Objective
Since the development of (monoclonal) antibody technology, there have been major efforts to (de)construct or engineer these biomolecules to obtain smaller binding units that retained the specificity and affinity of classical antibodies. Two-domain variable fragments (Fv) or single-chain variable fragments (scFv) have been introduced, but large scale production of these fragments often led to accumulation of insoluble protein. A further decrease in size has been achieved by generating minibodies or single-domain VH fragments. The low solubility and tendency to aggregate of many such fragments, probably due to exposure to solvent of the hydrophobic surface normally facing the VL domain and/or the constant domain, have impaired the scale production. The naturally occurring antibodies devoid of a light chain and of a CH1 domain discovered in camelids constitute a promising and novel alterative in this domain, and represent a potentially valuable tool for applications in biotechnology.
Following immunization of camels or llama and subsequent cloning of the variable domains of these heavy chain antibodies, antigen-specific recombinant heavy chain variable fragment (VHHs) can be produced as highly soluble monomers in heterologous organisms such as E. coli and yeast. Moreover, naive and/or dedicated synthetic libraries containing the VHH-repertoire can be constructed, from which specific binders can directly be selected. Recombinant VHHs have already been raised against both proteins and haptens, and these minimal-sized recognition units were characterised by a good stability and solubility and an affinity range of 10-7 to 10-8 M. Moreover, VHHs have been generated that were capable of specifically inhibiting the activity of their enzyme-antigen. Structural analysis of complexes of an anti-lysozyme-VHH with the enzyme revealed that the CDR3 region of the VHH, which can be up 20-30 amino acid of length, protrudes from the VHH-surface and penetrates deeply into the active site of the enzyme. This unique potential of camelid VHHs as highly specific and selective enzyme inhibitor constitutes an additional feature of great biotechnological promise.
The goal of this project is to fully characterise the structure/function relationships of this new class of immunoglobulin fragments. VHHs will be generated against different types of antigens, and the nature and structure of the antibody-antigen interfaces will be investigated in detail using X-ray and/or NMR methods. In addition, individual VHHs and complexes of VHHs with proteins and haptens will be further characterised in terms of stability, affinity and function (enzyme inhibition, scavenging of undesirable compounds,...). These characteristics will also be assessed under the harsh conditions of industrial processes and consumer products.
This approach should allow to draw important conclusions on the parameters that yield good stability, good expression and folding as well as high affinity, and as such make camelid-VHHs particularly suited for various biotechnological applications. The acquired insights can further be used to transplant selected features of camelid-VHH on the framework of other antibodies of interest (i.e. "camelization"). Protein engineering techniques and molecular evolution will be adopted to create bispecifc and/or multivalent constructs, as well as to generate disulphide-free fragments which can be used for intracellular expression. Finally, these fragments will be used as a tool to study protein folding. Indeed, VHHs have been generated that strongly interact with the region on Iysozyme, where the folding nucleation centre is located. This should allow to explore the role of VHHs as folding assistants, which may have important implications for those pathologies that result from a folding anomaly.
Fields of science (EuroSciVoc)
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques.
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques.
- medical and health sciencesbasic medicineimmunologyimmunisation
- natural sciencesbiological sciencesbiochemistrybiomoleculesproteinsprotein folding
- natural scienceschemical sciencesorganic chemistryamines
- natural sciencesbiological sciencesmolecular biologymolecular evolution
- natural sciencesbiological sciencesbiochemistrybiomoleculesproteinsenzymes
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Call for proposal
Data not availableFunding Scheme
CSC - Cost-sharing contractsCoordinator
1640 RHODE-ST-GENESE
Belgium