Structure, properties and biotechnology applications of VHH antibody fragments from camelids

Información del proyecto

Identificador del acuerdo de subvención: BIO4980048

Proyecto cerrado

Fecha de inicio 1 Octubre 1998

Fecha de finalización 30 Septiembre 2000

Financiado con arreglo a

• Specific research, technological development and demonstration programme in the field of biotechnology, 1994-1998

Coste total

Sin datos

Aportación de la UE

Sin datos

Coordinado por

FLANDERS INTERUNIVERSITY INSTITUTE FOR BIOTECHNOLOGY VZW
Belgium

Objetivo

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.

Ámbito científico (EuroSciVoc)

• ciencias médicas y de la salud › medicina básica › inmunología › inmunización
• ciencias naturales › ciencias biológicas › bioquímica › biomoléculas › proteínas › plegamiento de proteínas
• ciencias naturales › ciencias químicas › química orgánica › aminas
• ciencias naturales › ciencias biológicas › biología molecular › evolución molecular
• ciencias naturales › ciencias biológicas › bioquímica › biomoléculas › proteínas › enzima

Programa(s)

• FP4-BIOTECH 2 - Specific research, technological development and demonstration programme in the field of biotechnology, 1994-1998

Tema(s)

• 0601 - Structure-function relationships

Convocatoria de propuestas

Datos no disponibles

Régimen de financiación

CSC - Cost-sharing contracts

Coordinador

FLANDERS INTERUNIVERSITY INSTITUTE FOR BIOTECHNOLOGY VZW

• Aportación de la UE: Sin datos
• Dirección: 65,Paardenstraat 65 VUB ; 1640 RHODE-ST-GENESE ; Bélgica
• Coste total: Sin datos

Participantes (4)

CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE

Francia

• Aportación de la UE: Sin datos
• Dirección: 31,Chemin Joseph Aiguier 31 ; 13402 MARSEILLE
• Coste total: Sin datos

THE CHANCELLOR, MASTERS AND SCHOLARS OF THE UNIVERSITY OF OXFORD

Reino Unido

• Aportación de la UE: Sin datos
• Dirección: South Parks Road ; OX1 3QT OXFORD
• Coste total: Sin datos

Unilever Nederland BV

Países Bajos

• Aportación de la UE: Sin datos
• Dirección: 120,Olivier van Noortlaan ; 3133 AT Vlaardingen
• Coste total: Sin datos

Universität Zürich

Suiza

• Aportación de la UE: Sin datos
• Dirección: 190,Winterhurerstraße ; 8057 Zürich
• Coste total: Sin datos