Tuneable thiol exchange linkers for traceless drug release applications in prodrugs and ADCs.

We describe a versatile and tuneable thiol responsive linker system using thiovinylketones, which relies on the conjugate addition-elimination mechanism of Michael acceptors for the traceless release of therapeutics. In a proof-of-principle study, we translate our findings to exhibit potent thiol-cleavable antibiotic prodrugs and antibody-drug conjugates.

Red-light modulated ortho-chloro azobenzene photoswitch for peptide stapling via aromatic substitution.

The application of peptide stapling using photoswitchable linkers has gained notable interest for potential therapeutic applications. However, many existing methodologies of photoswitching still rely on the use of tissue-damaging and weakly skin-penetrating UV light. Herein, we describe the development of a tetra-ortho-chloro azobenzene linker that was successfully used for cysteine-selective peptide stapling via SNAr. This linker facilitates precise photocontrol of peptide structure via trans to cis isomerisation under red light irradiation. As a proof-of-concept, we applied the developed peptide stapling platform to a modified PMI peptide, targeting the inhibition of MDM2/p53 protein-protein interaction (PPI). Biophysical characterisation of the photoswitchable peptide by competitive fluorescence polarisation showed a significant difference in affinity between the trans and cis isomer for the p53-interacting domain of the human MDM2. Remarkably, the cis isomer displayed a >240-fold higher potency. To the best of our knowledge, this is the highest reported difference in binding affinity between isoforms of a photoswitchable therapeutic peptide. Overall, our findings demonstrate the potential of this novel photoswitchable peptide stapling system for tuneable, selective modulation of PPIs via visible-light isomerisation with deeply-tissue penetrating red light.

Peroxide-cleavable linkers for antibody-drug conjugates.

Antibody-drug conjugates containing peroxide-cleavable arylboronic acid linkers are described, which target the high levels of reactive oxygen species (ROS) in cancer. The arylboronic acid linkers rapidly release a payload in the presence of hydrogen peroxide, but remain stable in plasma. Anti-HER2 and PD-L1 peroxide-cleavable ADCs exhibited potent cytotoxicity in vitro.

Non-internalising antibody-drug conjugates.

Antibody-drug conjugates (ADCs) typically require internalisation into cancer cells to release their cytotoxic payload. However, this places stringent constraints on therapeutic development, requiring cancer targets that have high expression of internalising antigens and efficient intracellular processing. An alternative approach is emerging whereby the payloads can be released extracellularly from cleavable linkers upon binding to poorly-internalising antigens or other tumoral components. This removes the reliance on high antigen expression, avoids potentially inefficient internalisation, and can greatly expand the range of cancer targets to components of the extracellular tumour matrix. This review gives an overview of recent developments towards non-internalising ADCs, including emerging cancer-associated cell surface and extracellular proteins, cancer stromal targeting and the linking chemistry that enables extracellular payload release.

Antibody dual-functionalisation enabled through a modular divinylpyrimidine disulfide rebridging strategy.

Herein we report the development of a methodology for the dual-functionalisation of IgG antibodies. This is accomplished through the combination of disulfide rebridging divinylpyrimidine technology, with bicyclononyne and methylcyclopropene handles to facilitate sequential SPAAC and IEDDA reactions. Advantageously, the strategy does not require metal catalysis and avoids the need for purification between functionalisation steps.

A dual-enzyme cleavable linker for antibody-drug conjugates.

A novel enzyme cleavable linker for antibody-drug conjugates is reported. The 3-O-sulfo-ß-galactose linker is cleaved sequentially by two lysosomal enzymes - arylsulfatase A and ß-galactosidase - to release the payload in targeted cells. An α-HER2 antibody-drug conjugate synthesised using this highly hydrophilic dual-cleavable linker exhibited excellent cytotoxicity and selectivity.

Site-selective modification strategies in antibody-drug conjugates.

Antibody-drug conjugates (ADCs) harness the highly specific targeting capabilities of an antibody to deliver a cytotoxic payload to specific cell types. They have garnered widespread interest in drug discovery, particularly in oncology, as discrimination between healthy and malignant tissues or cells can be achieved. Nine ADCs have received approval from the US Food and Drug Administration and more than 80 others are currently undergoing clinical investigations for a range of solid tumours and haematological malignancies. Extensive research over the past decade has highlighted the critical nature of the linkage strategy adopted to attach the payload to the antibody. Whilst early generation ADCs were primarily synthesised as heterogeneous mixtures, these were found to have sub-optimal pharmacokinetics, stability, tolerability and/or efficacy. Efforts have now shifted towards generating homogeneous constructs with precise drug loading and predetermined, controlled sites of attachment. Homogeneous ADCs have repeatedly demonstrated superior overall pharmacological profiles compared to their heterogeneous counterparts. A wide range of methods have been developed in the pursuit of homogeneity, comprising chemical or enzymatic methods or a combination thereof to afford precise modification of specific amino acid or sugar residues. In this review, we discuss advances in chemical and enzymatic methods for site-specific antibody modification that result in the generation of homogeneous ADCs.