A general approach for the site-selective modification of native proteins, enabling the generation of stable and functional antibody-drug conjugates.

Stephen J Walsh,Marko Hyvönen,Terence T.-L Kwan,Hannah F Sore,David R Spring,Jason S Carroll,Warren R J D Galloway,Soleilmane Omarjee,Jeremy S Parker

doi:10.1039/c8sc04645j

Stephen J Walsh, Marko Hyvönen + Show 7 more

Open Access

https://doi.org/10.1039/c8sc04645j

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Abstract

Antibody-drug conjugates (ADCs) are a class of targeted therapeutics that utilize the specificity of antibodies to selectively deliver highly potent cytotoxins to target cells. Although recent years have witnessed significant interest in ADCs, problems remain with the standard linkage chemistries used for cytotoxin-antibody bioconjugation. These typically (1) generate unstable constructs, which may lead to premature cytotoxin release, (2) often give a wide variance in drug-antibody ratios (DAR) and (3) have poor control of attachment location on the antibody, resulting in a variable pharmacokinetic profile. Herein, we report a novel divinylpyrimidine (DVP) linker platform for selective bioconjugation via covalent re-bridging of reduced disulfide bonds on native antibodies. Model studies using the non-engineered trastuzumab antibody validate the utility of this linker platform for the generic generation of highly plasma-stable and functional antibody constructs that incorporate variable biologically relevant payloads (including cytotoxins) in an efficient and site-selective manner with precise control over DAR. DVP linkers were also used to efficiently re-bridge both monomeric and dimeric protein systems, demonstrating their potential utility for general protein modification, protein stabilisation or the development of other protein-conjugate therapeutics.

Highlights

Model studies using the non-engineered trastuzumab antibody validate the utility of this linker platform for the generic generation of highly plasma-stable and functional antibody constructs that incorporate variable biologically relevant payloads in an efficient and site-selective manner with precise control over drug–antibody ratios (DAR)
It was postulated that vinylpyridine bioconjugation would be too slow to enable efficient cross-linking but that replacement of the pyridine with a pyrimidine would enhance the reactivity to desirable levels by increasing the electron accepting capacity of the heteroaryl ring, without compromising the stability seen with vinylpyridine conjugates.[38]
We have developed a novel DVP linker platform for bioconjugation through the covalent re-bridging of cysteine residues generated by the reduction of native disul de bonds

Summary

Introduction

The emergence of biotherapeutics in recent decades has opened up vast new areas of research for the treatment of a range of grievous diseases,[1,2] with antibody–drug conjugates (ADCs) demonstrating considerable promise as anticancer agents.[3,4] ADCs utilize the impeccable cell-targeting ability of an antibody in combination with the highly potent nature of a cytotoxic payload to achieve cell-selective cytotoxicity[5] while overcoming the dose-limiting toxicity of classical non-targeted small molecule chemotherapy.[6,7] There are currently four ADCs on the market[8,9,10,11] and over 60 other ADCs in clinical trials.[12]. We report a novel divinylpyrimidine (DVP) linker platform for selective bioconjugation via covalent re-bridging of reduced disulfide bonds on native antibodies. Model studies using the non-engineered trastuzumab antibody validate the utility of this linker platform for the generic generation of highly plasma-stable and functional antibody constructs that incorporate variable biologically relevant payloads (including cytotoxins) in an efficient and site-selective manner with precise control over DAR.

Results

Conclusion