Engineering functional recombinant proteins based on antibody domains and fragments of C. botulinum neurotoxin

Genetic engineering allows the construction of many diverse antibody-related molecules. The focus of this study is single chain Fv (Fragment variable) and single domain antibodies. Recombinant botulinum neurotoxin fragments derived from serotype A, B, C and D are selected as backbones for an investigation into the feasibility and properties of novel recombinant targeted multidomain proteins. Single chain Fv’s consist of a VH region of heavy chain linked by a stretch of synthetic peptide to a VL region of the light chain. Fv is the region for binding to antigens as determined by immunoglobulin Ig hypervariable domains called complementary-determining regions (CDR’S). The selected scFv binds to CD117 that is a membrane tyrosine kinase receptor present on the surface of various tumour cells. The variable part of chains shows great variability in amino acid sequence among the chains. The unique sequence of amino acid residues for variable fragments leads to the large diversity of structure, which counts for antibody specificity. For the creation of single domain antibodies (sdAbs), where the antibody domain is engineered to comprise only the variable heavy or variable light chain (and therefore consist of three CDR’s), six of the anti-EGFR sdAbs have been chosen in order to obtain variety of data. All botulinum neurotoxin backbones include an activation site, which is cleaved by a protease such as EK (Enterokinase) or FXa (Factor Xa) at specific residues within the sequence. The contrast in molecule activation will distinguish the best candidates. It was decided that targeting the tyrosine kinase receptor family was most suited to this research because (i) there is significant knowledge in the literature regarding receptor biology; (ii) ligands such as epidermal growth factor (EGF) which bind to EGFR were of importance to Syntaxin Ltd, (iii) growth factors (including EGF) are involved in tumour maintenance and growth, therefore many tumour cells express high levels of EGFR; and (iv) antibodies to EGFR are promising leads in cancer biology. There are now 26 successfully cloned clostridial endopeptidase-antibody fragment/ antibody domain constructs and six full-length high quality proteins available for further investigation.

Grantor dc:publisher

Oxford Brookes University

Year dc:date

2010

Author dc:creator

• Tolkacz, J

Statement dc:rights

• All rights reserved

Language dc:language

en

Repository record dc:identifier

https://radar.brookes.ac.uk/radar/items/e0e6e5ad-c44b-42e9-b17b-cb420941575a/1/

OAI identifier oai:identifier

tle:e0e6e5ad-c44b-42e9-b17b-cb420941575a:d6bd9758-527a-46cd-bfe2-c433766e8fca:1