ANTIBODIES TO INFLIXIMAB IN PATIENTS WITH INFLAMMATORY BOWEL DISEASE: CLINICAL AND MOLECULAR TARGETS

Infliximab was the first biological therapy to be approved for use in inflammatory bowel disease (IBD). Similar to other biological therapies, secondary loss of response to infliximab occurs in up to 50% of patients at 1 year follow up. In the case of infliximab, antibodies to infliximab (ATI) are likely to contribute towards secondary loss of response. This PhD thesis aims to identify the associated clinical outcomes of ATI, assess a strategy of infliximab dose escalation (DE) and to identify epitopes on infliximab using phage display. In a retrospective review of 214 patients, ATI were detected in 64% of patients during the first year of infliximab treatment. They were significantly associated with loss of clinical response, reduced infliximab levels and adverse drug reactions. Assessment of infliximab DE in 92 patients with subtherapeutic drug levels, showed it was effective at significantly increasing infliximab levels in those with and without ATI. DE was also associated with a high rate of clinical remission at 6 months. Pooled sera from eight patients with positive ATI and differing clinical outcomes was used for biopanning of two phage display libraries. Three distinct linear epitopes were heavily enriched from a novel whole gene fragment library (GFL), all mapped to complementarity determining regions of infliximab. Using a random peptide library (RPL), three different peptides were enriched. Using Pepsurf, a mimotope prediction programme, three conformational epitopes were predicted using the enriched peptides. One of the predicted epitopes from the RPL overlapped with a linear epitope selected from the GFL. In increasing our understanding of immunogenicity to infliximab, this may lead to an identification of an epitope associated with loss of response, a biomarker, and will help aid treatment decisions when ATI are detected

Mineral binding peptides by phage display: experimental and bioinformatics studies

Phage display has attracted a great deal of interest in the identification of peptides specific to nanomaterials revealing distinctive binding behaviour. Though significant progress has been made in selecting and screening of biomolecule binding peptides, the accuracy of molecular recognition for inorganic materials is still challenging due to the limitations of phage display libraries and biopanning process. The study presented in this thesis is aimed at isolating mineral binding peptides by phage display and verifying them experimentally and/ or bioinformatically; exploring the role of electrostatic/ non-electrostatic interactions in the aqueous phase and the factors responsible for the adsorption or desorption of peptide or phage from the mineral surface. Firstly, silica binding peptides LPVRLDW, NDLMNRA, GQSEKHL and GASESYL have been identified using the phage display technique by varying experimental conditions including pH, detergent, washing and elution buffers to remove unique 7-mer peptide binding phages from amorphous hydrophilic silica nanoparticles via disruption of the molecular interactions between the phage attached peptides and the nanoparticles. A repanning method reported here, has experimentally reproduced the majority of the initially discovered silica binders; alongside identifying/ recovering additional peptide sequences HYIDFRW, KIAVIST and YSLKQYQ that may have been overlooked in the routine approach to biopanning. Secondly, an alternative three step elution method reported here, has eluted and recovered most target silica binders including ADIRHIK in the early panning rounds and removed the phage clones that are bound to silica by hydrophobic, hydrogen bonding and electrostatic attractions or repulsions; as opposed to one specific buffer being used for all panning rounds including elution steps in traditional biopanning experiments. Also, the phage clones that resist detection to single elution step have been eluted in the other successive elution steps, thereby recovering and improving the elution procedure for silica surfaces. In addition, these three different elution buffers have eluted phage clones that are interaction or charge specific subject to change in the elution buffer pH condition. The experimental results demonstrate that this sequential three step elution process was able to isolate tightly bound target silica binders in one or two biopanning rounds than the more typical four to five; thereby reducing biopanning rounds, cost and effort. Moreover, the bioinformatic analysis to cross check the authenticity/ quality of target binders has been reported. Furthermore, selected silica binding peptides isolated from phage display experiments were synthesized by a solid phase peptide synthesis approach and peptide-silica interactions explored in vitro, using quantitative and qualitative techniques. The fluorometric analysis of these peptides revealed that the peptide adsorption to silica surfaces would have more than one type of interactions (i.e. electrostatic/ hydrophobic/ H-bonding and Van der Waals) and could be influenced by the experimental conditions. More significantly, an increase in binding activity to negatively charged silica nanoparticles was noticed for the peptides (HYIDFRW, KIAVIST and YSLKQYQ) modified with an amide (NH2) group as opposed to a carboxyl group at the C-terminal end; driving an increase in overall charge or pI of the peptides. Insights from the studies presented may provide valuable information for designing and engineering of silica directed constructs for a range of biomedical and nanotechnological applications

Epitope Mapping of Metuximab on CD147 Using Phage Display and Molecular Docking

Metuximab is the generic name of Licartin, a new drug for radioimmunotherapy of hepatocellular carcinoma. Although it is known to be a mouse monoclonal antibody against CD147, the complete epitope mediating the binding of metuximab to CD147 remains unknown. We panned the Ph.D.-12 phage display peptide library against metuximab and got six mimotopes. The following bioinformatics analysis based on mimotopes suggested that metuximab recognizes a conformational epitope composed of more than 20 residues. The residues of its epitope may include T28, V30, K36, L38, K57, F74, D77, S78, D79, D80, Q81, G83, S86, N98, Q100, L101, H102, G103, P104, V131, P132, and K191. The homology modeling of metuximab and the docking of CD147 to metuximab were also performed. Based on the top one docking model, the epitope was predicted to contain 28 residues: AGTVFTTV (23–30), I37, D45, E84, V88, EPMGTANIQLH (92–102), VPP (131–133), Q164, and K191. Almost half of the residues predicted on the basis of mimotope analysis also appear in the docking result, indicating that both results are reliable. As the predicted epitopes of metuximab largely overlap with interfaces of CD147-CD147 interactions, a structural mechanism of metuximab is proposed as blocking the formation of CD147 dimer