K149C, over 50% of the drug remains conjugated even after seven days. In vivo efficacy studies demonstrate that this novel anti-CD22-DM1-ADC can induce tumor regression in a human lymphoma xenograft mouse model.
MD1 related products:
DM1-MCC-PEG3-Biotin | CAS:2183472-94-6
In the same year, this novel strategy was employed for ADCs using PBD as the payload. Compared to the maleimide peptide (Val-Cit)-PBD-ADC, the novel disulfide linker ADC exhibited similar activity in a human non-Hodgkin lymphoma xenograft mouse model. Additionally, the maximum tolerated dose (MTD) of this novel disulfide linker ADC was higher than that of the Val-Cit ADC (10 vs 2.5 mg/kg).
PBD-dimer related product：
Fe(II)-Cleavable Linkers
Abnormal iron metabolism can elevate levels of free ferrous ions, and based on this strategy, increasing unbound ferrous ion concentration has been utilized in prodrug design. In 2018, Spangler et al. reported a Fe(II)-reactive 1,2,4-trioxane scaffolds (TRX) linker and employed this linker for ADCs. β-elimination.
The linker undergoes Fenton reaction cleavage between TRX and Fe(II) O–O bond, providing a carbonyl intermediate and releasing the payload via β-elimination.
In vitro cytotoxicity studies showed that ADCs containing the TRX linker exhibited activity similar to classical ADCs containing Val-Cit linker in antigen-positive cells. However, in antigen-negative MDA-MB-468 cell lines, ADCs containing the TRX linker still displayed significant toxicity. This instability is attributed to nonspecific interactions between the adamantane moiety and nearby sites on the antibody.
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Novel Enzyme-Cleavable Linkers
Similar to cathepsins, phosphatases and phosphodiesterases are also selectively expressed hydrolases in lysosomes. In 2016, researchers at Merck designed linkers containing phosphate and phosphonate to pair with the Val-Cit-PABA sensitive to cathepsin B, aiming to deliver glucocorticoids.
The advantages of these hydrophilic and permanently charged moieties are solubility, enabling bioconjugation ( bioconjugation service ) with lipophilic glucocorticoid derivatives, and facilitating ADC purification, with residual linkers in the ADC being less than 0.10%. ADCs containing phosphate and phosphonate exhibited activity in vitro.
The same group of researchers at Merck also developed a unique linker based on phosphodiesterase for releasing hydroxy-containing payloads such as Dexamethasone and Fluticasone Propionate.
Both ADCs exhibited good stability in vitro and demonstrated strong activity against tumor cell lines.
In addition to the classic β-glucuronidase-cleavable linker developed for ADCs in 2006, it was discovered that β-galactosidase is overexpressed in tumor cells and possesses hydrolytic activity.
Recently, an ADC using a β-galactosidase-cleavable linker containing a PEG10 spacer has been reported. The spacer is nitro-substituted to enhance its self-degradation rate. Analogous to β-glucuronidase linkers, its dissociation mechanism involves hydrolysis by β-galactosidase, imparting hydrophilicity to the chemical precursor. Another advantage is that β-galactosidase is only present in lysosomes, whereas β-glucuronidase is expressed in lysosomes and also in the microenvironment of solid tumors. Studies have shown that ADCs containing β-galactosidase linkers are more effective in vitro and in vivo than T-DM1, which releases MMAE in the context of anti-HER2 ADCs.
Recently, sulfatase-cleavable linkers have emerged, as sulfatases are overexpressed in several types of cancer, exhibiting potential selectivity.
Research involving anti-Her2 antibodies carrying MMAE as the payload showed that compared to classic cleavable Val-Cit and Val-Ala linkers, sulfatase linkers demonstrated similar efficacy against Her2+ cell lines.
Light-responsive cleavable linkers
In recent years, strategies for payload release based on light-responsive cleavage have gradually emerged. Light-responsive cleavable linkers offer several advantages, including low toxicity, rapid response, high sensitivity, and specificity.
In 2015, Nani et al. first applied a near-infrared (NIR) light-locking strategy to ADCs. The light-responsive cleavage was based on a hepta-methine cyanine fluorophore scaffold. After irradiation with NIR light (λ=650–900nm), the ADC effectively released the small molecule cytotoxin CA-4 in a site-specific manner within the illuminated tumor area.
Read more about NIR:
The Ultimate Guide to Fluorescent Dye
Alexa Fluor series fluorescent dyes and equivalents
In vitro cytotoxicity experiments revealed that ADCs containing NIR light-cleavable linkers exhibited activity equivalent to CA-4 in EGFR+ cell lines post-irradiation while showing low activity without irradiation. However, the self-aggregation and light-instability characteristics of these linkers limited their further development and application as drugs.
NIR related products:
Sulfo-Cyanine7 carboxylic acid
Recently, a novel ultraviolet (UV) light-controlled ADC has been reported in research. The linker introduces a UV light-controlled ortho-nitrobenzyl moiety as a cleavage trigger. In stability and release studies, this linker containing MMAE released <1% under natural light within 6 days, and showed rapid MMAE release within 10 minutes post-irradiation, reaching a maximum plateau. In vitro cytotoxicity experiments demonstrated a significant increase in activity of the ADC containing the ortho-nitrobenzyl linker after irradiation with 365nm (40W) UV light, showing a 50-fold increase compared to the non-irradiated ADC.
In 2019, a light-reactive, self-cleaving linker utilizing the lightcage C40 as an oxidative alkaline site (PC4AP) was reported. This linker, compared to the two ADCs mentioned earlier, has a “double insurance” design. Under 365 nm radiation, the hydroxyl group of PC4AP undergoes intramolecular addition with an amine proximal to its antibody, followed by an elimination reaction leading to cleavage and payload release. ADCs containing peptide-PC4AP-DOX exhibit toxicity equivalent to the payload DOX in positive cells, with no cytotoxicity in the absence of radiation.
Bioorthogonal cleavable linkers
Bioorthogonal chemistry offers high selectivity, rapid processing, and non-toxic byproducts. In 2019, Wang et al. developed a bioorthogonal cleavable linker utilizing the classic bioorthogonal cleavage of Cu(I)-BTTAA and dsProc.
However, bioorthogonal cleavable linkers are currently mainly focused on in vitro exploration. Issues such as reaction efficiency, reaction rate, substrate stability, biocompatibility, and ease of operation still need to be addressed, indicating a long way from clinical applications. ( Know more about our Bioanalytical Service.)
In recent years, significant progress has been made in the optimization of ADC structures and the expansion of mechanisms. New cleavable linkers have been developed to achieve higher selectivity for tumors. Particularly, novel light-reactive cleavable linkers and bioorthogonal cleavable linkers have the potential to overcome the intracellular drug release limitations of traditional ADCs. The preliminary data on these new linkers are encouraging and are expected to greatly facilitate the rapid development of ADC therapeutics in the future.
References:
1.The Chemistry Behind ADCs. Pharmaceuticals (Basel). 2021 May; 14(5): 442.
2. Antibody-drugconjugates: Recent advances in linker chemistry. Acta Pharm Sin B. 2021Dec;11(12):3889-3907.