In the development of ADCs, the linker structure impacts stability, homogeneity, cytotoxic potency, tolerability, and pharmacokinetics (PK). Therefore, selecting the appropriate linker is crucial for optimizing the therapeutic potential and safety of ADCs. An ideal linker for ADCs should be stable in circulation and specifically release the cytotoxic payload to the tumor site. However, existing linkers often lead to nonspecific release of the payload in non-tumor tissues, resulting in off-target toxicity^[1]. Consequently, the development of novel linkers is essential to achieve ADCs with optimized therapeutic windows.

Linkers are typically classified as cleavable or non-cleavable based on their cleavage mechanism. More than 80% of the clinically approved ADCs employ cleavable linkers ^[2], such as Inotuzumab ozogamicin (Besponsa) and Brentuximab vedotin (Adcetris)^[3,4]. Various cleavable-linkers are currently utilized in ADCs, including cathepsin-cleavable linkers, acid-cleavable linkers, GSH-cleavable linkers, Fe(II)-cleavable linkers, and novel enzyme-cleavable linkers^[5]. Among them, cathepsin-cleavable linkers have been well studied and employed in approved ADCs. This article provides a comprehensive review of the enzymes involved in cleavable enzyme-linker systems.

Figure 1 The general structure of an ADC and the roles of the chemical trigger, the linker‒antibody attachment and the linker‒payload attachment^[5].

Cathepsins Cleavable Linkers

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, and more. However, only Cathepsin B is widely expressed in cancer cells, and its broad sensitivity to other cathepsins may lead to off-target toxicity of ADCs in normal cells.

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 studies of intracellular cleavage, the release of drugs linked via cBu-Cit was efficiently inhibited (by over 75%) by a Cathepsin B inhibitor, whereas a Cathepsin K inhibitor had no significant impact. Compared with ADC containing Val-Cit linker, ADC containing cBu-Cit linker showed greater tumor inhibition in vitro.

Studies have shown that even minimal structural modifications to peptide linkers can significantly impact their performance. Zheng et al. have developed a valine-alanine (Val-Ala) linker, and experiments have demonstrated that Val-Ala exhibits better hydrophilicity and stability compared to Val-Cit. Furthermore, both Reid et al. and Salomon et al. highlighted that ADCs incorporating (L,L)-dipeptide linkers demonstrate enhanced antitumor efficacy in both in vitro and in vitro settings^[5].

Figure 2 The structure of the cBu-Cit-PABC-containing ADCs^[5].

Matrix Metalloproteinase Family Cleavable Linkers

Matrix metalloproteinase-2 (MMP-2) is highly expressed in various tumor tissues, including breast, cervical, and ovarian cancers, etc^[6]. Studies suggest that MMP-2 plays a crucial role in promoting cancer cell metastasis, regulating tumor growth signaling pathways, and inhibiting apoptosis^[7]. In addition, within the tumor microenvironment, MMP-2 can degradation extracellular matrix (ECM) components, thereby enhancing tumor invasion and metastasis. Leveraging these characteristics, researchers have developed various enzyme-reactive drug delivery systems ^[8-10]. Unlike normal cells, the tumor microenvironment is characterized by hypoxia and weak acidity, making it an effective bio-trigger for controlled and targeted drug delivery in cancer diagnosis and therapy ^[11].

Mu et al. have introduced a groundbreaking antibody-drug conjugate (ADC), FMSN-Dox-H2-AE01, featuring human serum albumin (HSA) shelled mesoporous silica nanoparticles as a biocompatible drug carrier. Within this innovative construct, doxorubicin (DOX), a potent chemotherapeutic agent, resides within the mesoporous silica structure, while an anti-EGFR antibody (AE01) is strategically linked for precise tumor-specific targeting ^[12].

The presence of HSA and antibodies on the particle surface enhances biocompatibility and prevents premature drug leakage. Additionally, this system allows for selective biodegradation triggered by MMP-2, ensuring specific cytotoxicity against cancer cells while minimizing impacts on normal cells. In vitro studies have demonstrated the remarkable efficacy of this ADC, with cancer cell mortality rates reaching about 85-90%. Moreover, this system enables efficient control over drug release, modulated by variations in MMP-2 levels and pH conditions. These findings highlight the potential of this enzyme-responsive FMSN-Dox-H2-AE01 as a promising avenue for cancer therapy ^[12].

Figure 3 (A) Schematic description of anti-EGFR (AE01) and HSA functionalized mesoporous silica nanoparticles.(B) Mechanism diagram ofFMSN-DOX-H2-AE01

Katrin et al. devised a proteolytically activatable ADC targeting IgM for the treatment of IgM-positive B cell lymphoma. Through the integration of a masking unit derived from the IgM antigen into full-length antibodies, they engineered a sophisticated construct. The masking unit was intricately fused using a synthetic linker equipped with dual-protease sites identified by MMP-2/9 and matriptase, ensuring precise activation exclusively within the tumor microenvironment. This innovative design, based on a chicken anti-IgM antibody and conjugated with MMAE, enabled targeted binding, internalization, and controlled cytotoxic payload release, effectively inducing tumor cell death. The results demonstrated that the masked CH2-aIgM exhibited significantly reduced binding to IgM-positive cells compared to the unmasked aIgM. Upon protease treatment, the masked antibody regained activity, displaying strong binding to IgM.

Figure 4 Design and mode of action of masked aIgM ADC.

Novel Enzyme Cleavable Linkers

In addition to the classic β-glucuronidase-cleavable linkers that were developed for ADCs. The β-Galactosidase is over expressed in some tumors and possess hydrolytic activity. Kolodych et al. introduced ADCs with a β-galactosidase-cleavable linker, rapidly hydrolyzed in vitro by 10 U/mL β-galactosidase. The ADC, linking trastuzumab and MMAE through this linker, exhibited a lower IC50 than Val-Cit-linked ADCs and Kadcyla. In vivo, ADCs with the β-galactosidase-cleavable linker achieved a significant 57% and 58% reduction in tumor volumes in a xenograft mouse model at 1 mg/kg, outperforming Kadcyla at the same dose ^[14].

Similarly, Bargh et al. introduced a sulfatase-cleavable linker that targets sulfatase , analogous to β-galactosidase in tumor cells. This linker showed high susceptibility to sulfatase enzymes and improved cytotoxicity and selectivity in HER2+ cells over noncleavable and Val-Ala ADCs^[5].

Figure 5 Structures of glycosidase- and sulfatase-cleavable triggers. (A)The structure of a β-glucuronidase-cleavable, linker-containing ADC. (B)Release mechanism of b-glucuronidase and β-glucuronidase-cleavable linkercontaining ADCs. (C)The structure and release mechanism of sulfatase-cleavable linker-containing ADCs.

For the screening and validation of linkers for ADCs, we have developed a series of proteases specialized in linker cleavage, encompassing Cathepsin B, Cathepsin L, Cathepsin S, MMP-2, MMP-7, MMP-9, β-glucuronidase, β-galactosidase.

Product Features

• Natural conformation: HEK293 expressed protein to ensure the natural structure

• Robust and consistent enzyme activity, ensuring reproducibility across batches

• High purity: Verified by SDS-PAGE and SEC-MALS

Product Features

• Analysis and characterization for ADCs: Conducting linker screening and stability evaluation verification

• Assessment of drug release to guarantee efficient intracellular payload delivery

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• Enzyme Activity of Cathepsin B

• Enzyme Activity of MMP-9

>>> Click for Solutions for Antibody-Drug Conjugates (ADCs) Development

Reference:

[1]Zhi Xin Phuna, Prashanth Ashok Kumar, Elio Haroun, Dibyendu Dutta, Seah H. Lim, Antibody-drug conjugates: Principles and opportunities, Life Sciences, Volume 347, 2024, 122676, ISSN 0024-3205, https://doi.org/10.1016/j.lfs.2024.122676.

[2]Lyon R. Drawing lessons from the clinical development of antibody-drug conjugates. Drug Discov Today Technol 2018;30:105e9.

[3]Hamann PR, Hinman LM, Hollander I, Beyer CF, Lindh D, Holcomb R, et al. Gemtuzumab ozogamicin, a potent and selective anti-CD33 antibody-calicheamicin conjugate for treatment of acutemyeloid leukemia. Bioconjug Chem 2002;13:47e58. Na

[4]Perini GF, Pro B. Brentuximab vedotin in CD30þ lymphomas. Biol Ther 2013;3:15e23.

[5]Zheng Su, Dian Xiao, Fei Xie, Lianqi Liu, Yanming Wang, Shiyong Fan, Xinbo Zhou, Song Li, Antibody–drug conjugates: Recent advances in linker chemistry, Acta Pharmaceutica Sinica B, Volume 11, Issue 12, 2021, Pages 3889-3907, ISSN 2211-3835, https://doi.org/10.1016/j.apsb.2021.03.042.

[6]Roomi, M. W.; Monterrey, J. C.; Kalinovsky, T.; Rath, M.; Niedzwiecki, A. In vitro modulation of MMP-2 and MMP-9 in human cervical and ovarian cancer cell lines by cytokines, inducers and inhibitors. Oncol. Rep. 2010, 23 (3), 605−614.

[7]Kessenbrock, K.; Plaks, V.; Werb, Z. Matrix metalloproteinases: regulators of the tumor microenvironment. Cell 2010, 141 (1), 52−67.

[8]Chen, W. H.; Luo, G. F.; Lei, Q.; Jia, H. Z.; Hong, S.; Wang, Q.R.; Zhuo, R. X.; Zhang, X. Z. MMP-2 responsive polymeric micelles for cancer-targeted intracellular drug delivery. Chem. Commun. (Camb) 2015, 51 (3), 465−8.

[9]Peng, Z. H.; Kopecek, J. Enhancing Accumulation and Penetration of HPMA Copolymer-Doxorubicin Conjugates in 2D and 3D Prostate Cancer Cells via iRGD Conjugation with an MMP-2 Cleavable Spacer. J. Am. Chem. Soc. 2015, 137 (21), 6726−9.        [10]Taghizadeh, B.; Taranejoo, S.; Monemian, S. A.; Salehi Moghaddam, Z.; Daliri, K.; Derakhshankhah, H.; Derakhshani, Z. Classification of stimuli-responsive polymers as anticancer drug delivery systems. Drug Deliv 2015, 22 (2), 145−55.

[11]Ling, D.; Li, H.; Xi, W.; Wang, Z.; Bednarkiewicz, A.; Dibaba, S. T.; Shi, L.; Sun, L. Heterodimers made of metal-organic frameworks and upconversion nanoparticles for bioimaging and pH-responsive dual-drug delivery. J. Mater. Chem. B 2020, 8 (6), 1316−1325.

[12]Wu H, Ding X, Chen Y, Cai Y, Yang Z, Jin J. Constructed Tumor-Targeted and MMP-2 Biocleavable Antibody Conjugated Silica Nanoparticles for Efficient Cancer Therapy. ACS Omega. 2023 Mar 29;8(14):12752-12760. doi: 10.1021/acsomega.2c07949. PMID: 37065049; PMCID: PMC10099448.

[13]Schoenfeld, K., Harwardt, J., Habermann, J., Elter, A., & Kolmar, H. (2023). Conditional activation of an anti-IgM antibody-drug conjugate for precise B cell lymphoma targeting. Frontiers in Immunology, 14.

[14]Kolodych S, Michel C, Delacroix S, Koniev O, Ehkirch A, Eberova J, Cianférani S, Renoux B, Krezel W, Poinot P, Muller CD, Papot S, Wagner A. Development and evaluation of β-galactosidase-sensitive antibody-drug conjugates. Eur J Med Chem. 2017 Dec 15;142:376-382. doi: 10.1016/j.ejmech.2017.08.008. Epub 2017 Aug 4. PMID: 28818506.