The development of antibody-drug conjugates (ADCs) has progressed significantly over the past decade due to improvements in payloads, linkers, and conjugation methods. In particular, linker design plays a key role in regulating ADC stability in systemic circulation and payload release efficiency in tumors, thereby affecting ADC pharmacokinetics (PK), efficacy, and toxicity profiles.
Some key linker parameters, such as coupling chemistry, linker length, and linker steric hindrance, affect the PK and efficacy of ADC drugs. The ideal linker should remain stable in the circulation and release cytotoxic payloads in the tumor. However, existing linkers often unspecifically release payloads and inevitably lead to off-target toxicity.
Therefore, in the design of ADC drugs, it is necessary to correctly adjust these important parameters of the linker to achieve the balance between ADC stability and payload release efficiency, so as to achieve the desired effect of ADC drugs. In the past few years, many 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.
1. Cathepsin Cleavable Linker
In 2017, Caculitan et al. found that the valine-citrulline (Val-Cit) linker exhibits broad sensitivity to a variety of cathepsins, including cathepsin B, cathepsin K, cathepsin L, etc. To improve selectivity, Wei et al. designed a linker using the cyclobutane-1,1-dicarboxamide (cBu) structure, which is largely dependent on cathepsin B.
In cells, the cathepsin B inhibitor effectively inhibited (over 75%) the release of the cBu-Cit linker-containing drug, while the cathepsin K inhibitor had no significant effect. In contrast, traditional Val-Cit-containing linkers appear to be resistant to all single protease inhibitors (less than 15% inhibitors for cathepsin B, L, and K). compared with ADC containing val-CIT linker, ADC containing CBU-CIT linker showed greater tumor inhibition in vitro.
Furthermore, the optimization of peptide linkers is not limited to the development of new structures, peptide linkers can be optimized with minimal structural changes, such as the tpe of amino acid and structural chemistry. Several studies have compared the Val-Cit and Val-Ala dipeptide structures with MMAE payload connections. In the case of non-internalizing antibodies, both Val-Cit and Val-Ala linkers bound to engineered cysteines exhibited similar characteristics and better performance than Val-Lys and Val-Arg analogues. In the case of a random cysteine-conjugated anti-Her2 ADCs, Val-Ala showed less aggregation in the high DAR structure compared to Val-Cit. On the other hand, both linkers showed similar buffer stability, cathepsin B release efficiency, cellular viability and histopathological characteristics.
The tetrapeptide GLy-Gly-Phe-Gly exhibits all the properties of a stable and effective cleavable linker used in the marketed ADC drug Enhertu. Enhertu is a plasma-stable ADC with a DAR of 7.7 that undergoes protease degradation in the lysosome to release DX-8951f, a potent topoisomerase I inhibitor derived from exatecan. Since the linker does not contain a solubilized solvent, achieving such a high DAR is considerable because it contradicts the widely established principle that high DAR binders may have poor pharmacokinetic characteristics. The self-degrading spacer used here is a simple and compact hemiamination rather than the PABC used by the Val-Cit linker.
| CAS | Product Name | Type |
| 159858-33-0 | Val-Cit | Val-Cit linker |
| 159857-79-1 | Val-Cit-PAB-OH | Val-Cit linker |
| 159858-22-7 | Fmoc-Val-Cit-PAB-OH | Val-Cit linker |
| 863971-53-3 | Fmoc-Val-Cit-PAB-PNP | Val-Cit linker |
| 870487-08-4 | Boc-Val-Cit | Val-Cit linker |
| 870487-09-5 | Boc-Val-Cit-PAB | Val-Cit linker |
| 870487-10-8 | Boc-Val-Cit-PAB-PNP | Val-Cit linker |
| 2504147-59-3 | MC (C5)-Val-Cit | Val-Cit linker |
| 159857-80-4 | MC-Val-Cit-PAB-OH | Val-Cit linker |
| 159857-81-5 | MC-Val-Cit-PAB-PNP | Val-Cit linker |
| MC-Val-Cit-PAB-Gly | Val-Cit linker | |
| 2748039-76-9 | Alkyne-Val-Cit-PAB-OH | Val-Cit linker |
| 2748039-77-0 | Alkyne-Val-Cit-PAB-PNP | Val-Cit linker |
| SPDP-Val-Cit-PAB-OH | Val-Cit linker | |
| SPDP-Val-Cit-PAB-PNP | Val-Cit linker | |
| 1343476-44-7 | Val-Ala-PAB-OH | Val-Ala linker |
| 1342211-31-7 | MC-Val-Ala-OH | Val-Ala linker |
| 1870916-87-2 | Mal-Val-Ala-PAB | Val-Ala linker |
| 70396-18-8 | Boc-Val-Ala-OH | Val-Ala linker |
| 1884577-99-4 | Boc-Val-Ala-PAB | Val-Ala linker |
| 150114-97-9 | Fmoc-Val-Ala-OH | Val-Ala linker |
2. Acid Cleavable Linkers
Acid cleavable linkers selectively deliver payloads to tumor tissue by taking advantage of the pH difference between tumor tissue (4.0-5.0) and plasma (~ 7.4). This strategy had its earliest clinical success with Mylotarg and was later used in Besponsa. However, the lack of stability of acid cleavable linkers severely limits its application in ADC. The phenylketone-derived hydrazone linker hydrolyzed with a 2 days half-life in human and mouse plasma, and the serum stability of Sacituzumab- Govitecan (Trodelvy) was also not satisfactory with a 36 hours half-life.
3. Glutathione (GSH) Cleavable Linkers
GSH cleavable linkers depend on higher levels of glutathione (1-10 mmol/L) in the cytoplasm compared to plasma (~ 5 μmol/L). Disulfide bonds are most commonly used among these linkers, however, current disulfide bond structures cannot achieve a perfect combination of high cycling stability and efficient intracellular release. In 2017, Thomas et al. attempted to address this issue by directly linking small-molecule drugs to engineered cysteines in mAb. By directly linking the antibody, steric protection of the antibody will improve cycling stability.
In vivo stability studies have shown that when DM1 is attached to mAb K149C via a disulfide bond, more than 50% of the drugs remain connected even after seven days. In vivo pharmacodynamics studies have shown that this novel anti-CD22-DM1-ADC induces tumor regression in a human lymphoma-tumor xenograft mouse model.
In the same year, this new strategy was applied to the PBD as an ADC payload. Compared to maleimide peptide (Val-Cit) -PBD-ADC, the ADC of the novel disulfide bond exhibited similar activity in a human non-Hodgkin’s lymphoma xenograft mouse model. Meanwhile, the MTD of the new disulfide ADC is higher than that of the Val-Cit ADC.
4. Fe (II) Cleavable Linkers
Abnormal iron metabolism can increase the level of free iron, and based on this strategy, increasing the concentration of unbound iron has been used in prodrug design. In 2018, Spangler et al. reported an Fe (II) reactive 1,2, 4-trioxane (TRX) ligand for use in ADC.
Reference
Antibody-drug conjugates: Recent advances in linker chemistry. Acta Pharm Sin B. 2021Dec;11(12):3889-3907.