CO 054: AFFINITY MATURATION AND FINE FUNCTIONAL MAPPING OF AN ANTIBODY FRAGMENT AGAINST A NOVEL NEUTRALIZING EPITOPE ON HUMAN VASCULAR ENDOTHELIAL GROWTH FACTOR

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J Pharm Pharmacogn Res 2(Suppl. 1): S33, 2014 Special supplement with the abstract book of LATINFARMA 2013 Oral Communication CO 054: AFFINITY MATURATION AND FINE FUNCTIONAL MAPPING OF AN ANTIBODY FRAGMENT AGAINST A NOVEL NEUTRALIZING EPITOPE ON HUMAN VASCULAR ENDOTHELIAL GROWTH FACTOR Lamdan Ha, Gavilondo JVa, Muñoz Ya, Pupo Ab, Huerta Va, Musacchio Aa, Pérez … Continue reading CO 054: AFFINITY MATURATION AND FINE FUNCTIONAL MAPPING OF AN ANTIBODY FRAGMENT AGAINST A NOVEL NEUTRALIZING EPITOPE ON HUMAN VASCULAR ENDOTHELIAL GROWTH FACTOR

J Pharm Pharmacogn Res 2(Suppl. 1): S33, 2014

Special supplement with the abstract book of LATINFARMA 2013

Oral Communication

CO 054: AFFINITY MATURATION AND FINE FUNCTIONAL MAPPING OF AN ANTIBODY FRAGMENT AGAINST A NOVEL NEUTRALIZING EPITOPE ON HUMAN VASCULAR ENDOTHELIAL GROWTH FACTOR

Lamdan Ha, Gavilondo JVa, Muñoz Ya, Pupo Ab, Huerta Va, Musacchio Aa, Pérez La, Ayala Ma, Rojas Gb, Balint RFc, Larrick JWd.

aCenter for Genetic Engineering and Biotechnology, La Habana 10600, Cuba. Email: humberto.lamdan@cigb.edu.cu
bCenter of Molecular Immunology, La Habana 11600, Cuba.
cCytoDesign, Inc., Palo Alto, CA 94306, USA.
dPanorama Research Inc., Sunnyvale, CA 94089, USA.
Abstract

Vascular endothelial growth factor (VEGF) is a major mediator of pathological angiogenesis. VEGF antagonists represent attractive candidates as therapeutic agents in the treatment of tumors and other angiogenesis-dependent diseases. Despite the clinical success of Bevacizumab, a humanized monoclonal antibody that blocks the interaction between VEGF and its receptors, the search for new neutralizing antibodies targeting this molecule has continued until now. We used a human VEGF variant containing three mutations in the region recognized by Bevacizumab to direct antibody selection towards recognition of other epitopes. A total of seven phage-displayed antibody fragments were obtained from a human phage display library. All of them were able to recognize not only the selector mutated antigen, but also native VEGF. One of these phage-displayed antibody fragments, denominated 2H1, was shown to compete with the VEGF receptor 2 for VEGF binding. Soluble 2H1 inhibited VEGF biological activity but exhibited a moderate binding affinity. We performed the affinity maturation of 2H1 antibody fragment. Two phage-displayed libraries were constructed by diversification of the third complementarity-determining regions (CDRs) of the light (VL) and heavy (VH) chain variable domains of 2H1 using parsimonious mutagenesis. A competitive phage-selection strategy in the presence of 2H1 as a competitor was used to eliminate low affinity binders. High affinity variants were retrieved from both libraries. An optimized VL variant was constructed by combining recurrent replacements found among selected variants, resulting in an additional affinity increase. Further affinity improvements were achieved by combining this optimized VL with the best VH variants. The final variant, L3H6, showed an overall affinity improvement of 18-fold over the parental antibody and exhibited an enhanced potency to block the binding of VEGF to its receptor. Functional mapping studies of L3H6 using phage display and extensive mutagenesis of VEGF revealed a novel neutralizing epitope on human VEGF.

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