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Engineering Antibody Specificity Using Combinatorial Antibody Libraries

Jenny Boström (Institutionen för kemi- och bioteknik)
Göteborg : Chalmers University of Technology, 2009. ISBN: 978-91-7385-332-3.- 89 s.
[Doktorsavhandling]

The research presented in this thesis investigates antibody specificity, antibody-antigen and protein-protein interactions using combinatorial antibody libraries, phage-display and bacterial display technology. One goal was to develop a method to generate antibodies that bind two protein antigens with high affinity using a single-antigen binding site, and to investigate the molecular and thermodynamic requirements for antibody multispecificity. The thesis describes how naïve combinatorial antibody libraries with diversity in the light chain can be used to generate antibodies of different antigen-binding specificities. A variant of the HERCEPTIN® antibody, a therapeutic monoclonal antibody that binds the human epidermal growth factor receptor 2 (HER2), was isolated on the basis of its ability to simultaneously interact with vascular endothelial growth factor (VEGF). Crystallographic and mutagenesis studies revealed that distinct amino acids of this antibody, called bH1, engage HER2 and VEGF energetically, but there is extensive overlap between the antibody surface areas contacting the two antigens. By selectively mutating residues of the complementarity determining regions (CDRs), the binding affinity of bH1 was improved for both antigens 100-fold (KD = 0.2 nM and 3 nM for HER2 and VEGF, respectively). This affinity-improved version of bH1 specifically inhibits both HER2- and VEGF-mediated cell proliferation in vitro and tumor progression in mouse models with activity similar to the Herceptin® antibody and the anti-VEGF antibody bevacizumab, respectively. While the structural and mutagenesis studies highlight the differences of the interfaces between the dual specific antibody and its two antigens in surface topology and energetic engagement of CDR residues, the two interactions share a common thermodynamic signature: favorable entropy and enthalpy. The positive entropy change upon binding and large negative heat capacities suggests that the hydrophobic effect plays an important role in the dual specific interactions. Finally we show that by mutation of merely two residues in the CDRs, the high affinity dual specific antibodies can be converted back to essentially monospecific HER2 or VEGF antibodies. The described dual specific “two-in-one” antibodies challenge the paradigm of monoclonal antibodies being one binding site/one antigen, and they could provide new opportunities for antibody-based therapies.

Nyckelord: Dual Specific Antibodies, therapeutic Antibody Engineering, Synthetic Phagedisplayed Antibody Libraries, Vascular Endothelial Growth Factor, Human Epidermal Growth Factor Receptor 2, Herceptin, trastuzumab, antibody/Antigen Interactions, Thermodynamic Analysis, Bacterial Display



Denna post skapades 2009-11-11. Senast ändrad 2013-09-25.
CPL Pubid: 101474

 

Institutioner (Chalmers)

Institutionen för kemi- och bioteknik (2005-2014)

Ämnesområden

Molekylärbiologi

Chalmers infrastruktur

Examination

Datum: 2009-11-20
Tid: 09:15
Lokal: 10:an, Kemihuset, Kemigården 4
Opponent: Dr Adnane Achour, Karolinska Institutet, Stockholm

Ingår i serie

Doktorsavhandlingar vid Chalmers tekniska högskola. Ny serie 3013