Macrocyclic peptide ligands and probes for CD59: modulators of innate immunity

Abstract

CD59 is an immuno-regulatory protein, that plays a major role in protecting healthy cells from lysis by the complement system. Numerous studies have shown that CD59 is overexpressed in several cancer cell lines and tumour tissues. This overexpression was shown to play a role in the development of resistance against monoclonal antibody (mAb) therapies such as trastuzumab and rituximab. Potent inhibitors of CD59 thus have potential as adjuvants to mAbs to restore their effect on resistant cancer cells. CD59 is also involved in S. intermedius pathogenesis, which can lead to lung, liver, and brain abscesses. The main virulence agent of this bacteria, ILY, promotes the disease by hijacking CD59 to lyse human cells. Thus, potent inhibitors of CD59 have another potential as anti-virulence agents for S. intermedius infection. Despite the promise of CD59 as a drug target, no synthetic CD59 inhibitors have been reported to date. Previous work in our group has resulted in the identification of six macrocyclic peptides with nanomolar binding affinity for CD59 and determined the structure of one of these hits in complex with CD59. This thesis focuses on optimisation of the initial hits through structure-activity relationship (SAR) determination and development of new chemical probes for CD59 based on the parent peptides. Alanine scanning of the top hits enabled identification of key residues that are essential for binding as well as residues suitable for modification. The activity of the peptides was then determined in a novel cellular assay, reporting the first synthetic cell-active CD59 inhibitor, CP-06. With the aim of enhancing the potency and selectivity of CP-06, three optimisation strategies were explored: (1) Dimerisation, which resulted in a modest enhancement of cellular activity. (2) Conjugation to a lipid tail to enhance membrane association, which generated a peptide with cellular activity enhanced by orders of magnitude. (3) Antibody conjugation to direct association with target cancer cells. Further work will be required to fully investigate and validate the activities of each of these constructs. The results presented here represent promising progress towards the development of CD59 inhibitors that can have dual applications; as adjuvants to mAbs in cancer therapy and anti-virulence agent in S. intermedius infection.