Acid Cleavable Linkers/Hydrazone Linkers

Acid Cleavable Linkers/Hydrazone Linkers

Acid cleavable linkers/hydrazone linkers are widely used in antibody-drug conjugates (ADCs) because of their ability to increase the endosome-lysosome pathway acidity and the glutathione concentration inside cells. BOC Sciences provides an integrated platform with small molecule linker and cytotoxin development and manufacturing capabilities from preclinical to commercial stages. We are striving to offer a wide range of services including synthetic route development, process development and optimization, scale up, and commercial supply production.

Introduction

Acid-cleavable linkers are stable to alkaline environments but highly sensitive to acidic environments such as hydrazone. Acid-cleavable linkers take advantage of the low pH in the endosome and the lysosome to trigger the hydrolysis of the acid-labile hydrazone linker and subsequently release the payload. These linkers have been associated with non-specific release of the drug in clinical studies. The BR96-doxorubicin (BR96-Dox) as an excellent example is constructed by conjugating doxorubicin to the monoclonal antibody BR96 through an acid-cleavable hydrazone. After reaching and binding to the target tumor cells, BR96-Dox is internalized through the endocytosis into lysosomes. In clinical trials, BR96-Dox has been found to not be associated with the typical side-effect profile of native doxorubicin and could potentially deliver high doses of doxorubicin to antigen expressing tumors, which has been found to enable complete remission and cure subcutaneous human breast, lung and colon tumors.

Acid Cleavable Linkers/Hydrazone LinkersFig. 1. Cleavage of acyl hydrazone linker releases doxorubicin (Pharmaceuticals 2021, 14: 442).

Hydrazone linkers display a pH-dependent stability, being stable at neutral pH (blood stream) and being hydrolized in acidic media (pH < 6 for endosomes then pH < 5 for lysosome) to form the corresponding ketone and hydrazine. This approach has been applied with success in IMMU-110, containing a cleavable acyl hydrazone linker formed in the reaction of the hydrazide of 4-maleimidomethyl cyclohexane-1-carboxylate (MCC) and the keto group present in doxorubicin. Hydrazone linkers are also frequently associated with the calicheamicin payload family. In this context, the release is triggered by a two-step’s activation process: first the acid-sensitive hydrazone is hydrolyzed and in the second step the disulfide-bond is reduced by GSH, allowing cyclization of the sulfhydryl intermediate. In its development, the stabilities of a range of hydrazones were tested at pH 4.5 and pH 7.4. These hydrazone linkers were tested in ADCs in vitro and in vivo in mice and those that were stable at pH 7.4 and labile at pH 4.5 afforded the most potent and efficacious ADCs.

Research Applications

Acid Cleavable Linkers/Hydrazone Linkers1

Acid cleavable linkers aim to exploit the acidity of the endosomes (pH 5.5-6.2) and lysosomes (pH 4.5-5.0), whilst maintaining stability in circulation at pH 7.4. This strategy yielded the earliest clinical success in the field with gemtuzumab ozogamicin (Mylotargs) ADC. Moreover, the chimeric antibody BR96-doxorubicin conjugate (BR96-DOX) was developed with the hydrazone conjugation strategy. BR96-DOX was advanced to a Phase II human clinical trial in metastatic breast cancer. The toxicity profile of the conjugate was considerably improved compared to free doxorubicin administration. However, gastrointestinal toxicity was still prominent and clinical outcomes were not satisfying due to its low tolerability. Indeed, ADCs with the hydrazone linker undergo slow hydrolysis under physiological conditions (pH 7.4, 37 °C), resulting in a slow release of the toxic payload.

Our Services

  • Acid Cleavable Linker/Hydrazone Linker Design For Payload

Chemical linker is a critical component of ADC connecting mAb and cytotoxic payload. The linker facilitates ADC stability in circulation until the ADC reaches the target cell. BOC Sciences provides optimized acid cleavable linker/hydrazone linker design schemes to balance ADC stability and payload release kinetics so that payload release in tumor cells reaches a higher therapeutic threshold.

  • Acid Cleavable Linker/Hydrazone Linker Design For Antibody

The attachment site of antibodies is an important consideration for ADC design and assessment, which is largely attributed to the chemical groups on the linker. BOC Sciences provides custom-design services for ADC linkers. The acid cleavable linker/hydrazone linker designed by us with a wide range of chemical groups can meet the needs of various antibody attachment sites. Furthermore, our one-stop service platform supports a wide range of antibody modification and conjugation services to meet your research needs.

  • Acid Cleavable Linker/Hydrazone Linker Design For Target

The in-depth understanding of target molecular characteristics by BOC Sciences has promoted the recognition of potential antigen targets for ADC treatment. Armed with advanced technique platforms, BOC Sciences provides personalized acid cleavable linker/hydrazone linker development services for targeted therapy including but not limited to the target-specific antibody modification and conjugation, linker-payload complex custom synthesis, ADC linker analysis and characterization.

What Can We Do For You?

BOC Sciences has provided customized ADC development services for clients all over the world. Scientists from BOC Sciences are capable of designing and performing different ADC linker constructs. We have established the most comprehensive ADC linker development service process including integrated linker design for dose scheme design and ADC efficacy. We have been dedicating ourselves to helping every customer to facilitate their projects in a highly productive and cost-effective way.

Our Linker Development Workflow

Linker Development Workflow

References

  1. Kotschy, A. et al. The Chemistry Behind ADCs. Pharmaceuticals (Basel). 2021, 14(5): 422.
  2. Zhang, G. et al. Linkers Having a Crucial Role in Antibody-Drug Conjugate. Int. J. Mol. Sci. 2016, 17: 561.
  3. Tsuchikama, K. et al. Antibody-drug conjugates: recent advances in conjugation and linker chemistries. Protein Cell. 2018, 9: 33-46.
* Only for research. Not suitable for any diagnostic or therapeutic use.

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