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Title: Site-selective conjugation at native amino acids
Author: Peciak, K.
ISNI:       0000 0004 5352 0843
Awarding Body: University College London (University of London)
Current Institution: University College London (University of London)
Date of Award: 2014
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The efficacy of protein-based medicines is frequently compromised by rapid clearance from circulation. Achieving prolonged and optimal pharmacokinetics (PK) is necessary to avoid dose dumping while ensuring that a therapeutic concentration can be maintained between each time the the dose is administered. Optimised PK properties are important for therapeutic proteins, such as interferon alpha 2a (IFN) which is used to treat infections to ensure complete pathogen kill. The conjugation of poly(ethylene glycol) (PEG) to a protein, which is known as protein PEGylation, is a clinically proven strategy to increase the circulation half-life of proteins. However, most clinically used PEGylated proteins, including PEGylated IFN α-2, are heterogeneous mixtures comprised of positional conjugation isomers. There is a genuine need to develop efficient strategies for site-specific and site-selective PEGylation to obtain more homogeneous PEGylated products. The work described in this thesis examines the use of bis-alkylation PEGylation reagents (i) to site-specifically conjugate PEG to IFN consensus (IFN-con) at the two cysteine thiols in either of its two native disulfides and (ii) to site-selectively conjugate PEG at two histidines which have been added or substituted into the IFN structure. Initial work focused on PEGylation at the existing disulfides of IFN-con which is approved for the treatment of HCV patients who have failed first line treatment of PEG-IFN-α/ribovarin. IFN-con is more potent than IFN α-2 and PEGylated IFN-con is not clinically available. Disulfide PEGylation of IFN-con was then conducted using just 1 molar equivalent of the bis-alkylation PEG reagent (20 kDa PEG or 2×20 kDa PEG), which yielded 28–57% conversion to the mono-PEGylated products. Purified PEG-IFN-con displayed 3-fold higher activity than the clinically used PEG-IFN α-2a (Pegasys®). These results support continued investigation of disulfide PEGylated IFN-con as a potential long-acting treatment for HCV non-responders. While utilising the native disulfides for site specific conjugation is an improvement over the existing methods for PEGylating IFN, it was hypothesised that bis-alkylation PEGylation could be accomplished by conjugation to two histidines at a slightly acidic pH. This second strategy would therefore provide a means to engineer the site of conjugation within IFN (i.e. siteselective PEGylation). Bis-alkylation PEGylation was investigated with IFN alpha-2a that had been herein focused on confirmation by either trypsin or CNBr digestion that the site of PEGylation on IFN alpha-2a was achieved at the His8-tag. To achieve true site-selectivity it was hypothesised that two histidines as a conjugation site could be rationally added to a region of IFN alpha-2a distant from the receptor binding site. This method could potentially be applied to any protein and avoids challenges associated with engineering a free cysteine or incorporation of a non-native amino acid to achieve site-selectivePEG conjugation. Genes encoding for IFN alpha-2a analogs were constructed by site-directed mutagenesis and IFN alpha-2a variants were successfully expressed as soluble and active protein in identical yields as the production of native IFN alpha-2a. Site-selective PEGylation at the engineered histidines was as efficient as PEGylation at the N-terminal His8-tag. The use of the bis-alkylation PEG conjugation reagent proceeded with a similar efficiency that is generally observed for other amine conjugation methods. A good level of biological activity was retained for all conjugates produced (ranging from 10-75%). A IFN -2a variant that had been PEGylated at position 106 was produced in the highest yields (33%) and displayed the highest level of bioactivity (75%),which is greater than any reported PEGylated conjugate of IFN. In contrast, Pegasys® which is the most widely used PEG-IFN conjugate in the clinic has an activity of ~7%.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available