Discovery of novel macrocyclic peptide inhibitors of CD59 through chemical biology
File(s)
Author(s)
Bickel, Jasmine Kaileigh
Type
Thesis
Abstract
Immunotherapy has emerged as one of the most promising therapeutic strategies for various cancers, with several monoclonal antibodies (mAbs) having reached clinical trials or receiving FDA approval. mAbs function through several mechanisms of action, including complement dependent cytotoxicity (CdC). CdC activates the terminal pathway of the complement system to form membrane attack complex (MAC) pores on the tumour cell membrane, resulting in lysis. However, resistance or unresponsiveness to treatment remain serious barriers to full therapeutic benefit. One of the most prominent mechanisms of evasion is through overexpression of membrane bound complement regulatory proteins on the tumour cell surface, in particular CD59. Inhibitors of CD59 function therefore offer a promising therapeutic avenue to enhance the efficacy of mAb cancer therapies.
Streptococcus intermedius intermedilysin (ILY) is a bacterial pore forming toxin which hijacks CD59 binding to promote pore formation on host cell membranes. The recombinant domain 4 of ILY, rILYd4, has been proposed as an inhibitor of CD59 function, however its therapeutic potential is limited by immunogenicity and poor metabolic stability. Using a co crystal structure of the ILY CD59 complex, cyclic peptides were developed based on the primary binding interface of these two proteins. Strategies to improve membrane anchoring and beta-hairpin characteristics were therefore developed with the aim of improving efficacy.
Further, state of the art techniques to screen DNA encoded libraries of more than one trillion unique peptide sequences have emerged as highly powerful methodologies in ligand discovery. Two such screens, phage display and mRNA display, were therefore undertaken to identify novel cyclic peptide binders of CD59. Whilst the output of the phage display screen is yet to be fully determined, the mRNA display screen successfully identified a panel of high affinity cyclic peptide binders of CD59. One of the most promising peptides, mRNA 06, was identified with a KD of 11 nM and the co crystal structure was solved in complex with CD59. Structural studies provide unprecedented insight into the key binding residues and conformational characteristics of this peptide. In addition, functional studies show that mRNA 06 competes with ILY, and consequently rILYd4, for CD59 binding.
Inhibition of CD59 represents an attractive and tractable strategy to reactivate the CdC effect of cancer therapeutic mAbs. The chemical tools and methodology developed here provides a major advance in targeting of CD59 with cyclic peptides, providing a road map for future studies. mRNA 06 represents the best performing CD59 binder identified thus far, displaying genuine promise for optimisation as an adjuvant for mAb therapy.
Streptococcus intermedius intermedilysin (ILY) is a bacterial pore forming toxin which hijacks CD59 binding to promote pore formation on host cell membranes. The recombinant domain 4 of ILY, rILYd4, has been proposed as an inhibitor of CD59 function, however its therapeutic potential is limited by immunogenicity and poor metabolic stability. Using a co crystal structure of the ILY CD59 complex, cyclic peptides were developed based on the primary binding interface of these two proteins. Strategies to improve membrane anchoring and beta-hairpin characteristics were therefore developed with the aim of improving efficacy.
Further, state of the art techniques to screen DNA encoded libraries of more than one trillion unique peptide sequences have emerged as highly powerful methodologies in ligand discovery. Two such screens, phage display and mRNA display, were therefore undertaken to identify novel cyclic peptide binders of CD59. Whilst the output of the phage display screen is yet to be fully determined, the mRNA display screen successfully identified a panel of high affinity cyclic peptide binders of CD59. One of the most promising peptides, mRNA 06, was identified with a KD of 11 nM and the co crystal structure was solved in complex with CD59. Structural studies provide unprecedented insight into the key binding residues and conformational characteristics of this peptide. In addition, functional studies show that mRNA 06 competes with ILY, and consequently rILYd4, for CD59 binding.
Inhibition of CD59 represents an attractive and tractable strategy to reactivate the CdC effect of cancer therapeutic mAbs. The chemical tools and methodology developed here provides a major advance in targeting of CD59 with cyclic peptides, providing a road map for future studies. mRNA 06 represents the best performing CD59 binder identified thus far, displaying genuine promise for optimisation as an adjuvant for mAb therapy.
Version
Open Access
Date Issued
2020-01
Date Awarded
2020-03
Copyright Statement
Creative Commons Attribution NonCommercial Licence
License URL
Advisor
Tate, Edward
Bubeck, Doryen
Sponsor
Engineering and Physical Sciences Research Council
Publisher Department
Department of Chemistry
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)