Kozakowska, KA; (2015) Site-specific peptide conjugation to carrier molecules. Doctoral thesis , UCL (University College London).

Abstract

Clinically used peptides and antibodies can be modified to improve their therapeutic potential. As peptides are relatively small molecules, they display rapid renal clearance. Modifying a peptide by conjugation to poly(ethylene glycol) (PEG) or a protein carrier, such as an antibody are clinically valid examples to make peptides therapeutically more efficacious. Peptide modification using an efficient site-selective conjugation strategy is required to prepare homogenous conjugates that if efficacious, then have the potential for development. Mono-thiol specific conjugation can be accomplished using maleimide reagents. However, maleimide based conjugates are prone to de-conjugation or exchange reactions with plasma proteins. To address this limitation a mono-sulfone PEG reagent 1 that can undergo efficient conjugation to a single cysteine thiol in a therapeutic peptide that had been developed has been evaluated with peptides. In contrast to maleimide derived conjugates, any resulting conjugate from reagent 1 can be further stabilised with sodium borohydride to prevent PEG de-conjugation by a retro-Michael reaction. A dual-agonist peptide, PEP5 underwent conjugation with reagent 1 derived from a 40 kDa PEG with 90% conversion to give the PEG40-PEP5 conjugate. The pure conjugate was obtained in 50% yield after purification by ion exchange chromatography (IEX). An in vitro receptor binding assay showed that the PEG40-PEP5 conjugate displayed 0.11-4% and 0.08-2.2% binding affinities against two relevant receptors. A mouse PK study showed the PEG40-PEP5 conjugate had a 60-fold improved circulation time (15.9 h) compared to the non-modified PEP5 (0.26 h) while also reducing blood glucose levels in obese mice. Conjugation by bis-alkylation to the two thiols that are derived from a disulfide bond is another efficient way to prepare stable and homogenous PEG-peptide conjugates. The thiol bis-alkylating PEG reagent 3 can selectively alkylate both sulfurs derived from a naturally occurring disulfide bond to form a conjugate with a three-carbon bridge between the two cysteine thiols. Octreotide (OCT) is a cyclic octapeptide with a disulfide that underwent reaction at the two cysteine thiols with the 10 kDa PEG variant of reagent 3 at 78% conversion to give a PEG10-OCT conjugate. The PEG10-OCT conjugate maintained its selectivity towards somatostatin receptor 2 (sst2) and 5 (sst5) in an in vitro receptor-binding assay. A molecular dynamics (MD) study showed that insertion of the three-carbon bridge between the cysteine thiols maintained the active structure of OCT responsible for the interactions with the somatostatin receptors. PEG10-OCT also displayed a significant inhibitory effect on IGF-1 at 24 and 48 h in an in vivo study while displaying a longer in vivo circulation time than the non-modified OCT at the same 0.3 mg/kg dose. The PEG10-OCT was detected at average 43 ng/mL and 55 ng/mL at 4 h and 8 h respectively, while OCT was below the detection limit (<10 ng/mL) at both time points. Since peptides are much smaller relative to the PEG molecule, a bis-functional PEG reagent 5 was examined to conjugate two copies of OCT to prepare OCT-PEG10-OCT conjugate. OCT-PEG10-OCT inhibited IMR-32 cell growth in vitro and was more efficient than PEG10-OCT by 29% at 0.1 µg/mL. In the MD study less PEG shielding effect was observed in OCT-PEG10-OCT than in PEG10-OCT resulting in 13% greater solvent accessible surface area (SASA) of OCT. Conjugation of a peptide to an antibody to make an antibody drug conjugate (ADC), rather than to a large molecular weight PEG, is a clinically proven strategy for increasing the efficacy of cytotoxic peptides. Colleagues used the bis-alkylating PEG reagent 3 as a basis to design two new reagents 7 and 8 with the cytotoxic peptide, monomethyl auristatin E (MMAE). These reagents are being used for the selective conjugation of MMAE to the thiols derived from the four interchain disulfide bonds in an IgG1 to produce more homogenous and stable ADCs. The linker between the MMAE and the antibody must be stable in circulation to avoid systemic toxicity upon premature release of the payload. Strategies were developed to purify a model ADC, prepared by bis-alkylation to evaluate the antibody-drug ratio (DAR) and its in vivo stability. A protein precipitation method was developed that with LC-MS analysis enabled the detection of unconjugated MMAE reagent 7 species below 1% (wt) in the purified ADC at <1 mg scale. An affinity capture method was also developed to purify trastuzumab-based ADCs from animal serum from an in vivo study. Hydrophobic interaction chromatography (HIC) enabled analysis of the DAR profile of these ADCs on <2 µg scale. Finally HER2 which is a trastuzumab-specific antigen was efficiently biotinylated and coated onto magnetic beads with >99% efficiency. The functionalised beads allowed a 80% recovery of trastuzumab-PEG(24u)-val-cit-PAB-MMAE conjugate from mouse serum. The DAR profile of the purified ADC conjugate was assessed by HIC and showed there was drug loss (~30%) possibly being due to instability of the val-cit-PAB cleavable linker in blood circulation but not de-conjugation as shown by mass spectrometry analysis.

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