UCL Discovery
UCL home » Library Services » Electronic resources » UCL Discovery

Protein-Protein Conjugation using Interferon

Herrington-Symes, AP; (2015) Protein-Protein Conjugation using Interferon. Doctoral thesis , UCL (University College London). Green open access

[thumbnail of Herrington-Symes FINAL THESIS 24.5.15_AHS.pdf]
Preview
PDF
Herrington-Symes FINAL THESIS 24.5.15_AHS.pdf

Download (13MB)

Abstract

Therapeutic proteins are often potent and have rapid onsets of action. Unfortunately protein-based medicines can be immunogenic and have short half-lives. The circulation half-life of many proteins has been improved by the covalent conjugation of poly(ethylene)glycol (PEG) to the protein. For example, PEGylated interferon-2 (PEGASYS® and PEG-INTRON®) has become a first line treatment for hepatitis C. The aim of this thesis was to examine the possibility of using a homobifunctional PEG reagent to make protein dimers. Our group has developed PEGylation reagents that undergo conjugation by bis-alkylation to selectively conjugate either (i) two cysteine thiols from a native disulfide or (ii) two histidine residues in a polyhistidine tag. It was hypothesised that IFN dimers (IFN-PEG-IFN) could be prepared with higher retained activity than monoPEGylated IFN. It was also hypothesised that a heterodimer of IFN and an antibody fragment (Fab) could be made while retaining the activity of both proteins within the heterodimer. His8IFN-PEG-His8IFN and IFN-PEG-IFN homodimers were prepared by site-selective conjugation to the N-terminal 8-polyhistidine tag and to one of the disulfides of IFN respectively. These homodimer conjugates were characterised in terms of purity and in vitro activity. The in vitro cell based assays were optimised to accurately elucidate the specific activities of the IFN conjugates. The His8IFN-PEG20-His8IFN homodimer was found to retain greater activity than IFN-PEG20-IFN. The increased activity was thought to be due to conjugation to the polyhistidine tag, which is distal from the IFN binding surface. It was also found that IFN-PEG10-IFN homodimer retained greater activity than PEG10-IFN, which could be due to the presence of the second IFN molecule in the IFN-PEG10-IFN homodimer. Two different Fabs were used to prepare the IFN-PEG-Fab heterodimers. Conjugation was conducted at one disulfide of IFN and the accessible interchain disulfide of the Fab. One Fab was derived from a polyclonal antibody to albumin (Fabalb) and the rationale for this heterodimer was that a longer lasting form of IFN could be made (IFN-PEG20-Fabalb). The other Fab was derived from bevacizumab (Fabbeva) to give an IFN-PEG20-Fabbeva heterodimer that could, in principle, display antiangiogenic properties. Both heterodimers were evaluated using antiviral and antiproliferative assays to determine the activity of IFN in the conjugate. The IFN-PEG20-Fabalb conjugate displayed a 10-fold reduction in activity compared to IFN-PEG20-Fabbeva. It was thought that Fabalb underwent competitive binding with components of the media. Interestingly, the IFN-PEG20-Fabbeva heterodimer displayed greater activity than PEG20-IFN and IFN-PEG20-IFN in both the antiviral and antiproliferative assays. The binding properties of Fabbeva were determined by SPR. It was observed that the dissociation rate of IFN-PEG20-Fabbeva was similar to Fabbeva and PEG20-Fabbeva. IFN-PEG20-Fabalb was found to have a similar dissociation rate to Fabalb. However, PEG20-Fabalb was found to have a slower dissociation to both IFN-PEG20-Fabalb and Fabalb, this result requires further investigation but was thought to be due to the sample impurity. The association rates of heterodimers were found to similar to the PEG-Fab conjugates but slower than their native Fabs. This data suggests that the novel IFN-PEG20-Fabbeva and IFN-PEG20-Fabalb heterodimer conjugates have retained their binding affinities to their antigens. Overall, it was shown that a homobifunctional bis-alkylating conjugation reagent (e.g. PEG di(bis)sulfone 4) could be used successfully to prepare dimeric protein conjugates. This work highlighted the importance to ensure the homobifunctional conjugation reagent was pure, especially for the preparation of protein heterodimeric conjugates. To develop this work further, it would be important to investigate three broad areas: i) improving the purity of the starting homobifunctional reagents, ii) evaluate the in vivo efficacy of the resulting protein homo-/hetero-dimers and iii) determine the overall potential for efficient scaling of the process to make the desired protein homo-/hetero-dimers.

Type: Thesis (Doctoral)
Title: Protein-Protein Conjugation using Interferon
Open access status: An open access version is available from UCL Discovery
Language: English
UCL classification: UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > UCL School of Pharmacy
URI: https://discovery.ucl.ac.uk/id/eprint/1464509
Downloads since deposit
357Downloads
Download activity - last month
Download activity - last 12 months
Downloads by country - last 12 months

Archive Staff Only

View Item View Item