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|Title||Molecular evolution of a C5aR antagonist against inflammatory disease|
Department of Immunotechnology
|Full-text||Available as PDF|
|Defence place||Room Rune Grubbsalen, Sölvegatan 23, Lund University Faculty of Engineering|
New anti-inflammatory drugs with fewer adverse effects than existing drugs may prove to be useful in the treatment of several inflammatory disorders.
A critical step in acute inflammation is the infiltration of neutrophils into tissues. Therefore, molecules that target and inhibit this early inflammatory event are attractive to engineer to suit medical needs. The Chemotaxis Inhibitory Protein of Staphylococcus aureus (CHIPS) binds and blocks the C5a receptor (C5aR) and formylated peptide receptor (FPR) and is therefore a potent inhibitor of activation and migration of neutrophils. However, as the majority of the human population has been exposed to S. aureus, pre-existing antibodies against CHIPS will be present in human sera.
This thesis is based on four original papers, with the overall aim to decrease the interaction of CHIPS with pre-existing human IgG in order to tailor it for pharmaceutical purposes. Specific IgG epitopes on the CHIPS surface were mapped by the use of phage displayed random peptide libraries. This study showed that polyclonal CHIPS specific IgGs mainly recognize conformational epitopes exposed on the surface. In addition, amino acid residues in CHIPS that are involved in this interaction, as well as in C5aR inhibition were identified.
Directed evolution is a process commonly used to improve certain protein properties without the need for detailed prior knowledge of the protein structure. This can be performed by the use of in vitro DNA recombination, a procedure by which beneficial mutations from a randomly mutated library can be recombined to generate new protein variants. In this thesis, the DNA recombination technology Fragment INduced Diversity (FIND), was applied in combination with molecular modeling and site-directed mutagenesis to generate CHIPS variants with low interaction with human IgG and retained ability to inhibit the C5aR. One CHIPS variant, designated ADC-1004, was selected for further studies. This new CHIPS variant, has high affinity for the C5aR and inhibits it efficiently, despite the truncation and seven mutations that mediate low interaction with pre-existing human IgG.
Technology and Engineering