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Title:
METHOD FOR IMPROVING VEGF-RECEPTOR BLOCKING SELECTIVITY OF AN ANTI-VEGF ANTIBODY
Document Type and Number:
WIPO Patent Application WO/2019/129679
Kind Code:
A1
Abstract:
The present invention relates to methods for improving anti-VEGF antibodies in order to provide or improve antibodies that preferentially inhibit binding to VEGF to VEGF-R2 rather than VEGF binding to VEGF-R1; and antibodies provided by said methods.

Inventors:
BENZ, Joerg (4070 Basel, 4070, CH)
DENGL, Stefan (Penzberg, 82377, DE)
FENN, Sebastian (Penzberg, 82377, DE)
MOELLEKEN, Joerg (Penzberg DEUTSCHLAND, 82377, DE)
EHLER, Andreas (4070 Basel, 4070, CH)
Application Number:
EP2018/086468
Publication Date:
July 04, 2019
Filing Date:
December 21, 2018
Export Citation:
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Assignee:
F. HOFFMANN-LA ROCHE AG (Grenzacherstrasse 124, 4070 Basel, 4070, CH)
HOFFMANN-LA ROCHE INC. (Great Notch, 150 Clove Road 8th Floor Suite 8 - Legal Departmen, Little Falls New Jersey, 07424, US)
International Classes:
A61K39/395; C07K16/22
Domestic Patent References:
WO2009060198A12009-05-14
WO2000064946A22000-11-02
WO2009060198A12009-05-14
WO2012089176A12012-07-05
WO2000046251A22000-08-10
WO2002012437A22002-02-14
WO2005007696A22005-01-27
WO2006047367A22006-05-04
WO2008027986A22008-03-06
Foreign References:
EP2662388A12013-11-13
EP3006465A12016-04-13
EP3006465A12016-04-13
US20040101920A12004-05-27
US4816567A1989-03-28
US20070033661A12007-02-08
Other References:
YAN-DA LAI ET AL: "Generation of Potent Anti-Vascular Endothelial Growth Factor Neutralizing Antibodies from Mouse Phage Display Library for Cancer Therapy", INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, vol. 17, no. 2, 5 February 2016 (2016-02-05), XP055558844, DOI: 10.3390/ijms17020214
YANLAN YU ET AL: "A humanized anti-VEGF rabbit monoclonal antibody inhibits angiogenesis and blocks tumor growth in xenograft models", PLOS ONE, PUBLIC LIBRARY OF SCIENCE, vol. 5, no. 2, 5 February 2010 (2010-02-05), pages E9072 - 1, XP002674471, ISSN: 1932-6203, DOI: 10.1371/JOURNAL.PONE.0009072
JING YANG ET AL: "Potent anti-angiogenesis and anti-tumor activity of a novel human anti-VEGF antibody, MIL60", CELLULAR & MOLECULAR IMMUNOLOGY, vol. 11, no. 3, 10 March 2014 (2014-03-10), pages 285 - 293, XP055202027, ISSN: 1672-7681, DOI: 10.1038/cmi.2014.6
JIHONG YANG ET AL: "Comparison of Binding Characteristics and In Vitro Activities of Three Inhibitors of Vascular Endothelial Growth Factor A", MOLECULAR PHARMACEUTICS, vol. 11, no. 10, 6 October 2014 (2014-10-06), pages 3421 - 3430, XP055201786, ISSN: 1543-8384, DOI: 10.1021/mp500160v
LEI JIA ET AL: "Protein asparagine deamidation prediction based on structures with machine learning methods", PLOS ONE, vol. 12, no. 7, 21 July 2017 (2017-07-21), pages e0181347, XP055465148, DOI: 10.1371/journal.pone.0181347
GERMAINE FUH ET AL: "Structure-Function Studies of Two Synthetic Anti-vascular Endothelial Growth Factor Fabs and Comparison with the Avastin(TM) Fab", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 281, no. 10, 10 March 2006 (2006-03-10), US, pages 6625 - 6631, XP055572086, ISSN: 0021-9258, DOI: 10.1074/jbc.M507783200
HOLASH, J. ET AL., PROC NATL ACAD SCI USA., vol. 99, no. 17, 20 August 2002 (2002-08-20), pages 1 1393 - 8
ALDRIDGE, S.E. ET AL., BIOCHEM BIOPHYS RES COMMUN., vol. 335, no. 3, 30 September 2005 (2005-09-30), pages 793 - 8
BREKKEN, R. A. ET AL., CANCER RES., vol. 60, no. 18, 15 September 2000 (2000-09-15), pages 5117 - 24
SULLIVAN, L.A. ET AL., PLOS ONE, vol. 5, no. 8, 6 August 2010 (2010-08-06), pages el2031
FLATMAN ET AL., J. CHROMATOGR. B, vol. 848, 2007, pages 79 - 87
KINDT ET AL.: "Kuby Immunology", 2007, W.H. FREEMAN AND CO., pages: 91
KABAT ET AL.: "Sequences of Proteins of Immunological Interest", 1991, PUBLIC HEALTH SERVICE, NATIONAL INSTITUTES OF HEALTH
METH. MOL. BIOL., vol. 248, 2004, pages 443 - 463
PROT. SCI., vol. 9, 2000, pages 487 - 496
HARLOW; LANE: "Antibodies", COLD SPRING HARBOR PRESS
JUNGHANS ET AL., CANCER RES., vol. 50, 1990, pages 1495 - 1502
S SEEBER ET AL., PLOS ONE, vol. 9, no. 2, 4 February 2014 (2014-02-04), pages e86184
KABSCH, W. ACTA CRYST., vol. D66, 2010, pages 133 - 144
MCCOY, A.J; GROSSE-KUNSTLEVE, R.W; ADAMS, P.D.; STORONI, L.C.; READ, R.J., J. APPL. CRYST., vol. 40, 2007, pages 658 - 674
"Collaborative Computational Project", NUMBER 4 ACTA CRYST, vol. D50, 1994, pages 760 - 763
BRICOGNE, G.; BLANC, E.; BRANDL, M.; FLENSBURG, C.; KELLER, P.; PACIOREK, W.; ROVERSI, P.; SHARFF, A.; SMART, O.S.; VONRHEIN, C.: "Buster", 2011, GLOBAL PHASING LTD
EMSLEY, P.; LOHKAMP, B.; SCOTT, W.G.; COWTAN, K., ACTA CRYST, vol. D66, 2010, pages 486 - 501
Attorney, Agent or Firm:
MERTES, Maria (Roche Diagnostics GmbH Patent Department, Penzberg, 82372, DE)
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Claims:
PATENT CLAIMS

1. A method of improving VEGFR-blocking selectivity of an antibody that binds to VEGF comprising an antigen binding site formed by cognate pair of a VH and a VL domain, wherein the antibody binds to an epitope of VEGF that overlaps with the VEGF-R1 -binding region and the VEGF-R2 -binding region in the VEGF molecule, the method comprising the steps of

a) providing an analysis of the tertiary structure of a complex of a VEGF-dimer bound by a first and a second antigen binding site of said antibody that binds to VEGF (VEGF-dimer-antibody-complex);

b) identifying at least one amino acid residue located in the VH domain or VL domain of said antibody, wherein said amino acid residue within the first antigen binding site and said amino acid residue within the second antigen binding site are spatially arranged in close proximity in the VEGF-dimer- antigen-complex; and

c) substituting said at least one amino acid residue identified in step b) by an amino acid having a larger side chain volume.

2. The method of claim 1, wherein the antibody binds to the same or overlapping epitope than an antibody characterized by a VH of SEQ ID NO:Ol and a VL of SEQ ID NO:02.

3. The method of one of the preceding claims, wherein the antibody binds to a conformational epitope on a dimer of VEGF-A121, wherein VEGF-A121 comprises an amino acid sequence of SEQ ID NO: 36, wherein the epitope comprises

in one of the individual VEGF-A121 molecules within the VEGF dimer amino acids F17, M18, D19, Y21, Q22, R23, Y25, H27, P28, 129, E30, M55, N62, L66, N100, K101, C102, E103, C104, R105 and P106; and in the other one of the individual VEGF-A121 molecules within the VEGF dimer amino acids E30, K48, M81 and Q87.

4. The method of one of the preceding claims, wherein the amino acid with a larger side chain volume is an aromatic amino acid.

5. The method of one of the preceding claims, wherein the at least one substituted amino acid residue is located in the heavy chain variable domain of said antibody.

6. The method of one of the preceding claims, wherein the at least one substituted amino acid residue is located within a heavy chain CDR of said antibody.

7. The method of one of the preceding claims, wherein the at least one substituted amino acid residue is located within H-CDR2 of said antibody.

8. The method of one of the preceding claims, wherein the at least one amino acid residue replaced is located within a heavy chain FR of said antibody.

9. The method of one of the preceding claims, wherein the at least one amino acid residue replaced is located within H-FR3 of said antibody.

10. The method of one of the preceding claims, wherein the antibody comprises comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:03; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:04; (c) CDR- H3 comprising the amino acid sequence of SEQ ID NO:05; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:06; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:07; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:08.

11. The method of one of the preceding claims, wherein the antibody comprises a VH comprising SEQ ID NO:0l and a VL comprising SEQ ID NO:02.

12. An antibody that binds to VEGF provided by a method of one of the preceding claims.

13. An antibody that binds to VEGF, wherein binding of the antibody to VEGF significantly inhibits VEGF -binding to VEGF receptor VEGF-R2 without significantly inhibiting VEGF -binding to VEGF receptor VEGF-R1 , provided by a method of one of the preceding claims.

14. An isolated nucleic acid encoding for an antibody provided by a method of claims 1 to 13.

15. A host cell comprising the nucleic acid of claim 14.

Description:
METHOD FOR IMPROVING VEGF-RECEPTOR BLOCKING SELECTIVITY OF AN ANTI-VEGF ANTIBODY

FIELD OF THE INVENTION

The present invention relates to methods for improving anti-VEGF antibodies in order to provide or improve antibodies that preferentially inhibit binding to VEGF to VEGF-R2 rather than VEGF binding to VEGF-R1; and antibodies provided by said methods.

BACKGROUND OF THE INVENTION

Anti-VEGF antibodies that are approved for clinical application, such as Avastin® and Lucentis®, inhibit VEGF-binding to both receptors, VEGF-R1 (FLT- 1, fms-like tyrosine kinase) and VEGF-R2 (KDR/FLK-l, fetal liver kinase). VEGF- Rl and VEGF-R2 are closely related receptor tyrosine kinases (RTK). While VEGF- R2 is hypothized to be primarily responsible for VEGF -mediated angiogenesis (Holash, J. et a , Proc Natl Acad Sci U S A. 2002 Aug 20;99(l 7): 11393-8), VEGF- Rl is known to have other important biological roles unrelated to angiogenesis, e.g. in osteoclast differentiation (Aldridge, S.E. et ah, Biochem Biophys Res Commun. 2005 Sep 30;335(3):793-8).

A few anti-VEGF antibodies that preferentially inhibit VEGF binding to VEGF-R2 and but do not significantly inhibit VEGF-binding to VEGF-R1 have been reported, but were not yet clinically successfull (W0200064946 describing an antibody termed“2C3”, WO 2009060198 describing an antibody termed“r84”, and WO 2012089176 describing an antibody termed“L3H6”, EP3006465 describing antibodies termed“HF2-1, HF2-5, HF2-9, and HF2-11”). By blocking VEGF binding to VEGF-R2, but not VEGF-R1, the antibodies are described to have an improved safety profile and do not show common toxicity-related side effects associated with anti-VEGF therapy (Brekken, R. A., et ak, Cancer Res. 2000 Sep l5;60(l 8):5117-24; Sullivan, L.A., et ak, PLoS One, 2010 Aug 6;5(8):el203 l).

There is a need for methods for improving anti-VEGF antibodies, particularly the VEGFR-b locking selectivity, to provide promising clinical antibody candidates. SUMMARY OF THE INVENTION

The present invention relates to a method of improving VEGFR-blocking selectivity of an antibody that binds to VEGF comprising an antigen binding site formed by cognate pair of a VH and a VL domain, wherein the antibody binds to an epitope of VEGF that overlaps with the VEGF-R1- binding region and the VEGF- R2 -binding region in the VEGF molecule. The method of the invention comprises (a) providing an analysis of the tertiary structure of a complex of a VEGF-dimer bound by a first and a second antigen binding site of said antibody that binds to VEGF (VEGF-dimer-antibody-complex), (b) identifying at least one amino acid residue located in the VH domain or VL domain of said antibody, wherein said amino acid residue within the first antigen binding site and said amino acid residue within the second antigen binding site are spatially arranged in close proximity in the VEGF-dimer-antigen-complex; and (c) substituting said at least one amino acid residue identified in step b) by an amino acid having a larger side chain volume.

With the method of the invention the VEGFR-blocking selectivity of certain anti- VEGF antibodies may be improved by a few modifications in their amino acid sequence, particularly in regions that are not involved in antigen binding. Such antibodies may exhibit an improved safety profile, e.g. by avoiding or reducing side effects caused by blocking VEGF -signalling through VEGF-R1.

In one embodiment the antibody binds to a conformational epitope on a dimer of VEGF-A121, wherein VEGF-A121 comprises an amino acid sequence of SEQ ID NO: 36, wherein the epitope comprises in one of the individual VEGF-A121 molecules within the VEGF dimer amino acids F17, M18, D19, Y21, Q22, R23, Y25, H27, P28, 129, E30, M55, N62, L66, N100, K101, C102, E103, C104, R105 and P106; and in the other one of the individual VEGF-A121 molecules within the VEGF dimer amino acids E30, K48, M81 and Q87. In one embodiment the epitope is measured by x-ray crystallography.

In one embodiment the amino acid with a larger side chain volume is an aromatic amino acid.

In one embodiment the at least one substituted amino acid residue is located in the heavy chain variable domain of said antibody.

Another aspect of the invention is an antibody that binds to VEGF provided by a method of one of the invention. Another aspect of the invention is antibody that binds to VEGF, wherein binding of the antibody to VEGF significantly inhibits VEGF-binding to VEGF receptor VEGF-R2 without significantly inhibiting VEGF-binding to VEGF receptor VEGF-R1, provided by a method of the invention.

DESCRIPTION OF THE FIGURES

Figure 1: Crystal structure of VEGF dimer (purple) in complex with VEGF- Rl domain 2 (red) and with VEGF-R2 domain 2 and 3 (blue)

Figure 2: Inhibition of VEGF-binding to VEGF-R1 and VEGF-R2 in presence of antibody Fab fragments (VEGF:VEGF-R2/Rl inhibitition EFISA) as described in Example 2.

Figure 3: Crystal structure of VEGF dimer (purple) in complex with anti- VEGF antibody VEGF-0089 as determined by X ray crystallography according to Example 3. Red circle highlights regions in the VH domain of VEGF-0089 that are in close proximity.

Figure 4: Epitope amino acids bound by VEGF-0089 Fab fragment in a dimer of VEGF-A121 (SEQ ID NO: 36) as determined by X ray crystallography according to Example 3. Amino acid positions comprised in each one of the VEGF- A121 molecules in contact with VEGF-0089 Fab fragment within a distance of 5 A are highlighted in black.

Figure 5: Overlay of the crystal structures of a human VEGF- A 121 -dimer in complex with VEGF-R1 domain 2 and a human VEGF-Al2l-dimer in complex with VEGF-0089 Fab as measured in Example 3.

Figure 6: Overlay of the crystal structures of a human VEGF- A 121 -dimer in complex with VEGF-R2 domains 2 and 3 and a human VEGF-A121 -dimer in complex with VEGF-0089 Fab as measured in Example 3.

Figure 7: Inhibition of VEGF binding to VEGF-R1 in presence of anti- VEGF antibodies as described in Example 4 (0.34 nM VEGF).

Figure 8: Inhibition of VEGF binding to VEGF-R1 in presence of anti- VEGF antibodies as described in Example 4 (0.7 nM VEGF). Figure 9: Inhibition of VEGF binding to VEGF-R2 in presence of anti- VEGF antibodies as described in Example 4 (0.34 nM VEGF).

Figure 10: Inhibition of VEGF binding to VEGF-R2 in presence of anti- VEGF antibodies as described in Example 4 (0.7 nM VEGF).

DETAILED DESCRIPTION OF THE INVENTION

1. Definitions

Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The methods and techniques of the present disclosure are generally performed according to conventional methods well known in the art. Generally, nomenclatures used in connection with, and techniques of biochemistry, enzymology, molecular, and cellular biology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art.

Unless otherwise defined herein the term“comprising of’ shall include the term“consisting of’.

The term "antibody" herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.

Papain digestion of intact antibodies produces two identical antigen-binding fragments, called “Fab” fragments containing each the heavy- and light-chain variable domains (VH and VL, respectively) and also the constant domain of the light chain (CL) and the first constant domain of the heavy chain (CH1). The term “Fab fragment” thus refers to an antibody fragment comprising a light chain comprising a VL domain and a CL domain, and a heavy chain fragment comprising a VH domain and a CH1 domain.

An“isolated” antibody is one which has been separated from a component of its natural environment. In some embodiments, an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS- PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPFC) methods. For a review of methods for assessment of antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab’-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments.

The term“variable region” or“variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VF, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs), including the complementarity determining regions (CDRs) (see, e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)).

A“paratope” or an“antigen binding site”, as used interchangeably herein, refers to a part of an antibody which recognizes and binds to an antigen. An antigen binding site is formed by several individual amino acid residues from the antibody’s heavy and light chain variable domains arranged that are arranged in spatial proximity in the tertiary structure of the Fv region. In one embodiment, the antigen binding site is defined as a set of the six CDRs comprised in a cognate VH/VF pair.

The term“complementarity determining regions” or“CDRs” as used herein refers to each of the regions of an antibody variable domain which are hypervariable in sequence and contain antigen-contacting residues. Generally, antibodies comprise six CDRs: three in the VH domain (CDR-H1, CDR-H2, CDR-H3), and three in the VF domain (CDR-F1, CDR-F2, CDR-F3). Unless otherwise indicated, CDR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to the Rabat numbering system (Rabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991). An“acceptor human framework” for the purposes herein is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below.

“Framework” or“FR” as used herein refers to variable domain amino acid residues other than CDR residues. The framework of a variable domain generally consists of four framework domains: FR1, FR2, FR3, and FR4. Accordingly, the CDR and FR amino acid sequences generally appear in the following sequence in the (a) VH domain: FR1— CDR-H1— FR2— CDR-H2— FR3— CDR-H3— FR4; and (b) in the VL domain: FR 1— CDR-L 1— FR2— CDR-L2— FR3— CDR-L3— FR4.

Vascular endothelial growth factor (VEGF) is a homodimeric member of the cystine knot family of growth factors. The term“VEGF”, as used herein, refers to any native VEGF from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses“full-length”, unprocessed VEGF as well as any form of VEGF that results from processing in the cell. The term also encompasses naturally occurring variants of VEGF, e.g., splice variants or allelic variants. The amino acid sequence of an exemplary human VEGF is shown in SEQ ID NO:33. The term“VEGF-dimer” as used to herein refers to a homodimer of two identical VEGF -molecules. A complex formed by two identical antibody molecules that are bound to a VEGF- dimer is herein referred to as“VEGF-dimer-antibody-complex”.

A“first and a second antigen binding site” comprised in a VEGF-dimer- antibody-complex refers to the antigen binding site that is comprised in the VH/VL pair of each one of the two antibodies comprised in the VEGF-dimer-antibody- complex. For example, while the antigen binding site of one of the two anti- VEGF antibodies in the VEGF-dimer-antibody-complex is the“first antigen binding site”, the antigen binding site of other one of the two anti-VEGF antibodies is automatically the“second antigen binding site”.

VEGF stimulates cellular responses by binding to tyrosine kinase receptors (the VEGF -receptors, or“VEGFRs”) on the cell surface, causing them to dimerize and become activated through transphosphorylation, although to different sites, times, and extents. VEGF-R1 and VEGF-R2 are closely related receptor tyrosine kinases (RTK). VEGF-A binds to VEGFR-l (Flt-l), interacting with domain 2 of VEGF-R1, and VEGFR-2 (KDR/Flk-l), interacting with domains 2 and 3 of VEGF- R2 (see Figure 1). The“VEGF-R1 -binding region” and“VEGF-R2 -binding region” of a VEGF molecule or a VEGF-dimer as used herein refers to those amino acids on the VEGF that interact with domain 2 of VEGF-R1 or domains 2 or 3 of VEGF-R2, respectively.

The terms“anti- VEGF antibody” and“an antibody that binds to VEGF” refer to an antibody that is capable of binding VEGF with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting VEGF. In one embodiment, the extent of binding of an anti-VEGF antibody to an unrelated, non- VEGF protein is less than about 10% of the binding of the antibody to VEGF as measured, e.g., by surface plasmon resonance (SPR). In certain embodiments, an antibody that binds to VEGF has a dissociation constant (KD) of < 1 nM, or < 0.15 nM. An antibody is said to“specifically bind” to VEGF when the antibody has a KD of ImM or less.

By“VEGFR-blocking selectivity” is used herein as an abbreviative term when referred to the property of anti-VEGF antibodies that preferentially inhibit binding to VEGF to VEGF-R2 rather than VEGF binding to VEGF-R1 , when bound to a VEGF-dimer. Anti-VEGF antibodies that are capable of fully blocking VEGF- binding to VEGF-R2, but not fully block VEGF -binding to VEGF-R1, are condifered to selectively block VEGF-signalling through VEGF-R2 but not through VEGF-R1, i.e. exhibit“VEGFR-blocking selectivity”.

The“tertiary structure” of a proteinis the three dimensional shape of the protein. The tertiary structure exhibits a single polypeptide chain "backbone" with one or more protein secondary structures, the protein domains. Amino acid side chains may interact and bond in a number of ways. The interactions and bonds of side chains within a particular protein determine its tertiary structure. The“tertiary structure of a VEGF-dimer-antibody-complex” as used herein means the threedimensional shape of said complex.

Amino acid residues located“in close proximity” within a tertiary structure of a VEGF-dimer-antibody-complexes are amino acid residues derived from both anti-VEGF antibodies that are spatially arranged in the threedimensional shape of said complex in a way that their distance is up to 5Ά. This does not include amino acids adjacent to each other in the amino acid sequence of the individual domain, i.e. VH or VL, of the respective anti-VEGF antibody. “ Affinity” refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein,“binding affinity” refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described herein.

The term "epitope" denotes the site on an antigen, either proteinaceous or non-proteinaceous, to which an anti-VEGF antibody binds. Epitopes can be formed both from contiguous amino acid stretches (linear epitope) or comprise non contiguous amino acids (conformational epitope), e.g. coming in spatial proximity due to the folding of the antigen, i.e. by the tertiary folding of a proteinaceous antigen. Linear epitopes are typically still bound by an anti-VEGF antibody after exposure of the proteinaceous antigen to denaturing agents, whereas conformational epitopes are typically destroyed upon treatment with denaturing agents. An epitope comprises at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation.

Screening for antibodies binding to a particular epitope (i.e., those binding to the same epitope) can be done using methods routine in the art such as, e.g., without limitation, alanine scanning, peptide blots (see Meth. Mol. Biol. 248 (2004) 443- 463), peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of antigens (see Prot. Sci. 9 (2000) 487-496), and cross-blocking (see “Antibodies”, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY).

Antigen Structure -based Antibody Profiling (ASAP), also known as Modification- Assisted Profiling (MAP), allows to bin a multitude of monoclonal antibodies specifically binding to VEGF based on the binding profile of each of the antibodies from the multitude to chemically or enzymatically modified antigen surfaces (see, e.g., US 2004/0101920). The antibodies in each bin bind to the same epitope which may be a unique epitope either distinctly different from or partially overlapping with epitope represented by another bin.

Also competitive binding can be used to easily determine whether an antibody binds to the same epitope of VEGF as, or competes for binding with, a reference anti-VEGF antibody. For example, an“antibody that binds to the same epitope” as a reference anti-VEGF antibody refers to an antibody that blocks binding of the reference anti-VEGF antibody to its antigen in a competition assay by 50% or more, and conversely, the reference antibody blocks binding of the antibody to its antigen in a competition assay by 50% or more. Also for example, to determine if an antibody binds to the same epitope as a reference anti-VEGF antibody, the reference antibody is allowed to bind to VEGF under saturating conditions. After removal of the excess of the reference anti-VEGF antibody, the ability of an anti-VEGF antibody in question to bind to VEGF is assessed. If the anti-VEGF antibody is able to bind to VEGF after saturation binding of the reference anti-VEGF antibody, it can be concluded that the anti-VEGF antibody in question binds to a different epitope than the reference anti-VEGF antibody. But, if the anti-VEGF antibody in question is not able to bind to VEGF after saturation binding of the reference anti-VEGF antibody, then the anti-VEGF antibody in question may bind to the same epitope as the epitope bound by the reference anti-VEGF antibody. To confirm whether the antibody in question binds to the same epitope or is just hampered from binding by steric reasons routine experimentation can be used (e.g., peptide mutation and binding analyses using ELISA, RIA, surface plasmon resonance, flow cytometry or any other quantitative or qualitative antibody-binding assay available in the art). This assay should be carried out in two set-ups, i.e. with both of the antibodies being the saturating antibody. If, in both set-ups, only the first (saturating) antibody is capable of binding to VEGF, then it can be concluded that the anti-VEGF antibody in question and the reference anti-VEGF antibody compete for binding to VEGF.

In some embodiments two antibodies are deemed to bind to the same or an overlapping epitope if a 1-, 5-, 10-, 20- or lOO-fold excess of one antibody inhibits binding of the other by at least 50%, at least 75%, at least 90% or even 99% or more as measured in a competitive binding assay (see, e.g., Junghans et ah, Cancer Res. 50 (1990) 1495-1502).

In some embodiments two antibodies are deemed to bind to the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody also reduce or eliminate binding of the other. Two antibodies are deemed to have“overlapping epitopes” if only a subset of the amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.

An“isolated” nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

“Isolated nucleic acid encoding” an antibody refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present at one or more locations in a host cell.

The term“vector”, as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as“expression vectors”.

The terms“host cell”,“host cell line”, and“host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include“transformants” and“transformed cells”, which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

Amino acids may be grouped according to common side-chain properties: (1) hydrophobic side chains: Norleucine, Met (M), Ala (A), Val (V), Leu (L), Ile (I); uncharged hydrophilic side chains (also referred to in the art as“neutral” hydrophilic side chains): Cys (C), Ser (S), Thr (T), Asn (N), Gln (Q); negatively charged side chains (also referred to in the art as“acidic” side chains): Asp (D), Glu (E); positively charged side chains (also referred to in the art as“basic” side chains): His (H), Lys (K), Arg (R); aromatic side chains: Trp (W), Tyr (Y), Phe (F); and side chains that comprise residues that influence chain orientation: Gly (G), Pro (P).

When referring to amino acid modifications, amino acids“having a larger side chain volume” refer to amino acids that have a larger side chain volume than the original amino acid located at the position to be modified. In certain embodiments, amino acids having a larger side chain volume are aromatic amino acids, including tryptophane, tyrosine and phenylalanine. 2. Detailed description of the embodiments of the invention

The present invention relates to a method of improving VEGFR-blocking selectivity of an antibody that binds to VEGF comprising an antigen binding site formed by cognate pair of a VH and a VL domain, wherein the antibody binds to an epitope of VEGF that overlaps with the VEGF-R1 -binding region and the VEGF- R2 -binding region in the VEGF molecule. The method of the invention comprises (a) providing an analysis of the tertiary structure of a complex of a VEGF-dimer bound by a first and a second antigen binding site of said antibody that binds to VEGF (VEGF-dimer-antibody-complex), (b) identifying at least one amino acid residue located in the VH domain or VL domain of said antibody, wherein said amino acid residue within the first antigen binding site and said amino acid residue within the second antigen binding site are spatially arranged in close proximity in the VEGF-dimer-antigen-complex; and (c) substituting said at least one amino acid residue identified in step (b) by an amino acid having a larger side chain volume. With a method of the invention an antibody is provided that preferentially inhibits binding to VEGF to VEGF-R2 rather than VEGF binding to VEGF-R1 (VEGFR- blocking selectivity).

The method of the invention is for improving anti- VEGF antibodies that bind to an epitope of VEGF that overlaps with the VEGF-R1 -binding region and the VEGF-R2 -binding region in the VEGF molecule. In one embodiment said epitope comprises amino acids interacting with domain 2 of VEGF -Rl, when VEGF is bound to VEGF-R1. In one embodiment said epitope comprises amino acids interacting with domains 2 and 3 of VEGF-R2, when VEGF is bound to VEGF-R1. In one embodiment said anti- VEGF antibody binds to an epitope that overlaps with the epitope bound by an antibody characterized by a VH of SEQ ID NO:Ol and a VL of SEQ ID NO:02 (antibody VEGF-0089 as described herein). In one embodiment said anti- VEGF antibody binds to the same epitope than antibody VEGF-0089 as described herein, as measured by x-ray crystallography. In one embodiment said anti- VEGF antibody binds to the same epitope than antibody VEGF-0089 as described herein, as measured by x-ray crystallography as described in Example 3. In one embodiment, said epitope is a conformational epitope within a dimer of VEGF-A121, wherein VEGF-A121 comprises an amino acid sequence of SEQ ID NO: 36, wherein the epitope comprises in one of the individual VEGF-A121 molecules within the VEGF dimer amino acids F17, M18, D19, Y21, Q22, R23, Y25, H27, P28, 129, E30, M55, N62, L66, N100, K101, C102, E103, C104, R105 and P106; and in the other one of the individual VEGF-A121 molecules within the VEGF dimer amino acids E30, K48, M81 and Q87. The numbering is according to the position of the amino acid in the amino acid sequence of VEGF-A121 indicated in SEQ ID NO: 36 (see also Figure 4).

Within a method of the invention an analysis of the tertiary structure of a VEGF-dimer-antibody-complex is provided. The tertiary structure may be provided by methods known in the art. In one embodiment the tertiary structure is provided by x-ray crystallography, e.g. as described in Example 3 herein.

In a method of the invention amino acid residues in the VH domain and/or VL domain of said antibody are identified that are in close proximity within the VEGF-dimer-antibody-complex. In one embodiment“close proximity” refers to a distance of 10 A or less. In one embodiment“close proximity” refers to a distance of 5 A or less. This at least one amino acid residue is considered suitable for modification.

In one embodiment such at least one amino acid residue identified in step (b) is located in the heavy chain variable domain of said antibody. In one embodiment the at least one amino acid residue identified in step (b) is located within a heavy chain CDR of said antibody. In one embodiment the at least one amino acid residue identified in step (b) is located within H-CDR2 of said antibody. In one embodiment the at least one amino acid residue identified in step (b) is located within a heavy chain FR of said antibody. In one embodiment the at least one amino acid residue identified in step (b) is located within H-FR3 of said antibody.

In the method of the invention such amino acid residue is substituted by an amino acid having a larger side chain volume than the amino acid residue comprised in the anti-VEGF antibody to be improved. In one embodiment said amino acid having a larger side chain volume is an aromatic amino acid. In one embodiment said amino acid having a larger side chain volume is Trp (W), Tyr (Y), or Phe (F).

In one embodiment said anti-VEGF antibody of step a) binds to human VEGFA. In one embodiment said anti-VEGF antibody of step a) binds to human VEGF of SEQ ID NO:33.

In one aspect the anti-VEGF antibody improved by a method of the invention comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:03; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:04; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:05; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:06; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 07; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:08. One exemplary antibody comprising this set of CDR amino acid sequences is the antibody referred to herein as“VEGF-0089”. In one embodiment said antibody is characterized by a VH comprising SEQ ID NO:0l and a VL comprising SEQ ID NO:02.

In one embodiment of this aspect, the at least one amino acid residue identified in step (b) is located within H-CDR2 of said antibody. In one embodiment said at least one amino acid residue is selected from Rabat position 52a, 52c and 54. In one embodiment one, two or three amino acids of Rabat position 52a, 52c and 54 are modified. In one embodiment at least one amino acid residue is selected from Rabat position 52a, 52c and 54 is modified by substitutions with Trp (W), Tyr (Y), or Phe (F). In one embodiment step c) includes one or more of the following substitutions N52aS, G52cP and I54F.

In one embodiment of this aspect, the at least one amino acid residue identified in step (b) is located within H-FR3 of said antibody. In one embodiment said at least one amino acid residue is Rabat position 74. In one embodiment at least the amino acid residue at Rabat position 74 is modified by substitutions with Trp (W), Tyr (Y), or Phe (F). In one embodiment step c) includes a substitution S74W.

The present invention also relates to an anti-VEGF antibody provided by a method of the invention. The present invention further relates to an anti-VEGF antibody, wherein binding of the antibody to VEGF significantly inhibits VEGF- binding to VEGF receptor VEGF-R2 without significantly inhibiting VEGF -binding to VEGF receptor VEGF-R1, provided by a method of the invention. In one embodiment the antibody provided by a method of the invention is an isolated antibody.

Such anti-VEGF antibodies may be produced using recombinant methods known in the art, e.g., as described in US 4,816,567, e.g. recombinant expression in eukaryotic cell such as HER293 cells as described in Example 1. For these methods one or more isolated nucleic acid(s) encoding an antibody are provided.

In certain embodiments, an antibody provided herein is an antibody fragment. In one embodiment, the antibody fragment is a Fab, Fab’, Fab’-SH, or F(ab’)2 fragment, in particular a Fab fragment. In certain embodiments, an antibody provided herein is a full length antibody. In one embodiment the antibody is an IgGl antibody.

The present invention further relates to a nucleic acid encoding for an anti- VEGF antibody provided by a method of the invention. The present invention further relates to a host cell comprising the nucleic acid of the invention.

DESCRIPTION OF THE AMINO ACID SEQUENCES

EXAMPLES

The following examples are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.

Examnle T :

Generation of human anti-VEGF antibody (antibody VEGF-0089)

An overlay of the crystal structures of a human VEGF-dimer in complex with VEGF-R1 domain 2 and VEGF-R3 domains 2 and 3 is depicted in Figure 1. This superimposition illustrates that both VEGF receptors bind to a highly similar region on the VEGF dimer and that it therefore appears highly challenging to generate antibodies that bind to VEGF that do not inhibit VEGF-binding to both receptors, VEGF-R1 and VEGF-R2, in the same fashion. In line with this, among the plurality of anti-VEGF antibodies known in the art, only few antibodies were reported to selectively block VEGF-binding to VEGF-R2 rather than VEGF-binding to VEGF- Rl .

Antibody VEGF-0089 as described herein was derived from Roche proprietary transgenic rabbits, expressing a humanized antibody repertoire, upon immunization with a VEGF-derived antigen. Transgenic rabbits comprising a human immunoglobulin locus are reported in WO 2000/46251, WO 2002/12437, WO 2005/007696, WO 2006/047367, US 2007/0033661, and WO 2008/027986. The animals were housed according to the Appendix A“Guidelines for accommodation and care of animals” in an AAAL AC -accredited animal facility. All animal immunization protocols and experiments were approved by the Government of Upper Bavaria (permit number 55.2-1-54-2532-90-14) and performed according to the German Animal Welfare Act and the Directive 2010/63 of the European Parliament and Council.

Immunization of transgenic rabbits

Briefly, rabbits (n=3), 12-16 week old, were immunized with recombinant human VEGF-121 protein coupled to keyhole limpet hemocyanin (KLH) (produced in house). All animals were immunized with 400 pg protein, emulsified with complete Freund’s adjuvant (CFA), at day 0 by intradermal application, followed by 200 ug protein emulsion at weeks 1, 2, 6, 11 and 14, by alternating intramuscular and subcutaneous injections. Blood was taken at days 4, 5 and 6 post immunizations, starting from the 4th immunization onwards. Serum was prepared for immunogen- specific rabbit-, and human-specific immunoglobulin titer determination by ELISA, and peripheral mononuclear cells were isolated, which were used as a source of antigen- specific B cells in the B cell cloning process.

B cell cloning from transgenic rabbits

Isolation of rabbit peripheral blood mononuclear cells (PBMC): Blood samples were taken of immunized rabbits. EDTA containing whole blood was diluted twofold with lx PBS (PAA) before density centrifugation using lympholyte mammal (Cedarlane Laboratories) according to the specifications of the manufacturer. The PBMCs were washed twice with lx PBS.

EL-4 B5 medium: RPMI 1640 (Pan Biotech) supplemented with 10% FCS (Pan Biotech), 2 mM Glutamin, 1% penicillin/streptomycin solution (Gibco), 2 mM sodium pyruvate, 10 mM HEPES (PAN Biotech) and 0,05 mM b-mercaptoethanole (Invitrogen) was used.

Coating of plates: Sterile cell culture 6-well plates were coated with 2 pg/ml KLH in carbonate buffer (0,1 M sodium bicarbonate, 34 mM Disodiumhydrogencarbonate, pH 9,55) over night at 4°C. Plates were washed in sterile PBS three times before use.

Depletion of macrophages/monocytes or of human Fc binders: The

PBMCs were seeded on sterile 6-well plates (cell culture grade) to deplete macrophages and monocytes through unspecific adhesion. Each well was filled at maximum with 4 ml medium and up to 6 x l0e6 PBMCs from the immunized rabbit and were allowed to bind for 1 h at 37°C in the incubator. The cells in the supernatant (peripheral blood lymphocytes (PBLs)) were used for the antigen panning step.

Immune fluorescence staining and Flow Cytometry: The anti-IgG FITC (AbD Serotec) and the anti-huCk PE (Dianova) antibody was used for single cell sorting. For surface staining, cells from the depletion and enrichment step were incubated with the anti-IgG FITC and the anti-huCk PE antibody in PBS and incubated for 45 min in the dark at 4°C. After staining the PBMCs were washed two fold with ice cold PBS. Finally the PBMCs were resuspended in ice cold PBS and immediately subjected to the FACS analyses. Propidium iodide in a concentration of 5 pg/ml (BD Pharmingen) was added prior to the FACS analyses to discriminate between dead and live cells. A Becton Dickinson FACSAria equipped with a computer and the FACSDiva software (BD Biosciences) were used for single cell sort.

B-cell cultivation: The cultivation of the rabbit B cells was performed by a method described by Seeber et al. (S Seeber et al. PLoS One 9 (2), e86l84. 2014 Feb 04). Briefly, single sorted rabbit B cells were incubated in 96-well plates with 200 mΐ/wcll EL-4 B5 medium containing Pansorbin Cells (1 : 100000) (Calbiochem), 5% rabbit thymocyte supernatant (MicroCoat) and gamma-irradiated murine EL-4 B5 thymoma cells (5 x l0e5 cells/well) for 7 days at 37 °C in the incubator. The supernatants of the B-cell cultivation were removed for screening and the remaining cells were harvested immediately and were frozen at - 80 °C in 100 mΐ RLT buffer (Qiagen).

R A encoding the V domains of the antibodies was isolated. For recombinant expression of the antibody PCR-products coding for VH or VL were cloned as cDNA into expression vectors and transiently transformed into HEK-293 cells.

From the screening an antibody comprising a VH domain of SEQ ID NO:0l and a VL domain of SEQ ID NO:02 was selected. This antibody is herein also referred to as antibody“VEGF-0089”. For the subsequent analyses the antibody VEGF-0089 was generated as a Fab fragment (herein referred to as“VEGF-0089 Fab fragment” or simply“VEGF-0089 Fab”) having the human VH and VL domains and rabbit derived constant domains of the light chain (CLkappa) and heavy chain (CH1). The amino acid sequence of the heavy chain of VEGF-0089 Fab fragment is SEQ ID NO: 10. The amino acid sequence of the light chain of VEGF-0089 Fab fragment is SEQ ID NO: 11.

Examnle 2:

Characterisation of generated human anti-VEGF antibody (antibody VEGF- 0089)

VEGF-binding of antibody VEGF-0089 Fab fragment was assessed by surface plasmon resonance (SPR) as described below.

Determination of antibody binding affinity by surface plasmon resonance (SPR)

An anti-Elis capturing antibody (GE Healthcare 28995056) was immobilized to a Series S Sensor Chip Cl (GE Healthcare 29104990) using standard amine coupling chemistry resulting in a surface densitiy of 500 - 1000 resonance units (RU). As running and dilution buffer, HBS-P+ (10 mM HEPES, 150 mM NaCl pH 7.4, 0.05% Surfactant P20) was used, the measurement temperature was set to 25°C and 37°C, respectively. hVEGF-Al2l was captured to the surface with resulting capture levels ranging from 5 to 35 RU. Dilution series of anti-VEGF antibodies (0.37 - 30 nM) were injected for l20s, dissociation was monitored for at least 600s at a flow rate of 30 mΐ/min. The surface was regenerated by injecting 10 mM Glycine pH 1.5 for 60s. Bulk refractive index differences were corrected by subtracting blank injections and by subtracting the response obtained from the control flow cell without captured hVEGF-Al2l . Rate constants were calculated using the Langmuir 1 : 1 binding model within the Biacore Evaluation software.

As a result, the KD of the VEGF-0089 Fab fragment was determined to be 134 pM (at a temperature of 25 °C).

For further characterization of the antibody, inhibition of VEGF-binding to its receptors VEGF-R1 and VEGF-R2 in presence of VEGF-0089 Fab fragment was assessed as described below:

Inhibition of VEGF-binding to VEGF-R1 and VEGF-R2 in presence of antibody Fab fragments (VEGF:VEGF-R2/R1 inhibition ELISA)

384 well streptavidin plates (Nunc/Microcoat # 11974998001) were coated with 0.25 Lig/m 1 biotinylated VEGF-R1 or 0.5pg/ml biotinylated VEGF-R2 (inhouse production, each 25 mΐ/wcll in DPBS (lx) (PAN, #P04-36500)). Plates were incubated for 1 h at room temperature. In parallel, VEGF-l2l-His (inhouse production) at a concentration of 0.7 nM was incubated with antibodies in different dilutions (12c 1 :2 dilution steps, starting with a concentration of 500 nM). This pre incubation step was carried out in 384 well PP plates (Weidmann medical technology, # 23490-101) in lx OSEP buffer (bidest water, lOx, Roche, # 11 666 789 001 + 0.5% Bovine Serum Albumin Fraction V, fatty acid free, Roche, # 10 735 086 001 + 0,05% Tween 20). Plates were incubated for lh at room temperature. After washing VEGF-R1/VEGF-R2 coated streptavidin plates 3 times with 90 mΐ/wcll PBST-buffer (bidest water, lOxPBS Roche#l 1666789001 + 0.1% Tween 20), 25 mΐ of samples from the VEGF-antibody pre-incubation plate were transferred to coated strepavidin plates which were subsequenty incubated for lh at room temperature. After washing 3 times with 90 mΐ/wcll PBST-buffer, 25 mΐ/wcll detection antibody (anti His POD, Bethyl, # A190-114P, 1 : 12000) in lx OSEP was added. After incubation for lh at room temperature plates were washed 3 times with 90 mΐ PBST- buffer. 25 mΐ TMB (Roche, #11 835 033 001) was added to all wells simultaneously. After 10 min incubation at room temperature, signals were detected at 370nm/492nm on a Tecan Satire 2 Reader.

As a control and representative for a prior art anti-VEGF antibody that is used in the clinic, Lucentis® (ranibizumab, heavy chain amino acid sequence of SEQ ID NO:34, light chain amino acid sequence of SEQ ID NO:35) was assessed under the same conditions. The results are shown in Figure 2.

The results indicate that VEGF-0089 Fab fragment is capable of fully blocking VEGF-binding to VEGF-R2. VEGF-0089 Fab fragment did not fully block VEGF-binding to VEGF-R1. Consequently, VEGF-0089 Fab fragment is considered to selectively block VEGF-signalling through VEGF-R2 but not through VEGF-R1. As illustrated in Figure 2, prior art antibody Fucentis® is capable of fully blocking VEGF-binding to both receptors, VEGF-R2 and VEGF-R1.

Examnle 3:

X-ray crystallography of antibody VEGF-0089 in complex with VEGF-dimer and epitope determination

The crystal structure of VEGF-0089 Fab fragment as described above was analyzed according to standard methods known in the art.

X-ray crystallography of VEGF-0089 Fab fragment in complex with VEGF- A121 was performed as follows: Complex formation and purification of the dimeric complex VEGF-A121 - VEGF-0089 Fab. For complex formation the VEGF-0089 Fab fragment and human VEGF-A121 (Peprotech) were mixed in a 1.1 : 1 molar ratio. After incubation for 16 hours overnight at 4°C the complex was purified via gelfiltration chromatography on a Superdex200 (16/600) column in 20mM MES, l50mM NaCl, pH6.5. Fractions containing the dimeric complex were pooled and concentrated to 1.44 mg/ml.

Crystallization of dimeric VEGF-A121 - VEGF-0089 Fab complex. Initial crystallization trials were performed in sitting drop vapor diffusion setups at 2l°C at a protein concentration of 11.5 mg/ml. Crystals appeared within 1 day out of 0.1 M Tris pH 8.5, 0.2 M LiS04, 1.26 M (NH4) 2 S0 4 . Plate shaped crystals grew in a week to a final size of 150x100x30 pm. The crystals were directly harvested from the screening plate without any further optimization steps.

Data collection and structure determination. For data collection crystals were flash cooled at 100K in precipitant solution with addition of 15% ethylene glycol as cryoprotectant. Diffraction data were collected at a wavelength of 1.0000 A using a PILATUS 6M detector at the beamline X10SA of the Swiss Light Source (Villigen, Switzerland). Data have been processed with XDS (Kabsch, W. Acta Cryst. D66, 133-144 (2010)) and scaled with SADABS (BRUKER). The crystals belong to the space group C2 with cell axes of a= 227.61 A, b= 66.97 A, c= 218.31 A, b=104.54° and diffract to a resolution of 2.17A. The structure was determined by molecular replacement with PHASER (McCoy, A.J, Grosse-Kunstleve, R.W., Adams, P.D., Storoni, L.C., and Read, R.J. J. Appl. Cryst. 40, 658-674 (2007)) using the coordinates of a related in house structure of a Fab fragment and VEGF as search models. Programs from the CCP4 suite (Collaborative Computational Project, Number 4 Acta Cryst. D50, 760-763 (1994)) and Buster (Bricogne, G., Blanc, E., Brandl, M., Flensburg, C, Keller, P., Paciorek, W., Roversi, P., Sharff, A., Smart, O.S., Vonrhein, C., Womack, T.O . (2011). Buster version 2.9.5 Cambridge, United Kingdom : Global Phasing Ltd) have been used to subsequently refine the data. Manual rebuilding of protein using difference electron density was done with COOT (Emsley, P., Lohkamp, B., Scott, W.G. and Cowtan, K. Acta Cryst D66, 486-501 (2010)). Data collection and refinement statistics for both structures are summarized in the following Table. All graphical presentations were prepared with PYMOL (DeLano Scientific, Palo Alto, CA, 2002). Table: Data collection and structure refinement statistics

Data Collection

Wavelength (A) 1.0

Resolution 1 (A) 49.49 - 2.17 (2.27 - 2.17)

Space group C2

Unit cell (A, °) 227.61 66.97 218.31, 90.00 104.54

90.00

Unique reflections 168745 (21164)

Multiplicity 3.45 (3.43)

Completeness (%) 99.8 (99.6)

Mean I/s(I) 8.36 (0.71)

R-meas 0.073 (0.86)

CC1/2 0.999 (0.364)

Refinement

Resolution 1 (A) 49.49 - 2.17 (2.23 - 2.17)

Reflections used in refinement 168674 (12361)

Reflections used for R-free 8487 (617)

R-work 3 0.185 (0.262)

R-free 4 0.227 (0.287)

Number of atoms 16966

Protein residues 1466

RMS bonds (A) 0.010

RMS angles (°) 1.20

Ramachandran favored (%) 97.85

Ramachandran outliers (%) 0.15

Rotamer outliers (%) 3.47

Clashscore 2.39 Average B-factor (A 2 ) 65.89

protein 66.85

solvent 64.05

1 Values in parentheses refer to the highest resolution bins. intensity. F 0 is the observed and F c is the calculated structure factor amplitude.

4 R free was calculated based on 5% of the total data omitted during refinement.

A schematic illustration of the crystal structure of two VEGF-0089 Fab fragments in complex with a human VEGF-A121 dimer is shown in Figure 3. Amino acid residues in the VEGF-A121 dimer in contact within a distance of 5 A with antibody VEGF-0089 Fab fragment form the conformational epitope bound by VEGF-0089 Fab on the VEGF-A121 dimer. The amino acid sequence of VEGF- A121 is SEQ ID NO: 36. An illustration of the amino acids comprised in the epitope on both VEGF-A121 molecules in the VEGF dimer is highlighted in Figure 4.

Antibody VEGF-0089 Fab binds to the following epitope on the VEGF-A121 dimer: in one of the individual VEGF-A121 molecules within the VEGF dimer amino acids F17, M18, D19, Y21, Q22, R23, Y25, H27, P28, 129, E30, M55, N62, F66, N100, K101, C102, E103, C104, R105 and P106; and in the other one of the individual VEGF-A121 molecules within the VEGF dimer amino acids E30, K48, M81 and Q87.

An overlay of the crystal structures of a human VEGF -dimer in complex with VEGF-R1 domain 2 and VEGF-R3 domains 2 and 3 is depicted in Figure 1. This superimposition illustrates that both VEGF receptors bind to a highly similar region on the VEGF dimer.

An overlay of the crystal structures of a human VEGF -dimer in complex with VEGF-R1 domain 2 and in complex with VEGF-0089 Fab fragment is depicted in Figure 5. This superimposition illustrates that the light chain of the VEGF-0089 antibody of the invention superimposes with domain 2 of VEGF-R1. An overlay of the crystal structures of a human VEGF-dimer in complex with VEGF-R2 domains 2 and 3, and in complex with VEGF-0089 Fab fragment is depicted in Figure 6. This superimposition illustrates that the light chain of the VEGF-0089 antibody of the invention superimposes with domain 2 of VEGF-R2.

Consequently, the x-ray data show that the epitope bound by the VEGF-0089 antibody overlaps with the region of VEGF that binds to VEGF-R1 and VEGF-R2. From the structural information provided the expectation would be that binding of the VEGF-0089 antibody to VEGF would result in a similar inhibition of both VEGF-R1 and VEGF-R2 binding to VEGF. This is, surprisingly, not the case as demonstrated above in Example 2 and Figure 2. Rather, antibody VEGF-0089 preferentially inhibits binding of VEGF to VEGF-R2 rather than the binding of VEGF to VEGF-R1.

Without being bound to this theory, this observation may be resulting from different affinities of VEGF towards its two receptors, VEGF-R1 and VEGF-R2. While VEGF-R1 is known to be bound by VEGF with strong affinity, it is known that VEGF-R2 is known to be bound by VEGF with weak affinity. As VEGF exists in dimeric form, it is believed that the number of antibody molecules bound to the VEGF dimer influence binding to VEGF-R1. It is believed that in case only one antibody molecule is bound to the VEGF dimer, the VEGF dimer is still capable of binding to the strong affinity receptor VEGF-R1 while binding to the low affinity receptor VEGF-R2 is inhibited.

Examnle 4:

Provision of improved variants of VEGF-0089

Based on the theory described in Example 3, the VEGF-0089 antibody was modified in order to facilitate that preferentially only one antibody molecule rather than two antibody molecules are capable of simultaneously binding to the VEGF dimer. From the tertiary structure of the VEGF-dimer-antibody-complex shown in Figure 3 it can be seen that two regions within the antibody’s VH domain, i.e. H- CDR2 and H-FR3, are in close spatial proximity to each other. To avoid simultaneous binding of two antibody molecules to the VEGF-dimer those regions were modified by amino acid substitutions using large amino acids to replace amino acids with smaller side chain volume in this regions. In the examples aromatic amino acids were used to replace the amino acid comprised in the original VEGF-0089 amino acid sequence. The theory was, that in this case binding of two antibody molecules to a VEGF dimer would be sterically hindered and the observed preferential inhibition of VEGF -binding to VEGF-R2 rather than the VEGF -binding to VEGF-R1 would be even more prominent.

Based on this theory, the following antibody variants of antibody VEGF- 0089 were generated. Candidate antibodies VEGF-0013, VEGF-0014, VEGF-

P1AD8675, VEGF-P1AE3520 and VEGF-P1AE3521 comprise amino acid substitutions with aromatic amino acid residues within H-CDR2, H-FR3 or both. The listed control antibodies comprise amino acid substitutions that were expected to have no effect or to even have a detrimental effect on VEGFR-b locking selectivity. Table 1: Amino acid sequences and amino acid modifications in generated candidate and control antibodies

Fab fragments of the antibodies were cloned and expressed as described in Example 1. Amino acid sequences of heavy chains and light chains are shown in Table 2.

Table 2: Amino acid sequences of anti-VEGF antibodies

Examnle 4:

VEGFR-blocking selectivity of improved variants of VEGF-0089

Binding of VEGF to VEGF-R1 as well as VEGF-R2 in presence of improved antibody Fab fragments was tested as described in Example 2. All antibody Fab fragments listed in Table 2 were tested. Results are shown in Figures 7 to 10.

Examnle 5:

VEGF-binding affinity of improved variants of VEGF-0089

The affinity of the antibodies was determined by SPR using the same methods as described in Example 2. All antibody Fab fragments listed in Table 2 were tested. Results are shown in Table 3. Table 3: Affinities of anti-VEGF antibodies

Example 6:

Chemical stability of improved variants of VEGF-0089 Chemical stability of improved antibody F ab fragments was tested as follows :

Chemical degradation test:

Antibody samples were formulated in 20 mM His/HisCl, 140 mM NaCl, pH 6.0, and were split into three aliquots: one aliquot was re -buffered into PBS, respectively, and two aliquots were kept in the original formulation. The PBS aliquot and one His/HisCl aliquot were incubated for 2 weeks (2w) at 40°C (His/NaCl) or 37°C (PBS) in 1 mg/ml, the PBS sample was incubated further for total 4 weeks (4w). The third control aliquot sample was stored at -80°C. After incubation ended, samples were analyzed for relative active concentration (Biacore; active concentration of both stressed aliquots of each binder is normalized to unstressed 4°C aliquot), aggregation (SEC) and fragmentation (capillary electrophoresis or

SDS-PAGE) and compared with the untreated control.

For Size Exclusion UHPLC (= SEC), proteins were separated depending on their molecular size in solution using a chromatographic gel like TSKgel UP- SW3000. With this method, protein solution was analyzed regarding their relative content of monomer, high molecular species (e.g. aggregates, dimers, impurities) and low molecular species (e.g. degradation products, impurities). 0.2 M Potassium phosphate, 0.25 M KC1, pH 6.2 was used as mobile phase. Protein solutions were diluted such as ~50 m of protein was injected in a volume of 5 mΐ, and analyzed with a flow rate of 0.3 ml/min at 25°C. Protein detection was done at 280 nm. Peak definition and peak integration were performed as demonstrated in the typical chromatograms in the product specific information document.

All antibody Fab fragments listed in Table 2 were tested. Results are shown in Tables 4 and 5.

Table 4: YE GF -binding activity after stress of improved antibody Fab fragments

Table 5: Molecular integrity after stress (4 weeks, pH 7.4, 37°C) of improved antibody Fab fragments




 
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