Construction of synthetic antibodies against tumour necrosis factor alpha

Longin, Ondřej (2018) Construction of synthetic antibodies against tumour necrosis factor alpha. PhD thesis, University of Glasgow.

Abstract

This thesis describes efforts towards size reduction of a human tumour necrosis factor alpha (hTNFα)-targeting monoclonal antibody (mAb) infliximab (Remicade®) by means of construction of medium-sized antibody mimics, denoted as synthetic antibodies. The synthetic antibodies constructed by Cu(I) catalyzed azide-alkyne cycloaddition of complementarity determining region (CDR) mimics onto semi-orthogonally protected CTV scaffold derivatives have less than 4% of the molecular weight of the original monoclonal antibody infliximab (144 kDa).
The introductory Chapter 1 describes different approaches of targeting hTNFα with small, medium, and large molecules. Furthermore, it explains research undertaken in the Liskamp group which served as a basis for the research reported in this thesis.
Chapter 2 describes the development of a synthetic route for the synthesis of semi-orthogonally protected CTV scaffold derivatives. The synthetic route was applied in the synthesis of four semi-orthogonally protected CTV scaffold derivatives with different aqueous solubility-modifying spacer.
Chapter 3 focuses on the selection of CDR sequences for mimicry of the mAb infliximab. It further describes their synthesis, cyclisation, and sequential attachment to the CTV scaffold derivatives towards synthesis of synthetic antibodies.
Chapter 4 describes an evaluation of the capability of the prepared synthetic antibodies to mimic mAb infliximab. The evaluation was attempted by means of an MTT cell cytotoxicity assay, by affinity determination using isothermal titration calorimetry (ITC), and by surface plasmon resonance (SPR).
Chapter 5 focuses on the expression of a recombinant hTNFα using an E. coli expression system. Furthermore, the characterisation of the expressed hTNFα, which was used in the ITC and SPR experiments, is described.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Due to copyright issues an edited electronic version of the thesis will be made available when the embargo period expires, 20 December 2021.
• Keywords: synthetic antibody, TNF alpha, infliximab.
• Subjects: Q Science > Q Science (General); Q Science > QD Chemistry
• Colleges/Schools: College of Science and Engineering > School of Chemistry
• Supervisor's Name: Liskamp, Professor R.M.J.
• Date of Award: 2018
• Embargo Date: 20 December 2021
• Depositing User: Ing Ondřej/O Longin
• Unique ID: glathesis:2018-39025
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 04 Jan 2019 17:11
• Last Modified: 19 Mar 2019 10:08
• URI: https://theses.gla.ac.uk/id/eprint/39025