What Are ADC Linkers: Cleavable vs. Non-Cleavable Linkers

Antibody-drug conjugates (ADCs) are now established as the main therapies for clinical cancer treatment. ADC consists of linker, payload, and monoclonal antibody (mAb). It combines the advantages of highly specific targeting ability and potent killing action to achieve precise and efficient elimination of cancer cells. Since the first ADC drug, Mylotarg® (gemtuzumab ozogamicin), was approved by the FDA in 2000, there are currently 15 ADC drugs approved worldwide for hematologic malignancies and solid tumors. In addition, there are currently more than 100 ADC candidates in various stages of clinical trials.

The development of ADC involves a critical understanding of target antigen selection, conjugate internalization by tumor cells, drug potency, and stability of the linker between drug and antibody. One of the main challenges in developing safe and effective antibody-drug conjugates is the generation of suitable chemical linkers between cytotoxic drugs and monoclonal antibodies.

Linker has a very significant impact on the stability of ADCs, and payload release profile, and is critical for the ultimate efficacy of ADC drugs. The ideal linker should not induce ADC aggregation and can prevent premature release of payload in plasma and can be effectively released at desired targeted sites. Depending on the metabolic fate in the cell, two types of linkers are used in most ADC drugs, including cleavable and non-cleavable linkers.

cleavable-and-non-cleavable-linkers
Image resource: royalsocietypublishing.org

Cleavable Linkers in ADCs

Cleavable linkers play a key role in the success of antibody-drug conjugates. They use the inherent properties of tumor cells to selectively release cytotoxins from ADCs. They are stable in the blood circulation for a long period of time. There are three commonly used mechanisms: 1) protease sensitivity, 2) pH sensitivity, and 3) glutathione sensitivity.

1) The protease sensitivity strategy utilizes predominant proteases found in lysosomes of tumor cells to recognize and cleave specific peptide sequences in the linker. Dubowchik and Firestone et al. pioneered the discovery of the valine-citrulline (VC) dipeptide as an intracellular cleavage mechanism by cathepsin B. Cathepsin B are usually overexpressed in cancer cells, allowing the drug to be released precisely in the vicinity of the tumor. In addition, the linkers are usually stable in the systemic circulation due to the presence of protease inhibitors in the blood, and it reduces the risk of side effects. Nine of the 14 approved ADC drugs use peptide-based linkers. For example, brentuximab vedotin (Adcetris) uses a valine-citrulline linker.

2) The acid-sensitive strategy is to use a lower pH of the endosome (pH = 5-6) and lysosome (pH = 4.8) compartments compared to the cytoplasm (pH = 7.4) to trigger the hydrolysis of an acid-labile group within the linker, such as a hydrazone. However, hydrolysis of hydrazine bonds is not exclusively confined to lysosomes and occasionally occurs in plasma, leading to reduced targeting efficiency and off-target effects. For example, Sacituzumab govitecan-hziy (Trodelvy) combines a humanized monoclonal antibody with SN-38, which is conjugated to the antibody via an acid-sensitive hydrolysable linker called CL2A. PEG can increase the solubility of ADC linkers, we can offer a wide array of different ADC PEG Linkers to empower our customer's advanced research worldwide.

Trodelvy formula, resulting from anti-TROP-2 antibody conjugation to SN-38 via an acid-sensitive cleavable linker. image source: http://dx.doi.org/10.3390/ph13090245

3) The third release strategy utilizes higher intracellular glutathione (GSH) concentrations than in plasma. Disulfide bond-based linkers are sensitive to reduced glutathione. GSH plays a key role in maintaining intracellular redox balance during cell survival, cell proliferation and differentiation. The concentration of GSH in blood is considerably lower than the intracellular concentration in cancer cells. Therefore, this type of linker can remain stable in the blood system while releasing active payloads exclusively in cancer cells with elevated GSH levels.

Non-Cleavable Linkers in ADCs

Non-cleavable linkers are divided into two groups, namely thioether or maleimidocaproyl (MC). They have no obvious drug release mechanism, and the ADC prepared by this strategy relies on the complete lysosomal proteolytic degradation of the antibody that releases the antibody-drug after internalization. Through this degradation, the non-cleavable linker carrying the drug will also be coupled to the conjugated amino acid of the antibody. Therefore, ADCs with non-cleavable linkers are more dependent on the biology of the target cells than cleavable linkers. Ado-trastuzumab emtansine (T-DM1) demonstrates the successful application of thioether linker. The conjugate is the product of an anti-HER2 monoclonal antibody to DM1 (emtansine) via a succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker. This linker makes the conjugate more stable in the blood and releases the active metabolite of DM1, lysine-MCC-DM1, after the antibody molecule is digested by proteases inside the cancer cells.

Kadcyla Structure
Kadcyla formula, image source: http://dx.doi.org/10.3390/ph13090245

One of the advantages of non-cleavable linkers over cleavable linkers is their increased plasma stability, which can improve the therapeutic index. Studies have shown that non-cleavable linked ADCs generally perform better than their cleavable counterparts in vivo.

Moreover, due to the fact that payload derivatives from non-cleavable ADCs can kill target cells, non-cleavable linkers can potentially provide a larger therapeutic window compared to cleavable linkers. Finally, it is expected to reduce off-target toxicity compared to cleavable linker conjugates because non-cleavable ADCs can provide greater stability and tolerance.

Conclusion

Considerable efforts have been made in the design and selection of suitable linkers to conjugate monoclonal antibodies and cytotoxic drugs. These linkers can affect the stability, toxicity, PK properties and pharmacodynamics of ADCs. Each linker has its advantages and disadvantages and many factors must be considered in the selection and application to identified monoclonal antibodies and specific cytotoxic drugs. The appropriate linker must take into account the existing groups presented in the monoclonal antibody, the reactive groups in the cytotoxic drug, and the derivative functional groups. The perfect linker ensures adequate stability of the cytotoxic drug in the circulation, effectively prevents premature release of the drug, effectively promotes the release of the cytotoxic drug in the targeted tumor cells, and strongly promotes the efficacy and tolerability of a successful ADC.

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References:
[1] Fu, Z., Li, S., Han, S. et al. Antibody drug conjugate: the “biological missile” for targeted cancer therapy. Sig Transduct Target Ther 7, 93 (2022). https://doi.org/10.1038/s41392-022-00947-7
[2] Joubert, N.; Beck, A.; Dumontet, C.; Denevault-Sabourin, C. Antibody–Drug Conjugates: The Last Decade. Pharmaceuticals 2020, 13, 245. https://doi.org/10.3390/ph13090245
[3] Bargh, J. D. et al. Cleavable linkers in antibody-drug conjugates. Chem Soc Rev. 2019; 48(16):4361-4374. doi: 10.1039/c8cs00676h