The Development of Methods for Generating Fc-Conjugates and Antibody-Drug Conjugates

Abstract

Biotherapeutics, such as peptides and proteins, constitute a valuable class of drugs due to their high target specificity and their ability to modulate protein interactions (PPIs), which are often considered ‘undruggable’ by traditional small molecule drugs. Additionally, antibodies possess many beneficial characteristics, such as a long circulation half-life, the ability to target tumours and engaging the immune system; therefore, antibodies can be exploited as therapeutics by attaching small molecules or peptide drugs. Section I of this thesis describes the design, synthesis, and bioconjugation of small molecule linkers to Fc protein followed by post-functionalisation with therapeutic peptides to generate Fc-peptide conjugates. Many biologically active proteins and peptides suffer from an extremely short plasma half-life, which limits their therapeutic potential. A common half-life extension strategy employs the fusion of a biotherapeutic to the Fc fragment of an antibody, resulting in what is known as an Fc-fusion protein. Fc-fusion proteins are commonly obtained through recombinant DNA technology; however, this approach does not permit the incorporation of drugs that include numerous unnatural amino acids, unnatural cyclic peptides, oligonucleotides, or other non-proteinogenic features. The primary aim of this work was the establishment of a methodology to generate Fc-peptide conjugates semi-synthetically. The secondary aim of this work was to prove that the methodology afforded Fc-peptide conjugates with biological activity and a long circulation half-life. This report details the development of a series of trifunctional disulfide rebridging linkers based on the previously reported bis-divinylpyrimidine (BisDVP) linker. The ability of the linkers to cross-link Fc protein fragments was investigated and optimised. The synthesised Fc-linker conjugates were subsequently functionalised via click-chemistry with a therapeutic peptide and/or a fluorophore or a cytotoxic drug. An Fc-Exenatide conjugate was synthesised and demonstrated good biological activity in a cell-based potency assay. The Fc-Exenatide conjugate was also evaluated in an in vivo PK study and exhibited a long circulation half-life (27-35 hours) compared to the Exenatide peptide alone (8 minutes) in mice. A one-pot method of generating Fc-peptide conjugates was investigated. Lastly, a cytotoxin-containing dual-functional Fc-conjugate was evaluated in vitro and demonstrated cell-killing ability. Section II of this thesis describes the generation of antibody-drug conjugates (ADCs). The therapeutic area of ADCs is constantly evolving, and the specificity of drug release is significantly influenced by the linker connecting the drug to the antibody. This section describes the generation and evaluation of ADCs comprising a novel thiol-responsive linker system employing thiovinylketones, which relies on the conjugate addition-elimination mechanism of Michael acceptors for traceless drug release. Assessment of the biological activity of the thiol-responsive ADCs demonstrated dose-dependent cytotoxicity in breast cancer cells (SKBR3).