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Bispecific binding molecules binding to VEGF and Ang2

20220017642 · 2022-01-20

    Inventors

    Cpc classification

    International classification

    Abstract

    Bispecifc binding molecules binding to both VEGF and Ang2, preferably in the form of immunoglobulin single variable domains like VHHs and domain antibodies, pharmaceutical compositions containing the same and their use in the treatment of diseases that are associated with VEGF- and/or Ang2-mediated effects on angiogenesis are disclosed. Further, nucleic acids encoding bispecific binding molecules, host cells and methods for preparing same are also described.

    Claims

    1. A bispecific binding molecule comprising at least one VEGF-binding component, at least one Ang2-binding component and at least one serum albumin binding component, wherein said Ang2-binding component binds to Ang2 with a potency at least 5,000 times higher than to Ang1 or to Ang4.

    2. A bispecific binding molecule of claim 1, wherein said VEGF-binding component comprises at least a variable domain with four framework regions and three complementarity determining regions CDR1, CDR2 and CDR3, respectively, wherein said CDR3 has the amino acid sequence Ser Arg Ala Tyr Xaa Ser Xaa Arg Leu Arg Leu Xaa Xaa Thr Tyr Xaa Tyr as shown in SEQ ID NO: 1, wherein Xaa at position 5 is Gly or Ala; Xaa at position 7 is Ser or Gly; Xaa at position 12 is Gly, Ala or Pro; Xaa at position 13 is Asp or Gly; Xaa at position 16 is Asp or Glu; and wherein said VEGF-binding component is capable of blocking the interaction of human recombinant VEGF165 with the human recombinant VEGFR-2 with an inhibition rate of ≥60° %.

    3. A bispecific binding molecule of claim 2, wherein said CDR3 has a sequence selected from TABLE-US-00050 SEQ ID NO: 2 SRAYGSSRLRLGDTYDY, SEQ ID NO: 3 SRAYGSSRLRLADTYDY; SEQ ID NO: 4 SRAYGSSRLRLADTYEY; SEQ ID NO: 5 SRAYGSGRLRLADTYDY; SEQ ID NO: 6 SRAYASSRLRLADTYDY; SEQ ID NO: 7 SRAYGSSRLRLPDTYDY; SEQ ID NO: 8 SRAYGSSRLRLPGTYDY.

    4. A bispecific binding molecule of claim 3, wherein said VEGF-binding component comprises one or more immunoglobulin single variable domains each containing a. a CDR3 with an amino acid sequence selected from a first group of sequences shown in SEQ ID NO: 2 to 8; b. a CDR1 and a CDR2 with an amino acid sequences that is contained, as indicated in Table 3, in a sequence selected from a second group of sequences shown in SEQ ID NOs: 9 to 46, wherein said second sequence contains the respective CDR3 in said selected sequence according to a).

    5. A bispecific binding molecule of claim 4, wherein said one or more immunoglobulin single variable domains are VHHs.

    6. A bispecific binding molecule of claim 5, wherein said one or more VHHs have amino acid sequences selected from the amino acid sequences shown in SEQ ID NOs: 9-46.

    7. A bispecific binding molecule of claim 6, which comprises one or more VHHs having amino acid sequences selected from SEQ ID NO: 15, SEQ ID NO: 18 and SEQ ID NO: 25.

    8. A bispecific binding molecule, the VEGF-binding component of which has been obtained by affinity maturation and/or sequence optimization of a VHH defined in claim 7.

    9. A bispecific binding molecule according to claim 8, the VEGF-binding component of which has been obtained by sequence optimization of a VHH having an amino acid sequence shown in SEQ ID NO: 18.

    10. A bispecific binding molecule according to claim 9, the VEGF-binding component of which having an amino acid sequence selected from sequences shown in SEQ ID NOs: 47-57.

    11. A bispecific binding molecule according to claim 5, the VEGF-binding component of which comprising two or more VHHs, which are a. identical VHHs that are capable of blocking the interaction between recombinant human VEGF and the recombinant human VEGFR-2 with an inhibition rate of 60% or b. different VHHs that bind to non-overlapping epitopes of VEGF, wherein at least one VHH is capable of blocking the interaction between recombinant human VEGF and the recombinant human VEGFR-2 with an inhibition rate of ≥60% and wherein at least one VHH is capable of blocking said interaction with an inhibition rate of ≤60%.

    12. A bispecific binding molecule according to claim 11, wherein said identical VHHs a) are selected from VHHs having amino acid sequences shown in SEQ ID NOs: 9-46 or VHHs that have been obtained by affinity maturation and/or sequence optimization of such VHH.

    13. A bispecific binding molecule according to claim 12, wherein said VHH is selected from VHHs having the amino acid shown in SEQ ID NO: 18 or SEQ ID NO: 47-57.

    14. A bispecific binding molecule according to claim 13 comprising two VHHs each having the amino acid sequence shown in SEQ ID NO: 57.

    15. A bispecific binding molecule according to claim 14, wherein a. said one or more VHHs with an inhibition rate of ≥60% are selected from i. VHHs having an amino acid sequence selected from amino acid sequences shown in SEQ ID NOs: 9-46 or ii. VHHs that have been obtained by affinity maturation and/or sequence optimization of such VHHs, and wherein b. said one or more VHHs with an inhibition rate of ≤60% are selected from i. SEQ ID NOs: 58-124 or ii. VHHs that have been obtained by affinity maturation and/or sequence optimization of such VHH.

    16. A bispecific binding molecule according to claim 15, wherein two VHHs are contained in polypeptides with amino acid sequences shown in SEQ ID NOs: 128-168, separated by linker sequences as indicated in Table 13.

    17. A bispecific binding molecule according to claim 16, wherein said VHH a) i. has an amino acid sequence shown in SEQ ID NO: 18 and said VHH b) i. has an amino acid sequence shown in SEQ ID NO: 64.

    18. A bispecific binding molecule according to claim 17, wherein said VHHs according to a) ii) are selected from VHHs having an amino acid sequence shown in SEQ ID NOs: 47-57 and wherein said VHHs according to b) ii) are selected from VHHs having an amino acid sequence shown in SEQ ID NOs: 125-127.

    19. A bispecific binding molecule according to claim 18, comprising two VHHs, one of them having the amino acid shown in SEQ ID NO: 57 and one of them having the amino acid shown in SEQ ID NO: 127.

    20. The bispecific binding molecule of claim 1, comprising an Ang2-binding component comprising at least a variable domain with four framework regions and three complementarity determining regions CDR1, CDR2 and CDR3, respectively, wherein said CDR3 has an amino acid sequence selected from amino acid sequences shown in SEQ IDs NOs: 226, 229, 232, 235, 238, 241, 244, 247, 250, or 253.

    21. The bispecific binding molecule of claim 20, the Ang2-binding component of which is an isolated immunoglobulin single variable domain or a polypeptide containing one or more of said immunoglobulin single variable domains, wherein said immunoglobulin single variable domain consists of four framework regions and three complementarity determining regions CDR1, CDR2 and CDR3, respectively, and wherein said CDR3 has an amino acid sequence selected from amino acid sequences shown in SEQ IDs NOs: 226, 229, 232, 235, 238, 241, 244, 247, 250, or 253.

    22. The bispecific binding molecule of claim 21, wherein said one or more immunoglobulin single variable domain contain a. a CDR3 with an amino acid sequence selected from a first group of amino acid sequences shown in SEQ ID NOs: SEQ IDs NOs: 226, 229, 232, 235, 238, 241, 244, 247, 250, or 253 (Table 49); b. a CDR1 with an amino acid sequences that is contained, as indicated in Table 36-A, 38-A, 41-A, or 45-A, as partial sequence in a sequence selected from a second group of amino acid sequences shown SEQ ID NOs: 224, 227, 230, 233, 236, 239, 242, 245, 248, or 251 (Table 49); c. a CDR2 with an amino acid sequences that is contained, as indicated in Table 36-A, 38-A, 41-A, or 45-A, as partial sequence in a sequence selected from a second group of amino acid sequences shown SEQ ID NOs:225, 228, 231, 234, 237, 240, 243, 246, 249, or 252 (Table 49).

    23. The bispecific binding molecule of claim 20, wherein said one or more immunoglobulin single variable domains are VHHs.

    24. The bispecific binding molecule of claim 23, wherein said one or more VHHs have an amino acid sequence selected from amino acid sequences shown in SEQ ID NOs: 214, 215, 216, 217, 218, 219, 220, 221, 222, or 223.

    25. An immunoglobulin single variable domain which has been obtained by affinity maturation of an immunoglobulin single variable domain as defined in claim 22.

    26. A VHH which has been obtained by affinity maturation of a VHH as defined in claim 24.

    27. An Ang2-binding VHH with an amino acid sequence selected from acid sequences shown in SEQ ID NOs: 214, 215, 216, 217, 218, 219, 220, 221, 222, or 223.

    28. An immunoglobulin single variable domain which has been obtained by humanization of a VHH defined in claim 27.

    29. An immunoglobulin single variable domain which has been obtained by humanization of an immunoglobulin single variable domain as defined in claim 22.

    30. The binding molecule of claim 1, the serum albumin binding component of which is an isolated immunoglobulin single variable domain or a polypeptide containing one or more of said immunoglobulin single variable domains, wherein said immunoglobulin single variable domain consists of four framework regions and three complementarity determining regions CDR1, CDR2 and CDR3, respectively, and wherein said CDR3 has an amino acid sequence selected from amino acid sequences shown in SEQ ID NOs: 257, 260, 263, 266, 269, 272, or 275.

    31. The binding molecule of claim 30, wherein said one or more immunoglobulin single variable domain contain a. a CDR3 with an amino acid sequence selected from a first group of amino acid sequences shown in SEQ ID NOs: SEQ IDs NOs: 257, 260, 263, 266, 269, 272, or 275; b. a CDR1 with an amino acid sequences selected from a second group of amino acid sequences shown SEQ ID NOs: 255, 258, 261, 264, 267, 270, or 273; c. a CDR2 with an amino acid sequences selected from a second group of amino acid sequences shown SEQ ID NOs: 256, 259, 262, 265, 268, 271, or 274.

    32. The bispecific binding molecule of claim 30, wherein said one or more immunoglobulin single variable domains are VHHs.

    33. The bispecific binding molecule of claim 32, wherein said one or more VHHs have an amino acid sequence shown in SEQ ID NOs: 98 or 254.

    34. The bispecific binding molecule of claim 1 having the amino acid sequence selected from amino acid sequences shown in SEQ ID NOs: 180-213.

    35. A nucleic acid molecule encoding a bispecific binding molecule of claim 1 or a vector containing same.

    36. A host cell comprising a nucleic acid molecule of claim 35.

    37. A pharmaceutical composition comprising at least one bispecific binding molecule of claim 1 as the active ingredient.

    38. A method of treating a disease that is associated with VEGF- and/or Ang2-mediated effects on angiogenesis comprising administering to a patient an effective amount of a pharmaceutical composition according to claim 37.

    39. The method of claim 38 wherein the disease is selected from cancer and cancerous diseases.

    40. The method of claim 38 wherein the disease is eye diseases.

    Description

    BRIEF DESCRIPTION OF THE FIGURES

    [0316] FIG. 1: Purified monovalent VHHs block the hVEGF165/hVEGFR2-Fc interaction (ELISA)

    [0317] FIG. 2: Purified monovalent VHHs block the hVEGF165/hVEGFR1-Fc interaction (ELISA)

    [0318] FIG. 3: Purified monovalent VHHs block the hVEGF165/hVEGFR2-Fc interaction (AlphaScreen)

    [0319] FIG. 4: Purified monovalent VHHs block the hVEGF165/hVEGFR1-Fc interaction (AlphaScreen)

    [0320] FIG. 5: Binding of monovalent VHHs to recombinant human and mouse VEGF (ELISA)

    [0321] FIG. 6: Binding of monovalent VHHs to human VEGF121

    [0322] FIG. 7: Purified VHHs do not bind to VEGFB, VEGFC, VEGFD and PIGF

    [0323] FIG. 8: Formatted VHHs block hVEGF165/hVEGFR2-Fc interaction (ELISA)

    [0324] FIG. 9: Formatted VHHs block hVEGF165/hVEGFR1-Fc interaction (ELISA)

    [0325] FIG. 10: Formatted VHHs block hVEGF165/hVEGFR2-Fc interaction (AlphaScreen)

    [0326] FIG. 11:Formatted VHHs block hVEGF165/hVEGFR1-Fc interaction (AlphaScreen)

    [0327] FIG. 12: Formatted VHHs block mVEGF164/mVEGFR2-Fc interaction (AlphaScreen)

    [0328] FIG. 13: Formatted VHHs bind to mouse and human VEGF

    [0329] FIG. 14A-14H: Formatted VHHs do not bind to VEGFB, VEGFC, VEGFD and PIGF

    [0330] FIG. 15: Formatted VHHs bind to VEGF121

    [0331] FIG. 16: Sequence alignment of VHH VEGFBII23B04 (SEQ ID NO:18) with human VH3/JH (SEQ ID NO:179) [0332] germline consensus sequence

    [0333] FIG. 17: VHH variants of VEGFBII23B04 block hVEGF165/hVEGFR2-Fc interaction (AlphaScreen)

    [0334] FIG. 18: Sequence-optimized clones of VEGFBII23B04 block the hVEGF165/hVEGFR2-Fc interaction (AlphaScreen)

    [0335] FIG. 19: Sequence alignment of VHH VEGFBII5B05 (SEQ ID NO: 64) with human VH3/JH (SEQ ID NO: 179) [0336] germline consensus sequence

    [0337] FIG. 20 A-20C: Description bivalent Ang2 VHHs: A.discloses “9GS” and “9 GlySer” as SEQ ID NO: 170 and “40GS” and “40 GlySer” as SEQ ID NO: 171; B. Expression levels of all bivalent VHHs is depicted in FIG. 20 A; C. IC.sub.50 values (pM) in human Ang2/human Tie2, mouse Ang2/mouse Tie2, cyno Ang2/cyno Tie2 and hAng1/hTie2 competition ELISA.

    [0338] FIG. 21-1A to 21-1D; 21-2A to 21-2D; 21-3A to 21-3D: Purified bivalent Ang2 VHHs blocking hAng2-hTie2 (25-1), mAng2-mTie2 (25-2) and cAng2-cTie2 (25-3) interaction (ELISA)

    [0339] FIG. 22: Purified bivalent Ang2 VHHs blocking hAng1-hTie2 interaction (ELISA)

    [0340] FIG. 23A-23D: A. Description trivalent VEGFxAng2 bispecific VHHs (FIG. 23 discloses “9GS” and “9 GlySer” as SEQ ID NO: 170); B. Expression levels are indicated for all four bispecific VHHs depicted in FIG. 23 A; C. IC.sub.50 values (nM) in human VEGF165/human VEGFR2 competition AlphaScreen: D. IC.sub.50 values (pM) in human Ang2/human Tie2 competition ELISA * * *

    [0341] FIG. 24A-24D: Purified trivalent VEGFxAng2 Nanobodies blocking hVEGF-hVEGFR2 interaction (AlphaScreen)

    [0342] FIG. 25A-25D: Purified trivalent VEGFxAng2 VHHs blocking hAng2-hTie2 interaction (ELISA)

    [0343] FIG. 26A-26C: A. Description trivalent and tetravalent VEGFxAng2 bispecific VHHs (FIG. 26 discloses “9GS” and “9 GlySer” as SEQ ID NO: 170); B. Expression levels all ten bispecific VHHs as depicted in FIG. 26A are shown; C. IC50 values (nM) in human VEGF165/human VEGFR2 and human VEGF165/human VEGFR1 competition AlphaScreen

    [0344] FIG. 27-1A to 27-1H; 27-2A to 27-2H: Purified trivalent and tetravalent VEGFxAng2 VHHs blocking hVEGF-hVEGFR2 (31-1) and hVEGF-hVEGFR1 (31-2) interaction (AlphaScreen)

    [0345] FIG. 28-1A to 28-1H; 28-2A to 28-2G; 28-3A to 28-3G: Purified trivalent and tetravalent VEGFxAng2 VHHs blocking hAng2-hTie2 (32-1), mAng2-mTie2 (32-2) and cAng2-cTie2 (32-3) interaction (ELISA)

    [0346] FIG. 29A-29G: A-F. Purified trivalent and tetravalent VEGFxAng2 VHHs blocking hAng2 mediated HUVEC survival; G. IC.sub.50 values (pM) and % inhibition in human Ang2/human Tie2, mouse Ang2/mouse Tie2 and cyno Ang2/cyno Tie2 competition ELISA and IC.sub.50 (nM) values and % inhibition in hAng2 mediated HUVEC survival.

    [0347] FIG. 30A-30D: A. Description sequence optimized and affinity VEGFxAng2 bispecific VHHs (FIG. 30 discloses “9GS” and “9 GlySer” as SEQ ID NO: 170); B. Expression levels of all 14 bispecific VHHs as depicted in (A); C. IC.sub.50 values (nM) in human VEGF165/human VEGFR2 and human VEGF165/human VEGFR1 competition AlphaScreen; D. Overview of kinetic parameters of the bispecific VHHs on human VEGF165 in hVEGF165 Biacore assay.

    [0348] FIG. 31-1A to 31-1C; 31-2A to 31-2C: Purified VEGFANGBII00022-25-28 VEGFxAng2 VHHs blocking hVEGF-hVEGFR2 (35-1) and hVEGF-hVEGFR1 (35-2) interaction (AlphaScreen)

    [0349] FIG. 32A-32B: Purified VEGFANGBII00022-25-28 VEGFxAng2 VHHs binding to human VEGF165 (36-1) and hVEGF121 (36-2) (ELISA)

    [0350] FIG. 33A-33B: Purified VEGFANGBII00022-25-28 VEGFxAng2 VHHs binding to (A) mouse and (B) rat VEGF164 (ELISA)

    [0351] FIG. 34A-34D: Purified VEGFANGBII00022-25-28 VEGFxAng2 VHHs binding to (A) human VEGF-B, (B) human VEGF-C, (C) human VEGF-D and (D) human PIGF (ELISA)

    [0352] FIG. 35-1A to 35-1C; 35-2A to 35-2C; 35-3A to 35-3C: Purified VEGFANGBII00022-25-28 VEGFxAng2 VHHs blocking hAng2-hTie2 (39-1), mAng2-mTie2 (39-2) and cAng2-cTie2 (39-3) interaction (ELISA)

    [0353] FIG. 36A-36C: Purified VEGFANGBII00022-25-28 VEGFxAng2 VHHs blocking hAng1-hTie2 interaction (ELISA)

    [0354] FIG. 37A-37C: Purified VEGFANGBII00022-25-28 VEGFxAng2 VHHs blocking hAng2 mediated HUVEC survival.

    [0355] FIG. 38: A summary of IC.sub.50 values and % inhibition: 1050 values (pM) in human Ang2/human Tie2, mouse Ang2/mouse Tie2 and cyno Ang2/cyno Tie2 competition ELISA, hAng1 ELISA and IC50 values (nM) in hAng2 mediated HUVEC survival assay.

    [0356] Materials and Methods:

    [0357] a) Production and Functionality Testing of VEGF109

    [0358] A cDNA encoding the receptor binding domain of human vascular endothelial growth factor isoform VEGF165 (GenBank: AAM03108.1; AA residues 27-135) is cloned into pET28a vector (Novagen, Madison, Wis.) and overexpressed in E. coli (BL21 Star DE3) as a His-tagged insoluble protein. Expression is induced by addition of 1 mM IPTG and allowed to continue for 4 hours at 37° C. Cells are harvested by centrifugation and lysed by sonication of the cell pellet. Inclusion bodies are isolated by centrifugation. After a washing step with 1% Triton X 100 (Sigma-Aldrich), proteins are solubilized using 7.5M guanidine hydrochloride and refolded by consecutive rounds of overnight dialysis using buffers with decreasing urea concentrations from 6M till OM. The refolded protein is purified by ion exchange chromatography using a MonoQ5/50GL (Amersham BioSciences) column followed by gel filtration with a Superdex75 10/300 GL column (Amersheim BioSciences). The purity and homogeneity of the protein is confirmed by SDS-PAGE and Westen blot. In addition, binding activity to VEGFR1, VEGFR2 and Bevacizumab is monitored by ELISA. To this end, 1 μg/mL of recombinant human VEGF109 is immobilized overnight at 4° C. in a 96-well MaxiSorp plate (Nunc, Wiesbaden, Germany). Wells are blocked with a casein solution (1%). Serial dilutions of VEGFR1, VEGFR2 or Bevacizumab are added to the VEGF109 coated plate and binding is detected using alkaline phosphatase (AP) conjugated goat anti-human IgG, Fc specific (Jackson Immuno Research Laboratories Inc., West Grove, Pa., USA) and a subsequent enzymatic reaction in the presence of the substrate PNPP (p-nitrophenylphosphate) (Sigma-Aldrich). VEGF109 could bind to VEGFR1, VEGFR2 and Bevacizumab, indicating that the produced VEGF109 is active.

    [0359] b) KLH Conjugation of VEGF165 and Functionality Testing of KLH-Conjugated VEGF165

    [0360] Recombinant human VEGF165 (R&D Systems, Minneapolis, Minn., USA) is conjugated to mariculture keyhole limpet hemocyanin (mcKLH) using the Imject Immunogen EDC kit with mcKLH (Pierce, Rockford, Ill., USA) according to the manufacturer's instructions. Efficient conjugation of the polypeptide to mcKLH is confirmed by SDS-PAGE. Functionality of the conjugated protein is checked by ELISA: 2 μg/mL of KLH conjugated VEGF165 is immobilized overnight at 4° C. in a 96-well MaxiSorp plate (Nunc, Wiesbaden, Germany). Wells are blocked with a casein solution (1%). Serial dilutions of VEGFR1 or VEGFR2 are added and binding is detected using a horseradish peroxidase (HRP)-conjugated goat anti-human IgG, Fc specific (Jackson Immuno Research Laboratories Inc., West Grove, Pa., USA) and a subsequent enzymatic reaction in the presence of the substrate TMB (3,3′,5,5′-tetramentylbenzidine) (Pierce, Rockford, Ill., USA). The KLH conjugated protein could still interact with VEGFR1, VEGFR2 and Bevacizumab, confirming that the relevant epitopes onVEGF165 are still accessible.

    EXAMPLE 1

    [0361] Immunization with Different VEGF Formats Induces a Humoral Immune Response in Ilama

    [0362] 1.1 Immunizations

    [0363] After approval of the Ethical Committee of the faculty of Veterinary Medicine (University Ghent, Belgium), 4 llamas (designated No. 264, 265, 266, 267) are immunized according to standard protocols with 6 intramuscular injections (100 or 50 μg/dose at weekly intervals) of recombinant human VEGF109. The first injection at day 0 is formulated in Complete Freund's Adjuvant (Difco, Detroit, Mich., USA), while the subsequent injections are formulated in Incomplete Freund's Adjuvant (Difco, Detroit, Mich., USA). In addition, four llamas (designated No. 234, 235, 280 and 281) are immunized according to the following protocol: 5 intramuscular injections with KLH-conjugated human VEGH165 (100 or 50 μg/dose at biweekly intervals) followed by 4 intramuscular injections of human VEGF109 (first dose of 100 μg followed 2 weeks later with three 50 μg/dose at weekly interval).

    [0364] 1.2 Evaluation of VEGF-Induced Immune Responses in Ilama

    [0365] To monitor VEGF specific serum titers, an ELISA assay is set up in which 2 μg/mL of recombinant human VEGF165 or VEGF109 is immobilized overnight at 4° C. in a 96-well MaxiSorp plate (Nunc, Wiesbaden, Germany). Wells are blocked with a casein solution (1%). After addition of serum dilutions, bound total IgG is detected using horseradish peroxidase (HRP)-conjugated goat anti-llama immunoglobulin (Bethyl Laboratories Inc., Montgomery, TX, USA) and a subsequent enzymatic reaction in the presence of the substrate TMB (3,3′,5,5′-tetramentylbenzidine) (Pierce, Rockford, IL, USA). For llamas 264, 265, 266 and 267, an additional ELISA is performed in which the isotype-specific responses against VEGF165 and VEGF109 are evaluated. Isotype specific responses are detected using mouse mAbs specifically recognizing conventional llama IgG1 and the heavy-chain only llama IgG2 and IgG3 [Daley et al. (2005). Clin. Diagn. Lab. Imm. 12:380-386] followed by a rabbit anti-mouse-HRP conjugate (DAKO). ELISAs are developed using TMB as chromogenic substrate and absorbance is measured at 450nm. The serum titers for each llama are depicted in Table 1.

    TABLE-US-00004 TABLE 1 Antibody-mediated specific serum response against VEGF165 and VEGF109 ELISA (recombinant protein solid phase coated) Recombinant human Recombinant human EGF165 VEGF109 Llama Immunogen Total IgG IgG1 IgG2 IgG3 Total IgG IgG1 IgG2 IgG3 234 VEGF165-KLH + ++ n/d n/d n/d ++ n/d n/d n/d VEGF109 235 VEGF165-KLH + ++ n/d n/d n/d ++ n/d n/d n/d VEGF109 280 VEGF165-KLH + + n/d n/d n/d + n/d n/d n/d VEGF109 281 VEGF165-KLH + + n/d n/d n/d + n/d n/d n/d VEGF109 264 VEGF109 n/d ++ + + ++ ++ + + 265 VEGF109 n/d ++ + + + ++ + + 266 VEGF109 n/d ++ + +/− ++ ++ + +/− 267 VEGF109 n/d +/− − − +/− +/− − − n/d, not determined

    EXAMPLE 2

    [0366] Cloning of the Heavy-Chain Only Antibody Fragment Repertoires and Preparation of Phage

    [0367] Following the final immunogen injection, immune tissues as the source of B-cells that produce the heavy-chain antibodies are collected from the immunized llamas. Typically, two 150-mi blood samples, collected 4 and 8 days after the last antigen injection, and one lymph node biopsy, collected 4 days after the last antigen injection are collected per animal. From the blood samples, peripheral blood mononuclear cells (PBMCs) are prepared using Ficoll-Hypaque according to the manufacturer's instructions (Amersham Biosciences, Piscataway, N.J., USA). From the PBMCs and the lymph node biopsy, total RNA is extracted, which is used as starting material for RT-PCR to amplify the VHH encoding DNA segments, as described in W02005/044858. For each immunized llama, a library is constructed by pooling the total RNA isolated from all collected immune tissues of that animal. In short, the PCR-amplified VHH repertoire is cloned via specific restriction sites into a vector designed to facilitate phage display of the VHH library. The vector is derived from pUC119 and contains the LacZ promoter, a M13 phage gill protein coding sequence, a resistance gene for ampicillin or carbenicillin, a multiple cloning site and a hybrid gIII-pelB leader sequence (pAX050). In frame with the VHH coding sequence, the vector encodes a C-terminal c-myc tag and a His6 tag (SEQ ID NO: 283). Phage are prepared according to standard protocols and stored after filter sterilization at 4° C. for further use.

    EXAMPLE 3

    [0368] Selection of VEGF-Specific VHHs Via Phage Display

    [0369] VHH phage libraries are used in different selection strategies applying a multiplicity of selection conditions. Variables include i) the VEGF protein format (rhVEGF165, rhVEGF109 or rmVEGF164), ii) the antigen presentation method (solid phase: directly coated or via a biotin-tag onto Neutravidin-coated plates; solution phase: incubation in solution followed by capturing on Neutravidin-coated plates), iii) the antigen concentration and iv) the elution method (trypsin or competitive elution using VEGFR2). All selections are carried out in Maxisorp 96-well plates (Nunc, Wiesbaden, Germany).

    [0370] Selections are performed as follows: Phage libraries are incubated at RT with variable concentrations of VEGF antigen, either in solution or immobilized on a solid support. After 2hrs of incubation and extensive washing, bound phage are eluted. In case trypsin is used for phage elution, the protease activity is immediately neutralized by addition of 0.8 mM protease inhibitor AEBSF. Phage outputs that show enrichment over background are used to infect E. coli. Infected E. coli cells are either used to prepare phage for the next selection round (phage rescue) or plated on agar plates (LB+amp+glucose.sup.2%) for analysis of individual VHH clones. In order to screen a selection output for specific binders, single colonies are picked from the agar plates and grown in 1 mL 96-deep-well plates. The lacZ-controlled VHH expression is induced by adding IPTG (0.1-1 mM final). Periplasmic extracts (in a volume of ˜80 μL) are prepared according to standard methods.

    EXAMPLE 4

    [0371] Identification of VEGF-binding and VEGF receptor-blocking VHHs Periplasmic extracts are tested for binding to human VEGF165 by ELISA. In brief, 2 μg/mL of recombinant human VEGF165 is immobilized overnight at 4° C. in a 96-well MaxiSorp plate (Nunc, Wiesbaden, Germany). Wells are blocked with a casein solution (1%). After addition of typically a 10-fold dilution of the periplasmic extracts, VHH binding is detected using a mouse anti-myc (Roche) and an anti-mouse-HRP conjugate (DAKO). Clones showing ELISA signals of >3-fold above background are considered as VEGF binding VHHs.

    [0372] In addition, periplasmic extracts are screened in a human VEGF165/human VEGFR2 AlphaScreen assay (Amplified Luminescent Proximity Homogeneous Assay) to assess the blocking capacity of the VHHs. Human VEGF165 is biotinylated using Sulfo-NHS-LC-Biotin (Pierce, Rockford, Ill., USA). Human VEGFR2/Fc chimera (R&D Systems, Minneapolis, Minn., USA) is captured using an anti-humanFc VHH which is coupled to acceptor beads according to the manufacturer's instructions (Perkin Elmer, Waltham, Mass., US). To evaluate the neutralizing capacity of the VHHs, periplasmic extracts are diluted 1/25 in PBS buffer containing 0.03% Tween 20 (Sigma-Aldrich) and preincubated with 0.4 nM biotinylated human VEGF165 for 15 minutes at room temperature (RT). To this mixture the acceptor beads (10 μg/ml) and 0.4 nM VEGFR2-huFc are added and further incubated for 1 hour at RT in the dark. Subsequently donor beads (10 μg/ml) are added followed by incubation of 1 hour at RT in the dark. Fluorescence is measured by reading plates on the Envision Multi label Plate reader (Perkin Elmer, Waltham, Mass., USA) using an excitation wavelength of 680 nm and an emission wavelength between 520 nm and 620 nm. Periplasmic extract containing irrelevant VHH is used as negative control. Periplasmic extracts containing anti-VEGF165 VHHs which are able to decrease the fluorescence signal with more than 60% relative to the signal of the negative control are identified as a hit. All hits identified in the AlphaScreen are confirmed in a competition ELISA. To this end, 1 μg/mL of human VEGFR2 chimera (R&D Systems, Minneapolis, MN, USA) is coated in a 96-well MaxiSorp plate (Nunc, Wiesbaden, Germany). Fivefold dilutions of the periplasmic extracts are incubated in the presence of a fixed concentration (4nM) of biotinylated human VEGF165 in PBS buffer containing 0.1% casein and 0.05% Tween 20 (Sigma-Aldrich). Binding of these VHH/bio-VEGF165 complexes to the human VEGFR2 chimera coated plate is detected using horseradish peroxidase (HRP) conjugated extravidin (Sigma, St Louis, Mo., USA). VHH sequence IDs and the corresponding AA sequences of VEGF-binding (non-receptor-blocking) VHHs and inhibitory (receptor-blocking) VHHs are listed in Table 2 and Table 3, respectively.

    TABLE-US-00005 TABLE 2 Sequence IDs and AA sequences of monovalent “non-receptor-blocking” anti-VEGF VHHs  (FR, framework; CDR, complementary determining region) VHH ID/ CDR1/ SEQ ID FR1/SEQ ID SEQ ID FR2/SEQ CDR2/SEQ FR3/SEQ ID CDR3/SEQ ID FR4/SEQ NO: NO: NO: ID NO: ID NO: NO: NO: ID NO: VEGFBII EVQLVESGGG SYGMG/ WFRQSPG AISEYSNTYC RFTISRDNTKN SPTILLTTEQW WGQGTQVT 01C02/58 LVQAGGSLRL 284 KEREFVS/ SDSVRG/330 TVYLQMNSLTP YKY/389 VSS/603 SCTASGGSFS/ 517 DDTAIYYCAA/ 466 548 VEGFBII EVQLVESGGG ASDMG/ WFRQAPG AINWSGLSTF RFTISRDNDNG GRIPSSSRFSS WGQGTQVT 01E07/59 LVQAGDSLRL 285 KEREFVA/ YTDSVKG/331 ALYLQMNTLKP PAAYAS/390 VSS/603 SCVATGRTFR/ 518 EDTAVYSCAA/ 467 549 VEGFBII EVQLVESGGG ITVMA/ WFRQAPG AITWSAPTTY RFTISRDNAKN DRFKGRSIVTP WGQGTQVT 03D12/60 LVQAGGSLRL 286 KEREFVA/ YADSVKG/332 TVYLRMNSLKP SDYRY/391 VSS/603 SCTASTSIYT/ 518 EDSAIYYCAA/ 468 550 VEGFBII EVQLVESGGG DITVA/ WYRQAPG TITPSGYTYY RFTISRDNSKN QFY/392 WGQGTQVT 04B08/61 LVQPGGSLRL 287 IQRQLVA/ WDFVKG/333 IVYLQMNSLKP VSS/603 SCAASGSAVG/ 519 EDTAAYYCNT/ 469 551 VEGFBII EVQLVESGGG TDDVG/ WFRQAPG VIRWSTGGTY RFTLSRDNAKN RSRPLGAGAWY WGQGTQVT 05B02/62 LVQAGGSLRL 288 KEREFVA/ TSDSVKG/334 TMYLQMNSLKP SGEKHYNY/393 VSS/603 SCAASGRTFS/ 518 EDTAVYYCAA/ 470 552 VEGFBII EVQLVESGGG HYNMG/ WFRQAPG SIRGGGGSTT RFTISRENAKN TAFYRGPYDYD WGQGTQVT 05B03/63 LAQAGDSLRL 289 KEREFVA/ YANSVKD/335 TVYLQMNSLKP Y/394 VSS/603 SCAASGRSFS/ 518 EDTAVYYCAA/ 471 553 VEGFBII EVQLVESGGG SMA/ WYRQAPG RISSGGTTAY RFTISRDNSKN FSSRPNP/395 WGAGTQVT 05B05/64 LVQPGGSLRL 290 KHRELVA/ VDSVKG/336 TVYLQMNSLKA VSS/604 SCVASGIRFM/ 520 EDTAVYYCNT/ 472 554 VEGFBII EVQLVESGGG NNAMA/ WYRQAPG RISSGGGFTY RFTVSRDNAKN AYRTYNY/396 WGQGTQVT 06G02/65 LVQPGGSLRL 291 KQRELVA/ YLDSVKG/337 TVYLQMNSLKP VSS/603 SCAASGNIFS/ 521 EDTAVYYCNA/ 473 555 VEGFBII EVQLVESGGG ITVMA/ WFRQAPG AITWSAPSSY RFTISRDNAKN DRFKGRSIVTR WGQGTQVT 07A03/66 LVQAGGSLRL 286 KESEFVA/ YADSVKG/338 TVYLQMNSLKP SDYKY/397 VSS/603 SCAASTSIYS/ 522 EDSAIYYCAA/ 474 556 VEGFBII EVQLVESGGG ISVMA/ WFRQAPG AITWSAPTTY RFTISRDNAKN DRFKGRSIVTR WGQGTQVT 07A06/67 LVQAGGSLRL 292 KERAFVA/ YADSVKG/332 TVYLQTNSLKP SDYRY/398 VSS/603 SCAVSTSIYS/ 523 EDSAIYYCAA/ 475 557 VEGFBII EVQLVESGGG NYAMA/ WFRQAPG AINQRGSNTN RFTISRDSAKN STWYGYSTYAR WGQGTQVT 07D08/68 LVQTGGSLRL 293 KEREFVS/ YADSVKG/339 SVFLQMNSLKP REEYRY/399 VSS/603 SCAASGRTFS/ 524 EDTAVYYCAA/  476 558 VEGFBII EVQLVESGGG DNVMG/ WFRQAAG HISRGGSRTE RFTISRDNTKK SRSVALATARP WGQGTQVT 08D09/69 LVQAGGSLRL 294 KEREFVA/ YAESVKG/340 TMYLQMNSLKP  YDY/400 VSS/603 SCAASGRSFS/ 525 EDTAVYYCAA/ 477 559 VEGFBII EVQLVESGGG SYYMG/ WFRQAPG TISWNKISTI RFTVSRDNNKN DASRPTLRIPQ WGQGTPVT 08E07/70 LAQAGGSLRL 295 KEREFVA/ YTDSVKG/341 TVYLQMNNLKP Y/401 VSS/603 SCTTSGLTFS/ 518 EDTAVYYCAA/ 478 560 VEGFBII EVQLVESGGG SDVMG/ WYRQAPG FIRSLGSTYY RFTISRDDAAN  RFSGESY/402 WGQGTQVT 08F06/71 LVQPGGSLRL 296 KQRELVA/ AGSVKG/342 TVYLQMNSLKP VSS/605 SCAASGSIVR/ 521 EDTAVYYCNA/ 479 561  VEGFBII EVQLVESGGG LYAMG/ WFRQAPG AITWSAGDTQ RFTISRDNARN RQWGGTYYYHG WGQGTQVT 08F07/72 LVQAGGSLRL 297 REREFLS/ YADSVKG/343 TVNLQMNGLKP  SYAY/403 VSS/603 SCAVSGSTFG/ 526 EDTAVYYCAG/ 480 562 VEGFBII EVQLVESGGG SMA/ WYRQAPG RISSEGTTAY RFTISRDNSKN  FSSRPNP/395 WGAGTTVT 09A09/73 LVQPGGSLRL 290 KHRELVA/ VDSVKG/344 TVYLQMNSLKA  VSS/606 SCVASGIRFM/ 520 EDTAVYYCNT/ 472 554 VEGFBII EVQLVESGGG TDDVG/ WFRQAPG VIRWSTGGTY RFTLSRDNAKN RSRPLGAGAWY WGQGTQVT 09A12/74 LVQAGGSLRL 288 KEREFVA/ TSDSVAG/345 TMYLQMNSLKP TGETRYDS/405 VSS/603 SCAASGRTFS/ 518 EDTAVYYCAA/ 470 552 VEGFBII EVQLVESGGG RYGMG/ WFRQAPG AISEYDNVYT RFTISRDNSKS SPTILLSTDEW WGRGTQVT 09D05/75 LVQPGDSLRL 298 KEREFVI/ ADSVRG/346 TVYLQMNSLKS YKY/406 VSS/607 SCAASGLSFS/ 527 EDTAVYYCAA/ 481 563 VEGFBII EVQLVESGGG TDDVG/ WFRQAPG VIRWSTGGTY RFTLSRDNAKN  RSRPLGAGAWY WGQGTQVT 09F05/76 LVQAGGSLRL 288 KEREFVA/ TSDSVKG/334 TMYLQMNSLKP TGETRYNY/407 VSS/603 SCAASGRTFS/ 518 EDTAVYYCAA/ 470 552 VEGFBII EVQLVESGGG NYAMG/ WFRQVPG VITRSPSNTY RFTISRDNAKN HYWNSDSYTYT WGQGTQVT 10C07/77 LVQAGGSLRL 299 REREFVA/ TYDSVKG/347 IVYLQMNSLKP DSRWYNY/408 VSS/603 SCAASARAFS/ 528 EDTAVYYCAA/ 482 564 VEGFBII EVQLVESGGG NYAMG/ WFRQAPG DISSSGINTY RFTISRDNAKN SAWWYSQMARD WGQGTQVT 10E07/78 LVQAGGSLRL 299 KERVLVA/ VADAVKG/348 TVYLQMNSLKP NYRY/409 VSS/603 SCAASGRTFS/ 529 EDTAVYYCAA/ 470 565 VEGFBII EVQLVESGGG RYAMG/ WFRQAPG SINTSGKRTS RFAVSRDNAKN DRFFGSDSNEP WGQGTQVT 10G04/79 LVQAGGSLRL 300 KEREFVA/ YADSMKG/ TGYLQMNSLKL RAYRY/410 VSS/603 SCAASGDTLS/ 518 349 EDTATYYCAA/ 483 566 VEGFBII EVQLVESGGG NYNMG/ WFRQAPG TIRHHGYDTY RFTISRDNAKN KLFWDMDPKTG WGQGTQVT 10G05/80 LVQAGESLRL 301 KEREFVA/ YAESVKG/350 EVYLQMNSLKP FSS/411 VSS/603 SCVASGITFS/ 518 EDTALYSCAK/ 484 567 VEGFBII EVQLVESGGG SYGLG/ WFRQAPG AIGWSGSSTY RFTVSVDNAKN KVRNFNSDWDL WGQGTQVT 11C08/81 LVQAGGSLRL 302 KEREFVA/ YADSVKG/351 TVYLKMNSLEP LTSYNY/412 VSS/603 SCAASGRTLS/ 518 EDTAVYYCAA/ 485 568 VEGFBII EVQLVESGGG SYAIG/ WFRQAPG RISWSGANTY RFTISRGNAKN QTTSKYDNYDA WGQGTQVT 11C11/82 LVQAGGSLML 303 REREFVA/ YADSVKG/ TVYLQMNSLKP RAYGY/413 VSS/603 SCAASGRALS/ 530 352 EDTAAYYCAA/ 486 569 VEGFBII EEQLVESGGG SYAIG/ WFRQAPG RISWSGANTY RFTISRGNAKN QTTSKYDNYDA WGQGTQVT 11D09/83 LVQAGGSLML 303 REREFVA/ YADSVKG/352 TVYLQMNSLKP RAYGY/413 VSS/603 SCAASGRALS/ 530 EDTAAYYCAA/ 487 569 VEGFBII EVQLVESGGG SYAMG/ WFRQAPG TISQSGYSTY RFTISRDNAKN DPFYSYGSPSP WGQGTQVT 11E04/84 LVQAGGSLRL 304 KEREFVA/ YADSVKG/353 TVNLQMNSLKPP YRY/414 VSS/603 SCAASGRTFS/ 518 EDTAVYYCAA/ 470 570 VEGFBII EVQLVESGGG FSAMG/ WFRQAPG AFKWSGSTTY RFTISTDNAKN DRFYTGRYYSS WGQGTQVT 11E05/85 LVQPGGSLRL 305 KEREFVA/ TADYVKG/354 ILFLQMNSLKP DEYDY/415 VSS/603 SCASSGRLFS/ 518 EDTAIYYCAV/ 488 571 VEGFBII EVQLVESGGG ITVMA/ WFRQAPG AITWSAPSSY RFTISRDNAKN DRFKGRSIVTR WGQGTQVT 11F10/86 LVQAGGSLRL 286 KEREFVA/ YADSVKG/ TVYLQVNSLKP SDYRY/416 VSS/603 SCAASTSIYS/ 518 355 EDSAIYYCAA/ 474 572 VEGFBII EVQLVESGGG SLAMG/ WFRQVPG SISQSGITTS RFTISRDSAKN SVFYSTALTRP WGQGTQVT 11F12/87 LVQSGGSLRL 306 KDREFVA/ YADSVKS/356 TVYLQMNLLKP VDYRY/417 VSS/603 SCAASGRSFS/ 531 EDTAVYYCAT/ 489 573 VEGFBII EVQLVESGGG ITVMA/ WFRQAPG AITWSAPTTY RFTISRDNAKN DRFKGRSIVTR WGQGTQVT 11G09/88 LVQAGGSLRL 286 KEREFVA/ SADSVKG/ TVYLQMNSLKP SDYRY/416 VSS/603 SCAASTSIYS/ 518 357 EDSAIYYCAA/ 474 556 VEGFBII EVQLVESGGG KYVMG/ WFRQAPG AITSRDGPTY RFTISGDNTKN DEDLYHYSSYH WGQGTQVT 12A07/89 LVQAGGSLRL 307 NDREFVA/ YADSVKG/358 KIFLQMNSLMP FTRVDLYHY/ VSS/603 SCSVTGRTFN/ 532 EDTAVYYCAI/ 418 490 574 VEGFBII EVQLVESGGG SSWMY/ WVRQAPG RISPGGLFTY RFSVSTDNANN GGAPNYTP/419 RGRGTQVT 12B01/90 LVQPGGSLRL 308 KGLEWVS/ YVDSVKG/359 TLYLQMNSLKP VSS/608 ACAASGFTLS/ 533 EDTALYSCAK/ 491 575 VEGFBII EVQLVESGGG SDVMG/ WYRQAPG FIRSLGSTYY RFTISRDNAAN RFSGESY/402 WGQGTPVT 12C04/91 LVQPGGSLRL 296 KQRELVA/ AGSVKG/342 TVYLQMNNLKP VSS/605 SCAASGSIVR/ 521 EDTAVYYCNA/ 479 576 VEGFBII EVQLVESGGG NYVMG/ WFRQAPG AITSTNGPTY RFTISGDNTKN DEDLYHYSSYH WGQGTQVT 12E10/92 LAQAGGSLRL 309 NEREFVA/ YADSVKG/360 KVFLQMDSLRP YTRVALYHY/ VSS/603 SCTASGRTFN/ 534 EDTAVYYCAI/ 420 492 577 VEGFBII EVQLVESGGG LYAMG/ WFRQAPG AITWSAGDTQ RFTISRDNARN RQWGGTYYYHG WGQGTQVT 12G04/93 LVQSGDSLRL 297 REREFVS/ YADSVKG/343 TVNLQMNGLKP SYAW/421 VSS/603 SCAVSGNTFG/ 535 EDTAVYYCAG/ 493 562 VEGFBII EVQLVESEGG TDDVG/ WFRQAPG VIRWSTGGTY RFTLSRDNAKN RSRPLGAGAWY WGQGTQVT 16C03/94 LVQAGGSLRL 288 KEREFVA/ TSDSVKG/ TMYLQMNSLKP TGENYYNY/ VSS/603 SCAASGRTFS/ 518 334 EDTAVYYCAA/ 422 494 552 VEGFBII EVQLVESGGG GYDMG/ WFRQAPG AITWSGGSTY RFTISRDNAKN GRIWRSRDYDS WGHGTQVT 16F11/95 LVQAGGSLRL 310 KEREFVT/ SPDSVKG/361 TVYLQMNNLTP EKYYDI/423 VSS/609 SCAASGRTSS/ 536 EDTAVYYCAS/ 495 578 VEGFBII EVQLVESGGG AYDMG/ WFRQAPG VISWTNSMTY RFTISRDNAKN DRRRTYSRWRF WGQGTQVT 36C08/96 LVQAGGSLRL 311 KEREFVA/ YADSVKG/362 TVYLQMNSLKP YTGVNDYDY/ VSS/603 SCAASGRTFS/ 518 EDTAVYYCAV/ 424 470 579 VEGFBII EVQLVESGGG AYDMG/ WFRQAPG VISWSGGMTY RFTISRDNAKS DRRRAYSRWRY WGQGTQVT 37F09/97 LVQTGGSLRL 311 KEREFVA/ YADSVQG/363 TVYLQMNSPKP YTGVNDYEF/ VSS/603 SCAASGRTFS/ 518 EDTAVYYCAV/ 425 476 580 VEGFBII EVQLVESGGG AYDMG/ WFRQAPG VISWSGGMTY RFTISRDNAKN DRRRLYSRWRY WGQGTQVT 38A06/98 LVQAGGSLRL 311 KEREFVA/ YADSVKG/364 TVYLQMNSLKP YTGVNDYDY/ VSS/603 SCAASGRTFS/ 518 EDTAVYYCAV/ 426 470 579 VEGFBII EVQLVESGGG AYDMG/ WFRQAPG VISWTGGMTY RFTISRDKAKN DRRRTYSRWRY WGQGTQVT 39H11/99 LVQAGGSLRL 311 KEREFVA/ YADSVKG/365 TVSLQMNSLKP YTGVNEYEY/ VSS/603 SCAASGRTFS/ 518 EDTAVYYCAV/ 427 470 581 VEGFBII EVQLVESGGG AYDMG/ WFRQAPG VISWTGDMTY RFTISRDKAKN DRRRTYSRWRY WGQGTQVT 41B06/ LVQAGGSLRL 311 KEREFVA/ YADSVKG/ TVSLQMNSLKP YTGVNEYEY/ VSS/603 100 SCAASGRTFS/ 518 366 EDTAVYYCAA/ 427 470 582 VEGFBII EVQLVESGGG VYTMG/ WFRQAPG TISRTGDRTS RFTISRENAKN GPIAPSPRPRE WGQGTQVT 41C05/ LVQAGGSLRL 312 KEREFVA/ YANSVKG/367 TVYLQMNSLKP YYY/428 VSS/603 101 SCAASGRTFS/ 518 EDTAVYSCAA/ 470 583 VEGFBII EVQLMESGGG AYDMG/ WFRQAPG VISWTGGMTY RFTISRDKAKN DRRRTYSRWRY WGQGTQVT 41D11/ LVQAGGSLRL 311 KEREFVA/ YADSVKG/ TVSLQMNSLKP YTGVNEYEY/ VSS/603 102 SCAASGRTFS/ 518 365 EDTAVYYCAV/ 427 496 581 VEGFBII EVQLVESGGG AYDMG/ WFRQAPG VISWSGGMTD RFTISRENAKN GRRRAYSRWRY WGQGTQVT 42F10/ LVQAGGSLRL 311 KEREFVA/ YADSVKG/368 TQFLQMNSLKP YTGVNEYDY/ VSS/603 103 SCAASGRTFS/ 518 EDTAVYYCAV/ 429 470 584 VEGFBII EVQLVESGGG SYAMG/ WFRQAPG HINRSGSSTY RFTISRDNAKN GRYYSSDGVPS WGQGTQVT 86C11/ LVQAGDSLRL 304 KERESVA/ YADSVKG/ TVYLQLNSLKP ASFNY/430 VSS/603 104 SCTASGRTFN/ 537 369 EDTAVYYCAA/ 497 585 VEGFBII EVQLVESGGG TWAMA/ WFRQAPG AISWSGSMTY RFIISRDNAQN KTVDYCSAYEC WGRGAQVT 86F11/ LVQAGDSLRL 313 KEREFIS/ YTDSVKG/ TLFLQMNNTAP YARLEYDY/ VSS/610 105 SCFTSARTFD/ 538 370 EDTAVYYCAA/ 431 498 586 VEGFBII EVQLVESGGG STNMG/ WFRQGPG AITLSGTTYY RFTISRDNDKN DPSYYSTSRYT WGQGTQVT 86G08/ LMQTGDSLRL 314 KEREFVA/ AEAVKG/ TVALQMNSLKP KATEYDY/ VSS/603 106 SCAASGLRFT/ 539 371 EDTAVYYCGA/ 432 499 587 VEGFBII EVQLVESGGG TYTMG/ WFRQTPG AIRWTVNITY RFTISRDIVKN QTSAPRSLIRM WGQGTQVT 86G10/ LVQAGGSLRL 315 TEREFVA/ YADSVKG/ TVYLQMNSLKP SNEYPY/433 VSS/603 107 SCAASGRTFN/ 540 372 EDTAVYYCAA/ 500 588 VEGFBII EVQLVESGGG LYTVG/ WFRQAPG YISRSGSNRY RFTLSRDNAKN TSRGLSSLAGE WGRGTQVT 86G11/ LVQAGGSLRL 316 KEREFVA/ YVDSVKG/ TVDLQMNSLKT YNY/434 VSS/607 108 SCAASGLTFS/ 518 373 EDTAVYYCAA/ 501 589 VEGFBII EVQLVESGGG SYRMG/ WFRRTPG SISWTYGSTF RFTMSRDKAKN GAQSDRYNIRS WGQGTQVT 86H09/ LVQAGGSLRL 317 KEDEFVA/ YADSVKG/ AGYLQMNSLKP YDY/435 VSS/603 109 SCTASGSAFK/ 541 374 EDTALYYCAA/ 502 590 VEGFBII EVQLVESGGG TSWMH/ WVRQAPG SIPPVGHFAN RFTISRDNAKN DSAGRT/436 KGQGTQVT 87B07/ LVQPGGSLKL 318 KGLEWVS/ YAPSVKG/ TLFLQMNSLKS VSS/611 110 SCTASGFTFS/ 533 375 EDTAVYYCAK/ 503 591 VEGFBII KVQLVESGGG NYAMD/ WFRQAPG AITRSGGGTY RFTISRDNAKN TRSSTIVVGVG WGKGTLVT 88A01/ LVQAGGSLRL 319 KEREFVA/ YADSVKG/ TVYLQMNSLKP GMEY/437 VSS/612 111 SCAASERTFS/ 518 376 EDTAVYYCAA/ 504 565 VEGFBII EVQLVESGGG DYDIG/ WFRQAPG CITTDVGTTY RFTISSDNAKN DTQDLGLDIFC WGQGTQVT 88A02/ LVQAGGSLRL 320 NEREGVS/ YADSVKG/ TVYLQINDLKP RGNGPFDG/ VSS/603 112 SCAASGFTFG/ 542 377 EDTAIYYCAV/ 438 505 592 VEGFBII EVQLVESGGG DYAIG/ WFRQAPG CISSYDSVTY RFTISRDSAKN EREQLRRRESP SGKGTLVT 88B02/ LVQPGGSLRL 230 KEREGVS/ YADHVKG/ TLYLQMNSLSI HDELLRLCFYG VSS/613 113 SCTASGLNLD/ 543 378 EDTGVYYCAA/ MRY/439 506 593 VEGFBII EVQLVESGGG DYAIG/ WFRQAPG CISSSDTSID RFTFSRDNAKN AFRCSGYELRG WGQGTQVT 88E02/ LVQPGGSLRL 230 KEREAVS/ YTNSVKG/ TVYLQMNSLKP FPT/440 VSS/603 114 SCVASGFRLD/ 544 379 EDTAVYYCAA/ 507 594 VEGFBII EVQLVESGGG SLAVG/ WFRQAPG RITWSGATTY RFTISRDNAKN DRSPNIINVVT WGQGTQVT 88G03/ LVQAGGSLRL 321 KEREFVA/ YADAVKD/ TMYLQMNSLKP AYEYDY/441 VSS/603 115 SCAASGGTFS/ 518 380 EDTAVYYCAA/ 508 595 VEGFBII EVQLVESGGG LYNMG/ WFRQAPG AITSSPMSTY RFSISINNDKT PEGSFRRQYAD WGQGTQVT 88G05/ LVQPGASLRL 322 KEREFVA/ YADSVKG/ TGFLQMNVLKP RAMYDY/442 VSS/603 116 SCAASGDGFT/ 518 381 EDTGVYFCAA/ 509 596 VEGFBII EVQLVESGGG GSDMG/ WFRQSPG AIRLSGSITY RFTISRDNAKN RSTYSYYLALA WGQGTQVT 88G11/ LAQAGGSLRL 323 KEREIVA/ YPDSVKG/ TVYLQMNSLKP DRGGYDY VSS/603 117 SCAASGRTFS/ 545 382 EDTAVYYCAA/ /443 510 565 VEGFBII EVQLVESGGG TYAIG/ WFRQAPG CMSAGDSIPW RFTTSTDNARN ARYHGDYCYYE WGQGTQVT 88H01/ LVQAGGSLRL 324 KEREAVS/ YTASVKG/ TVYLQMNSLKP GYYPF/444 VSS/603 118 SCVASGFTLG/ 544 383 EDTAHYYCAA/ 511 597 VEGFBII EVQLVESGGG TNFMG/ WYRQAPG TITSSSITNY RFTISRDNAKN RWRWSDVEY/ WGKGTLVT 89B04/ LVQAGGSLRL 325 KQRELVA/ VDSVKG/ TVYLQMTSLKP 445 VSS/612 119 SCAASTSISS/ 521 384 EDTAVYYCHA/ 512 598 VEGFBII EVQLVESGGG IFAMR/ WYRQAPG SITRSSITTY RFTPSRDNAKN AIRPELYSVVN WGQGTQVT 89B08/ LVQPGGSLRL 326 KQRELVA/ ADSVKG/ TVSLQMNSLKP DY/446 VSS/603 120 SCAASGTTSS/ 521 385 EDTAVYYCNA/ 513 599 VEGFBII EVQLVESGGG DYNLG/ WFRQAPG VISWRDSFAY RFTISRDNAKN DRVSSRLVLPN WGQGTQVT 89D04/ LVQPGGSLRL 327 KERQFVA/ YAEPVKG/ TVYLQMNSLKP TSPDFGS/ VSS/603 121 SCATSGLTFS/ 546 386 EDTAVYYCAA/ 447 514 565 VEGFBII EVQLVESGGG NAIMG/ WFRQAPG AMNWRGGPTY RFTISGDNTKN DEDLYHYSSYH WGQGTQVT 89F09/ LVQAGDSLRL 328 QEREFVA/ YADSVKG/ TVFLQMNFLKP YSRVDLYHY/ VSS/603 122 SCAASGRTFN/ 547 387 EDTAVYYCAA/ 448 515 600 VEGFBII EVQLVESGGG IFAMR/ WYRQAPG SITRSSITTY RFTLSRDNAKN AIRPELYSVVN WGQGTQVT 89G09/ LVQPGGSLRL 326 KQRELVA/ ADSVKG/ TVSLQMNSLKP DY/446 VSS/603 123 SCAASGTTSS/ 521 385 EDTAVYYCNA/ 513 601 VEGFBII EVQLVESGGG SYAPG/ WFRQAPG AFTRSSNIPY RFTISRDNAHT NLGSTWSRDQR WGQGTQVT 89H08/ LVQAGGSLRL 329 KEREFVA/ YKDSVKG/ VYLQMNSLKPE TYDY/449 VSS/603 124 SCAASGGSFS/ 518 388 DTAIYYCAV/ 516 602

    TABLE-US-00006 TABLE 3 Sequence IDs and AA sequences of monovalent receptor-blocking anti-VEGF VHHs (FR, framework; CDR, complementary determining region) SEQ ID NO: 9-46 CDR1/ VHH ID/ FR1/SEQ SEQ FR2/SEQ CDR2/SEQ FR3/SEQ CDR3/SEQ FR4/SEQ SEQ ID NO: ID NO: ID NO: ID NO: ID NO: ID NO: ID NO: ID NO: VEGFBII EVQLVESGG SYSM WFRQAQGKE AISSSGGYI RFTISRDNTKNT SRAYGSSRLRL WGQGTQV 22A10/9 GLVQPGDSL G/450 REFVV/636 YDSVSLEG/ VYLQTPSLKPED ADTYDY/3 TVSS/603 KLSCAFSGR 453 TADYYCAA/638 TFS/614 VEGFBII EVQLVESGG SYSM WFRQAQGKE AISSGGFIY RFTISRDNTKNT SRAYGSSRLRL WGQGTQV 22A11/10 GLVQPGDSL A/451 REFVV/636 DAVSLEG/ VYLQTPSLKPED ADTYDY/3 TVSS/603 KLSCAFSGR 454 TAVYYCAA/639 TFS/614 VEGFBII EVQLVESGG SYSM WFRQAQGKE AISSSGGYI RFTISRDNTKNT SRAYGSSRLRL WGQGTQV 22B06/11 GLVQPGDSL G/450 REFVV/636 YDSVSLEG/ VYLQTPSLKPED ADTYDY/3 TVSS/603 KLSCAASGR 453 TAVYYCAA/639 TFS/615 VEGFBII EVQLVESGG SYSM WFRQAQGKE AISSSGNYK RFTISRDNTKNT SRAYGSSRLRL WGQGTQV 22B07/12 GLVQAGDSL G/450 REFVV/636 YDSVSLEG/ VYLQINSLKPED GDTYDY/2 TVSS/603 RLSCAASGR 455 TAVYYCAA/640 TFS/616 VEGFBII EVQLVESGG SYSM WFRQAQGKE AISSGGSIY RFTISRDNTKNT SRAYASSRLRL WGQGTQV 22E04/13 GLVQPGDSL G/450 REFVV/636 DSVSLQG/ VYLQTPSLKPED ADTYDY/6 TVSS/603 KLSCVASGR 456 TAVYYCAA/639 TSS/617 VEGFBII EVQLVESGG SYSM WFRQAQGKE AISSGGYIY RFTISRDNTKNT SRAYGSSRLRL WGQGTQV 23A03/14 GLVQPGDSL G/450 REFVV/636 DSVSLQG/ VYLQTPSLKPED ADTYDY/3 TVSS/603 KLSCVASGR 457 TAVYYCAA/639 TFS/618 VEGFBII EVQLVESGG SYSM WFRQAQGKE AISSGGFIY RFTISRDNTKNT SRAYGSSRLRL WGQGTQV 23A06/15 GLVQPGDSL G/450 REFVV/636 DAVSLEG/ VYLQTPSLKPED ADTYDY/3 TVSS/603 KLSCAFSGR 454 TAVYYCAA/639 TFS/619 VEGFBII EVQLVESGG SYSM WFRQAQGKE AISNGGYKY RFTISRDNTKNT SRAYGSSRLRL WGQGTQV 23A08/16 GLVQTGDSL G/450 REFVV/636 DSVSLEG/ VYLQINSLKPED ADTYDY/3 TVSS/603 RLSCVASGR 458 TAVYYCAA/640 TFS/620 VEGFBII EVQLVESGG SYSM WFRQAQGKE AISSSGGYI RFTISRDNSKNT SRAYGSSRLRL WGQGTQV 23A09/17 GLVQPGDSL G/450 REFVV/636 YDSVSLEG/ VYLQTPSLKPED PDTYDY/7 TVSS/603 KLSCAFSGR 453 TAVYYCAA/641 TFG/621 VEGFBII EVQLVESGG SYSM WFRQAQGKE AISKGGYKY RFTISKDNAKNT SRAYGSSRLRL WGQGTQV 23B04/18 GLVQTGDSL G/450 REFVV/636 DSVSLEG/ VYLQINSLKPED ADTYEY/4 TVSS/603 RLSCEVSGR 459 TAVYYCAS/642 TFS/622 VEGFBII EVQLVESGG SYSM WFRQAQGKE AISSGGFIY RFTISRDNTKNT SRAYGSSRLRL WGQGTQV 23D11/19 GLVQPGDSL A/451 REFVV/636 DAVSLEG/ VYLQTPSLKPED ADTYDY/3 TVSS/603 RLSCAFSGR 454 TAVYYCAA/639 TFS/623 VEGFBII EVQLVESEG SYSM WFRQAQGKE AISSGGYIY RFTISRDNTKNT SRAYGSSRLRL WGQGTQV 23E05/20 GLVQPGDSL G/450 REFVV/636 DSVSLQG/ VYLQTPSLKPED ADTYDY/3 TVSS/603 KLSCVASGR 457 TAVYYCAA/639 TSS/624 VEGFBII EMQLVESGG SYSM WFRQAQGKE AISSSGGYI RFTISRDNTKNT SRAYGSSRLRL WGQGTQV 23F02/21 GLVQPGDSL G/450 REFVV/636 YDSVSLEG/ VYLQTPSLKPED ADTYDY/3 TVSS/603 KLSCAFSGR 453 TADYYCAA/638 TFS/625 VEGFBII EVQLVESGG SYSM WFRQAQGKE AISSSGNYK RFTISRDNTKNT SRAYGSSRLRL WGQGTQV 23F05/22 GLVQAGDSL G/450 REFVV/636 YDSVSLEG/ VYLQINSLKPKD GDTYDY/2 TVSS/603 RLSCAASGR 455 TAVYYCAA/643 TFS/616 VEGFBII EVQLVESGG SYSM WFRQAQGKE AISSGGGYI RFTISRDNTKNT SRAYGSSRLRL WGQGTQV 23F11/23 GLVQPGDSL G/450 REFVV/636 YDSVSLEG/ VYLQTPSLKPED ADTYDY/3 TVSS/603 KLSCAFSGR 460 TADYYCAA/638 TFS/614 VEGFBII EVQLVESGG SYSM WFRQAQGKE AISSSGGYI RFTISRDNSKNT SRAYGSSRLRL WGQGTQV 23G03/24 GLVQPGDSL G/450 REFVV/636 YDSVSLEG/ VYLQTPSLKPED PGTYDY/8 TVSS/603 KLSCAFSGR 453 TAVYYCAA/641 TFG/621 VEGFBII EVQLVESGG SYSM WFRQAQGKE AISSGGYIY RFTISRDNTKNT SRAYGSSRLRL WGQGTQV 24C04/25 GLVQPGDSL G/450 REFVV/636 DSVSLQG/ VYLQTPSLKPED ADTYDY/3 TVSS/603 KLSCVASGR 457 TAVYYCAA/639 TSS/617 VEGFBII EVQLVESGG SYSM WFRQAQGKE AISSGGYKY RFTISRDNTQNT SRAYGSGRLRL WGQGTQV 27D08/26 GLVQTGDSL G/450 REFVV/636 DSVSLEG/ VYLQINSLKPED ADTYDY/5 TVSS/603 RLSCAASGR 461 TAVYYCAA/644 TFS/626 VEGFBII EVQLVESGG SYSM WFRQAQGQE AISSGGYIY RFTISRDNTKNT SRAYGSSRLRL WGQGTQV 27G07/27 GLVQPGDSL G/450 REFVV/637 DSVSLQG/ VYLQTPSLKPED ADTYDY/3 TVSS/603 KLSCVASGR 457 TAVYYCAA/639 TSS/617 VEGFBII EVQLVESGG SYSM WFRQAQGQE AISSGGYIY RFTISRDNTKNT SRAYGSSRLRL WGQGTQV 30C09/28 GLVQPGDSL G/450 REFVV/637 DSVSLQG/ VYLQTPSLKPED ADTYDY/3 TVSS/603 KLSCIASGR 457 TAVYYCAA/639 TSS/627 VEGFBII EVQLVESGG SYSM WFRQAQGKE AISSSGNYK RFTISRDNTKNT SRAYGSSRLRL WGQGTRV 30E07/29 GLVQAGDSL G/450 REFVV/636 YDSVSLEG/ VYLQINSLKPED GDTYDY/2 TVSS/652 RLSCAASGR 455 TAVYYCAA/640 TFS/616 VEGFBII EVQLVESGG SYSM WFRQAQGKE AISSSGGYI RFTISRDNTKNT SRAYGSSRLRL WGQGTQV 31C07/30 GLVQTGDSL G/450 REFVV/636 YDSVSLEG/ VYLQTPSLKPED ADTYDY/3 TVSS/603 RLSCAASGG 453 TADYYCAA/638 TFS/628 VEGFBII EVQLVESGG SYSM WFRQAQGKE AISSSGGYI RFTISRDNTKNT SRAYGSSRLRL WGQGTQV 39E02/31 GLVQPGDPL G/450 REFVV/636 YDSVSLEG/ VYLQTPSLKPED ADTYDY/3 TVSS/603 KLSCAFSGR 453 TADYYCAA/638 TFS/629 VEGFBII EVPLVESGG SYSM WFRQAQGKE AISSSGNYK RFTISRDNTKNT SRAYGSSRLRL WGQGTQV 39G04/32 GLVQAGDSL G/450 REFVV/636 YDSASLEG/ VYLQINSLKPED GDTYDY/2 TVSS/603 RLSCAASGR 462 TAVYYCAA/640 TFS/630 VEGFBII EVQLVESGG SYSM WFRQAQGKE AISSGGFIY RFTISRDNTKNT SRAYGSSRLRL WGQGTQV 40F02/33 GLVQPGDSL A/451 REFVV/636 DAVSLEG/ VYLQTPSLKPEG ADTYDY/3 TVSS/603 KLSCAFSGR 454 TAVYYCAA/645 TFS/614 VEGFBII EVQLVESGG SYSM WFRQAQGKE AISSSGGYI RFTISRDNTKNA SRAYGSSRLRL WGQGTQV 40G07/34 GLVQPGDSL G/450 REFVV/636 YDSVSLEG/ VYLQTPSLKPED ADTYDY/3 TVSS/603 KLSCAFSGR 453 TADYYCAA/646 TFS/614 VEGFBII EVQLMESGG SYSM WFRQAQGKE AISSSGGYI RFTISRDNTKNT SRAYGSSRLRL WGQGTQV 40H10/35 GLVQPGDSL G/450 REFVV/636 YDSVSLEG/ VYLQTPSLKPED ADTYDY/3 TVSS/603 KLSCAFSGR 453 TADYYCAA/638 TFS/631 VEGFBII EVQLVESGG SYSM WFRQAQGKE AISSGGFIY RFTISRDNTKNT SRAYGSSRLRL WGQGTQV 41B05/36 GLVQPGGSL G/450 REFVV/636 DAVSLEG/ VYLQTPSLKPED ADTYDY/3 TVSS/603 RLSCAFSGR 454 TAVYYCAA/639 TFS/632 VEGFBII EVQLVESGG SYSM WFRQAQGKE AISSGGFIY RFTISRENTKNT SRAYGSSRLRL WGQGTQV 41G03/37 GLVQPGDSL A/451 REFVV/636 DAVSLEG/ VYLQTPSLKPED ADTYDY/3 TVSS/603 KLSCAFSGR 454 TAVYYCAA/647 TFS/614 VEGFBII EVQLVESGG SYSM WFRQAQGKE AISSSGGYI RFTISRDNTKNT SRAYGSSRLRL WGQGTQV 42A05/38 GLVQPGDSL G/450 REFVV/636 YDSVSLEG/ VYLQMPSLKPED ADTYDY/3 TVSS/603 KLSCAFSGR 453 TADYYCAA/648 TFS/614 VEGFBII EVQLVESGG SYSM WFRQAQGKE AISSSGGYI RFTISRDNTKNT SRAYGSSRLRL WGQGTQV 42D05/39 GLVQPGDSL G/450 REFVV/636 YDSVSLEG/ VYLQTPSLKPED ADTYDY/3 TVSS/603 KLSCAFSGR 453 TAVYYCAA/639 TFS/614 VEGFBII EVQLVESGG SYSV WFRQAQGKE AISSGGYIY RFTISRDNTKNT SRAYGSSRLRL WGQGTQV 42F11/40 GLVQPGDSL G/452 REFVV/636 DSVSLQG/ VYLQTPSLKPED ADTYDY/3 TVSS/603 KLSCVASGR 457 TAVYYCAA/639 TSS/617 VEGFBII EVQLVESGG SYSM WFRQAQGKE AISSSGGYI RFTISRDNTKNT SRAYGSSRLRL WGQGTQV 56E11/41 GLVQPGDSL G/450 REFVV/636 YDSVSLEG/ VYLQTPSLKPED ADTYDY/3 TVSS/603 KLSCAFSGR 453 AADYYCAA/649 TFS/614 VEGFBII EVQLVESGG SYSM WFRQAQGKE AISSSGGYI RFTISRDNTRNT SRAYGSSRLRL WGQGTQV 60A09/42 GLVQPGDSL G/450 REFVV/636 YDSVSLEG/ VYLQTPSLKPED ADTYDY/3 TVSS/603 KLSCAFSGR 453 TADYYCAA/650 TFS/614 VEGFBII EVQLVESGG SYSM WFRQAQGKE AISSGGYKY RFTISRDNTKNT SRAYASSRLRL WGQGTQV 61A01/43 GLVQAGGSL G/450 REFVV/636 DAVSLEG/ VYLQTPSLKPED ADTYDY/6 TVSS/603 RLSCAFSGR 463 TAVYYCAA/639 TFS/633 VEGFBII EVQLVESGG SYSM WFRQAQGKE AISSSGGYI RFTISRDNTKNT SRAYGSSRLRL WGQGTQV 62A09/44 DLVQPGDSL G/450 REFVV/636 YDSVSLEG/ VYLQTPSLKPED ADTYDY/3 TVSS/603 KLSCAASGR 453 TAVYYCAA/639 TFS/634 VEGFBII EVQLVESEG SYSM WFRQAQGKE AISSSGNYK RFTISRDNTKNT SRAYGSSRLRL WGQGTQV 62D10/45 GLVQAGDSL G/450 REFVV/636 YDSVSLEG/ VYLQINSLKPED GDTYDY/2 TVSS/603 RLSCAASGR 455 TAVYYCAA/640 TFS/635 VEGFBII EVQLVESGG SYSM WFRQAQGKE AIASGGYIY RFTISRDNTKDT SRAYGSSRLRL WGQGTQV 62F02/46 GLVQPGDSL G/450 REFVV/636 DAVSLEG/ VYLQTPSLKPED ADTYDY/3 TVSS/603 KLSCAFSGR 464 TAVYYCAA/651 TFS/614

    [0373] Dissociation rates of inhibitory VHHs are analyzed on Biacore (Biacore T100 instrument, GE Healthcare). HBS-EP+buffer is used as running buffer and experiments are performed at 25.C. Recombinant human VEGF165 is irreversibly captured on a CM5 sensor chip via amine coupling (using EDC and NHS) up to a target level of +/−1500RU. After immobilization, surfaces are deactivated with 10 min injection of 1M ethanolamine pH8.5. A reference surface is activated and deactivated with respectively EDC/NHS and ethanolamine. Periplasmic extracts of VHHs are injected at a 10-fold dilution in running buffer for 2 min at 45 μl/min and allowed to dissociate for 10 or 15 min. Between different samples, the surfaces are regenerated with regeneration buffer. Data are double referenced by subtraction of the curves on the reference channel and of a blank running buffer injection. The of the processed curves is evaluated by fitting a two phase decay model in the Biacore T100 Evaluation software v2.0.1. Values for k.sub.d-fast, k.sub.d-slow and % fast are listed in Table 4.

    TABLE-US-00007 TABLE 4 Off-rate determination of anti-VEGF receptor-blocking VHHs with Biacore B- Bind- cell Unique ing line- sequence Representative % level age variant VHH ID k.sub.d(fast) k.sub.d(slow) fast (RU) 1  1 VEGFBII22B07 1.50E−02 7.80E−05 31 328 1  2 VEGFBII23A08 1.30E−02 5.00E−05 19 502 1  3 VEGFBII23B04 8.80E−03 4.00E−05 12 768 1  4 VEGFBII27D08 2.40E−02 8.10E−05 13 225 1  5 VEGFBII24C04 1.30E−02 3.40E−05 17 456 1  6 VEGFBII27G07 1.30E−02 3.80E−05 18 471 1  7 VEGFBII22E04 1.80E−02 1.10E−04 14 520 1  8 VEGFBII23A03 1.50E−02 3.20E−05 15 487 1  9 VEGFBII22B06 3.80E−02 9.00E−05 23 168 1 10 VEGFBII23A09 2.70E−02 4.60E−05 20 247 1 11 VEGFBII23G03 2.80E−02 8.60E−05 28 141 1 12 VEGFBII22A11 2.20E−02 4.70E−05 12 461 1 13 VEGFBII23A06 1.70E−02 3.70E−05 13 547 1 14 VEGFBII23F11 2.70E−02 1.30E−04 22 134 1 15 VEGFBII22A10 3.70E−02 4.00E−05 19 229 1 16 VEGFBII23F05 1.60E−02 1.30E−04 29 198 1 17 VEGFBII23D11 1.90E−02 5.80E−05 13 510 1 18 VEGFBII23F02 n/d n/d n/d n/d 1 19 VEGFBII23E05 1.50E−02 6.90E−05 18 275 1 20 VEGFBII31C07 3.70E−02 1.50E−04 25  77 1 21 VEGFBII30C09 1.50E−02 7.60E−05 19 264 1 22 VEGFBII30E07 1.70E−02 1.30E−04 29 226 1 23 VEGFBII39G04 1.40E−02 7.40E−04 40 210 1 24 VEGFBII41G03 1.20E−02 2.70E−04 20 332 1 25 VEGFBII41B05 1.90E−02 1.20E−04 16 324 1 26 VEGFBII40F02 1.20E−02 9.80E−05 20 258 1 27 VEGFBII39E02 1.90E−02 2.40E−04 13 181 1 28 VEGFBII42D05 3.30E−02 1.50E−04 26  77 1 29 VEGFBII40G07 1.80E−02 3.20E−04 19 139 1 30 VEGFBII42A05 1.60E−02 3.40E−04 25 118 1 31 VEGFBII42F11 9.10E−03 5.00E−04 46 100 1 32 VEGFBII40H10 1.40E−02 2.90E−04 17 200 1 33 VEGFBII62A09 4.10E−02 1.10E−04 23  84 1 34 VEGFBII60A09 3.70E−02 9.30E−05 20 106 1 35 VEGFBII62F02 1.40E−02 8.50E−05 21 205 1 36 VEGFBII62D10 1.90E−02 1.60E−04 40  94 1 37 VEGFBII61A01 7.40E−03 1.70E−04 21 275 1 38 VEGFBII56E11 3.30E−02 1.40E−04 24  76 n/d, not determined

    EXAMPLE 5

    [0374] Characterization of Purified Anti-VEGF VHHs

    [0375] Three inhibitory anti-VEGF VHHs are selected for further characterization as purified protein: VEGFBII23B04, VEGFBII24C4 and VEGFBII23A6. These VHHs are expressed in E. coli TG1 as c-myc, His6-tagged proteins (“His6” disclosed as SEQ ID NO: 283). Expression is induced by addition of 1 mM IPTG and allowed to continue for 4 hours at 37° C. After spinning the cell cultures, periplasmic extracts are prepared by freeze-thawing the pellets. These extracts are used as starting material for VHH purification via IMAC and size exclusion chromatography (SEC). Final VHH preparations show 95% purity as assessed via SDS-PAGE.

    [0376] 5.1 Evaluation of Human VEGF165/VEGFR2 Blocking VHHs in Human VEGF165/Human VEGFR2-Fc Blocking ELISA

    [0377] The blocking capacity of the VHHs is evaluated in a human VEGF165/human VEGFR2-Fc blocking ELISA. In brief, 1 μg/mL of VEGFR2-Fc chimera (R&D Systems, Minneapolis, Minn., USA) is coated in a 96-well MaxiSorp plate (Nunc, Wiesbaden, Germany). Dilution series (concentration range 1 mM-64 pM) of the purified VHHs in PBS buffer containing 0.1% casein and 0.05% Tween 20 (Sigma) are incubated in the presence of 4 nM biotinlyated VEGF165. Residual binding of bio-VEGF165 to VEGFR2 is detected using horseradish peroxidase (HRP) conjugated extravidin (Sigma, St Louis, Mo., USA) and TMB as substrate. As controls Bevacizumab (Avastin®) and Ranibizumab (Lucentis®) are taken along. Dose inhibition curves are shown in FIG. 1; the corresponding IC50 values and % inhibition are summarized in Table 5.

    TABLE-US-00008 TABLE 5 IC.sub.50 (nM) values and % inhibition for monovalent VHHs in hVEGF165/hVEGFR2-Fc competition ELISA IC.sub.50 % VHH ID (nM) inhibition VEGFBII23B04 2.1 100 VEGFBII23A06 3.0 100 VEGFBII24C04 2.5 100 Ranibizumab 1.6 100 Bevacizumab 1.7 100

    [0378] 5.2 Evaluation of Human VEGF165/VEGFR2 Blocking VHHs in Human VEGF165/Human VEGFR1-Fc Blocking ELISA

    [0379] VHHs are also evaluated in a human VEGF165/human VEGFR1-Fc blocking ELISA. In brief, 2 μg/mL of VEGFR1-Fc chimera (R&D Systems, Minneapolis, Minn., USA) is coated in a 96-well MaxiSorp plate (Nunc, Wiesbaden, Germany). Dilution series (concentration range 1 mM-64 pM) of the purified VHHs in PBS buffer containing 0.1% casein and 0.05% Tween 20 (Sigma) are incubated in the presence of 0.5nM biotinlyated VEGF165. Residual binding of bio-VEGF165 to VEGFR1 is detected using horseradish peroxidase (HRP) conjugated extravidin (Sigma, St Louis, Mo., USA) and TMB as substrate. As controls Bevacizumab, Ranibizumab and an irrelevant VHH (2E6) are taken along. Dose inhibition curves are shown in FIG. 2; the corresponding IC.sub.50 values and % inhibition are summarized in Table 6.

    TABLE-US-00009 TABLE 6 IC.sub.50 (nM) values and % inhibition of monovalent VHHs in hVEGF165/hVEGFR1-Fc competition ELISA VHH ID IC.sub.50 (nM) % inhibition VEGFBII23B04 0.5 64 VEGFBII23A06 0.9 55 VEGFBII24C04 0.8 71 Ranibizumab 1.2 91 Bevacizumab 1.5 96

    [0380] 5.3 Evaluation of the Anti-VEGF165 VHHs in the Human VEGF165/Human VEGFR2-Fc Blocking AlphaScreen

    [0381] The blocking capacity of the VHHs is also evaluated in a human VEGF165/human VEGFR2-Fc blocking AlphaScreen. Briefly, serial dilutions of purified VHHs (concentration range: 200 nM-0.7 pM) in PBS buffer containing 0.03% Tween 20 (Sigma) are added to 4 pM bio-VEGF165 and incubated for 15 min. Subsequently VEGFR2-Fc (0.4 nM) and anti-Fc VHH-coated acceptor beads (20 μg/ml) are added and this mixture is incubated for 1 hour in the dark. Finally, streptavidin donor beads (20 μg/ml) are added and after 1 hour of incubation in the dark, fluorescence is measured on the Envision microplate reader. Dose-response curves are shown in the FIG. 3. The IC.sub.50 values for VHHs blocking the human VEGF165-human VEGFR2-Fc interaction are summarized in Table 7.

    TABLE-US-00010 TABLE 7 IC.sub.50 (pM) values and % inhibition for VHHs in hVEGF165/hVEGFR2-Fc competition AlphaScreen VHH ID IC.sub.50 (pM) % inhibition VEGFBII23B04 160 100 VEGFBII23A06 250 100 VEGFBII24C04 250 100 Ranibizumab 860 100

    [0382] 5.4 Evaluation of the Anti-VEGF165 VHHs in the Human VEGF165/Human VEGFR1-Fc Blocking AlphaScreen

    [0383] The blocking capacity of the VHHs is also evaluated in a human VEGF165/human VEGFR1-Fc blocking AlphaScreen. Briefly, serial dilutions of purified VHHs (concentration range: 500 nM-1.8 pM)) in PBS buffer containing 0.03% Tween 20 (Sigma) are added to 0.4 nM bio-VEGF165 and incubated for 15 min. Subsequently VEGFR1-Fc (1 nM) and anti-Fc VHH-coated acceptor beads (20 μg/ml) are added and this mixture is incubated for 1 hour in the dark. Finally, streptavidin donor beads (20 μg/ml) are added and after 1 hour of incubation in the dark, fluorescence is measured on the Envision microplate reader. Dose-response curves are shown in the FIG. 4. The IC.sub.50 values and % inhibition for VHHs blocking the human VEGF165-human VEGFR1-Fc interaction are summarized in Table 8.

    TABLE-US-00011 TABLE 8 IC.sub.50 (nM) values for VHHs in hVEGF165/hVEGFR1-Fc competition AlphaScreen VHH ID IC.sub.50 (nM) % inhibition VEGFBII23B04 0.9 41 VEGFBII23A06 0.4 46 VEGFBII24C04 0.2 53 Ranibizumab 3.3 79

    [0384] 12-0332-US-4

    [0385] 5.5 Determination of the Affinity of the Human VEGF165-VHH Interaction

    [0386] Binding kinetics of VHH VEGFB1123B04 with hVEGF165 is analyzed by SPR on a Biacore T100 instrument. Recombinant human VEGF165 is immobilized directly on a CM5 chip via amine coupling (using EDC and NHS). VHHs are analyzed at different concentrations between 10 and 360 nM. Samples are injected for 2 min and allowed to dissociate up to 20 min at a flow rate of 45 μl/min. In between sample injections, the chip surface is regenerated with 100 mM HCl. HBS-EP+ (Hepes buffer pH7.4+EDTA) is used as running buffer. Binding curves are fitted using a Two State Reaction model by Biacore T100 Evaluation Software v2.0.1. The calculated affinities of the anti-VEGF VHHs are listed in Table 9.

    TABLE-US-00012 TABLE 9 Affinity K.sub.D (nM) of purified VHHs for recombinant human VEGF165 VEGF165 k.sub.a k.sub.a1 k.sub.a2 k.sub.d k.sub.d1 k.sub.d2 K.sub.D VHH ID (M.sup.−1 .Math. s.sup.−1) (M.sup.−1 .Math. s.sup.−1) (M.sup.−1 .Math. s.sup.−1) (s.sup.−1) (s.sup.−1) (s.sup.−1) (nM) VEGFBII23B04.sup.(a) — 2.1E+05 1.4E−02 — 8.6E−03 2.4E−04 0.7 VEGFBII23A06.sup.(a) — 4.2E+05 2.0E−02 — 5.7E−02 1.0E−04 0.7 VEGFBII24C04.sup.(a) — 3.2E+05 1.8E−02 — 2.6E−02 9.6E−05 0.4 .sup.(a)Heterogeneous binding curve resulting in no 1:1 fit, curves are fitted using a Two State Reaction model by Biacore T100 Evaluation Software v2.0.1

    [0387] 5.6 Binding to Mouse VEGF164

    [0388] Cross-reactivity to mouse VEGF164 is determined using a binding ELISA. In brief, recombinant mouse VEGF164 (R&D Systems, Minneapolis, MS, USA) is coated overnight at 4° C. at 1 μg/mL in a 96-well MaxiSorp plate (Nunc, Wiesbaden, Germany). Wells are blocked with a casein solution (1% in PBS). VHHs are applied as dilution series (concentration range: 500 nM-32 pM) in PBS buffer containing 0.1% casein and 0.05% Tween 20 (Sigma) and binding is detected using a mouse anti-myc (Roche) and an anti-mouse-HRP conjugate (DAKO) and a subsequent enzymatic reaction in the presence of the substrate TMB (3,3′,5,5′-tetramentylbenzidine) (Pierce, Rockford, Ill., USA) (FIG. 5). A mouse VEGF164 reactive mAb is included as positive control. As reference, binding to human VEGF165 is also measured. EC.sub.50 values are summarized in Table 10.

    TABLE-US-00013 TABLE 10 EC.sub.50 (pM) values for VHHs in a recombinant human VEGF165 and mouse VEGF164 binding ELISA rhVEGF165 rmVEGF164 VHH ID EC.sub.50 (pM) EC.sub.50 (pM) VEGFBII23B04 297 NB VEGFBII24C04 453 NB VEGFBII23A06 531 NB NB, no binding

    [0389] 5.7 Binding to VEGF121

    [0390] Binding to recombinant human VEGF121 is assessed via a solid phase binding ELISA. Briefly, recombinant human VEGF121 (R&D Systems, Minneapolis, MS, USA) is coated overnight at 4° C. at 1 μg/mL in a 96-well MaxiSorp plate (Nunc, Wiesbaden, Germany). Wells are blocked with a casein solution (1% in PBS). VHHs are applied as dilution series (concentration range: 500 nM-32 pM) in PBS buffer containing 0.1% casein and 0.05% Tween 20 (Sigma) and binding is detected using a mouse anti-myc (Roche) and an anti-mouse-HRP conjugate (DAKO) and a subsequent enzymatic reaction in the presence of the substrate TMB (3,3′,5,5′-tetramentylbenzidine) (Pierce, Rockford, Ill., USA) (FIG. 6). As positive control serial dilutions of the VEGFR2 is taken along. EC.sub.50 values are summarized in Table 11.

    TABLE-US-00014 TABLE 11 EC.sub.50 (pM) values for monovalent VHHs in a recombinant human VEGF121 binding ELISA VHH ID EC.sub.50 (pM) VEGFBII23B04 510 VEGFBII24C04 792 VEGFBII23A06 928

    [0391] 5.8 Binding to VEGF Family Members VEGFB, VEGFC, VEGFD and PIGF

    [0392] Binding to VEGFB, VEGFC, VEGFD and PIGF is assessed via a solid phase binding ELISA. In brief, VEGFB, VEGFC, VEGFD and PIGF (R&D Systems, Minneapolis, MS, USA) are coated overnight at 4° C. at 1 μg/mL in a 96-well MaxiSorp plate (Nunc, Wiesbaden, Germany). Wells are blocked with a casein solution (1% in PBS). VHHs are applied as dilution series (concentration range: 500 nM-32 pM) and binding is detected using a mouse anti-myc (Roche) and an anti-mouse-AP conjugate (Sigma, St Louis, Mo., USA). As positive controls serial dilutions of the appropriate receptors are taken along and detected with horseradish peroxidase (HRP)-conjugated goat anti-human IgG, Fc specific antibody (Jackson Immuno Research Laboratories Inc., West Grove, Pa., USA) and a subsequent enzymatic reaction in the presence of the substrate TMB (3,3′,5,5′-tetramentylbenzidine) (Pierce, Rockford, Ill., USA). Dose-response curves of VHHs and controls are shown in FIG. 7. The results show that there was no detectable binding of the selected VHHs to VEGFB, VEGFC, VEGFD or PIGF.

    [0393] 5.9 Epitope Binning

    [0394] Biacore-based epitope binning experiments are performed to investigate which VEGF binders bind to a similar or overlapping epitope as VEGFBII23B04. To this end, VEGFBII23B04 is immobilized on a CM5 sensor chip. For each sample, human VEGF165 is passed over the chip surface and reversibly captured by VEGFBII23B4. Purified VHHs (100 nM) or periplasmic extracts ( 1/10 diluted) are then injected with a surface contact time of 240 seconds and a flow rate of 10 uL/minute. Between different samples, the surface is regenerated with regeneration buffer (100 mM HCl). Processed curves are evaluated with Biacore T100 Evaluation software. VHHs could be divided within two groups: group one which gave additional binding to VEGFBII23B04 captured VEGF165 and a second group which is not able to simultaneously bind to VEGFBII23B04 captured VEGF165. Table 12-A summarizes the binding epitopes of the tested VHHs.

    [0395] The same assay set-up is used to assess whether VEGFR1, VEGFR2, Ranibizumab and Bevacizumab are able to bind to human VEGF-165 simultaneously with VEGFBII23B04. Table 12-B presents the additional binding responses to VEGFBII23B04-captured VEGF165. Only VEGFR2 is not able to bind to VEGFBII23B04-captured VEGF165, underscoring the blocking capacity of VEGFBII23B04 for the VEGF-VEGFR2 interaction. In addition, these data show that the VEGFBII23B04 epitope is different from the Bevacizumab and Ranibizumab epitope.

    TABLE-US-00015 TABLE 12-A Epitope binning of anti-VEGF VHHs—simultaneous binding with VEGFBII23604 No or low 1C02 1E07 4B08 8E07 8F07 12A07 12B01 86C11 86F11 86G08 additional 86G10 86G11 87B07 88A01 88A02 88B02 88E02 88G03 88G05 88G11 binding to 88H01 89B04 89D04 89F09 89G09 89H08 24C04 23A6 27G07 23B04 23B04- captured VEGF165* Additional 3D12 5B02 5B03 5B05 6G02 7D08 8D09 8F06 10C07 10E07 binding to 10G04 10G05 11C08 11D09 11E04 11E05 11F12 86H09 41C05 captured 23B04- VEGF165 *indicating same or overlapping epitopes

    TABLE-US-00016 TABLE 12-B Epitope binning of VEGFBII23B04-binding of benchmark inhibitors or cognate receptors on VEGFBII23B04 captured VEGF165 Injection Binding step Binding [sample] level (RU) 1 VEGF165 100 nM 1727 2 VEGFBII23B04 100 nM — 3 Ranibizumab 100 nM 763 4 Bevacizumab 100 nM 1349 5 VEGFR1 100 nM 1011 6 VEGFR2 100 nM —

    [0396] 5.10 Characterization of the Anti-VEGF VHHs in the HUVEC Proliferation Assay

    [0397] The potency of the selected VHHs is evaluated in a proliferation assay. In brief, primary HUVEC cells (Technoclone) are supplement-starved over night and then 4000 cells/well are seeded in quadruplicate in 96-well tissue culture plates. Cells are stimulated in the absence or presence of VHHs with 33 ng/mL VEGF. The proliferation rates are measured by [.sup.3H] Thymidine incorporation on day 4. The results of the HUVEC proliferation assay are shown in Table.

    TABLE-US-00017 TABLE 13 IC.sub.50 (nM) values and % inhibition of monovalent VEGFBII23B04, VEGFBII23A06 and VEGFBII24C04 in VEGF HUVEC proliferation assay VHH ID IC.sub.50 (nM) % inhibition VEGFBII23B04 0.36 91 Bevacizumab 0.21 92 VEGFBII23A06 4.29 73 VEGFBII24C04 3.8 79 Bevacizumab 0.78 78

    [0398] 5.11 Characterization of the Anti-VEGF VHHs in the HUVEC Erk Phosphorylation Assay

    [0399] The potency of the selected VHHs is assessed in the HUVEC Erk phosphorylation assay. In brief, primary HUVE cells are serum-starved over night and then stimulated in the absence or presence of VHHs with 10 ng/mL VEGF for 5 min. Cells are fixed with 4% Formaldehyde in PBS and ERK phosphorylation levels are measured by ELISA using phosphoERK-specific antibodies (anti-phosphoMAP Kinase pERK1&2, M8159, Sigma) and polyclonal Rabbit Anti-Mouse-Immunoglobulin-HRP conjugate (P0161, Dako). As shown in Table 14, VEGFBII23B04 and Bevacizumab inhibit the VEGF induced Erk phosphoryaltion by at least 90%, with IC.sub.50s<1 nM.

    TABLE-US-00018 TABLE 14 IC.sub.50 (nM) values and % inhibition of monovalent VEGFBII23B04 in VEGF HUVEC Erk phosphorylation assay VHH ID IC.sub.50 (nM) % inhibition VEGFBII23B04 0.37 90 Bevacizumab 0.63 98

    EXAMPLE 6

    [0400] Generation of Multivalent Anti-VEGF Blocking VHHs

    [0401] VHH VEGFBII23B04 is genetically fused to either VEGFBII23B04 resulting in a homodimeric VHH (AA sequence see Table 15) or different VEGF binding VHHs resulting in heterodimeric VHHs. To generate the heterodimeric VHHs, a panel of 10 unique VEGF binding VHHs are linked via a 9 or 40 Gly-Ser flexible linker (SEQ ID NOS 170 and 171, respectively) in two different orientations to VEGFBII23B04 (AA sequences see Table 15). Homodimeric VEGFBII23B04 (VEGFBII010) and the 40 heterodimeric bivalent' VHHs are expressed in E. coli TG1 as c-myc, His6-tagged proteins (“His6” disclosed as SEQ ID NO: 283). Expression is induced by addition of 1 mM IPTG and allowed to continue for 4 hours at 37° C. After spinning the cell cultures, periplasmic extracts are prepared by freeze-thawing the pellets. These extracts are used as starting material and VHHs are purified via IMAC and desalting resulting in 90% purity as assessed via SDS-PAGE.

    TABLE-US-00019 TABLE 15 Sequence ID, VHH ID and AA sequence of bivalent anti-VEGF VHHs (each of the used linkers is highlighted in one relevant sequence) Sequence ID/ SEQ ID NO: VHH ID AA sequence VEGFBII23B04- VEGFBII010 EVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVS 35GS-23B04/128 LEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSS GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQTGDSLRLSCE VSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVSLEGRFTISKDNAKNTVYLQINSLK PEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSS VEGFBII23B04- EVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVS 9GS-4B08/129 LEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSS GGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGSAVGDITVAWYRQAPGIQRQLVATITP SGYTYYWDFVKGRFTISRDNSKNIVYLQMNSLKPEDTAAYYCNTQFYWGQGTQVTVSS VEGFBII23B04- EVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVS 9GS-5B03/130 LEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSS GGGGSGGGSEVQLVESGGGLAQAGDSLRLSCAASGRSFSHYNMGWFRQAPGKEREFVASIR GGGGSTTYANSVKDRFTISRENAKNTVYLQMNSLKPEDTAVYYCAATAFYRGPYDYDYWGQG TQVTVSS VEGFBII23B04- VEGFBII022 EVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVS 9GS-5B05/131 LEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSS GGGGSGGGSEVQLVESGGGLVQPGGSLRLSCVASGIRFMSMAWYRQAPGKHRELVARISSG GTTAYVDSVKGRFTISRDNSKNTVYLQMNSLKAEDTAVYYCNTFSSRPNPWGAGTQVTVSS VEGFBII23B04- EVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVS 9GS-6G02/132 LEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSS GGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGNIFSNNAMAWYRQAPGKQRELVARIS SGGGFTYYLDSVKGRFTVSRDNAKNTVYLQMNSLKPEDTAVYYCNAAYRTYNYWGQGTQVTV SS VEGFBII23B04- EVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVS 9GS-10E07/133 LEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSS GGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTFSNYAMGWFRQAPGKERVLVADIS SSGINTYVADAVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAASAWWYSQMARDNYRYW GQGTQVTVSS VEGFBII23B04- EVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVS 9GS-12B01/134 LEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSS GGGGSGGGSEVQLVESGGGLVQPGGSLRLACAASGFTLSSSWMYWVRQAPGKGLEWVSRI SPGGLFTYYVDSVKGRFSVSTDNANNTLYLQMNSLKPEDTALYSCAKGGAPNYTPRGRGTQV TVSS VEGFBII23B04- EVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVS 9GS-86C11/135 LEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSS GGGGSGGGSEVQLVESGGGLVQAGDSLRLSCTASGRTFNSYAMGWFRQAPGKERESVAHIN RSGSSTYYADSVKGRFTISRDNAKNTVYLQLNSLKPEDTAVYYCAAGRYYSSDGVPSASFNYW GQGTQVTVSS VEGFBII23B04- EVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVS 9GS-86H09/136 LEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSS GGGGSGGGSEVQLVESGGGLVQAGGSLRLSCTASGSAFKSYRMGWFRRTPGKEDEFVASIS WTYGSTFYADSVKGRFTMSRDKAKNAGYLQMNSLKPEDTALYYCAAGAQSDRYNIRSYDYW GQGTQVTVSS VEGFBII23B04- EVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVS 9GS-87B07/137 LEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSS GGGGSGGGSEVQLVESGGGLVQPGGSLKLSCTASGFTFSTSWMHWVRQAPGKGLEWVSSIP PVGHFANYAPSVKGRFTISRDNAKNTLFLQMNSLKSEDTAVYYCAKDSAGRTKGQGTQVTVSS VEGFBII23B04- EVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVS 9GS-88A01/138 LEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSS GGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASERTFSNYAMDWFRQAPGKEREFVAAIT RSGGGTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAATRSSTIVVGVGGMEYW GKGTQVTVSS VEGFBII23B04- EVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVS 40GS-4B08/139 LEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSS GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGS LRLSCAASGSAVGDITVAWYRQAPGIQRQLVATITPSGYTYYWDFVKGRFTISRDNSKNIVYLQ MNSLKPEDTAAYYCNTQFYWGQGTQVTVSS VEGFBII23B04- EVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVS 40GS-5B03/140 LEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSS GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLAQAGDSL RLSCAASGRSFSHYNMGWFRQAPGKEREFVASIRGGGGSTTYANSVKDRFTISRENAKNTVY LQMNSLKPEDTAVYYCAATAFYRGPYDYDYWGQGTQVTVSS VEGFBII23B04- VEGFBII021 EVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVS 40GS-5B05/141 LEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSS GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGS LRLSCVASGIRFMSMAWYRQAPGKHRELVARISSGGTTAYVDSVKG RFTISRDNSKNTVYLQM NSLKAEDTAVYYCNTFSSRPNPWGAGTQVTVSS VEGFBII23B04- EVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVS 40GS-6G02/142 LEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSS GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGS LRLSCAASGNIFSNNAMAWYRQAPGKQRELVARISSGGGFTYYLDSVKGRFTVSRDNAKNTVY LQMNSLKPEDTAVYYCNAAYRTYNYWGQGTQVTVSS VEGFBII23B04- VEGFBII023 EVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVS 40GS-10E07/143 LEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSS GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGS LRLSCAASGRTFSNYAMGWFRQAPGKERVLVADISSSGINTYVADAVKGRFTISRDNAKNTVYL QMNSLKPEDTAVYYCAASAWWYSQMARDNYRYWGQGTQVTVSS VEGFBII23B04- EVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVS 40GS-12B01/144 LEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSS GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGS LRLACAASGFTLSSSWMYWVRQAPGKGLEWVSRISPGGLFTYYVDSVKGRFSVSTDNANNTL YLQMNSLKPEDTALYSCAKGGAPNYTPRGRGTQVTVSS VEGFBII23B04- EVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVS 40GS-86C11/145 LEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSS GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGDSL RLSCTASGRTFNSYAMGWFRQAPGKERESVAHINRSGSSTYYADSVKGRFTISRDNAKNTVYL QLNSLKPEDTAVYYCAAGRYYSSDGVPSASFNYWGQGTQVTVSS VEGFBII23B04- VEGFBII024 EVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVS 40GS-86H09/146 LEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSS GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGS LRLSCTASGSAFKSYRMGWFRRTPGKEDEFVASISWTYGSTFYADSVKGRFTMSRDKAKNAG YLQMNSLKPEDTALYYCAAGAQSDRYNIRSYDYWGQGTQVTVSS VEGFBII23B04- EVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVS 40GS-87B07/147 LEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSS GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGS LKLSCTASGFTFSTSWMHWVRQAPGKGLEWVSSIPPVGHFANYAPSVKGRFTISRDNAKNTLF LQMNSLKSEDTAVYYCAKDSAGRTKGQGTQVTVSS VEGFBII23B04- EVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVS 40GS-88A01/148 LEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSS GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGS LRLSCAASERTFSNYAMDWFRQAPGKEREFVAAITRSGGGTYYADSVKGRFTISRDNAKNTVY LQMNSLKPEDTAVYYCAATRSSTIVVGVGGMEYWGKGTQVTVSS VEGFBII4B08-9GS- EVQLVESGGGLVQPGGSLRLSCAASGSAVGDITVAWYRQAPGIQRQLVATITPSGYTYYWDFV 23B04/149 KGRFTISRDNSKNIVYLQMNSLKPEDTAAYYCNTQFYWGQGTQVTVSSGGGGSGGGSEVQLV ESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVSLEGRF TISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSS VEGFBII5B03-9GS- EVQLVESGGGLAQAGDSLRLSCAASGRSFSHYNMGWFRQAPGKEREFVASIRGGGGSTTYA 23B04/150 NSVKDRFTISRENAKNTVYLQMNSLKPEDTAVYYCAATAFYRGPYDYDYWGQGTQVTVSSGG GGSGGGSEVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKG GYKYDSVSLEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQ GTQVTVSS VEGFBII5B05-9GS- EVQLVESGGGLVQPGGSLRLSCVASGIRFMSMAWYRQAPGKHRELVARISSGGTTAYVDSVK 23B04/151 GRFTISRDNSKNTVYLQMNSLKAEDTAVYYCNTFSSRPNPWGAGTQVTVSSGGGGSGGGSE VQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVSL EGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSS VEGFBII6G02-9GS- EVQLVESGGGLVQPGGSLRLSCAASGNIFSNNAMAWYRQAPGKQRELVARISSGGGFTYYLD 23B04/152 SVKGRFTVSRDNAKNTVYLQMNSLKPEDTAVYYCNAAYRTYNYWGQGTQVTVSSGGGGSGG GSEVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDS VSLEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVS S VEGFBII10E07- EVQLVESGGGLVQAGGSLRLSCAASGRTFSNYAMGWFRQAPGKERVLVADISSSGINTYVAD 9GS-23B04/153 AVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAASAWWYSQMARDNYRYWGQGTQVTVS SGGGGSGGGSEVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAI SKGGYKYDSVSLEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYW GQGTQVTVSS VEGFBII12B01- EVQLVESGGGLVQPGGSLRLACAASGFTLSSSWMYWVRQAPGKGLEWVSRISPGGLFTYYV 9GS-23B04/154 DSVKGRFSVSTDNANNTLYLQMNSLKPEDTALYSCAKGGAPNYTPRGRGTQVTVSSGGGGS GGGSEVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYK YDSVSLEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQ VTVSS VEGFBII86C11- EVQLVESGGGLVQAGDSLRLSCTASGRTFNSYAMGWFRQAPGKERESVAHINRSGSSTYYAD 9GS-23B04/155 SVKGRFTISRDNAKNTVYLQLNSLKPEDTAVYYCAAGRYYSSDGVPSASFNYWGQGTQVTVS SGGGGSGGGSEVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAI SKGGYKYDSVSLEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYW GQGTQVTVSS VEGFBII86H09- EVQLVESGGGLVQAGGSLRLSCTASGSAFKSYRMGWFRRTPGKEDEFVASISWTYGSTFYAD 9GS-23B04/156 SVKGRFTMSRDKAKNAGYLQMNSLKPEDTALYYCAAGAQSDRYNIRSYDYWGQGTQVTVSS GGGGSGGGSEVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAIS KGGYKYDSVSLEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYW GQGTQVTVSS VEGFBII87B07- EVQLVESGGGLVQPGGSLKLSCTASGFTFSTSWMHWVRQAPGKGLEWVSSIPPVGHFANYA 9GS-23B04/157 PSVKGRFTISRDNAKNTLFLQMNSLKSEDTAVYYCAKDSAGRTKGQGTQVTVSSGGGGSGGG SEVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSV SLEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSS VEGFBII88A01- EVQLVESGGGLVQAGGSLRLSCAASERTFSNYAMDWFRQAPGKEREFVAAITRSGGGTYYAD 9GS-23B04/158 SVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAATRSSTIVVGVGGMEYWGKGTQVTVSS GGGGSGGGSEVQLVESGGGLVQTGDSLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAIS KGGYKYDSVSLEGRFTISKDNAKNTVYLQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYW GQGTQVTVSS VEGFBII4B08- EVQLVESGGGLVQPGGSLRLSCAASGSAVGDITVAWYRQAPGIQRQLVATITPSGYTYYWDFV 40GS-23B04/159 KGRFTISRDNSKNIVYLQMNSLKPEDTAAYYCNTQFYWGQGTQVTVSSGGGGSGGGGSGGG GSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQTGDSLRLSCEVSGRTFSSY SMGWFRQAQGKEREFVVAISKGGYKYDSVSLEGRFTISKDNAKNTVYLQINSLKPEDTAVYYC ASSRAYGSSRLRLADTYEYWGQGTQVTVSS VEGFBII5B03- EVQLVESGGGLAQAGDSLRLSCAASGRSFSHYNMGWFRQAPGKEREFVASIRGGGGSTTYA 40GS-23B04/160 NSVKDRFTISRENAKNTVYLQMNSLKPEDTAVYYCAATAFYRGPYDYDYWGQGTQVTVSSGG GGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQTGDSLRL SCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVSLEGRFTISKDNAKNTVYLQIN SLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSS VEGFBII5B05- EVQLVESGGGLVQPGGSLRLSCVASGIRFMSMAWYRQAPGKHRELVARISSGGTTAYVDSVK 40GS-23B04/161 GRFTISRDNSKNTVYLQMNSLKAEDTAVYYCNTFSSRPNPWGAGTQVTVSSGGGGSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQTGDSLRLSCEVSGRTF SSYSMGWFRQAQGKEREFVVAISKGGYKYDSVSLEGRFTISKDNAKNTVYLQINSLKPEDTAV YYCASSRAYGSSRLRLADTYEYWGQGTQVTVSS VEGFBII6G02- EVQLVESGGGLVQPGGSLRLSCAASGNIFSNNAMAWYRQAPGKQRELVARISSGGGFTYYLD 40GS-23B04/162 SVKGRFTVSRDNAKNTVYLQMNSLKPEDTAVYYCNAAYRTYNYWGQGTQVTVSSGGGGSGG GGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQTGDSLRLSCEVS GRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVSLEGRFTISKDNAKNTVYLQINSLKPE DTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSS VEGFBII10E07- VEGFBII025 EVQLVESGGGLVQAGGSLRLSCAASGRTFSNYAMGWFRQAPGKERVLVADISSSGINTYVAD 40GS-23B04/163 AVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAASAWWYSQMARDNYRYWGQGTQVTVS SGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQTGD SLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVSLEGRFTISKDNAKNTVY LQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSS VEGFBII12B01- EVQLVESGGGLVQPGGSLRLACAASGFTLSSSWMYWVRQAPGKGLEWVSRISPGGLFTYYV 40GS-23B04/164 DSVKGRFSVSTDNANNTLYLQMNSLKPEDTALYSCAKGGAPNYTPRGRGTQVTVSSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQTGDSLRLSCE VSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVSLEGRFTISKDNAKNTVYLQINSLK PEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSS VEGFBII86C11- EVQLVESGGGLVQAGDSLRLSCTASGRTFNSYAMGWFRQAPGKERESVAHINRSGSSTYYAD 40GS-23B04/165 SVKGRFTISRDNAKNTVYLQLNSLKPEDTAVYYCAAGRYYSSDGVPSASFNYWGQGTQVTVS SGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQTGD SLRLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVSLEGRFTISKDNAKNTVY LQINSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSS VEGFBI186H09- EVQLVESGGGLVQAGGSLRLSCTASGSAFKSYRMGWFRRTPGKEDEFVASISWTYGSTFYAD 40GS-23B04/166 SVKGRFTMSRDKAKNAGYLQMNSLKPEDTALYYCAAGAQSDRYNIRSYDYWGQGTQVTVSS GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQTGDSL RLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVSLEGRFTISKDNAKNTVYLQ INSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSS VEGFBII87B07- EVQLVESGGGLVQPGGSLKLSCTASGFTFSTSWMHWVRQAPGKGLEWVSSIPPVGHFANYA 40GS-23B04/167 PSVKGRFTISRDNAKNTLFLQMNSLKSEDTAVYYCAKDSAGRTKGQGTQVTVSSGGGGSGGG GSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQTGDSLRLSCEVSGR TFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVSLEGRFTISKDNAKNTVYLQINSLKPEDTA VYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSS VEGFBII88A01- EVQLVESGGGLVQAGGSLRLSCAASERTFSNYAMDWFRQAPGKEREFVAAITRSGGGTYYAD 40GS-23B04/168 SVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAATRSSTIVVGVGGMEYWGKGTQVTVSS GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQTGDSL RLSCEVSGRTFSSYSMGWFRQAQGKEREFVVAISKGGYKYDSVSLEGRFTISKDNAKNTVYLQ INSLKPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTQVTVSS

    [0402] The panel of 40 bivalent VHHs is tested in the VEGFR2 and VEGFR1 blocking AlphaScreen assay, as described in Example 5.3 and 5.4, respectively. Based on potency and maximum level of inhibition, the 5 best bivalent VHHs (VEGFBII021, VEGFBII022, VEGFBI023, VEGFBI024 and VEGFBII025) are chosen for further characterization. An overview of the screening results for the 5 selected bivalent VHHs in the competitive VEGFR2 and VEGFR1 AlphaScreen is shown in Table 16.

    TABLE-US-00020 TABLE 16 Potency and efficacy of 5 best bivalent VHHs in the VEGF/VEGFR1 and VEGF/VEGFR2 competition AlphaScreen assay VEGFR2 VEGFR1 VHH ID IC.sub.50 (pM) IC.sub.50 (pM) % inhibition VEGFBII021 9 16 100 VEGFBII022 7 8 100 VEGFBII023 38 44 91 VEGFBII024 12 46 100 VEGFBII025 51 39 82

    EXAMPLE 7

    [0403] Characterization of Formatted Anti-VEGF VHHs

    [0404] VHHs VEGFBII010, VEGFBII021, VEGFBII022, VEGFBII023, VEGFBII024 and VEGFBII025 are compared side-by-side in the VEGFR2 and VEGFR1 blocking ELISA (FIGS. 8 and 9, Table 17 and Table 18 respectively) and AlphaScreen assay (FIGS. 10 and 11, Table 19 and 20) as described in Examples 5.1, 5.2, 5.3 and 5.4, respectively.

    TABLE-US-00021 TABLE 17 IC.sub.50 (pM) values and % inhibition for formatted VHHs in hVEGF165/ hVEGFR2-Fc competition ELISA IC.sub.50 % VHH ID (pM) inhibition VEGFBII010 49 100 VEGFBII021 204 100 VEGFBII022 164 100 VEGFBII023 213 100 VEGFBII024 292 100 VEGFBII025 577 100 Bevacizumab 315 100 Ranibizumab 349 100

    TABLE-US-00022 TABLE 18 IC.sub.50 (pM) values and % inhibition of formatted VHHs in VEGF165/hVEGFR1-Fc competition ELISA IC.sub.50 % VHH ID (pM) inhibition VEGFBII010 73.5 67 VEGFBII021 254 97 VEGFBII022 225 89 VEGFBII023 279 91 VEGFBII024 326 92 VEGFBII025 735 91 Bevacizumab 484 91 Ranibizumab 594 96

    TABLE-US-00023 TABLE 19 IC.sub.50 (pM) values and % inhibition for formatted VHHs in hVEGF165/hVEGFR2-Fc competition AlphaScreen VHH ID IC.sub.50 (pM) % inhibition VEGFBII010 16 100 VEGFBII021  7 100 VEGFBII022  7 100 VEGFBII023 46 100 VEGFBII024 50 100 VEGFBII025 51 100 Ranibizumab 600  100

    TABLE-US-00024 TABLE 20 IC.sub.50 (pM) values and % inhibition of formatted VHHs in VEGF165/hVEGFR1-Fc competition AlphaScreen VHH ID IC.sub.50 (pM) % inhibition VEGFBII010  21 70 VEGFBII021  12 100  VEGFBII022   9 98 VEGFBII023  48 87 VEGFBII024  69 98 VEGFBII025  71 82 Ranibizumab 1300 87

    [0405] In addition, formatted VHHs are also tested for their capacity to block the mVEGF164/mVEGFR2-huFc interaction. In brief, serial dilutions of purified VHHs (concentration range: 4 μM-14.5 pM) in PBS buffer containing 0.03% Tween 20 (Sigma) are added to 0.1 nM biotinylated mVEGF164 and incubated for 15 min. Subsequently mouse VEGFR2-huFc (0.1 nM) and anti-huFc VHH-coated acceptor beads (20 μg/ml) are added and this mixture is incubated for 1 hour. Finally, streptavidin donor beads (20 μg/ml) are added and after 1 hour of incubation fluorescence is measured on the Envision microplate reader. Dose-response curves are shown in FIG. 12. The IC.sub.50 values for VHHs blocking the mouse VEGF164/VEGFR2-hFC interaction are summarized in Table 21.

    TABLE-US-00025 TABLE 21 IC.sub.50 (pM) values and % inhibition for formatted VHHs in mVEGF164/mVEGFR2-hFc competition AlphaScreen VHH ID IC.sub.50 (nM) % inhibition VEGFBII022 108 100 VEGFBII024 — — mVEGF164 0.05 100 Ranibizumab — —

    [0406] The formatted VHHs are also tested in ELISA for their ability to bind mVEGF164 and human VEGF165 (Example 5.6; FIG. 13; Table 22); VEGF121 (Example 5.7; FIG. 15; Table 23) and the VEGF family members VEGFB, VEGFC, VEGFD and PIGF (Example 5.8; FIG. 14). Binding kinetics for human VEGF165 are analyzed as described in Example 5.5. The KD values are listed in Table 24.

    TABLE-US-00026 TABLE 22 EC.sub.50 (pM) values for formatted VHHs in a recombinant human VEGF165 and mouse VEGF164 binding ELISA rhVEGF165 rmVEGF164 VHH ID EC.sub.50 (pM) EC.sub.50 (pM) VEGFBII010 428 — VEGFBII021 334 502 VEGFBII022 224 464 VEGFBII023 221 — VEGFBII024 320 — VEGFBII025 668 —

    TABLE-US-00027 TABLE 23 EC.sub.50 (pM) values for formatted VHHs in a recombinant human VEGF121 binding ELISA rhVEGF121 VHH ID EC.sub.50 (pM) VEGFBII010 920 VEGFBII022 540 VEGFBII024 325 VEGFBII025 475

    TABLE-US-00028 TABLE 24 Affinity KD (nM) of purified formatted VHHs for recombinant human VEGF165 K.sub.D VHH ID k.sub.a1 (1/Ms) k.sub.d1 (1/s) k.sub.a2 (1/s) k.sub.d2 (1/s) (nM).sup.(a) VEGFBII010.sup.(b) 4.5E + 05 1.7E − 02 2.9E − 02 1.3E − 04 0.16 VEGFBII021.sup.(b) 1.2E + 06 1.1E − 02 2.3E − 02 1.9E − 04 0.07 VEGFBII022.sup.(b) 1.2E + 06 9.1E − 03 1.4E − 02 2.6E − 04 0.14 VEGFBII023.sup.(b) 3.0E + 05 1.8E − 02 2.4E − 02 2.7E − 04 0.69 VEGFBII024.sup.(b) 3.0E + 05 1.3E − 02 2.6E − 02 2.8E − 04 0.47 VEGFBII025.sup.(b) 3.3E + 05 1.7E − 02 1.8E − 02 3.7E − 04 1.1  .sup.(a)K.sub.D = k.sub.d1/k.sub.a1.Math.(k.sub.d2/(k.sub.d2 + k.sub.a2)) .sup.(b)Curves are fitted using a Two State Reaction model by Biacore T100 Evaluation Software v2.0.1

    [0407] VHHs VEGFBII010, VEGFBII022, VEGFBII024 and VEGFBII025 are also tested in the VEGF-mediated HUVEC proliferation and Erk phosphorylation assay.

    [0408] The potency of the selected formatted VHHs is evaluated in a proliferation assay. In brief, primary HUVEC cells (Technoclone) are supplement-starved over night and then 4000 cells/well are seeded in quadruplicate in 96-well tissue culture plates. Cells are stimulated in the absence or presence of VHHs with 33 ng/mL VEGF. The proliferation rates are measured by [.sup.3H] Thymidine incorporation on day 4. The results shown in Table 25 demonstrate that the formatted VHHs and Bevacizumab inhibit the VEGF-induced HUVEC proliferation by more than 90%, with IC.sub.50s<1 nM.

    TABLE-US-00029 TABLE 25 IC.sub.50 (nM) values and % inhibition of formatted VHHs in VEGF HUVEC proliferation assay VHH ID IC.sub.50 (nM) % inhibition VEGFBII010 0.22 95 VEGFBII021 0.40 98 VEGFBII022 0.34 100  VEGFBII023 0.52 98 VEGFBII024 0.38 96 VEGFBII025 0.41 104  Bevacizumab 0.21 92

    [0409] The potency of the selected formatted VHHs is assessed in the HUVEC Erk phosphorylation assay. In brief, primary HUVE cells are serum-starved over night and then stimulated in the absence or presence of VHHs with 10 ng/mL VEGF for 5 min. Cells are fixed with 4% Formaldehyde in PBS and ERK phosphorylation levels are measured by ELISA using phosphoERK-specific antibodies (anti-phosphoMAP Kinase pERK1&2, M8159, Sigma) and polyclonal Rabbit Anti-Mouse-Immunoglobulin-HRP conjugate (PO161, Dako). As shown in Table 26, the formatted VHHs and Bevacizumab inhibit the VEGF induced Erk phosphoryaltion by more than 90%, with IC.sub.50s<1 nM.

    TABLE-US-00030 TABLE 26 IC.sub.50 (nM) values and % inhibition of formatted VHHs in VEGF HUVEC Erk phosphorylation assay VHH ID IC.sub.50 (nM) % inhibition VEGFBII010 0.19 92 VEGFBII021 0.21 103  VEGFBII022 0.18 94 VEGFBII023 0.25 100  VEGFBII024 0.23 94 VEGFBII025 0.23 99 Bevacizumab 0.63 98

    EXAMPLE 8

    [0410] Sequence Optimization

    [0411] 8.1 Sequence Optimization of VEGFBII23B04

    [0412] The amino acid sequence of VEGFBII23B04 is aligned to the human germline sequence VH3-23/JH5, see FIG. 16 (SEQ ID NO: 179)

    [0413] The alignment shows that VEGFBII23B04 contains 19 framework mutations relative to the reference germline sequence. Non-human residues at positions 14, 16, 23, 24, 41, 71, 82, 83 and 108 are selected for substitution with their human germline counterparts. A set of 8 VEGFBII23B04 variants is generated carrying different combinations of human residues at these positions (AA sequences are listed in Table 27). One additional variant is constructed in which the potential isomerization site at position D59S60 (CDR2 region, see FIG. 16, indicated as bold italic residues) is removed by introduction of a S60A mutation.

    TABLE-US-00031 TABLE 27 AA sequence of sequence-optimized variants of VHH VEGFBII23604 (FR, framework; CDR, complementary determining region) CDR1/ SEQ CDR2/ CDR3/ VHH ID/ FR1/SEQ ID ID SEQ ID SEQ ID FR4/SEQ ID SEQ ID NO: NO: NO: FR2/SEQ ID NO: NO: FR3/SEQ ID NO: NO: NO: VEGFBII EVQLVESGG SYSM WFRQAPGKERE AISKGGY RFTISRDNAKNTVYLQI SRAYGS WGQGTLVTV 111D05/47 GLVQTGGSL G/450 FVV/659 KYDSVSL NSLRPEDTAVYYCAS/ SRLRLAD SS/664 RLSCEASGRT EG/459 660 TYEY/4 FS/653 VEGFBII EVQLVESGG SYSM WFRQAPGKERE AISKGGY RFTISRDNAKNTVYLQ SRAYGS WGQGTLVTV 111G06/48 GLVQPGGSL G/450 FVV/659 KYDSVSL MNSLRPEDTAVYYCAS SRLRLAD/ SS/664 RLSCAASGRT EG/459 661 TYEY/4 FS/654 VEGFBII EVQLVESGG SYSM WFRQAPGKERE AISKGGY RFTISRDNAKNTVYLQI SRAYGS WGQGTLVTV 112D11/49 GLVQPGGSL G/450 FVV/659 KYDSVSL NSLRPEDTAVYYCAS/ SRLRLAD SS/664 RLSCEASGRT EG/459 660 TYEY/4 FS/655 VEGFBII EVQLVESGG SYSM WFRQAPGKERE AISKGGY RFTISKDNAKNTVYLQI SRAYGS WGQGTLVTV 113A08/50 GLVQTGGSL G/450 FVV/659 KYDSVSL NSLRPEDTAVYYCAS/ SRLRLAD SS/664 RLSCEVSGRT EG/459 662 TYEY4 FS/656 VEGFBII EVQLVESGG SYSM WFRQAQGKERE AISKGGY RFTISKDNAKNTVYLQ SRAYGS WGQGTLVTV 113E03/51 GLVQTGDSL G/450 FVV/691 KYDSVSL MNSLRPEDTAVYYCAS/ SRLRLAD SS/664 RLSCEVSGRT EG/459 663 TYEY/4 FS/690 VEGFBII EVQLVESGG SYSM WFRQAPGKERE AISKGGY RFTISKDNAKNTVYLQI SRAYGS WGQGTLVTV 114C09/52 GLVQPGDSL G/450 FVV/659 KYDSVSL NSLRPEDTAVYYCAS/ SRLRLAD SS/664 RLSCEVSGRT EG/459 662 TYEY/4 FS/657 VEGFBII EVQLVESGG SYSM WFRQAPGKERE AISKGGY RFTISRDNAKNTVYLQI SRAYGS WGQGTLVTV 114D02/53 GLVQTGGSL G/450 FVV/659 KYDSVSL NSLRPEDTAVYYCAS/ SRLRLAD SS/664 RLSCEVSGRT EG/459 660 TYEY/4 FS/656 VEGFBII EVQLVESGG SYSM WFRQAQGKERE AISKGGY RFTISKDNAKNTVYLQI SRAYGS WGQGTLVTV 114D03/54 GLVQTGDSL G/450 FVV/691 KYDSVSL NSLRPEDTAVYYCAS/ SRLRLAD SS/664 RLSCAVSGRT EG/459 662 TYEY/4 FS/658 VEGFBII EVQLVESGG SYSM WFRQAQGKERE AISKGGY RFTISKDNAKNTVYLQI SRAYGS WGQGTQVT 118E10/55 GLVQTGDSL G/450 FVV/691 KYDAVSL NSLKPEDTAVYYCAS/ SRLRLAD VSS/665 RLSCEVSGRT EG/465 692 TYEY/4 FS/690

    [0414] These variants are characterized as purified proteins in the VEGF165/VEGFR2 AlphaScreen (Example 5.3, FIG. 17). The melting temperature (T.sub.m) of each clone is determined in a thermal shift assay, which is based on the increase in fluorescence signal upon incorporation of Sypro Orange (Invitrogen) (Ericsson et al, Anal. Biochem. 357 (2006), pp 289-298). All variants displayed comparable IC.sub.50 when compared to VEGFBII23B04 and Tm values which are similar or higher when compared to the parental VEGFBII23B04. Table 28 summarizes the IC.sub.50 values and T.sub.m values at pH 7 for the 9 clones tested.

    TABLE-US-00032 TABLE 28 IC.sub.50 (pM) values, % inhibition and melting temperature (@pH 7) of sequence-optimized variants of VEGFBII23B04 VHH ID IC.sub.50 (pM) % inhibition T.sub.m @ pH 7 (° C.) VEGFBII23B04 (wt) 169 100 63 VEGFBII111D05 209 100 68 VEGFBII111G06 366 100 71 VEGFBII112D11 221 100 70 VEGFBII113A08 253 100 69 VEGFBII113E03 290 100 68 VEGFBII114C09 215 100 71 VEGFBII114D02 199 100 74 VEGFBII114D03 227 100 64 VEGFBII118E10 189 100 62

    [0415] In a second cycle, tolerated mutations from the humanization effort (VEGFBII111G06) and mutations to avoid potential posttranslational modification at selected sites (the D16G, the S60A substitution and an El D mutation) are combined resulting in a sequence-optimized clone derived from VEGFB1123B04: VEGFBI10037. One extra sequence-optimized variant (VEGFB11038) is anticipated which contains the same substitutions as VEGFBI10037, with the exception of the I82M mutation, as this mutation may be associated with a minor drop in potency. The sequences from both sequence-optimized clones are listed in Table 29. VEGFBII0037 and VEGFBII0038 are characterized in the VEGF165/VEGFR2 blocking AlphaScreen (Example 5.3, FIG. 18), the melting temperature is determined in the thermal shift assay as described above and the affinity for binding on VEGF165 is determined in Biacore (Example 5.5). An overview of the characteristics of the 2 sequence-optimized VHHs is presented in Table 30.

    TABLE-US-00033 TABLE 29 AA sequences of sequence-optimized variants of VHH VEGFBII23B04 CDR1/ VHH ID/ SEQ CDR 2/ FR SEQ ID FR 1/SEQ ID FR2/SEQ SEQ ID CDR 3/SEQ 4/SEQ NO: ID NO: NO: ID NO: NO: FR3/SEQ ID NO: ID NO: ID NO: VEGFBII0 DVQLVES SYSM WFRQAP AISKG RFTISRDNAKNT SRAYGSSR WGQG 37 GGGLVQP G/450 GKEREF GYKYD VYLQMNSLRPE LRLADTYE TLVTVS 56 GGSLRLS VV/659 AVSLE DTAVYYCAS/661 Y/4 S/664 CAASGRT G/465 FS/666 VEGFBII0 DVQLVES SYSM WFRQAP AISKG RFTISRDNAKNT SRAYGSSR WGQG 38 GGGLVQP G/450 GKEREF GYKYD VYLQINSLRPED LRLADTYE TLVTVS 57 GGSLRLS VV/659 AVSLE TAVYYCAS/660 Y/4 S/664 CAASGRT G/465 FS/666

    TABLE-US-00034 TABLE 30 IC.sub.50 (pM) values, % inhibition, melting temperature (@pH 7) and affinity (pM) of sequence-optimized clones VEGFBII037 and VEGFBII038 VHH ID IC.sub.50 (pM) % inhibition T.sub.m (° C.) @ pH 7 K.sub.D (pM) VEGFBII23B04 152 100 63 560 VEGFBII037 300 100 72 270 VEGFBII038 143 100 71 360

    [0416] 8.2 Sequence Optimization of VEGFBII5B05

    [0417] The amino acid sequence of VEGFBII5B05 is aligned to the human germline sequence VH3-23/JH5, see FIG. 19 (SEQ ID:NO: 179 The alignment shows that VEGFBII5B05 contains 15 framework mutations relative to the reference germline sequence. Non-human residues at positions 23, 60, 83, 105, 108 are selected for substitution with their human germline counterparts while the histidine at position 44 is selected for substitution by glutamine. One humanization variant is constructed carrying the 6 described mutations (AA sequence is listed in Table 31).

    TABLE-US-00035 TABLE 31 AA sequences of sequence-optimized variants of VHH VEGFBII5B05 (FR, framework; CDR, complementary determining region) CDR1/ SEQ CDR2/ CDR3/ VHH ID/ FR1/SEQ ID FR2/SEQ SEQ FR3/SEQ ID SEQ FR4/SEQ SEQ ID NO: ID NO: NO: ID NO: ID NO: NO: ID NO: ID NO: VEGFBII119 EVQLVES SM WYRQAP RISSG RFTISRDNS FSSR WGQGTL G11/ GGGLVQ A/ GKQRELV GTTA KNTVYLQMN PNP/ VTVSS/ 125 PGGSLRL 290 A/669 YADS SLRAEDTAV 395 664 SCAASGI VKG/ YYCNT/671 RFM/667 404 VEGFBII120E EVQLVES SM WYRQAP RISSG RFTISRDNS FSSR WGAGT 10/ GGGLVQ A/ GKHRELV GTTA KNTVYLQMN PNP/ QVTVSS/ 126 PGGSLRL 290 A/670 YVDS SLKAEDTAV 395 673 SCVASGI VKG/ YYCNT/672 RFI/668 336

    [0418] One additional variant is constructed in which the potential oxidation site at position M30 (CDR1 region, see FIG. 19 indicated as bold italic residue) is removed by introduction of a M30I mutation. Both variants are tested for their ability to bind hVEGF165 using the ProteOn. In brief, a GLC ProteOn Sensor chip is coated with human VEGF165. Periplasmic extracts of the variants are diluted 1/10 and injected across the chip coated with human VEGF165. Off-rates are calculated and compared to the off-rates of the parental VEGFBII5B05. Off-rates from the 2 variants are in the same range as the off-rates from the parental VEGFBII5B05 indicating that all mutations are tolerated (Table 32).

    TABLE-US-00036 TABLE 32 Off-rates sequence-optimized variants VEGFBII5605 VHH ID binding level (RU) k.sub.d (1/s) VEGFBII5B05 242 6.15E − 02 VEGFBII119G11 234 7.75E − 02 VEGFBII120E10 257 4.68E − 02

    [0419] In a second cycle, mutations from the humanization effort and the M30I substitution are combined resulting in a sequence-optimized clone of VEGFBII5B05, designated VEGFBII032. The sequence is listed in Table 33. Affinity of VEGFBII032 is determined by Biacore (see Example 5.5) and the melting temperature is determined in the thermal shift assay as described above. An overview of the characteristics of the sequence-optimized VHH VEGFBII032 is presented in Table 34.

    TABLE-US-00037 TABLE 33 AA sequence of sequence-optimized clone VEGFBII032 (FR, framework; CDR, complementary determining region) CDR1/ VHH ID/ SEQ CDR2/ CDR3/ SEQ ID FR1/SEQ ID FR2/SEQ SEQ FR3/SEQ ID SEQ FR4/SEQ NO: ID NO: NO: ID NO: ID NO: NO: ID NO: ID NO: VEGFBII032/ EVQLVES SM WYRQAP RISSG RFTISRDNS FSSR WGQGTL 127 GGGLVQ A/ GKQRELV GTTA KNTVYLQMN PNP/ VTVSS/ PGGSLRL 290 A/675 YADS SLRAEDTAV 395 664 SCAASGI VKG/ YYCNT/671 RFI/674 404

    TABLE-US-00038 TABLE 34 Melting temperature (@pH 7) and affinity (nM) of sequence-optimized clone VEGFBII032 VHH ID T.sub.m (° C.) @ pH 7 K.sub.D (nM) VEGFBII5B05(wt) 69 32 VEGFBII0032 71 44

    [0420] The potency of the sequence-optimized clones VEGFBVBII037 and VEGFBII038 is evaluated in a proliferation assay. In brief, primary HUVEC cells (Technoclone) are supplement-starved over night and then 4000 cells/well are seeded in quadruplicate in 96-well tissue culture plates. Cells are stimulated in the absence or presence of VHHs with 33 ng/mL VEGF. The proliferation rates are measured by [.sup.3H] Thymidine incorporation on day 4. The results shown in Table 35, demonstrate that the activity (potency and degree of inhibition) of the parental VHH VEGFBII23B04 is conserved in the sequence optimized clone VEGFBII038.

    TABLE-US-00039 TABLE 35 IC.sub.50 (nM) values and % inhibition of the sequence optimized clones VEGFBII037 and VEGFBII038 in VEGF HUVEC proliferation assay VHH ID IC.sub.50 (nM) % inhibition VEGFBII23B04 0.68 92 VEGFBII037 1.54 78 VEGFBII038 0.60 92 Bevacizumab 0.29 94

    EXAMPLE 9

    [0421] Construction, Production and Characterization of Bivalent VHHs Targeting Ang2

    [0422] VHHs 1D01 (SEQ ID No:214), 11B07, 00908 and 00027 (SEQ ID No:216) are genetically fused to 1D01 (SEQ ID No: 214), 11B07, 00908 and 00027 (SEQ ID No:216), respectively, resulting in homodimeric VHHs. The bivalent VHHs are linked via a 9-GlySer (SEQ ID NO: 170) or 40-GlySer (SEQ ID NO: 171) flexible linker. The encoding DNA sequences of the formatted VHHs are cloned in the expression vector pAX172. VHHs are expressed in Pichia pastoris as c-terminally myc-His6 tagged proteins (“His6” disclosed as SEQ ID NO: 283). In brief, BGCM cultures are started from a single colony streak incubated over weekend at 30° C. (250 rpm). After medium switch to BMCM, cultures are incubated until evening at 30° C. (250 rpm) and followed by an induction with 100% methanol. The next day the cultures are induced an additional 3 times (morning, afternoon, evening). The next day cultures are centrifuged for 20 min at 4° C. (1,500×g). The His6-tagged VHHs (“His6” disclosed as SEQ ID NO: 283) present in the supernatant are purified through immobilized metal affinity chromatography (IMAC) followed by desalting (DS) and finally gel filtration (GF) to remove any endotoxins/impurities. An overview of the format and sequence of all bivalent VHHs is depicted in FIG. 20 A and Table 36 (linker sequences underlined), SEQ ID Nos 180-185. Expression levels are indicated in FIG. 20B.

    [0423] To explore the anti-Ang2 blocking properties in comparison with the monovalent building blocks, bivalent VHHs are analyzed in a human Ang2/hTie2 (FIG. 21-1), mouse Ang2/mTie2 (FIG. 21-2), cyno Ang2/cTie2 (FIG. 21-3) and human Ang1/hTie2 (FIG. 22) competition ELISA. A summary of IC.sub.50 values is shown in FIG. 20C.

    TABLE-US-00040 TABLE 36 Sequences of bivalent VHH targeting Ang2 VHH ID AA sequence ANGBII00001 EVQLVESGGGLVQAGGSLRLSCAASGFTFDDYALGWFRQAAGKEREGVSCIRCSDGSTYYADSVKGR FTISSDNAKNTVYLQMNSLKPEDTAVYYCAASIVPRSKLEPYEYDAWGQGTLVTVSSGGGGSGGGSE VQLVESGGGLVQAGGSLRLSCAASGFTFDDYALGWFRQAAGKEREGVSCIRCSDGSTYYADSVKGRF TISSDNAKNTVYLQMNSLKPEDTAVYYCAASIVPRSKLEPYEYDAWGQGTLVTVSS (SEQ ID NO: 180) ANGBII00002 EVQLVESGGGLVQAGGSLRLSCAASGFTFDDYALGWFRQAAGKEREGVSCIRCSDGSTYYADSVKGR FTISSDNAKNTVYLQMNSLKPEDTAVYYCAASIVPRSKLEPYEYDAWGQGTLVTVSSGGGGSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGFTFDDYALGWF RQAAGKEREGVSCIRCSDGSTYYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAASIVPRS KLEPYEYDAWGQGTLVTVSS (SEQ ID NO: 181) ANGBII00003 EVQLVESGGGLVQVGDSLRLSCAASGRTFSTYLMVGWFRQAPGKEREFAAGIWSSGDTAYADSVRGR FTISRDNAKNTVYLQMNSLKTEDTAVYYCAGSYDGNYYIPGFYKDWGQGTLVTVSSGGGGSGGGSEV QLVESGGGLVQVGDSLRLSCAASGRTFSTYLMVGWFRQAPGKEREFAAGIWSSGDTAYADSVRGRFT ISRDNAKNTVYLQMNSLKTEDTAVYYCAGSYDGNYYIPGFYKDWGQGTLVTVSS (SEQ ID NO: 182) ANGBII00004 EVQLVESGGGLVQAGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSSIRDNDGSTYYADSVKGR FTISSDNDKNTVYLQMNSLKPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSSGGGGS GGGSEVQLVESGGGLVQAGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSSIRDNDGSTYYADS VKGRFTISSDNDKNTVYLQMNSLKPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSS (SEQ ID NO: 183) ANGBII00005 EVQLVESGGGLVQAGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSSIRDNDGSTYYADSVKGR FTISSDNDKNTVYLQMNSLKPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGFTLDDY AIGWFRQAPGKEREGVSSIRDNDGSTYYADSVKGRFTISSDNDKNTVYLQMNSLKPEDTAVYYCAAV PAGRLRFGEQWYPLYEYDAWGQGTLVTVSS (SEQ ID NO: 184) ANGBII00006 EVQLVESGGGLVQPGGSLRLSCAASGITLDDYAIGWFRQAPGKEREGVSSIRDNGGSTYYADSVKGR FTISSDNSKNTVYLQMNSLRPEDTAVYYCAAVPAGRLRYGEQWYPIYEYDAWGQGTLVTVSSGGGGS GGGSEVQLLESGGGLVQPGGSLRLSCAASGITLDDYAIGWFRQAPGKEREGVSSIRDNGGSTYYADS (SEQ ID NO: 185)

    EXAMPLE 10

    [0424] Construction, Production and Characterization of Trivalent Bispecific VHHs Targeting VEGF and Ang2 Using Anti-Serum Abumin as Half-Life Extension

    [0425] The anti-VEGF VHH VEGFBII00038 (US 2011/0172398 A1) and the anti-Ang2 VHH 00027 (SEQ ID No:216) are used as building blocks to generate bispecific VHHs VEGFANGBII00001-00004. A genetic fusion to a serum albumin binding VHH is used as half-life extension methodology. Building blocks are linked via a triple Ala or 9 Gly-Ser flexible linker (SEQ ID NO: 170). VHHs are produced and purified as described in Example 9. An overview of the format and sequence of all four bispecific VHHs is depicted in FIG. 23 A and Table 38 (linker sequences underlined), SEQ ID Nos 186-189. Expression levels are indicated in FIG. 23B.

    TABLE-US-00041 TABLE 38 Sequences of bispecific VHH targeting VEGF and Ang2 VHH ID AA sequence VEGFANGBII00001 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRF TISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGR FTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGG LVQAGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSSIRDNDGSTYYADSVKGRFTISSDNDKN TVYLQMNSLKPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSS (SEQ ID NO: 186) VEGFANGBII00002 EVQLVESGGGLVQAGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSSIRDNDGSTYYADSVKGR FTISSDNDKNTVYLQMNSLKPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSSGGGGS GGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADS VKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSDVQLVE SGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRFTISRDN AKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSS (SEQ ID NO: 187) VEGFANGBII00003 EVQLVESGGGLVQAGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSSIRDNDGSTYYADSVKGR FTISSDNDKNTVYLQMNSLKPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSSGGGGS GGGSDVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSL EGRFTISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGS GGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADS VKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS (SEQ ID NO: 188) VEGFANGBII00004 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRF TISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSAAAEVQLVE SGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRD NAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSAAAEVQLVESGGGLVQAGGSLRLSC AASGFTLDDYAIGWFRQAPGKEREGVSSIRDNDGSTYYADSVKGRFTISSDNDKNTVYLQMNSLKPE DTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSS (SEQ ID NO: 189)

    [0426] To explore the anti-VEGF blocking properties in comparison with the monovalent building block VEGFB1100038, all four bispecific VHHs are analyzed in the VEGF/VEGFR2-Fc (FIG. 22) competition AlphaScreen. The assay is slightly adjusted compared to Example 12.3 described in patent US 2011/0172398 A1. Both human VEGF165 and human VEGFR2-Fc are added at 0.05 nM. This competition assay is also performed after preincubation of the VHH with 25 μM human serum albumin. A summary of IC.sub.50 values and % inhibition is shown in FIG. 23C.

    [0427] To explore the anti-Ang2 blocking properties in comparison with the monovalent building block 00027 (SEQ ID No:216) , all four bispecific VHHs are analyzed in a human Ang2/hTie2-Fc (FIG. 25) competition ELISA. This assay is also performed after incubation of the VHH with 0.5 μM human serum albumin. A summary of IC.sub.50 values is shown in FIG. 23D.

    EXAMPLE 11

    [0428] Construction, Production and Characterization of Trivalent and tetravalent Bispecific VHHs Targeting VEGF and Ang2 Using Anti-Serum Albumin Binding as Half-Life Extension

    [0429] Ten bispecific VHHs targeting VEGF and Ang2 are constructed (VEGFANGBII00005-00015). In these constructs monovalent and bivalent 1D01 (SEQ ID NO:214), monovalent and bivalent 7G08 (SEQ ID NO:215) and bivalent 00027 (SEQ ID NO:216) anti-Ang2 building blocks are included. A genetic fusion to a serum albumin binding VHH is used as half-life extension methodology. Building blocks are linked via a 9 Gly-Ser flexible linker (SEQ ID NO: 170). VHHs are produced and purified as described in Example 8. An overview of the format and sequence of all ten bispecific VHHs is depicted in FIG. 26A and Table 41(linker sequences underlined), SEQ ID Nos 190-199. Expression levels are indicated in FIG. 26B.

    TABLE-US-00042 TABLE 41 Sequences of bispecific VHH targeting VEGF and Ang2 VHH ID AA sequence VEGFANGBII00005 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRF TISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS EVQLVESGGGLVQPGGSLRLSCAASGFALDYYAIGWFRQVPGKEREGVSCISSSDGITYYVDSVKGR FTISRDNAKNTVYLQMNSLKPEDTAVYYCATDSGGYIDYDCMGLGYDYWGQGTLVTVSSGGGGSGGG SEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKG RFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS (SEQ ID NO: 190) VEGFANGBII00006 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRF TISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGR FTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGG LVQPGGSLRLSCAASGFALDYYAIGWFRQVPGKEREGVSCISSSDGITYYVDSVKGRFTISRDNAKN TVYLQMNSLKPEDTAVYYCATDSGGYIDYDCMGLGYDYWGQGTLVTVSS (SEQ ID NO: 191) VEGFANGBII00007 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRF TISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS EVQLVESGGGLVQPGGSLRLSCAASGFALDYYAIGWFRQVPGKEREGVSCISSSDGITYYVDSVKGR FTISRDNAKNTVYLQMNSLKPEDTAVYYCATDSGGYIDYDCMGLGYDYWGQGTLVTVSSGGGGSGGG SEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKG RFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGG GLVQPGGSLRLSCAASGFALDYYAIGWFRQVPGKEREGVSCISSSDGITYYVDSVKGRFTISRDNAK NTVYLQMNSLKPEDTAVYYCATDSGGYIDYDCMGLGYDYWGQGTLVTVSS (SEQ ID NO: 192) VEGFANGBII00008 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRF TISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS EVQLVESGGGLVQAGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSSIRDNDGSTYYADSVKGR FTISSDNDKNTVYLQMNSLKPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSSGGGGS GGGSEVQLVESGGGLVQAGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSSIRDNDGSTYYADS VKGRFTISSDNDKNTVYLQMNSLKPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSSG GGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTL YADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS (SEQ ID NO: 193) VEGFANGBII00009 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRF TISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGR FTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGG LVQAGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSSIRDNDGSTYYADSVKGRFTISSDNDKN TVYLQMNSLKPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSSGGGGSGGGSEVQLVE SGGGLVQAGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSSIRDNDGSTYYADSVKGRFTISSD NDKNTVYLQMNSLKPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSS (SEQ ID NO: 194) VEGFANGBII00010 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRF TISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS EVQLVESGGGLVQAGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSSIRDNDGSTYYADSVKGR FTISSDNDKNTVYLQMNSLKPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSSGGGGS GGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADS VKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVE SGGGLVQAGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSSIRDNDGSTYYADSVKGRFTISSD NDKNTVYLQMNSLKPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSS (SEQ ID NO: 195) VEGFANGBII00011 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRF TISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS EVQLVESGGGLVQAGGSLRLSCAASGFTFDDYALGWFRQAAGKEREGVSCIRCSDGSTYYADSVKGR FTISSDNAKNTVYLQMNSLKPEDTAVYYCAASIVPRSKLEPYEYDAWGQGTLVTVSSGGGGSGGGSE VQLVESGGGLVQAGGSLRLSCAASGFTFDDYALGWFRQAAGKEREGVSCIRCSDGSTYYADSVKGRF TISSDNAKNTVYLQMNSLKPEDTAVYYCAASIVPRSKLEPYEYDAWGQGTLVTVSSGGGGSGGGSEV QLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFT ISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS (SEQ ID NO: 196) VEGFANGBII00012 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRF TISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGR FTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGG LVQAGGSLRLSCAASGFTFDDYALGWFRQAAGKEREGVSCIRCSDGSTYYADSVKGRFTISSDNAKN TVYLQMNSLKPEDTAVYYCAASIVPRSKLEPYEYDAWGQGTLVTVSSGGGGSGGGSEVQLVESGGGL VQAGGSLRLSCAASGFTFDDYALGWFRQAAGKEREGVSCIRCSDGSTYYADSVKGRFTISSDNAKNT VYLQMNSLKPEDTAVYYCAASIVPRSKLEPYEYDAWGQGTLVTVSS (SEQ ID NO: 197) VEGFANGBII00013 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRF TISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS EVQLVESGGGLVQAGGSLRLSCAASGFTFDDYALGWFRQAAGKEREGVSCIRCSDGSTYYADSVKGR FTISSDNAKNTVYLQMNSLKPEDTAVYYCAASIVPRSKLEPYEYDAWGQGTLVTVSSGGGGSGGGSE VQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRF TISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGL VQAGGSLRLSCAASGFTFDDYALGWFRQAAGKEREGVSCIRCSDGSTYYADSVKGRFTISSDNAKNT VYLQMNSLKPEDTAVYYCAASIVPRSKLEPYEYDAWGQGTLVTVSS (SEQ ID NO: 198) VEGFANGBII00014 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRF TISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGR FTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGG LVQAGGSLRLSCAASGFTFDDYALGWFRQAAGKEREGVSCIRCSDGSTYYADSVKGRFTISSDNAKN TVYLQMNSLKPEDTAVYYCAASIVPRSKLEPYEYDAWGQGTLVTVSS (SEQ ID NO: 199)

    [0430] To explore the anti-VEGF blocking properties in comparison with the monovalent building block VEGFBII00038, all ten bispecific VHHs are analyzed in the VEGF/VEGFR2-Fc (Example 10; FIG. 27-1) and VEGF/VEGFR1 (FIG. 27-2) competition AlphaScreen. The VEGFR1 assay is slightly adjusted compared to Example 12.4 as described in patent US 2011/0172398 A1. Human VEGF165 and human VEGFR1-Fc are added at 0.05 nM. These competition assays are also performed after preincubation of the VHH with 25 μM human serum albumin. A summary of IC.sub.50 values is shown in FIG. 26C.

    [0431] To explore the anti-Ang2 blocking properties in comparison with their respective monovalent building block 7G08 (SEQ ID No:215), 1D01 (SEQ ID No:214) and 00027 (SEQ ID No:216), all ten bispecific VHHs are analyzed in the human Ang2/hTie2-Fc (see Example 5.1; FIG. 28-1), mouse Ang2/mTie2-Fc (see Example 5.2; FIG. 28-2) and cyno Ang2/cTie2-Fc (see Example 5.2; FIG. 28-3) competition ELISA. The human assay is also performed after incubation of the VHH with 0.5 μM human serum albumin. Additionally, a hAng2 mediated HUVEC survival assay is performed (see Example 5.5; FIG. 29A-F). A summary of IC.sub.50 values and % inhibition is shown in FIG. 29G.

    [0432] Affinities of for human serum albumin have been determined and are shown in Table 44. Briefly, human serum albumin (Sigma, St Louis, Mo., USA) is immobilized on a CM5 chip via amine coupling. A multicycle kinetic approach is used: increasing concentrations of VHH (2-8-31-125-500 nM) are injected and allowed to associate for 2 min and to dissociate for 10 min at a flow rate of 100 μL/min. Between VHH injections, the surfaces are regenerated with a 10 sec pulse of 10 mM Glycine-HCl pH 1.5 and 60 sec stabilization period. Association/dissociation data are evaluated by fitting a 1:1 interaction model (Langmuir binding) or Heterogeneous Ligand model. The affinity constant KD is calculated from resulting association and dissociation rate constants k.sub.a and k.sub.d (Table 44)

    TABLE-US-00043 TABLE 44 Affinity K.sub.D of purified VHHs for human (HSA), cyno (CSA) and mouse serum albumin (MSA) HSA CSA MSA k.sub.a k.sub.d K.sub.D k.sub.a k.sub.d K.sub.D k.sub.a k.sub.d K.sub.D (1/Ms) (1/s) (nM) (1/Ms) (1/s) (nM) (1/Ms) (1/s) (nM) ALB11 4.5E+05 1.8E−03  4 4.3E+05 1.6E−03  4 6.6E+05 3.2E−02  49 VEGFANGBII00001 2.3E+05 4.8E−03 22 1.8E+05 4.3E−03 24 n.d. n.d. n.d. VEGFANGBII00005 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. VEGFANGBII00006 2.0E+05 4.6E−03 22 1.5E+05 4.5E−03 30 1.7E+05 6.0E−02 360 VEGFANGBII00007 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. VEGFANGBII00008 1.3E+05 4.3E−03 34 n.d. n.d. n.d. n.d. n.d. n.d. VEGFANGBII00009 1.5E+05 4.6E−03 30 1.1E+05 4.2E−03 39 1.2E+05 4.0E−02 340 VEGFANGBII00010 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. VEGFANGBII00011 1.3E+05 4.0E−03 31 n.d. n.d. n.d. n.d. n.d. n.d. VEGFANGBII00012 1.5E+05 4.3E−03 31 1.2E+05 4.2E−03 24 1.0E+05 2.5E−02 240 VEGFANGBII0013 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. VEGFANGBII0014 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d., not determined

    Example 12

    [0433] Construction, Production and Characterization of Sequence Optimized and Affinity Matured Bispecific VHHs Targeting VEGF and Ang2 Using Anti-Serum Albumin Binding as Half-Life Extension

    [0434] 14 bispecific VHHs targeting VEGF and Ang2 are constructed (VEGFANGBII00015-00028). In these constructs bivalent 00921 (a sequence optimized 1D01 variant) (SEQ ID No:220), monovalent VHHs 00908-00932-00933-00934-00935-0093600937-00938 (sequence optimized/affinity matured 28D10 variants) (SEQ ID No:222), bivalent 00956 (SEQ ID NO:223) (sequence optimized 28D10 variant) and monovalent 00928 (SEQ ID NO:221) (sequence optimized 37F02 variant) anti-Ang2 building blocks are included. A genetic fusion to a serum albumin binding VHH is used as half-life extension methodology. Building blocks are linked via a 9 Gly-Ser flexible linker (SEQ ID NO: 170). An overview of the format and sequence of all 14 bispecific VHHs is depicted in FIG. 30A and Table 45 (linker sequences underlined), SEQ ID Nos 200-213.

    [0435] Expression levels are indicated in FIG. 30B.

    TABLE-US-00044 TABLE 45 Sequences of bispecific VHH targeting VEGF and Ang2 VHH ID AA sequence VEGFANGBII00015 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRF TISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGR FTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGG LVQPGGSLRLSCAASGITLDDYAIGWFRQAPGKEREGVSSIRDNGGSTYYADSVKGRFTISSDNSKN TVYLQMNSLRPEDTAVYYCAAVPAGRLRYGEQWYPIYEYDAWGQGTLVTVSS (SEQ ID NO: 200) VEGFANGBII00016 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRF TISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGR FTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGG LVQPGGSLRLSCAVSGITLDDYAIGWFRQAPGKEREGVSSIRDNGGSTYYADSVKGRFTISSDNSKN TVYLQMNSLRPEDTAVYYCAAVPAGRLRYGEQWYPIYEYDAWGQGTLVTVSS SEQ ID NO: 201) VEGFANGBII00017 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRF TISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGR FTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGG LVQPGGSLRLSCAASGITLDDYAIGWFRQAPGKEREGVSAIRDNGGSTYYADSVKGRFTISSDNSKN TVYLQMNSLRPEDTAVYYCAAVPAGRLRYGEQWYPIYEYDAWGQGTLVTVSS SEQ ID NO: 202) VEGFANGBII00018 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRF TISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGR FTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGG LVQPGGSLRLSCAASGITLDDYAIGWFRQAPGKEREGVSAIRESGGSTYYADSVKGRFTISSDNSKN TVYLQMNSLRPEDTAVYYCAAVPAGRLRYGEQWYPIYEYDAWGQGTLVTVSS SEQ ID NO: 203) VEGFANGBII00019 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRF TISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGR FTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGG LVQPGGSLRLSCAASGITLDDYAIGWFRQAPGKEREGVSAIRSSGGSTYYADSVKGRFTISSDNSKN TVYLQMNSLRPEDTAVYYCAAVPAGRLRYGEQWYPIYEYDAWGQGTLVTVSS SEQ ID NO: 204) VEGFANGBII00020 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRF TISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGR FTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGG LVQPGGSLRLSCAVSGITLDDYAIGWFRQAPGKEREGVSAIRDNGGSTYYADSVKGRFTISSDNSKN TVYLQMNSLRPEDTAVYYCAAVPAGRLRYGEQWYPIYEYDAWGQGTLVTVSS SEQ ID NO: 205) VEGFANGBII00021 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRF TISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGR FTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGG LVQPGGSLRLSCAVSGITLDDYAIGWFRQAPGKEREGVSAIRESGGSTYYADSVKGRFTISSDNSKN TVYLQMNSLRPEDTAVYYCAAVPAGRLRYGEQWYPIYEYDAWGQGTLVTVSS SEQ ID NO: 206) VEGFANGBII00022 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRF TISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGR FTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGG LVQPGGSLRLSCAVSGITLDDYAIGWFRQAPGKEREGVSAIRSSGGSTYYADSVKGRFTISSDNSKN TVYLQMNSLRPEDTAVYYCAAVPAGRLRYGEQWYPIYEYDAWGQGTLVTVSS SEQ ID NO: 207) VEGFANGBII00023 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRF TISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGR FTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGG LVQPGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSAIRDNGGSTYYADSVKGRFTISSDNSKN TVYLQMNSLRPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSSGGGGSGGGSEVQLVE SGGGLVQPGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSAIRDNGGSTYYADSVKGRFTISSD NSKNTVYLQMNSLRPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSS SEQ ID NO: 208) VEGFANGBII00024 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRF TISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS EVQLVESGGGLVQPGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSAIRDNGGSTYYADSVKGR FTISSDNSKNTVYLQMNSLRPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSSGGGGS GGGSEVQLVESGGGLVQPGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSAIRDNGGSTYYADS VKGRFTISSDNSKNTVYLQMNSLRPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSSG GGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTL YADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS SEQ ID NO: 209) VEGFANGBII00025 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRF TISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGR FTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGG LVQPGGSLRLSCAASGFTFDDYALGWFRQAPGKEREGVSCIRCSGGSTYYADSVKGRFTISSDNSKN TVYLQMNSLRPEDTAVYYCAASIVPRSKLEPYEYDAWGQGTLVTVSSGGGGSGGGSEVQLVESGGGL VQPGGSLRLSCAASGFTFDDYALGWFRQAPGKEREGVSCIRCSGGSTYYADSVKGRFTISSDNSKNT VYLQMNSLRPEDTAVYYCAASIVPRSKLEPYEYDAWGQGTLVTVSS SEQ ID NO: 210) VEGFANGBII00026 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRF TISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYALGWFRQAPGKEREGVSCIRCSGGSTYYADSVKGR FTISSDNSKNTVYLQMNSLRPEDTAVYYCAASIVPRSKLEPYEYDAWGQGTLVTVSSGGGGSGGGSE VQLVESGGGLVQPGGSLRLSCAASGFTFDDYALGWFRQAPGKEREGVSCIRCSGGSTYYADSVKGRF TISSDNSKNTVYLQMNSLRPEDTAVYYCAASIVPRSKLEPYEYDAWGQGTLVTVSSGGGGSGGGSEV QLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFT ISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS SEQ ID NO: 211) VEGFANGBII00027 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRF TISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGR FTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGG LVQPGGSLRLSCAASGFALDYYAIGWFRQAPGKEREGVSCISSSGGITYYADSVKGRFTISRDNSKN TVYLQMNSLRPEDTAVYYCATDSGGYIDYDCSGLGYDYWGQGTLVTVSS SEQ ID NO: 212) VEGFANGBII00028 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRF TISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGR FTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGG LVQPGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSAIRSSGGSTYYADSVKGRFTISSDNSKN TVYLQMNSLRPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSSGGGGSGGGSEVQLVE SGGGLVQPGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSAIRSSGGSTYYADSVKGRFTISSD NSKNTVYLQMNSLRPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSS SEQ ID NO: 213)

    [0436] To explore the anti-VEGF blocking properties in comparison with the monovalent building block VEGFBII00038, the bispecific VHHs are analyzed in the VEGF/VEGFR2-Fc (Example 10; FIG. 31-1) and VEGF/VEGFR1 (Example 11; FIG. 31-2) competition AlphaScreen. These competition assays are also performed after preincubation of the VHH with 25 μM human serum albumin. A summary of IC.sub.50 values is shown in FIG. 30C.

    [0437] Binding kinetics of the bispecific VHHs on human VEGF165 is analyzed by SPR on a Biacore T100 instrument (see Example 12.5 described in patent US 2011/0172398 A1). Monovalent Nanobody VEGFBII00038 is taken along as reference (FIG. 30D).

    TABLE-US-00045 TABLE 46-B Overview of kinetic parameters in hVEGF165 Biacore assay. ka1 kd1 ka2 kd2 KD1 (1/Ms) (1/s) (1/s) (1/s) (M) VEGFBII00038 2.6E + 05 1.3E − 02 1.3E − 02 1.9E − 04 7.5E − 10 VEGFANG- 1.6E + 05 1.4E − 02 1.4E − 02 2.2E − 04 1.4E − 09 BII00022 VEGFANG- 1.1E + 05 1.4E − 02 1.4E − 02 2.1E − 04 1.9E − 09 BII00025 VEGFANG- 1.7E + 05 1.3E − 02 1.3E − 02 2.1E − 04 1.1E − 09 BII00028

    [0438] The ability of the VHHs to bind to human isoform VEGF121 is determined in a binding ELISA. Binding of a dilution series of VHH to 1 μg/mL directly coated human VEGF121 (R&D) (human VEGF165 as reference) is detected using biotinylated anti-VHH 1A4 followed by extravidin-HRP. 1A4 is a anti-VHH VHH (generated in-house by Ablynx N V). The benchmark Avastin serves as positive control and is detected using a HRP conjugated anti-human Fc antibody. An irrelevant VHH serves as negative control. Representative binding response curves on VEGF165 and VEGF121 are shown in FIG. 46 corresponding EC.sub.50 values are summarized in Table 46-C.

    TABLE-US-00046 TABLE 46-C Overview of EC.sub.50 values in hVEGF165 and hVEGF121 binding ELISA. hVEGF165 hVEGF121 EC.sub.50 (M) EC.sub.50 (M) VEGFANGBII00022 1.4E − 09 2.3E − 09 VEGFANGBII00025 1.5E − 09 2.5E − 09 VEGFANGBII00028 1.2E − 09 2.1E − 09

    [0439] Binding to rat and mouse VEGF164 is assessed in a binding ELISA. VHHs binding to 1 μg/mL directly coated murine or rat VEGF164 (R&D) are detected using biotinylated anti-VHH 1A4 followed by extravidin-HRP. As positive control the mouse/rat cross-reactive monoclonal antibody B20-4.1 (Genentech) is titrated and detected with an HRP conjugated anti-human Fc antibody. An irrelevant VHH serves as negative control. Results are shown in FIG. 33. All 3 bispecific VHH are not cross-reactive to mouse and rat VEGF.

    [0440] Binding to human VEGF-B, VEGF-C, VEGF-D and PIGF is assessed via a binding ELISA. Binding of VHHs to 1 μg/mL directly coated VEGF-B (R&D), VEGF-C (R&D), VEGF-D (R&D) and PIGF (R&D) was detected using biotinylated anti-VHH 1A4 followed by extravidin-HRP. As positive controls a series of dilutions of the appropriate receptors (hVEGFR1-Fc for hVEGF-B and hPIGF, hVEGFR2-Fc for hVEGF-C, anti-hVEGF-D mAb (R&D) for hVEGF-D) are taken along. An irrelevant VHH serves as negative control. Results are shown in FIG. 34. All 3 bispecific VHH are not binding to VEGF family members.

    [0441] To explore the anti-Ang2 blocking properties in comparison with their respective monovalent building block 00921 (SEQ ID NO:220) and 00938 (SEQ ID NO:222), all 3 bispecific VHHs are analyzed in the human Ang2/hTie2-Fc (see Example 5.1; FIG. 35-1), mouse Ang2/mTie2-Fc (see Example 5.2; FIG. 35-2) and cyno Ang2/cTie2-Fc (see Example 5.2; FIG. 35-3) competition ELISA. The human assay is also performed after incubation of the VHH with 0.5 μM human serum albumin. Additionally, bispecific VHHs are tested in the hAng1/hTie2 competition ELISA (see Example 5.3; FIG. 36) and the Ang2 mediated HUVEC survival assay (see Example 5.5; FIG. 37). A summary of IC50 values and % inhibition is shown in FIG. 38.

    [0442] Affinities of VEGFANGBII00022-25-28 for human, mouse, cyno and rat Ang2 (see Example 5.4) have been determined and are shown in Table 47.

    TABLE-US-00047 TABLE 47 Affinity KD of purified VHHs for recombinant human, cyno, mouse and rat Ang2 human Ang2-FLD cyno Ang2-FLD k.sub.a k.sub.d K.sub.D k.sub.a k.sub.d K.sub.D (1/Ms) (1/s) (M) (1/Ms) (1/s) (M) VEGFANGBII00022 9.7E+05 1.5E−05 1.6E−11 1.5E+06 1.3E−05 8.1E−12 VEGFANGBII00025 2.7E+06 1.2E−02 4.5E−09 4.3E+06 1.1E−02 2.7E−09 VEGFANGBII00028 5.9E+05 9.6E−04 1.6E−09 8.4E+05 8.7E−04 1.0E−09 mouse Ang2-FLD rat Ang2-FLD k.sub.a k.sub.d K.sub.D k.sub.a k.sub.d K.sub.D (1/Ms) (1/s) (M) (1/Ms) (1/s) (M) VEGFANGBII00022 5.5E+05 2.8E−05 5.1E−11 3.9E+05 3.8E−05 9.9E−11 VEGFANGBII00025 1.3E+06 1.4E−02 1.1E−08 8.7E+05 2.9E−02 3.3E−08 VEGFANGBII00028 3.6E+05 2.0E−03 5.6E−09 2.5E+05 3.1E−03 1.2E−08

    [0443] Affinities of VEGFANGBII00022-25-28 for human, mouse and cyno serum albumin have been determined (Example 11) and are shown in Table 48. The affinity constant K.sub.D is calculated from resulting association and dissociation rate constants k.sub.a and k.sub.d (Table 48).

    TABLE-US-00048 TABLE 48 Affinity KD (nM) of purified VHHs for recombinant human, mouse and cyno serum albumin using (A) 1:1 interaction model or (B) heterogeneous ligand model HSA CSA k.sub.a k.sub.d K.sub.D k.sub.a k.sub.d K.sub.D (A) (1/Ms) (1/s) (nM) (1/Ms) (1/s) (nM) ALB11 5.6E+05 1.9E−03 4 4.5E+05 1.7E−03 4 VEGFANGBII00022 6.7E+05 6.0E−03 9 6.2E+05 5.4E−03 9 VEGFANGBII00025 5.6E+05 5.6E−03 12 4.3E+05 5.1E−03 12 VEGFANGBII00028 5.6E+05 5.8E−03 10 5.2E+05 5.3E−03 10 MSA k.sub.a k.sub.d K.sub.D (1/Ms) (1/s) (nM) ALB11 5.9E+05 3.0E−02 51 VEGFANGBII00022 5.2E+05 5.4E−03 150 VEGFANGBII00025 — — — VEGFANGBII00028 — — — MSA k.sub.a1 K.sub.d1 k.sub.a2 K.sub.d2 K.sub.D1 K.sub.D2 (B) (1/Ms) (1/s) (1/s) (1/s) (nM) (nM) VEGFANGBII00025 6.2E+05 9.9E−02 4.7E+04 5.7E−04 160 * 12 VEGFANGBII00028 5.9E+04 6.9E−04 5.7E+05 9.4E−02 12 160 * * describes 70% or more of the interaction

    TABLE-US-00049 TABLE 49 Ang2-binding components (1D01 (SEQ ID No: 214 (CDRs 1-3 disclosed as SEQ ID NOS 224-226, respectively) (FRs 1-4 disclosed as SEQ ID NOS 689, 676, 677 and 603, respectively)); 7G08 (SEQ ID No: 215 (CDRs 1-3 disclosed as SEQ ID NOS 227-229, respectively) (FRs 1-4 disclosed as SEQ ID NOS 678, 679, 680 and 603, respectively)); 027 (SEQ ID No: 216 (CDRs 1-3 disclosed as SEQ ID NOS 230-232, respectively) (FRs 1-4 disclosed as SEQ ID NOS 681, 682, 683 and 603, respectively)); 00042 (SEQ ID No: 217 (CDRs 1-3 disclosed as SEQ ID NOS 233-235, respectively) (FRs 1-4 disclosed as SEQ ID NOS 684, 694, 685 and 664, respectively)); 00050 (SEQ DI No: 218 (CDRs 1-3 disclosed as SEQ ID NOS 224, 237, and 226, respectively) (FRs 1-4 disclosed as SEQ ID NOS 686, 694, 685 and 664, respectively); 00045 (SEQ ID No: 219 (CDRs 1-3 disclosed as SEQ ID NOS 227, 240, and 241, respectively) (FRs 1-4 disclosed as SEQ ID NOS 693, 694, 687 and 664, respectively)); 00921 (SEQ ID No: 220 (CDRs 1-3 disclosed as SEQ ID NOS 242-244, respectively) (FRs 1-4 disclosed as SEQ ID NOS 686, 694, 685 and 664, respectively)); 00928 (SEQ ID No: 221 (CDRs 1-3 disclosed as SEQ ID NOS 245-247, respectively) (FRs 1-4 disclosed as SEQ ID NOS 693, 694, 687 and 664, respectively)); 00938 (SEQ ID No: 222 (CDRs 1-3 disclosed as SEQ ID NOS 248-250, respectively) (FRs 1-4 disclosed as SEQ ID NOS 688, 694, 685 and 664, respectively)); 00956 (SEQ ID No: 223 (CDRs 1-3 disclosed as SEQ ID NOS 251-253, respectively) (FRs 1-4 disclosed as SEQ ID NOS 684, 694, 685 and 664, respectively)). FR1 CDR1 FR2 CDR2 1D01 EVQLVESGGGLVQAGGSLRLSCAASGFTFD DYALG WFRQAAGKEREGVS CIRCSDGSTYYADSVKG 7G08 EVQLVESGGGLVQPGGSLRLSCAASGFALD YYAIG WFRQVPGKEREGVS CISSSDGITYYVDSVKG 027 EVQLVESGGGLVQAGGSLRLSCAASGFTLD DYAIG WFRQAPGKEREGVS CIRDSDGSTYYADSVKG FR3 CDR3 FR4 1D01 RFTISSDNAKNTVYLQMNSLKPEDTAVYYCAA SIVPRSKLEPYEYDA WGQGTQVTVSS 7G08 RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAT DSGGYIDYDCMGLGYDY WGQGTQVTVSS 027 RFTISSDNDKNTVYLQMNSLKPEDTAVYYCAA VPAGRLRFGEQWYPLYEYDA WGQGTQVTVSS FR1 CDR1 FR2 CDR2 00042 EVQLVESGGGLVQPGGSLRLSCAASGFTLD DYAIG WFRQAPGKEREGVS SIRDNDGSTYYADSVKG 00050 EVQLVESGGGLVQPGGSLRLSCAASGFTFD DYALG WFRQAPGKEREGVS CIRCSDGSTYYADSVKG 00045 EVQLVESGGGLVQPGGSLRLSCAASGFALD YYAIG WFRQAPGKEREGVS CISSSDGITYYADSVKG FR3 CDR3 FR4 00042 RFTISSDNSKNTVYLQMNSLRPEDTAVYYCAA VPAGRLRFGEQWYPLYEYDA WGQGTLVTVSS 00050 RFTISSDNSKNTVYLQMNSLRPEDTAVYYCAA SIVPRSKLEPYEYDA WGQGTLVTVSS 00045 RFTISRDNSKNTVYLQMNSLRPEDTAVYYCAT DSGGYIDYDCMGLGYDY WGQGTLVTVSS FR1 CDR1 FR2 CDR2 00921 EVQLVESGGGLVQPGGSLRLSCAASGFTFD DYALG WFRQAPGKEREGVS CIRCSGGSTYYADSVKG 00928 EVQLVESGGGLVQPGGSLRLSCAASGFALD YYAIG WFRQAPGKEREGVS CISSSGGITYYADSVKG 00938 EVQLVESGGGLVQPGGSLRLSCAVSGITLD DYAIG WFRQAPGKEREGVS AIRSSGGSTYYADSVKG 00956 EVQLVESGGGLVQPGGSLRLSCAASGFTLD DYAIG WFRQAPGKEREGVS AIRSSGGSTYYADSVKG FR3 CDR3 FR4 00921 RFTISSDNSKNTVYLQMNSLRPEDTAVYYCAA SIVPRSKLEPYEYDA WGQGTLVTVSS 00928 RFTISRDNSKNTVYLQMNSLRPEDTAVYYCAT DSGGYIDYDCSGLGYDY WGQGTLVTVSS 00938 RFTISSDNSKNTVYLQMNSLRPEDTAVYYCAA VPAGRLRYGEQWYPIYEYDA WGQGTLVTVSS 00956 RFTISSDNSKNTVYLQMNSLRPEDTAVYYCAA VPAGRLRFGEQWYPLYEYDA WGQGTLVTVSS