Enzymatically Cleavable Linkers

Enzymatically Cleavable Linkers

Enzyme-cleavable linkers have emerged as a particularly effective ADC linker type due to their ability to selectively release payloads in the lysosomes of target cells. BOC Sciences is committed to providing flexible ADC enzymatically cleavable linkers at competitive prices. In BOC Sciences, we support various ADC linkers development and bioconjugation services for global customers to achieve new drug discovery milestones with comprehensive and advanced platforms.

Introduction

Enzymatically cleavable linkers have attracted significant attention in ADC development due to their superior plasma stability and release mechanisms. Typically, the advantage of using enzyme-cleavable linkers refers to their ability to selectively induce drug release at target cells rather than in circulation. Therefore, the only clinically explored enzyme-cleavable linkers are peptides sensitive to cleavage by lysosomal cathepsin proteases. Generally, linkers containing Val-Cit or Val-Ala sequences are most widely employed due to high stability in human plasma and efficient drug release toward the lysosomes of target cells. Moreover, a self-immolative para-aminobenzoyl carbamate (PABC) spacer is also required to ensure the cathepsin-mediated cleavage is unimpeded by the payload.

Enzymatically cleavable linkers for ADCFig. 1. Enzymatically cleavable linkers for ADC (Diss. Universidad de La Rioja, 2019).

Enzymatically cleavable linkers provide antibody-drug conjugates with plasma stabilities comparable to non-cleavable linkers while boasting a more defined drug release method than disulfide-linked or acid-labile linkers. The most popular enzymatic cleavage sequence is the dipeptide valine-citrulline, combined with a self-immolative linker p-aminobenzyl alcohol (PAB). Mechanistically, cleavage of an amide-linked PAB triggers a 1,6-elimination of carbon dioxide as well as the concomitant release of free drugs in the parental amine form. In order to limit the release of payloads before internalization, preventing or minimizing degradation outside the target cell, the proteome of the lysosome became a logical place to search for enzymes capable of ADC degradation and present in high concentration.

Designing Process Requirements

Enzymatically Cleavable Linkers

Effective enzyme-cleavable ADC linkers require high stability in the circulatory conditions but high lability upon entry to the lysosomes of target cells, thus efficiently releasing payloads. Enzyme-labile linkers can be cleaved under advantageous and characteristically mild reaction conditions because enzymatic transformations often occur at pH 6-8, and between room temperature and 40°C. In addition, enzymes often combine high chemo-, regio- and stereoselectivity for the recognized substrates and tolerance for secondary structures. Furthermore, the ability to pair these linkers with self-immolative chemical groups bestows the release of free drugs with minimal derivation, and the main constraint regards the requirement for drugs to bear amine or hydroxyl groups when conjugating with PAB.

Our Services

Enzymatically Cleavable Linkers 3

Cathepsin B, a cysteine protease presents in the late endosome and lysosome compartments in mammals, is also overexpressed in many cancer cells. In fact, cleavable dipeptides were explored as cathepsin B substrates for doxorubicin prodrugs, demonstrating antigen-driven cellular activity with Val-Cit dipeptide linkers. BOC Sciences offers customized-design services for cathepsin B cleavable linkers according to the antibody, payload, and target. We design and select the most suitable linker based on the efficacy and toxicity of an individual ADC module to deliver high-quality and low-cost linker products in time.

Enzymatically Cleavable Linkers 4

Phosphatase is a hydrolase exhibiting selective expression in the lysosome. In 2016, researchers first designed phosphate and pyrophosphate-containing linkers coupled with the well-established, cathepsin B sensitive, Val-Cit-PABA moiety aiming to deliver glucocorticoids. Within studies, researchers demonstrated that efficient payload release is achieved by induction with lysosomal extracts, and both phosphate and pyrophosphate-containing ADC are active in vitro. BOC Sciences is experienced in supporting phosphatase cleavable linkers customization services for specific antibodies, payloads, and targets. We also offer optimized linker design schemes to balance ADC stability and payload release kinetics so that the release of payloads within tumor cells reaches its highest therapeutic threshold.

Enzymatically Cleavable Linkers 5

β-Glucuronidases are a class of glycosidase enzymes, which catalyze β-glucuronic residues hydrolysis. The abundance of β-glucuronidases in lysosomes and tumor interstitium is associated with the hydrophilicity of its substrates, which is also the reason of interest for designing safe and efficient cleavable linkers for ADC. A seminal work published in 2006 described the anti-CD70 ADCs releasing amine-containing MMAE, MMAF, and doxorubicin payloads, an original linker containing a β-glucuronic moiety attached to a self-immolative spacer. BOC Sciences offers customized-design services for β-glucuronidases cleavable linkers according to our client's project needs. We have established the most comprehensive ADC linker development services, including integrated linker design for dose scheme design and ADC efficacy.

Enzymatically Cleavable Linkers 6

β-Galactosidase is overexpressed in certain tumor tissues and cleaves lysosomal linkers via hydrolysis. Mechanistically, when the β-galactosidase cleavable linker was conjugated with trastuzumab and MMAE, it demonstrated higher potency than in conjugation with Val-Cit-PABC. Using the β-glucuronidase linker analogy, the cleavage mechanism involves the hydrolysis of the β-galactosidase moiety, which confers hydrophilicity to the chemical precursor. Another advantage is that the β-galactosidase enzyme is present only in the lysosome, whereas β-glucuronidase is expressed in lysosomes and also in the microenvironment of solid tumors. BOC Sciences aims to provide our global clients with β-galactosidase cleavable linker design services to support your most advanced research. Our highly skilled Ph.D. and M.S. synthetic chemists will solve the linker development challenges you encounter, from biological to chemical linker development.

Enzymatically Cleavable Linkers 7

Sulfatases offer an opportunity for selective payload release because they reserve high activity within the lysosomes but low activity in human and rodent plasma. There are various sulfatases that reside in the lysosome, catalyzing the hydrolysis of alkylsulfate esters into alcohols. Moreover, sulfatases are overexpressed in a number of different cancer types, thereby offering the possibility of additional selectivity for arylsulfate-containing ADCs towards tumors. BOC Sciences also provides customized-design services for sulfatase cleavable linkers to adjust ADC stability and payload release, and we are capable of constructing viable linkers and other ADC product developments at your request.

What Can We Do For You?

BOC Sciences is committed to developing cutting-edge ADC technology. We have established a one-stop ADC technology development service platform, including mature ADC linkers development process, purification process, determining quality standards and key process parameters, as well as subsequent formulation development of products. BOC Sciences provides ADC linkers development service to customers worldwide to promote your ADC research projects.

Our Linker Development Workflow

Linker Development Workflow

References

  1. Owen, S.C. Antibody Drug Conjugates: Design and Selection of Linker, Payload and Conjugation Chemistry. The AAPS Journal, 2015, 17(2): 339-351.
  2. Kotschy, A. et al. The Chemistry Behind ADCs. Pharmaceuticals (Basel). 2021, 14(5): 422.
* Only for research. Not suitable for any diagnostic or therapeutic use.

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