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In the quest to discover new research tools and to develop better agents in the fight against cancer, two antibodies, G6 and B20-4, were isolated from synthetic antibody phage libraries. Unlike the AVASTIN™ antibody, a recently approved agent for the treatment of patients with colorectal cancer, B20-4 and G6 bind and block both human and murine vascular endothelial growth factor (VEGF). Here we have analyzed and compared the binding epitopes on VEGF for these three antibodies using alanine-scanning mutagenesis and structural analyses. The epitopes recognized by both synthetic antibodies are conserved between human and mouse VEGF, and they match closely to the receptor epitopes both structurally and functionally. In contrast, the Avastin epitope overlaps minimally with the receptor binding surface and centers around a residue that is not conserved in mouse. Our structural and functional analyses elucidate the cross-species reactivity of all three antibodies and emphasize the potential advantages of antibody generation using phage display as the resulting antibodies do not depend on sequence differences across species and preferentially target natural protein-protein interaction surfaces. In the quest to discover new research tools and to develop better agents in the fight against cancer, two antibodies, G6 and B20-4, were isolated from synthetic antibody phage libraries. Unlike the AVASTIN™ antibody, a recently approved agent for the treatment of patients with colorectal cancer, B20-4 and G6 bind and block both human and murine vascular endothelial growth factor (VEGF). Here we have analyzed and compared the binding epitopes on VEGF for these three antibodies using alanine-scanning mutagenesis and structural analyses. The epitopes recognized by both synthetic antibodies are conserved between human and mouse VEGF, and they match closely to the receptor epitopes both structurally and functionally. In contrast, the Avastin epitope overlaps minimally with the receptor binding surface and centers around a residue that is not conserved in mouse. Our structural and functional analyses elucidate the cross-species reactivity of all three antibodies and emphasize the potential advantages of antibody generation using phage display as the resulting antibodies do not depend on sequence differences across species and preferentially target natural protein-protein interaction surfaces. Angiogenesis, the process of new blood vessel formation, is not only essential for many physiologically important events but is also critical in a number of diseases such as tumor progression, diabetic retinopathy, and rheumatoid arthritis (1Folkman J. Nat. Med. 1995; 1: 27-31Crossref PubMed Scopus (7235) Google Scholar), (2Ferrara N. Curr. Opin. Biotechnol. 2000; 11: 617-624Crossref PubMed Scopus (356) Google Scholar). Starvation of tumors by inhibition of angiogenesis has been discussed for a number of years as a strategy in the fight against cancer, and dozens of anti-angiogenesis drugs are currently being tested in clinical trials (3Marx J. Science. 2003; 301: 452-454Crossref PubMed Scopus (79) Google Scholar). Vascular endothelial growth factor (VEGF) 3The abbreviations used are: VEGF, vascular endothelial grown factor; VEGFR, VEGF receptor; h-VEGF, human VEGF; m-VEGF, mouse VEGF; CDR, complement-determining region. is one of the key players in angiogenesis (4Ferrara N. Semin. Oncol. 2002; 29: 10-14Crossref PubMed Google Scholar). Blocking the activity of VEGF using specific antibodies or other protein entities to prevent VEGF binding and signaling through its receptors has been one of many approaches to reduce tumor growth (5Kim K.J. Li B. Winer J. Armanini M. Gillett N. Phillips H.S. Ferrara N. Nature. 1993; 362: 841-844Crossref PubMed Scopus (3363) Google Scholar, 6Prewett M. Huber J. Li Y. Santiago A. O'Connor W. King K. Overholser J. Hooper A. Pytowski B. Witte L. Bohlen P. Hicklin D.J. Cancer Res. 1999; 59: 5209-5218PubMed Google Scholar, 7Gerber H.P. Kowalski J. Sherman D. Eberhard D.A. Ferrara N. Cancer Res. 2000; 60: 6253-6258PubMed Google Scholar, 8Konner J. Dupont J. Clin. Colorectal Cancer. 2004; 4: S81-S85Abstract Full Text PDF PubMed Scopus (82) Google Scholar). Recently, the AVASTIN™ antibody (Bevacizumab), an antibody that binds human VEGF with high affinity, was approved for treating colorectal cancer patients (9Hurwitz H. Clin. Colorectal Cancer. 2004; 4: S62-S68Abstract Full Text PDF PubMed Scopus (142) Google Scholar). This study for the first time validated an approach by which tumor starvation is induced through inhibition of VEGF. VEGF (also called VEGF-A) and the members of the VEGF family such as VEGF-B (10Olofsson B. Pajusola K. Kaipainen A. von Euler G. Joukov V. Saksela O. Orpana A. Pettersson R.F. Alitalo K. Eriksson U. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2576-2581Crossref PubMed Scopus (629) Google Scholar), VEGF-C (11Joukov V. Pajusola K. Kaipainen A. Chilov D. Lahtinen I. Kukk E. Saksela O. Kalkkinen N. Alitalo K. EMBO J. 1996; 15: 1751Crossref PubMed Scopus (393) Google Scholar), VEGF-D (12Achen M.G. Jeltsch M. Kukk E. Makinen T. Vitali A. Wilks A.F. Alitalo K. Stacker S.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 548-553Crossref PubMed Scopus (1020) Google Scholar), and PlGF (13Maglione D. Guerriero V. Viglietto G. Delli-Bovi P. Persico M.G. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9267-9271Crossref PubMed Scopus (843) Google Scholar) mediate their biological function by binding with different specificities to the three receptor tyrosine kinases VEGFR1, (also called flt-1) (14Shibuya M. Yamaguchi S. Yamane A. Ikeda T. Tojo A. Matsushime H. Sato M. Oncogene. 1990; 5: 519-524PubMed Google Scholar), VEGFR2 (also called KDR) (15Terman B.I. Dougher-Vermazen M. Carrion M.E. Dimitrov D. Armellino D.C. Gospodarowicz D. Bohlen P. Biochem. Biophys. Res. Commun. 1992; 187: 1579-1586Crossref PubMed Scopus (1405) Google Scholar), and VEGFR3 (16Pajusola K. Aprelikova O. Korhonen J. Kaipainen A. Pertovaara L. Alitalo R. Alitalo K. Cancer Res. 1992; 52: 5738-5743PubMed Google Scholar). Structural and functional studies have identified the receptor binding domain of VEGF (residues 8-109 fragment) as an anti-parallel homodimer (17Muller Y.A. Christinger H.W. Keyt B.A. de Vos A.M. Structure. 1997; 5: 1325-1338Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 18Muller Y.A. Li B. Christinger H.W. Wells J.A. Cunningham B.C. de Vos A.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7192-7197Crossref PubMed Scopus (368) Google Scholar) that binds to VEGFR1 and VEGFR2 with two binding sites that are located on the opposite poles of the homodimer and are separated by >40 Å (19Wiesmann C. Fuh G. Christinger H.W. Eigenbrot C. Wells J.A. de Vos A.M. Cell. 1997; 91: 695-704Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar). The extracellular domain of the VEGF receptors consists of 7 immunoglobulin (Ig)-like domains with domains 2 and 3 being responsible for high affinity binding of VEGF. Alanine-scanning analyses and the crystal structures of VEGF in complex with the Fab fragment of the Avastin antibody (18Muller Y.A. Li B. Christinger H.W. Wells J.A. Cunningham B.C. de Vos A.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7192-7197Crossref PubMed Scopus (368) Google Scholar) and with the second domain of VEGFR1 (19Wiesmann C. Fuh G. Christinger H.W. Eigenbrot C. Wells J.A. de Vos A.M. Cell. 1997; 91: 695-704Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar) showed that the binding epitopes on VEGF for the receptor and for the Avastin Fab are distinct and only partially overlapping. The most likely mechanism by which active Avastin antibodies inhibit receptor binding is steric hindrance (18Muller Y.A. Li B. Christinger H.W. Wells J.A. Cunningham B.C. de Vos A.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7192-7197Crossref PubMed Scopus (368) Google Scholar). Highly selective, neutralizing antibodies play an essential role in the quest to decipher the specific role of VEGF among its family members with overlapping activities. Therefore, besides its use as a drug in fighting cancer, Avastin could be a very important reagent to investigate angiogenic processes and the exact function of VEGF and its receptors. However, Avastin is specific to human VEGF and does not bind, for example, murine VEGF; thus its use for studies in mouse models is rather limited. The Avastin antibody is the humanized version of the monoclonal antibody A.4.6.1, which was derived using hybridoma technology by immunizing mice with human VEGF (5Kim K.J. Li B. Winer J. Armanini M. Gillett N. Phillips H.S. Ferrara N. Nature. 1993; 362: 841-844Crossref PubMed Scopus (3363) Google Scholar). This traditional approach excludes the possibility of producing antibodies that bind to murine VEGF, as the immune response in the mouse prohibits the generation of self-reactive antibodies. Thus, with this approach antibodies can be developed that only target binding areas with sequence differences in the proteins between host and target. One solution to this limitation of the traditional method of raising monoclonal antibodies is a new technique that utilizes phage display of synthetic antibody libraries to find antibodies with high against of Eigenbrot C. Fuh G. J. 2004; PubMed Scopus Google Scholar). The synthetic antibody used in the approach in Eigenbrot C. Fuh G. J. 2004; PubMed Scopus Google Scholar) was on the of a humanized antibody against human growth factor receptor 2 C. M. L. P. J. 1993; PubMed Scopus Google Scholar). In this process a number of antibodies that bind murine VEGF with high affinity were of these antibodies, G6 and B20-4, with in their complement-determining bind human and mouse VEGF with high W. C. A. H. and N. J. This both antibodies research tools as they do not the of the Avastin antibody, which is to block the function of murine VEGF. G6 and B20-4 are to bind both mouse and human VEGF the Avastin antibody is we alanine-scanning mutagenesis of VEGF to the functional binding sites of both antibodies and to with the Avastin binding In we the crystal structures of VEGF in complex with the and the to structural for high affinity binding of both antibodies. Our that G6 and B20-4 VEGF a that is by the of this surface for drug This epitope only partially overlaps with the binding of the Avastin binding is conserved in the protein of human and mouse VEGF. have that G6 a in its but a in the of binding of the or VEGF VEGF were using VEGF phage as (18Muller Y.A. Li B. Christinger H.W. Wells J.A. Cunningham B.C. de Vos A.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7192-7197Crossref PubMed Scopus (368) Google Scholar). human VEGF (residues in was using the mutagenesis and the were on of phage VEGF were to that were with B20-4, murine of the Avastin or domain of The phage were using to an antibody on this a of phage was to with of G6 or B20-4 Fab in binding with and for the were to and for to VEGF The phage to the were using the antibody as were as the of Fab resulting in The of the of VEGF the of the binding for structural of VEGF with binding a of the tested antibodies, we the with a binding of binding as in the VEGF have that binding G6 or B20-4 and we the structural of these by their to bind or and receptor binding fragment of human VEGF 8-109 were and as H.W. Y.A. Keyt B.A. Cunningham B.C. Ferrara N. de Vos A.M. 1996; PubMed Scopus Google Scholar). G6 and were in and as Y.A. Y. Christinger H.W. Li B. Cunningham B.C. de Vos A.M. Structure. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). between Fab and were by the in a and as Y.A. Y. Christinger H.W. Li B. Cunningham B.C. de Vos A.M. Structure. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). of the and the were using the method in of and was in with protein solution 3 and to with of a Å and crystal one complex a VEGF and two Fab in the were and in were of the of the with and was in with protein solution in and to The were in and 2 and in to Å the on of the in its were from protein that was using a in and The protein was and used in used for the were grown in by of protein solution and and to with a and were in and in the by the for the of the to Å were the of the and were using and W. 1997; PubMed Scopus Google Scholar). The of the and the were using J. A. Scopus Google Scholar), using the of VEGF from a complex and Fab the domains or the domains of the complex The of in its was using of the as and were using D. PubMed Scopus Google Scholar) and M. A. 1991; PubMed Scopus Google Scholar), of of the in its is not its in the crystal is The of the crystal Fab the and the domains of all but the of the and from to and around and In the crystal the domains of the three around a in the of this is the but all other of both the and the to this as The resulting has the of a rather with the domains in different of these one of a the complex has a of and the of the is to a with a of Å of VEGF the and binds and with a of and Eigenbrot C. Fuh G. J. 2004; PubMed Scopus Google Scholar). first the functional binding epitope of VEGF the using alanine-scanning mutagenesis of of (17Muller Y.A. Christinger H.W. Keyt B.A. de Vos A.M. 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J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) The binds and with a of and Alanine-scanning mutagenesis that the important binding compared with the G6 as only 7 to a in binding affinity to However, of binding and in a of in binding are located on the of VEGF and residue is on the The functional binding epitopes for B20-4 and G6 as by alanine-scanning mutagenesis very with of the against G6 being also very important for B20-4 binding the structural binding and to binding of G6 and to of VEGF binding Y.A. Y. Christinger H.W. Li B. Cunningham B.C. de Vos A.M. Structure. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, Y. C. Fuh G. Li B. Christinger H.W. P. de Vos A.M. J. 1999; PubMed Scopus Google Scholar), we the crystal structures of the and the in complex with the receptor binding domain of human VEGF (residues The and of in the complex to investigate the of the in its of the and the complex to of and and for the in its to Å three structures were to and with The of the complex have one VEGF to two in the The of the two VEGF that VEGF of of these binds to one The crystal of G6 in its in the that a the and in the three crystal are located in the of the J. 1993; Google all but 2 of these are to in the which is in the of the in all in the three crystal structures and in to the is the of of a of of VEGF 2 VEGF 2 of of of of of is as for of the from all is the of of a in a new is as for of the from all The structures of both display VEGF in the as in structures W. L. (17Muller Y.A. Christinger H.W. Keyt B.A. de Vos A.M. Structure. 1997; 5: 1325-1338Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 18Muller Y.A. Li B. 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The binding on VEGF its two tyrosine receptors has been by for VEGFR1 and VEGFR2 (18Muller Y.A. Li B. Christinger H.W. Wells J.A. Cunningham B.C. de Vos A.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7192-7197Crossref PubMed Scopus (368) Google Scholar, B. Li B. J. 2002; PubMed Scopus Google Scholar, B. Fuh G. G. M.E. Cunningham B. de Vos A.M. J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) and structurally for VEGFR1 (19Wiesmann C. Fuh G. Christinger H.W. Eigenbrot C. Wells J.A. de Vos A.M. Cell. 1997; 91: 695-704Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar). studies that both receptors bind epitopes of VEGF, which are located on the two opposite poles of the growth factor and across the by the anti-parallel the VEGF The crystal of the two binding to the VEGF poles an which the complex a with the of of surface is in the the of the binding epitope between G6 and VEGF is not and the in other or protein-protein L. C. J. J. 1999; PubMed Scopus Google Scholar). in many other of the are by the of the only of the is The crystal of the complex the binding to an epitope that overlaps to a with binding epitopes for the VEGFR1 and G6 However, B20-4 approaches the growth factor from a different compared with The of its epitope has compared with the G6 binding epitope and is on the of VEGF. the two bind to the of VEGF; the resulting complex has a with the of the two separated by The binding between the and VEGF is the in the a of surface is in the and of the of surface on the VEGF is from by the of Structural of B20-4 and epitope recognized by the in the crystal is in with the functional epitope by alanine-scanning all VEGF that in binding affinity to have a of their surface in the with the antibody and This structural epitope of G6 is to the epitope responsible for binding and from the the and the of one VEGF as as from the and the of the second In 3 the and from the of VEGF with and can be identified as the of the structural binding epitope for for the B20-4 Fab structural and functional binding epitopes of VEGF are in 7 important VEGF are in the or of surface to the and The epitopes of VEGF B20-4 and G6 to a but are important The of the VEGF is in both from this the interaction between B20-4 and VEGF. Alanine-scanning mutagenesis that with one all that the affinity of VEGF B20-4 by or are located on the of VEGF and and of VEGF, are in the and for of the surface in the The most residue in this binding is its an in the complex and is in a by from and with the of in the structural the of with the binding affinity of VEGF by G6 an to and the bind VEGF they approach the growth factor from different In is important in the binding of both B20-4 to VEGF a complex with all of B20-4 In contrast, of to VEGF, a and In Fab structures against and a of the antibody that is of the and B. A.M. C. J. 1997; PubMed Scopus Google Scholar). to VEGF, of G6 is in a very different and against and the time the does not its and in its against The of this in the of G6 a number of with or in their B. A.M. C. J. 1997; PubMed Scopus Google Scholar) as in were to this of G6 is a of VEGF binding or an of the this we the in its The resulting of the in the with Fab all in In the to the in B. A.M. C. J. 1997; PubMed Scopus Google Scholar) 3 and of the in its and that to of their in the with the in the of the the of Å This in to be for VEGF as the in the steric in the VEGF of the with VEGF and of the the functional of this structural The G6 antibody was isolated from an antibody to the in natural the sequence of its is not In the crystal all VEGF binding are in of the in both the and the these are three and which are likely to the to such a is the most for and and in for human antibodies G. Res. 2000; PubMed Scopus Google Scholar), that this of of could a binding that is by other antibodies as The in the of G6 that the of the binding be an for antibodies to as for other antibodies that P. Science. 2003; PubMed Scopus Google Scholar). The VEGF their binding with a of or were as a of that in a in binding affinity their binding T. Wells J.A. Science. 1995; PubMed Scopus Google Scholar). proteins have also been to a to with binding de Vos A.M. Wells J.A. Science. 2000; PubMed Scopus Google Scholar). The exact of are not but that they are and in to sequence conserved de Vos A.M. Wells J.A. Science. 2000; PubMed Scopus Google Scholar) I. H. R. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). of the functional and structural VEGF epitopes G6 and B20-4 that a but of on the VEGF surface is responsible for binding of both of these are also important for binding to VEGFR1 In the of the and and a that from the of VEGF in the with VEGFR1, and this as a for binding all three This of VEGF is in is a surface with the important located in the of the structural binding are structural on the complex between VEGF and the of binding to this receptor is by (18Muller Y.A. Li B. Christinger H.W. Wells J.A. Cunningham B.C. de Vos A.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7192-7197Crossref PubMed Scopus (368) Google Scholar) that the in the of this is important for binding to this receptor as of B20-4, and Avastin for and of the human and mouse VEGF receptor binding domain (residues in differences are not only the they are also most of the VEGF surface However, all that the of the binding the receptor VEGFR1 and the derived antibodies G6 and B20-4 are conserved between human and mouse. 3 the of the between the two species three and rather sequence and in the of the binding that can be by B20-4, and the of this is conserved between mouse and these three not only bind human VEGF but are also of binding murine VEGF with high The is different for the Avastin The binding epitope for Avastin has been structurally and is in 3 to the epitopes for B20-4, and the Avastin binding centers on and not on the conserved by VEGFR1, and one of the 3 the of the receptor binding epitope that in the sequence between human and mouse VEGF; is with a residue in mouse. This two the of the of the The by this in the very between the Fab and human VEGF be by the Avastin its version is to bind with high affinity and In one the of as in the binding of the the of the in with is to a VEGF that binds with high affinity not of using B20-4 and which were using a synthetic antibody phage A.4.6.1, the antibody of is a of immune response against in mouse. The of thus on sequence differences between human and murine VEGF. The of VEGF, responsible for binding its receptor tyrosine kinases and the two synthetic antibodies G6 and B20-4, is conserved in the sequence of mouse and antibodies that target the exact receptor binding epitope be using the traditional method of raising antibodies in In that and areas that are in protein-protein interaction to be better conserved across species other S. J. Sci. 2004; PubMed Scopus Google Scholar, I. H. R. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, Phillips Sci. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). Therefore, traditional of raising antibodies the of binding antibodies that are to block the protein function as they depend on the of sequence differences in the protein of host and the target In contrast, that are important for protein binding to binding most likely also all the that as binding epitopes for antibodies. The generation of antibodies using phage display does not on sequence differences between host and target proteins but only on the of the protein Therefore, this new technique the of antibodies high affinity that block protein as they are likely to target surface areas that important protein-protein interaction The two derived antibodies analyzed are important to study the exact role of VEGF in mouse and they block the epitopes that are important for biological function of VEGF, they potential drug In we have identified a on the surface of VEGF that is responsible for binding of VEGFR1 and two antibodies. This is conserved between murine and human VEGF. of antibodies using the phage display approach is of the of sequence of the target Therefore, this study a of antibodies using phage display the traditional approach of raising antibodies using the hybridoma have that Avastin binds to a epitope to the conserved of VEGF and Avastin is of binding murine VEGF. de Vos for were the which is by the of of of the of and the which is by the of of and and by the of for and the of with
Fuh et al. (Fri,) studied this question.