VEGF-BINDING MOLECULES

Abstract

VEGF-binding molecules, preferably VEGF-binding immunoglobulin single variable domains like VHHs and domain antibodies, pharmaceutical compositions containing same and their use in the treatment of diseases that are associated with VEGF-mediated effects on angiogenesis. Nucleic acids encoding VEGF-binding molecules, host cells and methods for preparing same.

Claims

1. VEGF-binding molecule comprising 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 molecule is capable of blocking the interaction of human recombinant VEGF165 with the human recombinant VEGFR-2 with an inhibition rate of ≥60%.

2. A VEGF-binding molecule of claim 1, wherein said CDR3 has a sequence selected from TABLE-US-00040 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.

3. A VEGF-binding molecule of claim 2, which 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).

4. A VEGF-binding molecule of claim 3, wherein said one or more immunoglobulin single variable domains are VHHs.

5. A VEGF-binding molecule of claim 4, wherein said one or more VHHs have amino acid sequences selected from the amino acid sequences shown in SEQ ID NOs: 9-46.

6. A VEGF-binding molecule of claim 5, 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.

7. A VEGF-binding molecule which has been obtained by affinity maturation and/or sequence optimization of a VHH defined in claim 6.

8. A VEGF-binding molecule of claim 7 which has been obtained by sequence optimization of a VHH having an amino acid sequence shown in SEQ ID NO: 18.

9. A VEGF-binding molecule of claim 8 having an amino acid sequence selected from sequences shown in SEQ ID NOs: 47-57.

10. A VEGF-binding molecule of claim 4, 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%.

11. A VEGF-binding molecule of claim 10, 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.

12. A VEGF-binding molecule of claim 11, wherein said VHH is selected from VHHs having the amino acid shown in SEQ ID NO: 18 or SEQ ID NO: 47-57.

13. The VEGF-binding molecule of claim 12 comprising two VHHs each having the amino acid sequence shown in SEQ ID NO: 57.

14. A VEGF-binding molecule of claim 13, 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.

15. A VEGF-binding molecule of claim 14, 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.

16. A VEGF-binding molecule of claim 15, 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.

17. A VEGF-binding molecule of claim 16, 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.

18. A VEGF-binding molecule of claim 17, 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.

19. A nucleic acid molecule encoding a VEGF-binding molecule of claim 1 or a vector containing same.

20. A host cell comprising a nucleic acid molecule of claim 19.

21. A pharmaceutical composition comprising at least one VEGF-binding molecule of claim 1 as the active ingredient.

22. A method of treating a disease associated with VEGF-mediated effects on angiogenesis comprising administering a pharmaceutical composition of claim 21 to a patient in need thereof.

23. The method of claim 22 wherein the disease is selected from cancer and cancerous dieases.

24. The method of claim 22 wherein the disease is selected from eye diseases.

Description

BRIEF DESCRIPTION OF THE FIGURES:

[0257] FIG. 1: Purified monovalent VHHs block the hVEGF165/hVEGFR2-Fc interaction (ELISA)

[0258] FIG. 2: Purified monovalent VHHs block the hVEGF165/hVEGFR1-Fc interaction (ELISA)

[0259] FIG. 3: Purified monovalent VHHs block the hVEGF165/hVEGFR2-Fc interaction (AlphaScreen)

[0260] FIG. 4: Purified monovalent VHHs block the hVEGF165/hVEGFR1-Fc interaction (AlphaScreen)

[0261] FIGS. 5-1 and 5-2: Binding of monovalent VHHs to recombinant human and mouse VEGF (ELISA)

[0262] FIG. 6: Binding of monovalent VHHs to human VEGF121

[0263] FIG. 7-1 through 7-4: Purified VHHs do not bind to VEGFB, VEGFC, VEGFD and PIGF

[0264] FIGS. 8-1 and 8-2: Formatted VHHs block hVEGF165/hVEGFR2-Fc interaction (ELISA)

[0265] FIGS. 9-1 and 9-2: Formatted VHHs block hVEGF165/hVEGFR1-Fc interaction (ELISA)

[0266] FIG. 10: Formatted VHHs block hVEGF165/hVEGFR2-Fc interaction (AlphaScreen)

[0267] FIG. 11:Formatted VHHs block hVEGF165/hVEGFR1-Fc interaction (AlphaScreen)

[0268] FIG. 12: Formatted VHHs block mVEGF164/mVEGFR2-Fc interaction (AlphaScreen)

[0269] FIGS. 13-1 and 13-2: Formatted VHHs bind to mouse and human VEGF

[0270] FIG. 14-1 through 14-8:Formatted VHHs do not bind to VEGFB, VEGFC, VEGFD and PIGF

[0271] FIG. 15: Formatted VHHs bind to VEGF121

[0272] FIG. 16: Sequence alignment of VHH VEGFBII23B04 with human VH3/JH germline consensus sequence

[0273] FIG. 17: VHH variants of VEGFBII12B04 block hVEGF165/hVEGFR2-Fc interaction (AlphaScreen)

[0274] FIG. 18: Sequence-optimized clones of VEGFBII23B04 block the hVEGF165/hVEGFR2-Fc interaction (AlphaScreen)

[0275] FIG. 19: Sequence alignment of VHH VEGFBII5B05 with human VH3/JH germline consensus sequence

MATERIALS AND METHODS

[0276] a) Production and Functionality Testing of VEGF109

[0277] 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, WI) 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 3TC. 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.

[0278] b) KLH Conjugation of VEGF165 and Functionality Testing of KLH-conjugated VEGF165

[0279] 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

[0280] Immunization with Different VEGF Formats Induces a Humoral Immune Response in Llama

[0281] 1.1 Immunizations

[0282] 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).

[0283] 1.2 Evaluation of VEGF-induced immune responses in llama

[0284] 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, Tex., USA) and a subsequent enzymatic reaction in the presence of the substrate TMB (3,3′,5,5′-tetramentylbenzidine) (Pierce, Rockford, Ill., 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 450 nm. 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 EGF165 Total Recombinant human VEGF109 Llama Immunogen 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

[0285] Cloning of the Heavy-Chain Only Antibody Fragment Repertoires and Preparation of Phage

[0286] 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-ml 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 WO05/044858. 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 gill-pelB leader sequence (pAX050). In frame with the VHH coding sequence, the vector encodes a C-terminal c-myc tag and a His6 tag. Phage are prepared according to standard protocols and stored after filter sterilization at 4° C. for further use.

EXAMPLE 3

[0287] Selection of VEGF-Specific VHHs Via Phage Display

[0288] 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).

[0289] 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 2 hrs 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.

[0290] 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 (0.1-1mM final). Periplasmic extracts (in a volume of ˜80 μL) are prepared according to standard methods.

EXAMPLE 4

[0291] Identification of VEGF-Binding and VEGF Receptor-Blocking VHHs

[0292] 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.

[0293] 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, Minn., 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 (4 nM) 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/ SEQ ID NO: FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 VEGFBII EVQLVES SYGM WFRQS AISEYSN RFTISRDNTK SPTILLTTEQ WGQG 01C02/ GGGLVQA G PGKER TYCSDSV NTVYLQMNSL WYKY TQVT 58 GGSLRLS EFVS RG TPDDTAIYYC VSS CTASGGS AA FS VEGFBII EVQLVES ASDM WFRQA AINWSGL RFTISRDNDN GRIPSSSRFS WGQG 01E07/ GGGLVQA G PGKER STFYTDS GALYLQMNTL SPAAYAS TQVT 59 GDSLRLS EFVA VKG KPEDTAVYSC VSS CVATGRT AA FR VEGFBII EVQLVES ITVM WFRQA AITWSAP RFTISRDNAK DRFKGRSIVT WGQG 03D12/ GGGLVQA A PGKER TTYYADS NTVYLRMNSL PSDYRY TQVT 60 GGSLRLS EFVA VKG KPEDSAIYYC VSS CTASTS AA YT VEGFBII EVQLVES DITV WYRQA TITPSGY RFTISRDNSK QFY WGQG 04B08/ GGGLVQP A PGIQR TYYWDFV NIVYLQMNSL TQVT 61 GGSLRLS QLVA KG KPEDTAAYYC VSS CAASGSA NT VG VEGFBII EVQLVES TDDV WFRQA VIRWSTG RFTLSRDNAK RSRPLGAGAW WGQG 05B02/ GGGLVQA G PGKER GTYTSDS NTMYLQMNSL YSGEKHYNY TQVT 62 GGSLRLS EFVA VKG KPEDTAVYYC VSS CAASGRT AA FS VEGFBII EVQLVES HYNM WFRQA SIRGGGG RFTISRENAK TAFYRGPYDY WGQG 05B03/ GGGLAQA G PGKER STTYANS NTVYLQMNSL DY TQVT 63 GDSLRLS EFVA VKD KPEDTAVYYC VSS CAASGRS AA FS VEGFBII EVQLVES SMA WYRQA RISSGGT RFTISRDNSK FSSRPNP WGAG 05B05/ GGGLVQP PGKHR TAYVDSV NTVYLQMNSL TQVT 64 GGSLRLS ELVA KG KAEDTAVYYC VSS CVASGIR NT FM VEGFBII EVQLVES NNAM WYRQA RISSGGG RFTVSRDNAK AYRTYNY WGQG 06G02/ GGGLVQP A PGKQR FTYYLDS NTVYLQMNSL TQVT 65 GGSLRLS ELVA VKG KPEDTAVYYC VSS CAASGNI NA FS VEGFBII EVQLVES ITVM WFRQA AITWSAP RFTISRDNAK DRFKGRSIVT WGQG 07A03/ GGGLVQA A PGKES SSYYADS NTVYLQMNSL RSDYKY TQVT 66 GGSLRLS EFVA VKG KPEDSAIYYC VSS CAASTSI AA YS VEGFBII EVQLVES ISVM WFRQA AITWSAP RFTISRDNAK DRFKGRSIVT WGQG 07A06/ GGGLVQA A PGKER TTYYADS NTVYLQTNSL RSDYRY TQVT 67 GGSLRLS AFVA VKG KPEDSAIYYC VSS CAVSTSI AA YS VEGFBII EVQLVES NYAM WFRQA AINQRGS RFTISRDSAK STWYGYSTYA WGQG 07D08/ GGGLVQT A PGKER NTNYADS NSVFLQMNSL RREEYRY TQVT 68 GGSLRLS EFVS VKG KPEDTAVYYC VSS CAASGRT AA FS VEGFBII EVQLVES DNVM WFRQA HISRGGS RFTISRDNTK SRSVALATAR WGQG 08D09/ GGGLVQA G AGKER RTEYAES KTMYLQMNSL PYDY TQVT 69 GGSLRLS EFVA VKG KPEDTAVYYC VSS CAASGRS AA FS VEGFBII EVQLVES SYYM WFRQA TISWNKI RFTVSRDNNK DASRPTLRIP WGQG 08E07/ GGGLAQA G PGKER STIYTDS NTVYLQMNSL QY TQVT 70 GGSLRLS EFVA VKG KPEDTAVYYC VSS CTTSGLT AA FS VEGFBII EVQLVES SDVM WYRQA FIRSLGS RFTISRDDAA RFSGESY WGQG 08F06/ GGGLVQP G PGKQR TYYAGSV NTVYLQMNNL TPVT 71 GGSLRLS ELVA KG KPEDTAVYYC VSS CAASGSI NA VR VEGFBII EVQLVES LYAM WFRQA AITWSAG RFTISRDNAR RQWGGTYYYH WGQG 08F07/ GGGLVQA G PGRER DTQYADS NTVNLQMNGL GSYAY TQVT 72 GGSLRLS EFLS VKG KPEDTAVYYC VSS CAVSGST AG FG VEGFBII EVQLVES SMA WYRQA RISSEGT RFTISRDNSK FSSRPNP WGAG 09A09/ GGGLVQP PGKHR TAYVDSV NTVYLQMNSL TTVT 73 GGSLRLS ELVA KG KAEDTAVYYC VSS CVASGIR NT FM VEGFBII EVQLVES TDDV WFRQA VIRWSTG RFTLSRDNAK RSRPLGAGAW WGQG 09A12/ GGGLVQA G PGKER GTYTSDS NTMYLQMNSL YTGETRYDS TQVT 74 GGSLRLS EFVA VAG KPEDTAVYYC VSS CAASGRT AA FS VEGFBII EVQLVES RYGM WFRQA AISEYDN RFTISRDNSK SPTILLSTDE WGRG 09D05/ GGGLVQP G PGKER VYTADSV STVYLQMNSL WYKY TQVT 75 GDSLRLS EFVI RG KSEDTAVYYC VSS CAASGLS AA FS VEGFBII EVQLVES TDDV WFRQA VIRWSTG RFTLSRDNAK RSRPLGAGAW WGQG 09F05/ GGGLVQA G PGKER GTYTSDS NTMYLQMNSL YTGETRYNY TQVT 76 GGSLRLS EFVA VKG KPEDTAVYYC VSS CAASGRT AA FS VEGFBII EVQLVES NYAM WFRQV VITRSPS RFTISRDNAK HYWNSDSYTY WGQG 10C07/ GGGLVQA G PGRER NTYYTDS NIVYLQMNSL TDSRWYNY TQVT 77 GGSLSLS EFVA VKG KPEDTAVYYC VSS CAASARA AA FS EVQLVES NYAM WFRQA DISSSGI RFTISRDNAK SAWWYSQMAR WGQG VEGFBII GGGLVQA G PGKER NTYVADA NTVYLQMNSL DNYRY TQVT 10E07/ GGSLRLS VLVA VKG KPEDTAVYYC VSS 78 CAASGRT AA FS EVQLVES RYAM WFRQA SINTSGK RFAVSRDNAK DRFFGSDSNE WGQG VEGFBII GGGLVQA G PGKER RTSYADS NTGYLQMNSL PRAYRY TQVT 10G04/ GGSLRLS EFVA MKG KLEDTATYYC VSS 79 CAASGDT AA LS EVQLVES NYNM WFRQA TIRHHGY RFTISRDNAK KLFWDMDPKT WGQG VEGFBII GGGLVQA G PGKER DTYYAES NTVYLQMNSL GFSS TQVT 10G05/ GESLRLS EFVA VKG KPEDTALYSC VSS 80 CVASGIT AK FS VEGFBII EVQLVES SYGL WFRQA AIGWSGS RFTVSVDNAK KVRNFNSDWD WGQG 11C08/ GGGLVQA G PGKER STYYADS NTVYLKMNSL LLTSYNY TQVT 81 GGSLRLS EFVA VKG EPEDTAVYYC VSS CAASGRT AA LS VEGFBII EVQLVES SYAI WFRQA RISWSGA RFTISRGNAK QTTSKYDNYD WGQG 11C11/ GGGLVQA G PGRER NTYYADS NTVYLQMNSL ARAYGY TQVT 82 GGSLMLS EFVA VKG KPEDTAAYYC VSS CAASGRA AA LS VEGFBII EEQLVES SYAI WFRQA RISWSGA RFTISRGNAK QTTSKYDNYD WGQG 11D09/ GGGLVQA G PGRER NTYYADS NTVYLQMNSL ARAYGY TQVT 83 GGSLMLS EFVA VKG KPEDTAAYYC VSS CAASGRA AA LS VEGFBII EVQLVES SYAM WFRQA TISQSGY RFTISRDNAK DPFYSYGSPS  WGQG 11E04/ GGGLVQA G PGKER STYYADS NTVNLQMNSL PYRY TQVT 84 GGSLRLS EFVA VKG KPEDTAVYYC VSS CAASGRT AA FS VEGFBII EVQLVES FSAM WFRQA AFKWSGS RFTISTDNAK DRFYTGRYYS WGQG 11E05/ GGGLVQP G PGKER TTYYADY NILFLQMNSL SDEYDY TQVT 85 GGSLRLS EFVA VKG KPEDTAIYYC VSS CASSGRL AV FS VEGFBII EVQLVES ITVM WFRQA AITWSAP RFTISRDNAK DRFKGRSIVT WGQG 11F10/ GGGLVQA A PGKER SSYYADS NTVYLQVNSL RSDYRY TQVT 86 GGSLRLS EFVA VKG KPEDSAIYYC VSS CAASTS AA YS VEGFBII EVQLVES SLAM WFRQV SISQSGI RFTISRDSAK SVFYSTALTR WGQG 11F12/ GGGLVQS G PGKDR TTSYADS NTVYLQMNLL PVDYRY TQVT 87 GGSLRLS EFVA VKS KPEDTAVYYC VSS CAASGRS AT FS VEGFBII EVQLVES ITVM WFRQA AITWSAP RFTISRDNAK DRFKGRSIVT WGQG 11G09/ GGGLVQA A PGKER TTYSADS NTVYLQMNSL RSDYRY TQVT 88 GGSLRLS EFVA VKG KPEDSAIYYC VSS CAASTS AA YS VEGFBII EVQLVES KYVM WFRQA AITSRDG RFTISGDNTK DEDLYHYSSY WGQG 12A07/ GGGLVQA G PGNDR PTYYADS NKIFLQMNSL HFTRVDLYHY TQVT 89 GGSLRLS EFVA VKG MPEDTAVYYC VSS CSVTGRT AI FN VEGFBII EVQLVES SSWM WVRQA RISPGGL RFSVSTDNAN GGAPNYTP RGRG 12B01/ GGGLVQP Y PGKGL FTYYVDS NTLYLQMNSL TQVT 90 GGSLRLA EWVS VKG KPEDTALYSC VSS CAASGFT AK LS VEGFBII EVQLVES SDVM WYRQA FIRSLGS RFTISRDNAA RFSGESY WGQG 12C04/ GGGLVQP G PGKQR TYYAGSV NTVYLQMNNL TPVT 91 GGSLRLS ELVA KG KPEDTAVYYC VSS CAASGSI NA VR VEGFBII EVQLVES NYVM WFRQA AITSTNG RFTISGDNTK DEDLYHYSSY WGQG 12E10/ GGGLAQA G PGNER PTYYADS NKVFLQMDSL HYTRVALYHY TQVT 92 GGSLRLS EFVA VKG RPEDTAVYYC VSS CTASGRT AI FN VEGFBII EVQLVES LYAM WFRQA AITWSAG RFTISRDNAR RQWGGTYYYH WGQG 12G04/ GGGLVQS G PGRER DTQYADS NTVNLQMNGL GSYAW TQVT 93 GDSLRLS EFVS VKG KPEDTAVYYC VSS CAVSGNT AG FG VEGFBII EVQLVES TDDV WFRQA VIRWSTG RFTLSRDNAK RSRPLGAGAW WGQG 16C03/ EGGLVQA G PGKER GTYTSDS NTMYLQMNSL YTGENYYNY TQVT 94 GGSLRLS EFVA VKG KPEDTAVYYC VSS CAASGRT AA FS VEGFBII EVQLVES GYDM WFRQA AITWSGG RFTISRDNAK GRIWRSRDYD WGHG 16F11/ GGGLVQA G PGKER STYSPDS NTVYLQMNNL SEKYYDI TQVT 95 GGSLRLS EFVT VKG TPEDTAVYYC VSS CAASGRT AS SS VEGFBII EVQLVES AYDM WFRQA VISWTNS RFTISRDNAK DRRRTYSRWR WGQG 36C08/ GGGLVQA G PGKER MTYYADS NTVYLQMNSL FYTGVNDYDY TQVT 96 GGSLRLS EFVA VKG KPEDTAVYYC VSS CAASGRT AV FS VEGFBII EVQLVES AYDM WFRQA VISWSGG RFTISRDNAK DRRRAYSRWR WGQG 37F09/ GGGLVQT G PGKER MTYYADS STVYLQMNSP YYTGVNDYEF TQVT 97 GGSLRLS EFVA VQG KPEDTAVYYC VSS CAASGRT AV FS VEGFBII EVQLVES AYDM WFRQA VISWSGG RFTISRDNAK DRRRLYSRWR WGQG 38A06/ GGGLVQA G PGKER MTYYADS NTVYLQMNSL YYTGVNDYDY TQVT 98 GGSLRLS EFVA VKG KPEDTAVYYC VSS CAASGRT AV FS VEGFBII EVQLVES AYDM WFRQA VISWTGG RFTISRDKAK DRRRTYSRWR WGQG 39H11/ GGGLVQA G PGKER MTYYADS NTVSLQMNSL YYTGVNEYEY TQVT 99 GGSLRLS EFVA VKG KPEDTAVYYC VSS CAASGRT AV FS VEGFBII EVQLVES AYDM WFRQA VISWTGD RFTISRDKAK DRRRTYSRWR WGQG 41B06/ GGGLVQA G PGKER MTYYADS NTVSLQMNSL YYTGVNEYEY TQVT 100 GGSLRLS EFVA VKG KPEDTAVYYC VSS CAASGRT AA FS VEGFBII EVQLVES VYTM WFRQA TISRTGD RFTISRENAK GPIAPSPRPR WGQG 41C05/ GGGLVQA G PGKER RTSYANS NTVYLQMNSL EYYY TQVT 101 GGSLRLS EFVA VKG KPEDTAVYSC VSS CAASGRT AA FS VEGFBII EVQLMES AYDM WFRQA VISWTGG RFTISRDKAK DRRRTYSRWR WGQG 41D11/ GGGLVQA G PGKER MTYYADS NTVSLQMNSL YYTGVNEYEY TQVT 102 GGSLRLS EFVA VKG KPEDTAVYYC VSS CAASGRT AV FS VEGFBII EVQLVES AYDM WFRQA VISWSGG RFTISRENAK GRRRAYSRWR WGQG 42F10/ GGGLVQA G PGKER MTDYADS NTQFLQMNSL YYTGVNEYDY TQVT 103 GGSLRLS EFVA VKG KPEDTAVYYC VSS CAASGRT AV FS VEGFBII EVQLVES SYAM WFRQA HINRSGS RFTISRDNAK GRYYSSDGVP WGQG 86C11/ GGGLVQA G PGKER STYYADS NTVYLQLNSL SASFNY TQVT 104 GDSLRLS ESVA VKG KPEDTAVYYC VSS CTASGRT AA FN VEGFBII EVQLVES TWAM WFRQA AISWSGS RFIISRDNAQ KTVDYCSAYE WGRG 86F11/ GGGLVQA A PGKER MTYYTDS NTLFLQMNNT CYARLEYDY AQVT 105 GDSLRLS EFIS VKG APEDTAVYYC VSS CFTSART AA FD VEGFBII EVQLVES STNM WFRQG AITLSGT RFTISRDNDK DPSYYSTSRY WGQG 86G08/ GGGLMQT G PGKER TYYAEAV NTVALQMNSL TKATEYDY TQVT 106 GDSLRLS EFVA KG KPEDTAVYYC VSS CAASGLR GA FT VEGFBII EVQLVES TYTM WFRQT AIRWTVN RFTISRDIVK QTSAPRSLIR WGQG 86G10/ GGGLVQA G PGTER ITYYADS NTVYLQMNSL MSNEYPY TQVT 107 GGSLRLS EFVA VKG KPEDTAVYYC VSS CAASGRT AA FN VEGFBII EVQLVES LYTV WFRQA YISRSGS RFTLSRDNAK TSRGLSSLAG WGRG 86G11/ GGGLVQA G PGKER NRYYVDS NTVDLQMNSL EYNY TQVT 108 GGSLRLS EFVA VKG KTEDTAVYYC VSS CAASGLT AA FS VEGFBII EVQLVES SYRM WFRRT SISWTYG RFTMSRDKAK GAQSDRYNIR WGQG 86H09/ GGGLVQA G PGKED STFYADS NAGYLQMNSL SYDY TQVT 109 GGSLRLS EFVA VKG KPEDTALYYC VSS CTASGSA AA FK VEGFBII EVQLVES TSWM WVRQA SIPPVGH RFTISRDNAK DSAGRT KGQG 87B07/ GGGLVQP H PGKGL FANYAPS NTLFLQMNSL TQVT 110 GGSLKLS EWVS VKG KSEDTAVYYC VSS CTASGFT AK FS VEGFBII KVQLVES NYAM WFRQA AITRSGG RFTISRDNAK TRSSTIVVGV WGKG 88A01/ GGGLVQA D PGKER GTYYADS NTVYLQMNSL GGMEY TLVT 111 GGSLRLS EFVA VKG KPEDTAVYYC VSS CAASERT AA FS VEGFBII EVQLVES DYDI WFRQA CITTDVG RFTISSDNAK DTQDLGLDIF WGQG 88A02/ GGGLVQA G PGNER TTYYADS NTVYLQINDL CRGNGPFDG TQVT 112 GGSLRLS EGVS VKG KPEDTAIYYC VSS CAASGFT AV FG VEGFBII EVQLVES DYAI WFRQA CISSYDS RFTISRDSAK EREQLRRRES SGKG 88B02/ GGGLVQP G PGKER VTYYADH NTLYLQMNSL PHDELLRLCF TLVT 113 GGSLRLS EGVS VKG SIEDTGVYYC YGMRY VSS CTASGLN AA LD VEGFBII EVQLVES DYAI WFRQA CISSSDT RFTFSRDNAK AFRCSGYELR WGQG 88E02/ GGGLVQP G PGKER SIDYTNS NTVYLQMNSL GFPT TQVT 114 GGSLRLS EAVS VKG KPEDTAVYYC VSS CVASGFR AA LD VEGFBII EVQLVES SLAV WFRQA RITWSGA RFTISRDNAK DRSPNIINVV WGQG 88G03/ GGGLVQA G PGKER TTYYADA NTMYLQMNSL TAYEYDY TQVT 115 GGSLRLS EFVA VKD KPEDTAVYYC VSS CAASGGT AA FS VEGFBII EVQLVES LYNM WFRQA AITSSPM RFSISINNDK PEGSFRRQYA WGQG 88G05/ GGGLVQP G PGKER STYYADS TTGFLQMNVL DRAMYDY TQVT 116 GASLRLS EFVA VKG KPEDTGVYFC VSS CAASGDG AA FT VEGFBII EVQLVES GSDM WFRQS AIRLSGS RFTISRDNAK RSTYSYYLAL WGQG 88G11/ GGGLAQA G PGKER ITYYPDS NTVYLQMNSL ADRGGYDY TQVT 117 GGSLRLS EIVA VKG KPEDTAVYYC VSS CAASGRT AA FS VEGFBII EVQLVES TYAI WFRQA CMSAGDS RFTTSTDNAR ARYHGDYCYY WGQG 88H01/ GGGLVQA G PGKER IPWYTAS NTVYLQMNSL EGYYPF TQVT 118 GGSLRLS EAVS VKG KPEDTAHYYC VSS CVASGFT AA LG VEGFBII EVQLVES TNFM WYRQA TITSSSI RFTISRDNAK RWRWSDVEY WGKG 89B04/ GGGLVQA G PGKQR TNYVDSV NTVYLQMTSL TLVT 119 GGSLRLS ELVA KG KPEDTAVYYC VSS CAASTSI HA SS VEGFBII EVQLVES IFAM WYRQA SITRSSI RFTPSRDNAK AIRPELYSVV WGQG 89B08/ GGGLVQP R PGKQR TTYADSV NTVSLQMNSL NDY TQVT 120 GGSLRLS ELVA KG KPEDTAVYYC VSS CAASGTT NA SS VEGFBII EVQLVES DYNL WFRQA VISWRDS RFTISRDNAK DRVSSRLVLP WGQG 89D04/ GGGLVQP G PGKER FAYYAEP NTVYLQMNSL NTSPDFGS TQVT 121 GGSLRLS QFVA VKG KPEDTAVYYC VSS CATSGLT AA FS VEGFBII EVQLVES NAIM WFRQA AMNWRGG RFTISGDNTK DEDLYHYSSY WGQG 89F09/ GGGLVQA G PGQER PTYYADS NTVFLQMNFL HYSRVDLYHY TQVT 122 GDSLRLS EFVA VKG KPEDTAVYYC VSS CAASGRT AA FN VEGFBII EVQLVES IFAM WYRQA SITRSSI RFTLSRDNAK AIRPELYSVV WGQG 89G09/ GGGLVQP R PGKQR TTYADSV NTVSLQMNSL NDY TQVT 123 GGSLRLS ELVA KG KPEDTAVYYC VSS CAASGTT NA SS VEGFBII EVQLVES SYAP WFRQA AFTRSSN RFTISRDNAH NLGSTWSRDQ WGQG 89H08/ GGGLVQA G PGKER IPYYKDS TVYLQMNSLK RTYDY TQVT 124 GGSLRLS EFVA VKG PEDTAIYYCA VSS CAASGGS V FS

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 VHH ID/ SEQ ID NO: FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 VEGFBI EVQL SYSM WFRQ AISS RFTI SRAY WGQG I VESG G AQGK SGGY SRDN GSSR TQVT 22A10/ GGLV EREF IYDS TKNT LRLA VSS 9 QPGD VV VSLE VYLQ DTYD SLKL G TPSL Y SCAF KPED SGRT TADY FS YCAA VEGFBI EVQL SYSM WFRQ AISS RFTI SRAY WGQG I VESG A AQGK GGFI SRDN GSSR TQVT 22A11/ GGLV EREF YDAV TKNT LRLA VSS 10 QPGD VV SLEG VYLQ DTYD SLKL TPSL Y SCAF KPED SGRT TAVY FS YCAA VEGFBI EVQL SYSM WFRQ AISS RFTI SRAY WGQG I VESG G AQGK SGGY SRDN GSSR TQVT 22B06/ GGLV EREF IYDS TKNT LRLA VSS II QPGD VV VSLE VYLQ DTYD SLKL G TPSL Y SCAA KPED SGRT TAVY FS YCAA VEGFBI EVQL SYSM WFRQ AISS RFTI SRAY WGQG I VESG G AQGK SGNY SRDN GSSR TQVT 22B07/ GGLV EREF KYDS TKNT LRLG VSS 12 QAGD VV VSLE VYLQ DTYD SLRL G INSL Y SCAA KPED SGRT TAVY FS YCAA VEGFBI EVQL SYSM WFRQ AISS RFTI SRAY WGQG I VESG G AQGK GGSI SRDN ASSR TQVT 22E04/ GGLV EREF YDSV TKNT LRLA VSS 13 QPGD VV SLQG VYLQ DTYD SLKL TPSL Y SCVA KPED SGRT TAVY SS YCAA VEGFBI EVQL SYSM WFRQ AISS RFTI SRAY WGQG I VESG G AQGK GGYI SRDN GSSR TQVT 23A03/ GGLV EREF YDSV TKNT LRLA VSS 14 QPGD VV SLQG VYLQ DTYD SLKL TPSL Y SCVA KPED SGRT TAVY FS YCAA VEGFBI EVQL SYSM WFRQ AISS RFTI SRAY WGQG I VESG G AQGK GGFI SRDN GSSR TQVT 23A06/ GGLV EREF YDAV TKNT LRLA VSS 15 QPGD VV SLEG VYLQ DTYD SLKL TPSL Y SCAF KPED SGRT TAVY FS YCAA VEGFBI EVQL SYSM WFRQ AISN RFTI SRAY WGQG I VESG G AQGK GGYK SRDN GSSR TQVT 23A08/ GGLV EREF YDSV TKNT LRLA VSS 16 QTGD VV SLEG VYLQ DTYD SLRL INSL Y SCVA KPED SGRT TAVY FS YCAA VEGFBI EVQL SYSM WFRQ AISS RFTI SRAY WGQG I VESG G AQGK SGGY SRDN GSSR TQVT 23A09/ GGLV EREF IYDS SKNT LRLP VSS 17 QPGD VV VSLE VYLQ DTYD SLKL G TPSL Y SCAF KPED SGRT TAVY FG YCAA VEGFBI EVQL SYSM WFRQ AISK RFTI SRAY WGQG I VESG G AQGK GGYK SKDN GSSR TQVT 23B04/ GGLV EREF YDSV AKNT LRLA VSS 18 QTGD VV SLEG VYLQ DTYE SLRL INSL Y SCEV KPED SGRT TAVY FS YCAS VEGFBI EVQL SYSM WFRQ AISS RFTI SRAY WGQG I VESG A AQGK GGFI SRDN GSSR TQVT 23D11/ GGLV EREF YDAV TKNT LRLA VSS 19 QPGD VV SLEG VYLQ DTYD SLRL TPSL Y SCAF KPED SGRT TAVY FS YCAA VEGFBI EVQL SYSM WFRQ AISS RFTI SRAY WGQG I VESE G AQGK GGYI SRDN GSSR TQVT 23E05/ GGLV EREF YDSV TKNT LRLA VSS 20 QPGD VV SLQG VYLQ DTYD SLKL TPSL Y SCVA KPED SGRT TAVY SS YCAA VEGFBI EMQL SYSM WFRQ AISS RFTI SRAY WGQG I VESG G AQGK SGGY SRDN GSSR TQVT 23F02/ GGLV EREF IYDS TKNT LRLA VSS 21 QPGD VV VSLE VYLQ DTYD SLKL G TPSL Y SCAF KPED SGRT TADY FS YCAA VEGFBI EVQL SYSM WFRQ AISS RFTI SRAY WGQG I VESG G AQGK SGNY SRDN GSSR TQVT 23F05/ GGLV EREF KYDS TKNT LRLG VSS 22 QAGD VV VSLE VYLQ DTYD SLRL G INSL Y SCAA KPKD SGRT TAVY FS YCAA VEGFBI EVQL SYSM WFRQ AISS RFTI SRAY WGQG I VESG G AQGK GGGY SRDN GSSR TQVT 23F11/ GGLV EREF IYDS TKNT LRLA VSS 23 QPGD VV VSLE VYLQ DTYD SLKL G TPSL Y SCAF KPED SGRT TADY FS YCAA VEGFBI EVQL SYSM WFRQ AISS RFTI SRAY WGQG I VESG G AQGK SGGY SRDN GSSR TQVT 23G03/ GGLV EREF IYDS SKNT LRLP VSS 24 QPGD VV VSLE VYLQ GTYD SLKL G TPSL Y SCAF KPED SGRT TAVY FG YCAA VEGFBI EVQL SYSM WFRQ AISS RFTI SRAY WGQG I VESG G AQGK GGYI SRDN GSSR TQVT 24C04/ GGLV EREF YDSV TKNT LRLA VSS 25 QPGD VV SLQG VYLQ DTYD SLKL TPSL Y SCVA KPED SGRT TAVY SS YCAA VEGFBI EVQL SYSM WFRQ AISS RFTI SRAY WGQG I VESG G AQGK GGYK SRDN GSGR TQVT 27D08/ GGLV EREF YDSV TQNT LRLA VSS 26 QTGD VV SLEG VYLQ DTYD SLRL INSL Y SCAA KPED SGRT TAVY FS YCAA VEGFBI EVQL SYSM WFRQ AISS RFTI SRAY WGQG I VESG G AQGQ GGYI SRDN GSSR TQVT 27G07/ GGLV EREF YDSV TKNT LRLA VSS 27 QPGD VV SLQG VYLQ DTYD SLKL TPSL Y SCVA KPED SGRT TAVY SS YCAA VEGFBI EVQL SYSM WFRQ AISS RFTI SRAY WGQG I VESG G AQGQ GGYI SRDN GSSR TQVT 30C09/ GGLV EREF YDSV TKNT LRLA VSS 28 QPGD VV SLQG VYLQ DTYD SLKL TPSL Y SCIA KPED SGRT TAVY SS YCAA VEGFBI EVQL SYSM WFRQ AISS RFTI SRAY WGQG I VESG G AQGK SGNY SRDN GSSR TRVT 30E07/ GGLV EREF KYDS TKNT LRLG VSS 29 QAGD VV VSLE VYLQ DTYD SLRL G INSL Y SCAA KPED SGRT TAVY FS YCAA VEGFBI EVQL SYSM WFRQ AISS RFTI SRAY WGQG I VESG G AQGK SGGY SRDN GSSR TQVT 31C07/ GGLV EREF IYDS TKNT LRLA VSS 30 QTGD VV VSLE VYLQ DTYD SLRL G TPSL Y SCAA KPED SGGT TADY FS YCAA VEGFBI EVQL SYSM WFRQ AISS RFTI SRAY WGQG I VESG G AQGK SGGY SRDN GSSR TQVT 39E02/ GGLV EREF IYDS TKNT LRLA VSS 31 QPGD VV VSLE VYLQ DTYD PLKL G TPSL Y SCAF KPED SGRT TADY FS YCAA VEGFBI EVPL SYSM WFRQ AISS RFTI SRAY WGQG I VESG G AQGK SGNY SRDN GSSR TQVT 39G04/ GGLV EREF KYDS TKNT LRLG VSS 32 QAGD VV ASLE VYLQ DTYD SLRL G INSL Y SCAA KPED SGRT TAVY FS YCAA VEGFBI EVQL SYSM WFRQ AISS RFTI SRAY WGQG I VESG A AQGK GGFI SRDN GSSR TQVT 40F02/ GGLV EREF YDAV TKNT LRLA VSS 33 QPGD VV SLEG VYLQ DTYD SLKL TPSL Y SCAF KPEG SGRT TAVY FS YCAA VEGFBI EVQL SYSM WFRQ AISS RFTI SRAY WGQG I VESG G AQGK SGGY SRDN GSSR TQVT 40G07/ GGLV EREF IYDS TKNA LRLA VSS 34 QPGD VV VSLE VYLQ DTYD SLKL G TPSL Y SCAF KPED SGRT TADY FS YCAA VEGFBI EVQL SYSM WFRQ AISS RFTI SRAY WGQG I MESG G AQGK SGGY SRDN GSSR TQVT 4 OHIO/ GGLV EREF IYDS TKNT LRLA VSS 35 QPGD VV VSLE VYLQ DTYD SLKL G TPSL Y SCAF KPED SGRT TADY FS YCAA VEGFBI EVQL SYSM WFRQ AISS RFTI SRAY WGQG I VESG G AQGK GGFI SRDN GSSR TQVT 41B05/ GGLV EREF YDAV TKNT LRLA VSS 36 QPGG VV SLEG VYLQ DTYD SLRL TPSL Y SCAF KPED SGRT TAVY FS YCAA VEGFBI EVQL SYSM WFRQ AISS RFTI SRAY WGQG I VESG A AQGK GGFI SREN GSSR TQVT 41G03/ GGLV EREF YDAV TKNT LRLA VSS 37 QPGD VV SLEG VYLQ DTYD SLKL TPSL Y SCAF KPED SGRT TAVY FS YCAA VEGFBI EVQL SYSM WFRQ AISS RFTI SRAY WGQG I VESG G AQGK SGGY SRDN GSSR TQVT 42A05/ GGLV EREF IYDS TKNT LRLA VSS 38 QPGD VV VSLE VYLQ DTYD SLKL G MPSL Y SCAF KPED SGRT TADY FS YCAA VEGFBI EVQL SYSM WFRQ AISS RFTI SRAY WGQG I VESG G AQGK SGGY SRDN GSSR TQVT 42D05/ GGLV EREF IYDS TKNT LRLA VSS 39 QPGD VV VSLE VYLQ DTYD SLKL G TPSL Y SCAF KPED SGRT TAVY FS YCAA VEGFBI EVQL SYSV WFRQ AISS RFTI SRAY WGQG I VESG G AQGK GGYI SRDN GSSR TQVT 42F11/ GGLV EREF YDSV TKNT LRLA VSS 40 QPGD VV SLQG VYLQ DTYD SLKL TPSL Y SCVA KPED SGRT TAVY SS YCAA VEGFBI EVQL SYSM WFRQ AISS RFTI SRAY WGQG I VESG G AQGK SGGY SRDN GSSR TQVT 56E11/ GGLV EREF IYDS TKNT LRLA VSS 41 QPGD VV VSLE VYLQ DTYD SLKL G TPSL Y SCAF KPED SGRT AADY FS YCAA VEGFBI EVQL SYSM WFRQ AISS RFTI SRAY WGQG I VESG G AQGK SGGY SRDN GSSR TQVT 60A09/ GGLV EREF IYDS TRNT LRLA VSS 42 QPGD VV VSLE VYLQ DTYD SLKL G TPSL Y SCAF KPED SGRT TADY FS YCAA VEGFBI EVQL SYSM WFRQ AISS RFTI SRAY WGQG I VESG G AQGK GGYK SRDN ASSR TQVT 61A01/ GGLV EREF YDAV TKNT LRLA VSS 43 QAGG VV SLEG VYLQ DTYD SLRL TPSL Y SCAF KPED SGRT TAVY FS YCAA VEGFBI EVQL SYSM WFRQ AISS RFTI SRAY WGQG I VESG G AQGK SGGY SRDN GSSR TQVT 62A09/ GDLV EREF IYDS TKNT LRLA VSS 44 QPGD VV VSLE VYLQ DTYD SLKL G TPSL Y SCAA KPED SGRT TAVY FS YCAA VEGFBI EVQL SYSM WFRQ AISS RFTI SRAY WGQG I VESE G AQGK SGNY SRDN GSSR TQVT 62D10/ GGLV EREF KYDS TKNT LRLG VSS 45 QAGD VV VSLE VYLQ DTYD SLRL G INSL Y SCAA KPED SGRT TAVY FS YCAA VEGFBI EVQL SYSM WFRQ AIAS RFTI SRAY WGQG I VESG G AQGK GGYI SRDN GSSR TQVT 62F02/ GGLV EREF YDAV TKDT LRLA VSS 46 QPGD VV SLEG VYLQ DTYD SLKL TPSL Y SCAF KPED SGRT TAVY FS YCAA

[0294] 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 μI/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 Ica-fast, kd-slow and % fast are listed in Table 4.

TABLE-US-00007 TABLE 4 Off-rate determination of receptor-blocking VHHs with Biacore B-cell Unique Representative % Binding level lineage sequence 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 VEGFBI24004 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 VEGFBII31007 3.70E-02 1.50E-04 25 77 1 21 VEGFBII30009 1.50E-02 7.60E-05 19 264 1 22 VEGFBII30E07 1.70E-02 1.30E-04 29 226 1 23 VEGFBI39G04 1.40E-02 7.40E-04 40 210 1 24 VEGFBI41G03 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 VEGFBI42D05 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

[0295] Characterization of purified VHHs

[0296] 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. Expression is induced by addition of 1 mM IPTG and allowed to continue for 4 hours at 3TC. 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. 5.1 Evaluation of Human VEGF165/VEGFR2 Blocking VHHs in Human VEGF165/Human VEGFR2-Fc Blocking ELISA

[0297] 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 VHH ID IC.sub.50 (nM) % inhibition VEGFBII23B04 2.1 100 VEGFBII23A06 3.0 100 VEGFBII24C04 2.5 100 Ranibizumab 1.6 100 Bevacizumab 1.7 100

[0298] 5.2 Evaluation of Human VEGF165/VEGFR2 Blocking VHHs in Human VEGF165/Human VEGFR1-Fc Blocking ELISA

[0299] 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 0.5 nM 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

[0300] 5.3 Evaluation of the Anti-VEGF165 VHHs in the Human VEGF165/Human VEGFR2-Fc Blocking AlphaScreen

[0301] 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

[0302] 5.4 Evaluation of the Anti-VEGF165 VHHs in the Human VEGF165/Human VEGFR1-Fc Blocking AlphaScreen

[0303] 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

[0304] 5.5 Determination of the Affinity of the Human VEGF165-VHH Interaction

[0305] 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 360nM. Samples are injected for 2 min and allowed to dissociate up to 20 min at a flow rate of 45 μI/min. In between sample injections, the chip surface is regenerated with 100 mM HCI. 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.S.sup.−1) (M.sup.−1.S.sup.−1) (M.sup.−1.S.sup.−1) (M.sup.−1.S.sup.−1) (M.sup.−1.S.sup.−1) (M.sup.−1.S.sup.−1) (nM) VEGFB1123B04.sup.(a) — 2.1E+05 1.4E-02 — 8.6E-03 2.4E-04 0.7 VEGFB1123A06.sup.(a) — 4.2E+05 2.0E-02 — 5.7E-02 1.0E-04 0.7 VEGFB1124004.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

[0306] 5.6 Binding to Mouse VEGF164

[0307] Cross-reactivity to mouse VEGF164 is determined using a binding ELISA. In brief, recombinant mouse VEGF164 (R&D Systems, Minneapolis, Miss., 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: 500nM-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) (FIGS. 5-1 and 5-2). 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

[0308] 5.7 Binding to VEGF121

[0309] Binding to recombinant human VEGF121 is assessed via a solid phase binding ELISA. Briefly, recombinant human VEGF121 (R&D Systems, Minneapolis, Miss., 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

[0310] 5.8 Binding to VEGF Family Members VEGFB, VEGFC, VEGFD and PIGF

[0311] 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, Miss., 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 FIGS. 7-1 through 7-4. The results show that there was no detectable binding of the selected VHHs to VEGFB, VEGFC, VEGFD or PIGF.

[0312] 5.9 Epitope Binning

[0313] 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 HCI). 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.

[0314] 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 VEGFBII23B04 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 891304 89D04 89F09 89G09 89H08 24004 23A6 27G07 231304 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 23B04- captured 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 Binding Injection level step Binding [sample] (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 —

[0315] 5.10 Characterization of the Anti-VEGF VHHs in the HUVEC Proliferation Assay

[0316] 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

[0317] 5.11 Characterization of the Anti-VEGF VHHs in the HUVEC Erk Phosphorylation Assay

[0318] 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

[0319] Generation of Multivalent Anti-VEGF Blocking VHHs

[0320] 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 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. Expression is induced by addition of 1 mM IPTG and allowed to continue for 4 hours at 3TC. 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 bold and underlined in one relevant sequence) Sequence ID/ SEQ ID NO: VHH ID AA sequence VEGFBII23B04- VEGFBII010 EVQLVESGGGLVQTG 35GS-23B04/128 DSLRLSCEVSGRTFS SYSMGWFRQAQGKER EFVVAISKGGYKYDS VSLEGRFTISKDNAK NTVYLQINSLKPEDT AVYYCASSRAYGSSR LRLADTYEYWGQGTQ VTVSSGGGGSGGGGS GGGGSGGGGSGGGGS GGGGSGGGGSEVQLV ESGGGLVQTGDSLRL SCEVSGRTFSSYSMG WFRQAQGKEREFVVA ISKGGYKYDSVSLEG RFTISKDNAKNTVYL QINSLKPEDTAVYYC ASSRAYGSSRLRLAD TYEYWGQGTQVTVSS VEGFBII23B04- EVQLVESGGGLVQTG 9GS-4B08/129 DSLRLSCEVSGRTFS SYSMGWFRQAQGKER EFVVAISKGGYKYDS VSLEGRFTISKDNAK NTVYLQINSLKPEDT AVYYCASSRAYGSSR LRLADTYEYWGQGTQ VTVSSGGGGSGGGSE VQLVESGGGLVQPGG SLRLSCAASGSAVGD ITVAWYRQAPGIQRQ LVATITPSGYTYYWD FVKGRFTISRDNSKN IVYLQMNSLKPEDTA AYYCNTQFYWGQGTQ VTVSS VEGFBII23B04- EVQLVESGGGLVQTG 9GS-5B03/130 DSLRLSCEVSGRTFS SYSMGWFRQAQGKER EFVVAISKGGYKYDS VSLEGRFTISKDNAK NTVYLQINSLKPEDT AVYYCASSRAYGSSR LRLADTYEYWGQGTQ VTVSSGGGGSGGGSE VQLVESGGGLAQAGD SLRLSCAASGRSFSH YNMGWFRQAPGKERE FVASIRGGGGSTTYA NSVKDRFTISRENAK NTVYLQMNSLKPEDT AVYYCAATAFYRGPY DYDYWGQGTQVTVSS VEGFBII23B04- VEGFBII022 EVQLVESGGGLVQTG 9GS-5B05/131 DSLRLSCEVSGRTFS SYSMGWFRQAQGKER EFVVAISKGGYKYDS VSLEGRFTISKDNAK NTVYLQINSLKPEDT AVYYCASSRAYGSSR LRLADTYEYWGQGTQ VTVSSGGGGSGGGSE VQLVESGGGLVQPGG SLRLSCVASGIRFMS MAWYRQAPGKHRELV ARISSGGTTAYVDSV KGRFTISRDNSKNTV YLQMNSLKAEDTAVY YCNTFSSRPNPWGAG TQVTVSS VEGFBII23B04- EVQLVESGGGLVQTG 9GS-6G02/132 DSLRLSCEVSGRTFS SYSMGWFRQAQGKER EFVVAISKGGYKYDS VSLEGRFTISKDNAK NTVYLQINSLKPEDT AVYYCASSRAYGSSR LRLADTYEYWGQGTQ VTVSSGGGGSGGGSE VQLVESGGGLVQPGG SLRLSCAASGNIFSN NAMAWYRQAPGKQRE LVARISSGGGFTYYL DSVKGRFTVSRDNAK NTVYLQMNSLKPEDT AVYYCNAAYRTYNYW GQGTQVTVSS VEGFBII23B04- EVQLVESGGGLVQTG 9GS-10E07/133 DSLRLSCEVSGRTFS SYSMGWFRQAQGKER EFVVAISKGGYKYDS VSLEGRFTISKDNAK NTVYLQINSLKPEDT AVYYCASSRAYGSSR LRLADTYEYWGQGTQ VTVSSGGGGSGGGSE VQLVESGGGLVQAGG SLRLSCAASGRTFSN YAMGWFRQAPGKERV LVADISSSGINTYVA DAVKGRFTISRDNAK NTVYLQMNSLKPEDT AVYYCAASAWWYSQM ARDNYRYWGQGTQVT VSS VEGFBII23B04- EVQLVESGGGLVQTG 9GS-12B01/134 DSLRLSCEVSGRTFS SYSMGWFRQAQGKER EFVVAISKGGYKYDS VSLEGRFTISKDNAK NTVYLQINSLKPEDT AVYYCASSRAYGSSR LRLADTYEYWGQGTQ VTVSSGGGGSGGGSE VQLVESGGGLVQPGG SLRLACAASGFTLSS SWMYWVRQAPGKGLE WVSRISPGGLFTYYV DSVKGRFSVSTDNAN NTLYLQMNSLKPEDT ALYSCAKGGAPNYTP RGRGTQVTVSS VEGFBII23B04- EVQLVESGGGLVQTG 9GS-86C11/135 DSLRLSCEVSGRTFS SYSMGWFRQAQGKER EFVVAISKGGYKYDS VSLEGRFTISKDNAK NTVYLQINSLKPEDT AVYYCASSRAYGSSR LRLADTYEYWGQGTQ VTVSSGGGGSGGGSE VQLVESGGGLVQAGD SLRLSCTASGRTFNS YAMGWFRQAPGKERE SVAHINRSGSSTYYA DSVKGRFTISRDNAK NTVYLQLNSLKPEDT AVYYCAAGRYYSSDG VPSASFNYWGQGTQV TVSS VEGFBII23B04- EVQLVESGGGLVQTG 9GS-86H09/136 DSLRLSCEVSGRTFS SYSMGWFRQAQGKER EFVVAISKGGYKYDS VSLEGRFTISKDNAK NTVYLQINSLKPEDT AVYYCASSRAYGSSR LRLADTYEYWGQGTQ VTVSSGGGGSGGGSE VQLVESGGGLVQAGG SLRLSCTASGSAFKS YRMGWFRRTPGKEDE FVASISWTYGSTFYA DSVKGRFTMSRDKAK NAGYLQMNSLKPEDT ALYYCAAGAQSDRYN IRSYDYWGQGTQVTV SS VEGFBII23B04- EVQLVESGGGLVQTG 9GS-87B07/137 DSLRLSCEVSGRTFS SYSMGWFRQAQGKER EFVVAISKGGYKYDS VSLEGRFTISKDNAK NTVYLQINSLKPEDT AVYYCASSRAYGSSR LRLADTYEYWGQGTQ VTVSSGGGGSGGGSE VQLVESGGGLVQPGG SLKLSCTASGFTFST SWMHWVRQAPGKGLE WVSSIPPVGHFANYA PSVKGRFTISRDNAK NTLFLQMNSLKSEDT AVYYCAKDSAGRTKG QGTQVTVSS VEGFBII23B04- EVQLVESGGGLVQTG 9GS-88A01/138 DSLRLSCEVSGRTFS SYSMGWFRQAQGKER EFVVAISKGGYKYDS VSLEGRFTISKDNAK NTVYLQINSLKPEDT AVYYCASSRAYGSSR LRLADTYEYWGQGTQ VTVSSGGGGSGGGSE VQLVESGGGLVQAGG SLRLSCAASERTFSN YAMDWFRQAPGKERE FVAAITRSGGGTYYA DSVKGRFTISRDNAK NTVYLQMNSLKPEDT AVYYCAATRSSTIVV GVGGMEYWGKGTQVT VSS VEGFBII23B04- EVQLVESGGGLVQTG 40GS-4B08/139 DSLRLSCEVSGRTFS SYSMGWFRQAQGKER EFVVAISKGGYKYDS VSLEGRFTISKDNAK NTVYLQINSLKPEDT AVYYCASSRAYGSSR LRLADTYEYWGQGTQ VTVSSGGGGSGGGGS GGGGSGGGGSGGGGS GGGGSGGGGSGGGGS EVQLVESGGGLVQPG GSLRLSCAASGSAVG DITVAWYRQAPGIQR QLVATITPSGYTYYW DFVKGRFTISRDNSK NIVYLQMNSLKPEDT AAYYCNTQFYWGQGT QVTVSS VEGFBII23B04- EVQLVESGGGLVQTG 40GS-5B03/140 DSLRLSCEVSGRTFS SYSMGWFRQAQGKER EFVVAISKGGYKYDS VSLEGRFTISKDNAK NTVYLQINSLKPEDT AVYYCASSRAYGSSR LRLADTYEYWGQGTQ VTVSSGGGGSGGGGS GGGGSGGGGSGGGGS GGGGSGGGGSGGGGS EVQLVESGGGLAQAG DSLRLSCAASGRSFS HYNMGWFRQAPGKER EFVASIRGGGGSTTY ANSVKDRFTISRENA KNTVYLQMNSLKPED TAVYYCAATAFYRGP YDYDYWGQGTQVTVS S VEGFBII23B04* VEGFBII021 EVQLVESGGGLVQTG 40GS-5B05/141 DSLRLSCEVSGRTFS SYSMGWFRQAQGKER EFVVAISKGGYKYDS VSLEGRFTISKDNAK NTVYLQINSLKPEDT AVYYCASSRAYGSSR LRLADTYEYWGQGTQ VTVSSGGGGSGGGGS GGGGSGGGGSGGGGS GGGGSGGGGSGGGGS EVQLVESGGGLVQPG GSLRLSCVASGIRFM SMAWYRQAPGKHREL VARISSGGTTAYVDS VKGRFTISRDNSKNT VYLQMNSLKAEDTAV YYCNTFSSRPNPWGA GTQVTVSS VEGFBII23B04* EVQLVESGGGLVQTG 40GS-6G02/142 DSLRLSCEVSGRTFS SYSMGWFRQAQGKER EFVVAISKGGYKYDS VSLEGRFTISKDNAK NTVYLQINSLKPEDT AVYYCASSRAYGSSR LRLADTYEYWGQGTQ VTVSSGGGGSGGGGS GGGGSGGGGSGGGGS GGGGSGGGGSGGGGS EVQLVESGGGLVQPG GSLRLSCAASGNIFS NNAMAWYRQAPGKQR ELVARISSGGGFTYY LDSVKGRFTVSRDNA KNTVYLQMNSLKPED TAVYYCNAAYRTYNY WGQGTQVTVSS VEGFBII23B04- VEGFBII023 EVQLVESGGGLVQTG 40GS-10E07/143 DSLRLSCEVSGRTFS SYSMGWFRQAQGKER EFVVAISKGGYKYDS VSLEGRFTISKDNAK NTVYLQINSLKPEDT AVYYCASSRAYGSSR LRLADTYEYWGQGTQ VTVSSGGGGSGGGGS GGGGSGGGGSGGGGS GGGGSGGGGSGGGGS EVQLVESGGGLVQAG GSLRLSCAASGRTFS NYAMGWFRQAPGKER VLVADISSSGINTYV ADAVKGRFTISRDNA KNTVYLQMNSLKPED TAVYYCAASAWWYSQ MARDNYRYWGQGTQV TVSS VEGFBII23B04- EVQLVESGGGLVQTG 40GS-12B01/144 DSLRLSCEVSGRTFS SYSMGWFRQAQGKER EFVVAISKGGYKYDS VSLEGRFTISKDNAK NTVYLQINSLKPEDT AVYYCASSRAYGSSR LRLADTYEYWGQGTQ VTVSSGGGGSGGGGS GGGGSGGGGSGGGGS GGGGSGGGGSGGGGS EVQLVESGGGLVQPG GSLRLACAASGFTLS SSWMYWVRQAPGKGL EWVSRISPGGLFTYY VDSVKGRFSVSTDNA NNTLYLQMNSLKPED TALYSCAKGGAPNYT PRGRGTQVTVSS VEGFBII23B04- EVQLVESGGGLVQTG 40GS-86C11/145 DSLRLSCEVSGRTFS SYSMGWFRQAQGKER EFVVAISKGGYKYDS VSLEGRFTISKDNAK NTVYLQINSLKPEDT AVYYCASSRAYGSSR LRLADTYEYWGQGTQ VTVSSGGGGSGGGGS GGGGSGGGGSGGGGS GGGGSGGGGSGGGGS EVQLVESGGGLVQAG DSLRLSCTASGRTFN SYAMGWFRQAPGKER ESVAHINRSGSSTYY ADSVKGRFTISRDNA KNTVYLQLNSLKPED TAVYYCAAGRYYSSD GVPSASFNYWGQGTQ VTVSS VEGFBII23B04- VEGFBII024 EVQLVESGGGLVQTG 40GS-86H09/146 DSLRLSCEVSGRTFS SYSMGWFRQAQGKER EFVVAISKGGYKYDS VSLEGRFTISKDNAK NTVYLQINSLKPEDTA VYYCASSRAYGSSRL RLADTYEYWGQGTQV TVSSGGGGSGGGGSG GGGSGGGGSGGGGSG GGGSGGGGSGGGGSE VQLVESGGGLVQAGG SLRLSCTASGSAFKS YRMGWFRRTPGKEDE FVASISWTYGSTFYA DSVKGRFTMSRDKAK NAGYLQMNSLKPEDT ALYYCAAGAQSDRYN IRSYDYWGQGTQVTV SS VEGFBII23B04- EVQLVESGGGLVQTG 40GS-87B07/147 DSLRLSCEVSGRTFS SYSMGWFRQAQGKER EFVVAISKGGYKYDS VSLEGRFTISKDNAK NTVYLQINSLKPEDT AVYYCASSRAYGSSR LRLADTYEYWGQGTQ VTVSSGGGGSGGGGS GGGGSGGGGSGGGGS GGGGSGGGGSGGGGS EVQLVESGGGLVQPG GSLKLSCTASGFTFS TSWMHVWRQAPGKGL EWVSSIPPVGHFANY APSVKGRFTISRDNA KNTLFLQMNSLKSED TAVYYCAKDSAGRTK GQGTQVTVSS VEGFBII23B04* EVQLVESGGGLVQTG 40GS-88A01/148 DSLRLSCEVSGRTFS SYSMGWFRQAQGKER EFVVAISKGGYKYDS VSLEGRFTISKDNAK NTVYLQINSLKPEDT AVYYCASSRAYGSSR LRLADTYEYWGQGTQ VTVSSGGGGSGGGGS GGGGSGGGGSGGGGS GGGGSGGGGSGGGGS EVQLVESGGGLVQAG GSLRLSCAASERTFS NYAMDWFRQAPGKER EFVAAITRSGGGTYY ADSVKGRFTISRDNA KNTVYLQMNSLKPED TAVYYCAATRSSTIV VGVGGMEYWGKGTQV TVSS VEGFBII4B08- EVQLVESGGGLVQPG 9GS-23B04/149 GSLRLSCAASGSAVG DITVAWYRQAPGIQR QLVATITPSGYTYYW DFVKGRFTISRDNSK NIVYLQMNSLKPEDT AAYYCNTQFYWGQGT QVTVSSGGGGSGGGS EVQLVESGGGLVQTG DSLRLSCEVSGRTFS SYSMGWFRQAQGKER EFVVAISKGGYKYDS VSLEGRFTISKDNAK NTVYLQINSLKPEDT AVYYCASSRAYGSSR LRLADTYEYWGQGTQ VTVSS VEGFBII5B03- EVQLVESGGGLAQAG 9GS-23B04/150 DSLRLSCAASGRSFS HYNMGWFRQAPGKER EFVASIRGGGGSTTY ANSVKDRFTISRENA KNTVYLQMNSLKPED TAVYYCAATAFYRGP YDYDYWGQGTQVTVS SGGGGSGGGSEVQLV ESGGGLVQTGDSLRL SCEVSGRTFSSYSMG WFRQAQGKEREFVVA ISKGGYKYDSVSLEG RFTISKDNAKNTVYL QINSLKPEDTAVYYC ASSRAYGSSRLRLAD TYEYWGQGTQVTVSS VEGFBII5B05- EVQLVESGGGLVQPG 9GS-23B04/151 GSLRLSCVASGIRFM SMAWYRQAPGKHREL VARISSGGTTAYVDS VKGRFTISRDNSKNT VYLQMNSLKAEDTAV YYCNTFSSRPNPWGA GTQVTVSSGGGGSGG GSEVQLVESGGGLVQ TGDSLRLSCEVSGRT FSSYSMGWFRQAQGK EREFVVAISKGGYKY DSVSLEGRFTISKDN AKNTVYLQINSLKPE DTAVYYCASSRAYGS SRLRLADTYEYWGQG TQVTVSS VEGFBII6G02- EVQLVESGGGLVQPG 9GS-23B04/152 GSLRLSCAASGNIFS NNAMAWYRQAPGKQR ELVARISSGGGFTYY LDSVKGRFTVSRDNA KNTVYLQMNSLKPED TAVYYCNAAYRTYNY WGQGTQVTVSSGGGG SGGGSEVQLVESGGG LVQTGDSLRLSCEVS GRTFSSYSMGWFRQA QGKEREFVVAISKGG YKYDSVSLEGRFTIS KDNAKNTVYLQINSL KPEDTAVYYCASSRA YGSSRLRLADTYEYW GQGTQVTVSS VEGFBII10E07- EVQLVESGGGLVQAG 9GS-23B04/153 GSLRLSCAASGRTFS NYAMGWFRQAPGKER VLVADISSSGINTYV ADAVKGRFTISRDNA KNTVYLQMNSLKPED TAVYYCAASAWWYSQ MARDNYRYWGQGTQV TVSSGGGGSGGGSEV QLVESGGGLVQTGDS LRLSCEVSGRTFSSY SMGWFRQAQGKEREF VVAISKGGYKYDSVS LEGRFTISKDNAKNT VYLQINSLKPEDTAV YYCASSRAYGSSRLR LADTYEYWGQGTQVT VSS VEGFBII12B01- EVQLVESGGGLVQPG 9GS-23B04/154 GSLRLACAASGFTLS SSWMYWVRQAPGKGL EWVSRISPGGLFTYY VDSVKGRFSVSTDNA NNTLYLQMNSLKPED TALYSCAKGGAPNYT PRGRGTQVTVSSGGG GSGGGSEVQLVESGG GLVQTGDSLRLSCEV SGRTFSSYSMGWFRQ AQGKEREFVVAISKG GYKYDSVSLEGRFTI SKDNAKNTVYLQINS LKPEDTAVYYCASSR AYGSSRLRLADTYEY WGQGTQVTVSS VEGFBII86C11- EVQLVESGGGLVQAG 9GS-23B04/155 DSLRLSCTASGRTFN SYAMGWFRQAPGKER ESVAHINRSGSSTYY ADSVKGRFTISRDNA KNTVYLQLNSLKPED TAVYYCAAGRYYSSD GVPSASFNYWGQGTQ VTVSSGGGGSGGGSE VQLVESGGGLVQTGD SLRLSCEVSGRTFSS YSMGWFRQAQGKERE FVVAISKGGYKYDSV SLEGRFTISKDNAKN TVYLQINSLKPEDTA VYYCASSRAYGSSRL RLADTYEYWGQGTQV TVSS VEGFBII86H09* EVQLVESGGGLVQAG 9GS-23B04/156 GSLRLSCTASGSAFK SYRMGWFRRTPGKED EFVASISWTYGSTFY ADSVKGRFTMSRDKA KNAGYLQMNSLKPED TALYYCAAGAQSDRY NIRSYDYWGQGTQVT VSSGGGGSGGGSEVQ LVESGGGLVQTGDSL RLSCEVSGRTFSSYS MGWFRQAQGKEREFV VAISKGGYKYDSVSL EGRFTISKDNAKNTV YLQINSLKPEDTAVY YCASSRAYGSSRLRL ADTYEYWGQGTQVTV SS VEGFBII87B07- EVQLVESGGGLVQPG 9GS-23B04/157 GSLKLSCTASGFTFS TSWMHWVRQAPGKGL EWVSSIPPVGHFANY APSVKGRFTISRDNA KNTLFLQMNSLKSED TAVYYCAKDSAGRTK GQGTQVTVSSGGGGS GGGSEVQLVESGGGL VQTGDSLRLSCEVSG RTFSSYSMGWFRQAQ GKEREFVVAISKGGY KYDSVSLEGRFTISK DNAKNTVYLQINSLK PEDTAVYYCASSRAY GSSRLRLADTYEYWG QGTQVTVSS VEGFBII88A01* EVQLVESGGGLVQAG 9GS-23B04/158 GSLRLSCAASERTFS NYAMDWFRQAPGKER EFVAAITRSGGGTYY ADSVKGRFTISRDNA KNTVYLQMNSLKPED TAVYYCAATRSSTIV VGVGGMEYWGKGTQV TVSSGGGGSGGGSEV QLVESGGGLVQTGDS LRLSCEVSGRTFSSY SMGWFRQAQGKEREF VVAISKGGYKYDSVS LEGRFTISKDNAKNT VYLQINSLKPEDTAV YYCASSRAYGSSRLR LADTYEYWGQGTQVT VSS VEGFBII4B08* EVQLVESGGGLVQPG 40GS-23B04/159 GSLRLSCAASGSAVG DITVAWYRQAPGIQR QLVATITPSGYTYYW DFVKGRFTISRDNSK NIVYLQMNSLKPEDT AAYYCNTQFYWGQGT QVTVSSGGGGSGGGG SGGGGSGGGGSGGGG SGGGGSGGGGSGGGG SEVQLVESGGGLVQT GDSLRLSCEVSGRTF SSYSMGWFRQAQGKE REFVVAISKGGYKYD SVSLEGRFTISKDNA KNTVYLQINSLKPED TAVYYCASSRAYGSS RLRLADTYEYWGQGT QVTVSS VEGFBII5B03- EVQLVESGGGLAQAG 40GS-23B04/160 DSLRLSCAASGRSFS HYNMGWFRQAPGKER EFVASIRGGGGSTTY ANSVKDRFTISRENA KNTVYLQMNSLKPED TAVYYCAATAFYRGP YDYDYWGQGTQVTVS SGGGGSGGGGSGGGG SGGGGSGGGGSGGGG SGGGGSGGGGSEVQL VESGGGLVQTGDSLR LSCEVSGRTFSSYSM GWFRQAQGKEREFVV AISKGGYKYDSVSLE GRFTISKDNAKNTVY LQINSLKPEDTAVYY CASSRAYGSSRLRLA DTYEYWGQGTQVTVS S VEGFBII5B05- EVQLVESGGGLVQPG 40GS-23B04/161 GSLRLSCVASGIRFM SMAWYRQAPGKHREL VARISSGGTTAYVDS VKGRFTISRDNSKNT VYLQMNSLKAEDTAV YYCNTFSSRPNPWGA GTQVTVSSGGGGSGG GGSGGGGSGGGGSGG GGSGGGGSGGGGSGG GGSEVQLVESGGGLV QTGDSLRLSCEVSGR TFSSYSMGWFRQAQG KEREFVVAISKGGYK YDSVSLEGRFTISKD NAKNTVYLQINSLKP EDTAVYYCASSRAYG SSRLRLADTYEYWGQ GTQVTVSS VEGFBII6G02- EVQLVESGGGLVQPG 40GS-23B04/162 GSLRLSCAASGNIFS NNAMAWYRQAPGKQR ELVARISSGGGFTYY LDSVKGRFTVSRDNA KNTVYLQMNSLKPED TAVYYCNAAYRTYNY WGQGTQVTVSSGGGG SGGGGSGGGGSGGGG SGGGGSGGGGSGGGG SGGGGSEVQLVESGG GLVQTGDSLRLSCEV SGRTFSSYSMGWFRQ AQGKEREFVVAISKG GYKYDSVSLEGRFTI SKDNAKNTVYLQINS LKPEDTAVYYCASSR AYGSSRLRLADTYEY WGQGTQVTVSS VEGFBII10E07- VEGFBII025 EVQLVESGGGLVQAG 40GS-23B04/163 GSLRLSCAASGRTFS NYAMGWFRQAPGKER VLVADISSSGINTYV ADAVKGRFTISRDNA KNTVYLQMNSLKPED TAVYYCAASAWWYSQ MARDNYRYWGQGTQV TVSSGGGGSGGGGSG GGGSGGGGSGGGGSG GGGSGGGGSGGGGSE VQLVESGGGLVQTGD SLRLSCEVSGRTFSS YSMGWFRQAQGKERE FVVAISKGGYKYDSV SLEGRFTISKDNAKN TVYLQINSLKPEDTA VYYCASSRAYGSSRL RLADTYEYWGQGTQV TVSS VEGFBII12B01- EVQLVESGGGLVQPG 40GS-23B04/164 GSLRLACAASGFTLS SSWMYWVRQAPGKGL EWVSRISPGGLFTYY VDSVKGRFSVSTDNA NNTLYLQMNSLKPED TALYSCAKGGAPNYT PRGRGTQVTVSSGGG GSGGGGSGGGGSGGG GSGGGGSGGGGSGGG GSGGGGSEVQLVESG GGLVQTGDSLRLSCE VSGRTFSSYSMGWFR QAQGKEREFVVAISK GGYKYDSVSLEGRFT ISKDNAKNTVYLQIN SLKPEDTAVYYCASS RAYGSSRLRLADTYE YWGQGTQVTVSS VEGFBII86C11- EVQLVESGGGLVQAG 40GS-23B04/165 DSLRLSCTASGRTFN SYAMGWFRQAPGKER ESVAHINRSGSSTYY ADSVKGRFTISRDNA KNTVYLQLNSLKPED TAVYYCAAGRYYSSD GVPSASFNYWGQGTQ VTVSSGGGGSGGGGS GGGGSGGGGSGGGGS GGGGSGGGGSGGGGS EVQLVESGGGLVQTG DSLRLSCEVSGRTFS SYSMGWFRQAQGKER EFVVAISKGGYKYDS VSLEGRFTISKDNAK NTVYLQINSLKPEDT AVYYCASSRAYGSSR LRLADTYEYWGQGTQ VTVSS VEGFBII86H09- EVQLVESGGGLVQAG 40GS-23B04/166 GSLRLSCTASGSAFK SYRMGWFRRTPGKED EFVASISWTYGSTFY ADSVKGRFTMSRDKA KNAGYLQMNSLKPED TALYYCAAGAQSDRY NIRSYDYWGQGTQVT VSSGGGGSGGGGSGG GGSGGGGSGGGGSGG GGSGGGGSGGGGSEV QLVESGGGLVQTGDS LRLSCEVSGRTFSSY SMGWFRQAQGKEREF VVAISKGGYKYDSVS LEGRFTISKDNAKNT VYLQINSLKPEDTAV YYCASSRAYGSSRLR LADTYEYWGQGTQVT VSS VEGFBII87B07- EVQLVESGGGLVQPG 40GS-23B04/167 GSLKLSCTASGFTFS TSWMHWVRQAPGKGL EWVSSIPPVGHFANY APSVKGRFTISRDNA KNTLFLQMNSLKSED TAVYYCAKDSAGRTK GQGTQVTVSSGGGGS GGGGSGGGGSGGGGS GGGGSGGGGSGGGGS GGGGSEVQLVESGGG LVQTGDSLRLSCEVS GRTFSSYSMGWFRQA QGKEREFVVAISKGG YKYDSVSLEGRFTIS KDNAKNTVYLQINSL KPEDTAVYYCASSRA YGSSRLRLADTYEYW GQGTQVTVSS VEGFBII88A01- EVQLVESGGGLVQAG 40GS-23B04/168 GSLRLSCAASERTFS NYAMDWFRQAPGKER EFVAAITRSGGGTYY ADSVKGRFTISRDNA KNTVYLQMNSLKPED TAVYYCAATRSSTIV VGVGGMEYWGKGTQV TVSSGGGGSGGGGSG GGGSGGGGSGGGGSG GGGSGGGGSGGGGSE VQLVESGGGLVQTGD SLRLSCEVSGRTFSS YSMGWFRQAQGKERE FVVAISKGGYKYDSV SLEGRFTISKDNAKN TVYLQINSLKPEDTA VYYCASSRAYGSSRL RLADTYEYWGQGTQV TVSS

[0321] 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

[0322] Characterization of Formatted VHHs

[0323] VHHs VEGFBII010, VEGFBII021, VEGFBII022, VEGFBII023, VEGFBII024 and VEGFBII025 are compared side-by-side in the VEGFR2 and VEGFR1 blocking ELISA (FIGS. 8-1 and 8-2 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 VHH ID IC.sub.50 (pM) % inhibition VEGFBII010 49 100 VEGFBII021 204 100 VEGFBII022 164 100 VEGFBII023 213 100 VEGFBII024 292 100 VEGFBII025 677 100 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 VHH ID IC.sub.50 (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

[0324] 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.

[0325] 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 IC50 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 — —

[0326] The formatted VHHs are also tested in ELISA for their ability to bind mVEGF164 and human VEGF165 (Example 5.6; FIGS. 13-1 and 13-2; Table 22); VEGF121 (Example 5.7; FIG. 15; Table 23) and the VEGF family members VEGFB, VEGFC, VEGFD and PIGF (Example 5.8; FIGS. 14-1 through 14-8). Binding kinetics for human VEGF165 are analyzed as described in Example 5.5. The K.sub.D 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 K.sub.D (nM) of purified formatted VHHs for recombinant human VEGF165 VHH ID k.sub.a1 (1/Ms) k.sub.d1 (1/s) k.sub.a2 (1/s) k.sub.d2 (1/s) K.sub.D (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)KD = kd1/ka1 * (kd2/(kd2 + ka2)) .sup.(b)Curves are fitted using a Two State Reaction model by Biacore T100 Evaluation Software v2.0.1

[0327] VHHs VEGFBII010, VEGFBII022, VEGFBII024 and VEGFBII025 are also tested in the VEGF-mediated HUVEC proliferation and Erk phosphorylation assay.

[0328] 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

[0329] 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 (P0161, 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

[0330] Sequence Optimization

[0331] 8.1 Sequence Optimization of VEGFBII23B04

[0332] The amino acid sequence of VEGFBII23B04 is aligned to the human germline sequence VH3-23/JH5, see FIG. 16 (SEQ ID NO: 179)

[0333] 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 VEGFBII23B04 (FR, framework; CDR, complementary determining region) VHH ID/ SEQ ID NO: FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 VEGFBII EVQL SYSM WFRQ AISK RFTI SRAY WGQG 111D05/47 VESG G APGK GGYK SRDN GSSR TLVT GGLV EREF YDSV AKNT LRLA VSS QTGG VV SLEG VYLQ DTYE SLRL INSL Y SCEA RPED SGRT TAVY FS YCAS VEGFBII EVQL SYSM WFRQ AISK RFTI SRAY WGQG 111G06/48 VESG G APGK GGYK SRDN GSSR TLVT GGLV EREF YDSV AKNT LRLA VSS QPGG VV SLEG VYLQ DTYE SLRL MNSL Y SCAA RPED SGRT TAVY FS YCAS VEGFBII EVQL SYSM WFRQ AISK RFTI SRAY WGQG 112D11/49 VESG G APGK GGYK SRDN GSSR TLVT GGLV EREF YDSV AKNT LRLA VSS QPGG VV SLEG VYLQ DTYE SLRL INSL Y SCEA RPED SGRT TAVY FS YCAS VEGFBII EVQL SYSM WFRQ AISK RFTI SRAY WGQG 113A08/50 VESG G APGK GGYK SKDN GSSR TLVT GGLV EREF YDSV AKNT LRLA VSS QTGG VV SLEG VYLQ DTYE SLRL INSL Y SCEV RPED SGRT TAVY FS YCAS VEGFBII EVQL SYSM WFRQ AISK RFTI SRAY WGQG 113E03/51 VESG G AQGK GGYK SKDN GSSR TLVT GGLV EREF YDSV AKNT LRLA VSS QTGD VV SLEG VYLQ DTYE SLRL MNSL Y SCEV RPED SGRT TAVY FS YCAS VEGFBII EVQL SYSM WFRQ AISK RFTI SRAY WGQG 114C09/52 VESG G APGK GGYK SKDN GSSR TLVT GGLV EREF YDSV AKNT LRLA VSS QPGD VV SLEG VYLQ DTYE SLRL INSL Y SCEV RPED SGRT TAVY FS YCAS VEGFBII EVQL SYSM WFRQ AISK RFTI SRAY WGQG 114D02/53 VESG G APGK GGYK SRDN GSSR TLVT GGLV EREF YDSV AKNT LRLA VSS QTGG VV SLEG VYLQ DTYE SLRL INSL Y SCEV RPED SGRT TAVY FS YCAS VEGFBII EVQL SYSM WFRQ AISK RFTI SRAY WGQG 114D03/54 VESG G AQGK GGYK SKDN GSSR TLVT GGLV EREF YDSV AKNT LRLA VSS QTGD VV SLEG VYLQ DTYE SLRL INSL Y SCAV RPED SGRT TAVY FS YCAS VEGFBII EVQL SYSM WFRQ AISK RFTI SRAY WGQG 118E10/55 VESG G AQGK GGYK SKDN GSSR TQVT GGLV EREF YDAV AKNT LRLA VSS QTGD VV SLEG VYLQ DTYE SLRL INSL Y SCEV KPED SGRT TAVY FS YCAS

[0334] These variants are characterized as purified proteins in the VEGF165/VEGFR2 AlphaScreen (Example 5.3, FIG. 17). The melting temperature (Tm) 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), pp289-298). 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 Tm 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) optimized variants of sequence-of VEGFBII23604 T.sub.m @ pH 7 VHH ID IC.sub.50 (pM) % inhibition (° C.) VEGFBII23804 (wt) 169 100 63 VEGFBII111D05 209 100 68 VEGFBII111G06 366 100 71 VEGFBII112D11 221 100 70 VEGFBII113A08 253 100 69 VEGFBII113E03 290 100 68 VEGFBII114009 215 100 71 VEGFBII114D02 199 100 74 VEGFBII114D03 227 100 64 VEGFBII118E10 189 100 62

[0335] 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: VEGFBII0037. One extra sequence-optimized variant (VEGFBII038) is anticipated which contains the same substitutions as VEGFBII0037, 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 VHH ID/ CDR CDR CDR SEQ ID NO: FR 1 1 FR2 2 FR3 3 FR 4 VEGFBII037 DVQL SYSM WFRQ AISK RFTI SRAY WGQG 56 VESG G APGK GGYK SRDN GSSR TLVT GGLV EREF YDAV AKNT LRLA VSS QPGG VV SLEG VYLQ DTYE SLRL MNSL Y SCAA RPED SGRT TAVY FS YCAS VEGFBII038 DVQL SYSM WFRQ AISK RFTI SRAY WGQG 57 VESG G APGK GGYK SRDN GSSR TLVT GGLV EREF YDAV AKNT LRLA VSS QPGG VV SLEG VYLQ DTYE SLRL INSL Y SCAA RPED SGRT TAVY FS YCAS

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 T.sub.m (° C.) VHH ID IC.sub.50 (pM) % inhibition @ pH 7 K.sub.D (pM) VEGFBII23804 152 100 63 560 VEGFBII037 300 100 72 270 VEGFBII038 143 100 71 360

[0336] 8.2 Sequence optimization of VEGFBII5B05

[0337] The amino acid sequence of VEGFBII5B05 is aligned to the human germline sequence VH3-23/JHS, 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) VHH ID/ SEQ ID NO: FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 VEGFBII119G11/ EVQL SMA WYRQ RISS RFTI FSSR WGQG 125 VESG APGK GGTT SRDN PNP TLVT GGLV QREL AYAD SKNT VSS QPGG VA SVKG VYLQ SLRL MNSL SCAA RAED SGIR TAVY FM YCNT VEGFBII120E10/ EVQL SMA WYRQ RISS RFTI FSSR WGAG 126 VESG APGK GGTT SRDN PNP TQVT GGLV HREL AYVD SKNT VSS QPGG VA SVKG VYLQ SLRL MNSL SCVA KAED SGIR TAVY FI YCNT

[0338] 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 M301 mutation. Both variants are tested for their ability to bind hVEGF165 using the PrateOn. In brief, a GLC PrateOn 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) VEGFBII5805 242 6.15E−02 VEGFBII119G11 234 7.75E−02 VEGFBII120E10 257 4.68E−02

[0339] In a second cycle, mutations from the humanization effort and the M301 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) VHH ID/ SEQ ID NO: FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 VEGFBII032/ EVQL SMA WYRQ RISS RFTIS FSS WGQG 127 VESG APGK GGTT RDNSK RPN TLVT GGLV QREL AYAD NTVYL P VSS QPGG VA SVKG QMNSL SLRL RAEDT SCAA AVYYC SGIR NT FI

TABLE-US-00038 TABLE 34 Melting temperature (@pH 7) and affinity (nM) of sequence-optimized clone VEGFBII032 T.sub.m (° C.) VHH ID @ pH 7 K.sub.D (nM) VEGFBII5805(wt) 69 32 VEGFBII0032 71 44

[0340] The potency of the sequence-optimized clones VEGFBII037 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