Single-Payload Bioconjugation to Native Antibodies And A General Method for the Self-Immolative Release of Amide-Containing Molecules

Abstract

Antibody-drug conjugates (ADCs) have emerged as a powerful form of targeted cancer therapy that can deliver drugs with a high level of selectivity towards a specific cell type, reducing off-target effects and increasing the therapeutic window compared to small molecule therapeutics. However, creating ADCs that are stable, homogeneous, and with controlled drug-to-antibody ratio (DAR) remains a significant challenge. Chapter 1 describes the bioconjugation optimisation of the third generation of tetra-divinylpyrimidine (TetraDVP) linkers. These linkers allow for the creation of single-payload antibody conjugates by re-bridging all four interchain disulphide bonds in IgG1 antibodies with a single construct. Unlike earlier generations, the TetraDVPs used in this work contain the payload before the bioconjugation occurs; this means the entire construct is added in a single step ‘all-at-once’, reducing the antibody manipulation required. The optimised bioconjugation includes a modular reagent addition cycle approach that enabled constructs with varied payloads to be conjugated by >93% in all cases by simply adapting the number of addition cycles. This enabled the creation of protein catcher SpyTag, cytotoxic MMAE, and fluorophore BODIPY single-payload conjugates of trastuzumab. Flow cytometry and SpyTag-SpyCatcher studies carried out by Claudia Driscoll, Howarth group, showed that the ‘all-at-once’ TetraDVP concept can be used to make single-payload antibody constructs that maintain the activity and binding of the antibody and the payload. The controlled release of drug molecules is of interest across various chemical fields. It allows for the masking of a molecule's properties and precise deployment upon a single controllable release event. Although numerous methodologies have been developed for amines, alcohols, and thiols, approaches for utilising amides as payload-release handles have seldom been reported despite the prevalence of amides in therapeutic compounds and materials. Chapter 2 describes the development of a general amide release method utilising aminomethyl carbamate linkages. The linkage was shown to be stable in physiological conditions but would efficiently allow the liberation of the parent amide drug upon a specific trigger event. The approach was successfully used to release the secondary amide linezolid, primary amide levetiracetam, and aryl amide lidocaine. Nitrophenyl, glycoside, dipeptide, and cephalosporin-based triggers were all successfully in utilised to release the FDA-approved antibiotic linezolid, showing the method to be compatible with commonly used self-immolative systems. The sulphonamide sulfamethizole was successfully released, however, the linkage proved instable preventing further exploitation of the method for sulphonamides. Urea and thioamide release was also attempted but proved synthetically challenging. The utility of amide release was demonstrated by its successful application in the targeted release of linezolid from a prodrug using the cathepsin-cleavable dipeptide trigger valine-citrulline. Significantly, in its prodrug state, no activity against Mycobacterium tuberculosis was exhibited. Linezolid's full potential was achieved only upon controlled release, where an equipotent efficacy to a free linezolid control was demonstrated. With encouraging biological results, a DAR 4 linezolid-antibody conjugate was synthesised using an M. tb targeting antibody. This is the first reported linezolid ADC and preliminary data shows it is active against M. tb; further biological characterisation is ongoing.