VEGFA-BINDING MOLECULES

Abstract

VEGFA-binding molecules are disclosed. Also disclosed are nucleic acids and expression vectors encoding, compositions comprising, and methods using, the VEGFA-binding molecules.

Claims

1. An antigen-binding molecule, optionally isolated, which binds to VEGFA, wherein the antigen-binding molecule comprises a single domain antibody sequence incorporating the following CDRs: CDR1 having the amino acid sequence of SEQ ID NO:45 CDR2 having the amino acid sequence of SEQ ID NO:46 CDR3 having the amino acid sequence of SEQ ID NO:48.

2. The antigen-binding molecule according to claim 1, wherein the antigen-binding molecule comprises, or consists of, an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:49.

3. The antigen-binding molecule according to claim 1 or claim 2, wherein the antigen-binding molecule comprises a single domain antibody sequence incorporating the following FRs: FR1 having the amino acid sequence of SEQ ID NO:40 FR2 having the amino acid sequence of SEQ ID NO:41 FR3 having the amino acid sequence of SEQ ID NO:47 FR4 having the amino acid sequence of SEQ ID NO:44.

4. The antigen-binding molecule according to any one of claims 1 to 3, wherein the antigen-binding molecule comprises a single domain antibody sequence incorporating the following CDRs: CDR1 having the amino acid sequence of SEQ ID NO:50 CDR2 having the amino acid sequence of SEQ ID NO:51 CDR3 having the amino acid sequence of SEQ ID NO:52.

5. The antigen-binding molecule according to any one of claims 1 to 4, wherein the antigen-binding molecule comprises, or consists of, an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:53.

6. The antigen-binding molecule according to any one of claims 1 to 5, wherein the antigen-binding molecule comprises: (i) a single domain antibody sequence incorporating the following CDRs: CDR1 having the amino acid sequence of SEQ ID NO:17 CDR2 having the amino acid sequence of SEQ ID NO:18 CDR3 having the amino acid sequence of SEQ ID NO:19; or (ii) a single domain antibody sequence incorporating the following CDRs: CDR1 having the amino acid sequence of SEQ ID NO:6 CDR2 having the amino acid sequence of SEQ ID NO:7 CDR3 having the amino acid sequence of SEQ ID NO:8; or (iii) a single domain antibody sequence incorporating the following CDRs: CDR1 having the amino acid sequence of SEQ ID NO:13 CDR2 having the amino acid sequence of SEQ ID NO:14 CDR3 having the amino acid sequence of SEQ ID NO:15.

7. The antigen-binding molecule according to any one of claims 1 to 6, wherein the antigen-binding molecule comprises, or consists of, an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:16, 5 or 12.

8. The antigen-binding molecule according to any one of claims 1 to 3, wherein the antigen-binding molecule comprises a single domain antibody sequence incorporating the following CDRs: CDR1 having the amino acid sequence of SEQ ID NO:54 CDR2 having the amino acid sequence of SEQ ID NO:55 CDR3 having the amino acid sequence of SEQ ID NO:56.

9. The antigen-binding molecule according to any one of claims 1 to 3 or claim 8, wherein the antigen-binding molecule comprises, or consists of, an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:57.

10. The antigen-binding molecule according to any one of claims 1 to 3, claim 8 or claim 9, wherein the antigen-binding molecule comprises: (i) a single domain antibody sequence incorporating the following CDRs: CDR1 having the amino acid sequence of SEQ ID NO:21 CDR2 having the amino acid sequence of SEQ ID NO:22 CDR3 having the amino acid sequence of SEQ ID NO:23; or (ii) a single domain antibody sequence incorporating the following CDRs: CDR1 having the amino acid sequence of SEQ ID NO:25 CDR2 having the amino acid sequence of SEQ ID NO:26 CDR3 having the amino acid sequence of SEQ ID NO:27; or (iii) a single domain antibody sequence incorporating the following CDRs: CDR1 having the amino acid sequence of SEQ ID NO:29 CDR2 having the amino acid sequence of SEQ ID NO:30 CDR3 having the amino acid sequence of SEQ ID NO:31; or (iv) a single domain antibody sequence incorporating the following CDRs: CDR1 having the amino acid sequence of SEQ ID NO:33 CDR2 having the amino acid sequence of SEQ ID NO:34 CDR3 having the amino acid sequence of SEQ ID NO:35; or (v) a single domain antibody sequence incorporating the following CDRs: CDR1 having the amino acid sequence of SEQ ID NO:37 CDR2 having the amino acid sequence of SEQ ID NO:38 CDR3 having the amino acid sequence of SEQ ID NO:39.

11. The antigen-binding molecule according to any one of claims 1 to 3 or claims 8 to 10, wherein the antigen-binding molecule comprises, or consists of, an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:20, 24, 28, 32 or 36.

12. The antigen-binding molecule according to any one of claims 1 to 3, wherein the antigen-binding molecule comprises a single domain antibody sequence incorporating the following CDRs: CDR1 having the amino acid sequence of SEQ ID NO:2 CDR2 having the amino acid sequence of SEQ ID NO:3 CDR3 having the amino acid sequence of SEQ ID NO:4.

13. The antigen-binding molecule according to any one of claims 1 to 3 or claim 12, wherein the antigen-binding molecule comprises, or consists of, an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:1.

14. The antigen-binding molecule according to any one of claims 1 to 3, wherein the antigen-binding molecule comprises a single domain antibody sequence incorporating the following CDRs: CDR1 having the amino acid sequence of SEQ ID NO:2 CDR2 having the amino acid sequence of SEQ ID N0:10 CDR3 having the amino acid sequence of SEQ ID NO:11.

15. The antigen-binding molecule according to any one of claims 1 to 3 or claim 14, wherein the antigen-binding molecule comprises, or consists of, an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:9.

16. The antigen-binding molecule according to any one of claims 1 to 13, wherein the antigen-binding molecule comprises a single domain antibody sequence incorporating the following FRs: FR1 having the amino acid sequence of SEQ ID NO:40 FR2 having the amino acid sequence of SEQ ID NO:41 FR3 having the amino acid sequence of SEQ ID NO:42 FR4 having the amino acid sequence of SEQ ID NO:44.

17. The antigen-binding molecule according to any one of claims 1 to 3, claim 15 or claim 16, wherein the antigen-binding molecule comprises a single domain antibody sequence incorporating the following FRs: FR1 having the amino acid sequence of SEQ ID NO:40 FR2 having the amino acid sequence of SEQ ID NO:41 FR3 having the amino acid sequence of SEQ ID NO:43 FR4 having the amino acid sequence of SEQ ID NO:44.

18. The antigen-binding molecule according to any one of claims 1 to 17, wherein the antigen-binding molecule inhibits interaction between VEGFA and VEGFR.

19. The antigen-binding molecule according to any one of claims 1 to 18, wherein the antigen-binding molecule is a multispecific antigen-binding molecule, further comprising an antigen-binding domain specific for a target antigen other than VEGFA.

20. A chimeric antigen receptor (CAR) comprising an antigen-binding molecule according to any one of claims 1 to 19.

21. A nucleic acid, optionally isolated, encoding an antigen-binding molecule according to any one of claims 1 to 19, or a CAR according to claim 20.

22. An expression vector comprising a nucleic acid according to claim 21.

23. A cell comprising an antigen-binding molecule according to any one of claims 1 to 19, a CAR according to claim 20, a nucleic acid according to claim 21, or an expression vector according to claim 22.

24. A method for producing an antigen-binding molecule which binds to VEGFA, comprising culturing a cell according to claim 23 under conditions suitable for expression of an antigen-binding molecule or CAR by the cell.

25. A composition comprising an antigen-binding molecule according to any one of claims 1 to 19, a CAR according to claim 20, a nucleic acid according to claim 21, an expression vector according to claim 22, or a cell according to claim 23, and a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.

26. An antigen-binding molecule according to any one of claims 1 to 19, a CAR according to claim 20, a nucleic acid according to claim 21, an expression vector according to claim 22, a cell according to claim 23, or a composition according to claim 25, for use in a method of medical treatment or prophylaxis.

27. An antigen-binding molecule according to any one of claims 1 to 19, a CAR according to claim 20, a nucleic acid according to claim 21, an expression vector according to claim 22, a cell according to claim 23, or a composition according to claim 25, for use in a method of treatment or prevention of a disease in which VEGFA/VEGFR-mediated signalling is pathologically-implicated.

28. Use of an antigen-binding molecule according to any one of claims 1 to 19, a CAR according to claim 20, a nucleic acid according to claim 21, an expression vector according to claim 22, a cell according to claim 23, or a composition according to claim 25, in the manufacture of a medicament for treating or preventing a disease in which VEGFA/VEGFR-mediated signalling is pathologically-implicated.

29. A method of treating or preventing a disease in which VEGFA/VEGFR-mediated signalling is pathologically-implicated, comprising administering to a subject a therapeutically- or prophylactically-effective amount of an antigen-binding molecule according to any one of claims 1 to 19, a CAR according to claim 20, a nucleic acid according to claim 21, an expression vector according to claim 22, a cell according to claim 23, or a composition according to claim 25.

30. The antigen-binding molecule, CAR, nucleic acid, expression vector, cell or composition for use according to claim 27, the use according to claim 28, or the method according to claim 29, wherein the disease is selected from: a disease characterised by pathological angiogenesis, a cancer, a VEGFA-expressing cancer, a VEGFR-expressing cancer, an ocular disease, retinopathy, diabetic retinopathy, macular degeneration, age-related macular degeneration, wet age-related macular degeneration, retinal vein occlusion, myopic choroidal neovascularisation, retinopathy of prematurity, neovascular glaucoma, central serous retinopathy, ocular tumor, corneal neovascularisation, an inflammatory disease, an autoimmune disease, arthritis, rheumatoid arthritis, osteoarthritis, psoriasis, multiple sclerosis, sepsis, motor neuron disease and amyotrophic lateral sclerosis.

31. An in vitro complex, optionally isolated, comprising an antigen-binding molecule according to any one of claims 1 to 19 bound to VEGFA.

32. A method for detecting VEGFA in a sample, comprising contacting a sample containing, or suspected to contain, VEGFA with an antigen-binding molecule according to any one of claims 1 to 19, and detecting the formation of a complex of the antigen-binding molecule with VEGFA.

33. Use of an antigen-binding molecule according to any one of claims 1 to 19 in a method for detecting, localizing or imaging VEGFA, or cells comprising or expressing VEGFA.

34. A method of selecting or stratifying a subject for treatment with a VEGFA-targeted agent, the method comprising contacting, in vitro, a sample from the subject with an antigen-binding molecule according to any one of claims 1 to 19, and detecting the formation of a complex of the antigen-binding molecule with VEGFA.

35. Use of an antigen-binding molecule according to any one of claims 1 to 19 as an in vitro or in vivo diagnostic or prognostic agent.

36. Use of an antigen-binding molecule according to any one of claims 1 to 19 in a method for detecting, localizing or imaging a disease/condition characterised by expression of VEGFA.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0459] Embodiments and experiments illustrating the principles of the present disclosure will now be discussed with reference to the accompanying figures.

[0460] FIG. 1. Tables summarising amino acid differences in CDR1, CDR2 and CDR3 regions of Library 1 and Library 2, 4D5 (trastuzumab) and the starting template. Xaa denotes randomized amino acid.

[0461] FIGS. 2A to 2K. Sensorgrams showing binding of (2A) 13A6, (2B) 16A2.1, (2C) 16A6.1 and (2D) 16A2.1x2, (2E) 20A2.1, (2F) 20A3.1, (2G) 21A1.1, (2H) 21A8.1, (2I) 21D9.1, (2J) 21E6.1 and (2K) 23D5.1 to human VEGFA, as measured by biolayer interferometry (BLI). The concentrations tested for each DotBody are stated below the binding curves, in nM.

[0462] FIGS. 3A to 3J. Sensorgrams showing binding of (3A) 13A6, (3B) 16A2.1, (3C) 16A6.1, (3D) 20A2.1, (3E) 20A3.1, (3F) 21A1.1, (3G) 21A8.1, (3H) 21D9.1, (31) 21E6.1 and (3J) 23D5.1 to mouse VEGFA, as measured by biolayer interferometry (BLI). The concentrations tested for each DotBody are stated below the binding curves, in nM.

[0463] FIG. 4. Graph showing inhibition of the interaction between human VEGFA and VEGFR1 by 13A6, 16A2.1, 16A6.1 and Ranibizumab, in competitive ELISA.

[0464] FIGS. 5A to 51. Graphs showing inhibition of the interaction between human VEGFA and VEGFR1 by (5A) 16A2.1, (5B) 20A2.1, (5C) 20A3.1, (5D) 21A1.1, (5E) 21A8.1, (5F) 21D9.1, (5G) 21E6.1, (5H) 23D5.1 and (5I) Ranibizumab, in competitive ELISA.

[0465] FIGS. 6A to 6H. Sensorgrams showing binding of anti-VEGFA DotBodies (6A) 16A2.1, (6B) 20A2.1, (6C) 20A3.1, (6D) 21A1.1, (6E) 21A8.1, (6F) 21D9.1, (6G) 21E9.1 and (6H) 23D5.1 to human VEGFA, at a single concentration of 250 nM, after incubation for 1 h at room temperature, 60? C., 70? C. or 80? C. Measurements were performed by BLI as described in Example 10.

EXAMPLES

Example 1: Generation of Na?ve Synthetic DotBody Phage Display Libraries

[0466] Two DotBody phage display libraries were used for the identification of anti-VEGF DotBodies. These libraries were based on a humanized, stabilized and autonomous VH domain template derived from the trastuzumab VH domain (DotBody scaffold patents, described e.g. in WO 2016/072938A1).

[0467] Library 1 was based on the following VH domain template sequence (positions mutated for library creation are underlined):

TABLE-US-00003 SEVQLVESGGGLVQPGGSLRLSSAISGFSISSTSIDWVRQAPGKGLEWVA RISPSSGSTSYADSVKGRFTISADTSKNTVYLQMNSLRAEDTAVYYTGRS SSAMDYRGQGTLVTVSS

[0468] Library 2 was based on the following VH domain template sequence (positions mutated for library creation are underlined):

TABLE-US-00004 SEVQLVESGGGLVQPGGSLRLSCAISGFSISSTSIDWVRQAPGKGLEWVA RISPSSGSTSYADSVKGRFTISADTSKNTVYLQMNSLRAEDTAVYYCGRS SSAMDYRGQGTLVTVSS

[0469] CDR-1, CDR-2 and CDR-3 of the VH domain template were randomized according to the design shown in FIG. 1, by Kunkel mutagenesis according to procedures by Bostrom J. et al. (14,15) and Tonikian R. et al. (16). The primers employed for Library 1 are shown in Table 1, while those employed for Library 2 are shown in Table 2.

[0470] Library 1 contained approximately 2.87?10.sup.10 clones, while Library 2 contained approximately 1.37?10.sup.10 clones with all CDRs mutated. The libraries were assessed by serial dilution and colony counting after library transformation.

TABLE-US-00005 TABLE1 PrimersemployedtocreateLibrary1.Primer H1andH2weremixedwithprimersH3.6-H3.20 beforeperformingKunkelmutagenesisontheVH templatesequence,tocreatefifteensub- librariesthatwerelatercombinedtoformthe finallibrary.Codon[XYZ]havenucleotide frequenciesasfollows:X=0.2G+0.2A+ 0.5T+0.1C,Y=0.4A+0.2T+0.4CandZ= 0.1G+0.9C. Primer name PrimerSequence H1 CCTCTGCAATTTCTGGCTTC[XYZ]NTT[XYZ][XYZ]ACT [XYZ]ATAGACTGGGTGCGTCAGG H2 CTGGAATGGGTTGCAAGGATT[XYZ]CCT[XYZ][XYZ]GG T[XYZ]ACT[XYZ]TATGCCGATAGCGTCAAGGG H3.6 GCCGTCTATTATACTGKCCGC[XYZ][XYZ][XYZ][XYZ] [XYZ][XYZ]GSTNTKGACTACCGGGGTCAAG H3.7 GCCGTCTATTATACTGKCCGC[XYZ][XYZ][XYZ][XYZ] [XYZ][XYZ][XYZ]GSTNTKGACTACCGGGGTCAAG H3.8 GCCGTCTATTATACTGKCCGC[XYZ][XYZ][XYZ][XYZ] [XYZ][XYZ][XYZ][XYZ]GSTNTKGACTACCGGGGTCAA G H3.9 GCCGTCTATTATACTGKCCGC[XYZ][XYZ][XYZ][XYZ] [XYZ][XYZ][XYZ][XYZ][XYZ]GSTNTKGACTACCGGG GTCAAG H3.10 GCCGTCTATTATACTGKCCGC[XYZ][XYZ][XYZ][XYZ] [XYZ][XYZ][XYZ][XYZ][XYZ][XYZ]GSTNTKGACTA CCGGGGTCAAG H3.11 GCCGTCTATTATACTGKCCGC[XYZ][XYZ][XYZ][XYZ] [XYZ][XYZ][XYZ][XYZ][XYZ][XYZ][XYZ]GSTNTK GACTACCGGGGTCAAG H3.12 GCCGTCTATTATACTGKCCGC[XYZ][XYZ][XYZ][XYZ] [XYZ][XYZ][XYZ][XYZ][XYZ][XYZ][XYZ][XYZ]G STNTKGACTACCGGGGTCAAG H3.13 GCCGTCTATTATACTGKCCGC[XYZ][XYZ][XYZ][XYZ] [XYZ][XYZ][XYZ][XYZ][XYZ][XYZ][XYZ][XYZ] [XYZ]GSTNTKGACTACCGGGGTCAAG H3.14 GCCGTCTATTATACTGKCCGC[XYZ][XYZ][XYZ][XYZ] [XYZ][XYZ][XYZ][XYZ][XYZ][XYZ][XYZ][XYZ] [XYZ][XYZ]GSTNTKGACTACCGGGGTCAAG H3.15 GCCGTCTATTATACTGKCCGC[XYZ][XYZ][XYZ][XYZ] [XYZ][XYZ][XYZ][XYZ][XYZ][XYZ][XYZ][XYZ] [XYZ][XYZ][XYZ]GSTNTKGACTACCGGGGTCAAG H3.16 GCCGTCTATTATACTGKCCGC[XYZ][XYZ][XYZ][XYZ] [XYZ][XYZ][XYZ][XYZ][XYZ][XYZ][XYZ][XYZ] [XYZ][XYZ][XYZ][XYZ]GSTNTKGACTACCGGGGTCAA G H3.17 GCCGTCTATTATACTGKCCGC[XYZ][XYZ][XYZ][XYZ] [XYZ][XYZ][XYZ][XYZ][XYZ][XYZ][XYZ][XYZ] [XYZ][XYZ][XYZ][XYZ][XYZ]GSTNTKGACTACCGGG GTCAAG H3.18 GCCGTCTATTATACTGKCCGC[XYZ][XYZ][XYZ][XYZ] [XYZ][XYZ][XYZ][XYZ][XYZ][XYZ][XYZ][XYZ] [XYZ][XYZ][XYZ][XYZ][XYZ][XYZ]GSTNTKGACTA CCGGGGTCAAG H3.19 GCCGTCTATTATACTGKCCGC[XYZ][XYZ][XYZ][XYZ] [XYZ][XYZ][XYZ][XYZ][XYZ][XYZ][XYZ][XYZ] [XYZ][XYZ][XYZ][XYZ][XYZ][XYZ][XYZ]GSTNTK GACTACCGGGGTCAAG H3.20 GCCGTCTATTATACTGKCCGC[XYZ][XYZ][XYZ][XYZ] [XYZ][XYZ][XYZ][XYZ][XYZ][XYZ][XYZ][XYZ] [XYZ][XYZ][XYZ][XYZ][XYZ][XYZ][XYZ][XYZ]G STNTKGACTACCGGGGTCAAG

TABLE-US-00006 TABLE2 PrimersemployedtocreateLibrary2.Primer H1andH2weremixedwithprimersH3.6-H3.20 beforeperformingKunkelmutagenesisonthe VHtemplatesequence,tocreatefifteensub- librariesthatwerelatercombinedtoform thefinallibrary.Codon[XYZ]havenucleotide frequenciesasfollows:X=0.2G+0.2A+ 0.5T+0.1C,Y=0.4A+0.2T+0.4CandZ= 0.1G+0.9C. Primer name Primersequence H1 CCTGTGCAATTTCTGGCTTC[XYZ]NTT[XYZ][XYZ]ACT [XYZ]ATAGACTGGGTGCGTCAGG H2 CTGGAATGGGTTGCAAGGATT[XYZ]CCT[XYZ][XYZ]GG T[XYZ]ACT[XYZ]TATGCCGATAGCGTCAAGGG H3.6 GCCGTCTATTATTGTGKCCGC[XYZ][XYZ][XYZ][XYZ] [XYZ][XYZ]GSTNTKGACTACCGGGGTCAAG H3.7 GCCGTCTATTATTGTGKCCGC[XYZ][XYZ][XYZ][XYZ] [XYZ][XYZ][XYZ]GSTNTKGACTACCGGGGTCAAG H3.8 GCCGTCTATTATTGTGKCCGC[XYZ][XYZ][XYZ][XYZ] [XYZ][XYZ][XYZ][XYZ]GSTNTKGACTACCGGGGTCAA G H3.9 GCCGTCTATTATTGTGKCCGC[XYZ][XYZ][XYZ][XYZ] [XYZ][XYZ][XYZ][XYZ][XYZ]GSTNTKGACTACCGGG GTCAAG H3.10 GCCGTCTATTATTGTGKCCGC[XYZ][XYZ][XYZ][XYZ] [XYZ][XYZ][XYZ][XYZ][XYZ][XYZ]GSTNTKGACTA CCGGGGTCAAG H3.11 GCCGTCTATTATTGTGKCCGC[XYZ][XYZ][XYZ][XYZ] [XYZ][XYZ][XYZ][XYZ][XYZ][XYZ][XYZ]GSTNTK GACTACCGGGGTCAAG H3.12 GCCGTCTATTATTGTGKCCGC[XYZ][XYZ][XYZ][XYZ] [XYZ][XYZ][XYZ][XYZ][XYZ][XYZ][XYZ][XYZ]G STNTKGACTACCGGGGTCAAG H3.13 GCCGTCTATTATTGTGKCCGC[XYZ][XYZ][XYZ][XYZ] [XYZ][XYZ][XYZ][XYZ][XYZ][XYZ][XYZ][XYZ] [XYZ]GSTNTKGACTACCGGGGTCAAG H3.14 GCCGTCTATTATTGTGKCCGC[XYZ][XYZ][XYZ][XYZ] [XYZ][XYZ][XYZ][XYZ][XYZ][XYZ][XYZ][XYZ] [XYZ][XYZ]GSTNTKGACTACCGGGGTCAAG H3.15 GCCGTCTATTATTGTGKCCGC[XYZ][XYZ][XYZ][XYZ] [XYZ][XYZ][XYZ][XYZ][XYZ][XYZ][XYZ][XYZ] [XYZ][XYZ][XYZ]GSTNTKGACTACCGGGGTCAAG H3.16 GCCGTCTATTATTGTGKCCGC[XYZ][XYZ][XYZ][XYZ] [XYZ][XYZ][XYZ][XYZ][XYZ][XYZ][XYZ][XYZ] [XYZ][XYZ][XYZ][XYZ]GSTNTKGACTACCGGGGTCAA G H3.17 GCCGTCTATTATTGTGKCCGC[XYZ][XYZ][XYZ][XYZ] [XYZ][XYZ][XYZ][XYZ][XYZ][XYZ][XYZ][XYZ] [XYZ][XYZ][XYZ][XYZ][XYZ]GSTNTKGACTACCGGG GTCAAG H3.18 GCCGTCTATTATTGTGKCCGC[XYZ][XYZ][XYZ][XYZ] [XYZ][XYZ][XYZ][XYZ][XYZ][XYZ][XYZ][XYZ] [XYZ][XYZ][XYZ][XYZ][XYZ][XYZ]GSTNTKGACTA CCGGGGTCAAG H3.19 GCCGTCTATTATTGTGKCCGC[XYZ][XYZ][XYZ][XYZ] [XYZ][XYZ][XYZ][XYZ][XYZ][XYZ][XYZ][XYZ] [XYZ][XYZ][XYZ][XYZ][XYZ][XYZ][XYZ]GSTNTK GACTACCGGGGTCAAG H3.20 GCCGTCTATTATTGTGKCCGC[XYZ][XYZ][XYZ][XYZ] [XYZ][XYZ][XYZ][XYZ][XYZ][XYZ][XYZ][XYZ] [XYZ][XYZ][XYZ][XYZ][XYZ][XYZ][XYZ][XYZ]G STNTKGACTACCGGGGTCAAG

Example 2: Phage Display Selections from Na?ve Synthetic DotBody Phage Display Libraries

[0471] Human VEGF-121 (Acro Biosystems) was immobilized onto Maxisorp Immuno Tubes (Thermo Scientific) at 20 ?g in 1 mL PBS for round 1, and 10 ?g in 1 mL PBS for subsequent rounds, overnight at 4? C. The tubes were washed twice in PBS, and blocked for 1 h at room temperature (RT) in Milk Block Buffer (MBB: 1% skimmed milk in PBST i.e. PBS containing 0.05% Tween-20). Negative selection tubes were prepared in the same way as described for VEGF-121, but PBS was used in place of the VEGF-121 protein. 500 ?L of library 1 and library 2 (at ?2?10.sup.13 pfu/mL) were precipitated with PEG/NaCl buffer (20% PEG 8,000, 2.5M NaCl), and resuspended in MBB, before being transferred to the negative selection tube for incubation for 1 h at RT. The phages were transferred to the immuno tube coated with VEGF-121 and incubated for 2 h at RT. The tube was then washed 3 times with MBB, 3 times with PBST and twice with PBS, to remove non-bound phages. The bound phages were eluted with trypsin at 1 mg/mL in trypsin buffer (TBS+2 mM CaCl.sub.2). The eluted phages were used to infect 5 mL of TG1 bacterial cell culture (in 2YT media) in exponential growth phase (OD.sub.600 0.5) for 30 min at 37? C. From round 2 onwards, 1.2 mL of infected TG1 cells were stored with 20% glycerol at ?80? C., to be used for monoclonal screening (glycerol stocks for monoclonal screening). The remaining infected TG1 cells were transferred to 50 mL 2YT. The culture was incubated at 37? C. with shaking until OD.sub.600?0.5, before infection with 1?10.sup.10 pfu/mL M13K07 helper phage for 30 min at 37? C. The infected TG1 cells were pelleted at 3,900 g for 20 min at 4? C., resuspended in 500 ?L 2YT broth and plated onto 2?15 cm round 2YT agar plates supplemented with 100 ?g/mL carbenicillin and 50 ?g/mL kanamycin. After overnight incubation at 30? C., the bacterial-lawn was resuspended in 25 mL TBS. The phage produced were purified by precipitation with PEG/NaCl buffer. After two rounds of PEG/NaCl precipitation, the phages were resuspended in PBS+10% glycerol. The purified phages were used for the subsequent round of selection.

[0472] Four rounds of selections were performed against VEGF-121. From the second selection round onwards, the number of washes was increased as follows: [0473] Round 2: 4 times with MBB, 4 times with PBST, and 2 times with PBS. [0474] Round 3: 6 times with MBB, 6 times with PBST, 2 times with PBS [0475] Round 4: 7? with MBB, 7 times with PBST, 2 times with PBS From the second selection round onwards, 1 mL of phages purified from the previous round of selection at a final concentration of 1?10.sup.12 pfu/mL was used. The rest of the panning procedure was identical.

Example 3: Identification of Unique Binders by Monoclonal Phage ELISA

[0476] Monoclonal phage ELISA was used to identify unique binding DotBodies selected from the na?ve libraries, as well as the affinity maturation library. The glycerol stocks for monoclonal screening were plated onto 2YT agar plates supplemented with 100 ?g/mL Carbenicillin and incubated overnight at 37? C. Individual colonies were grown in 1 mL 2YT broth supplemented with 100 ?g/mL Carbenicillin for 2 hours before infection with 1?10.sup.10 pfu/mL M13K07 helper phage. The cultures were further supplemented with 50 ?g/mL Kanamycin and incubated at 30? C. overnight.

[0477] The cells were pelleted by centrifugation at 1,100 g for 10 mins at 4? C., and the supernatant was used for phage monoclonal ELISA. In the phage monoclonal ELISA, VEGF-121 was immobilized at a concentration of 1 ?g/mL onto a Maxisorp 96-wells plate (Thermo Scientific) overnight at 4? C., washed twice with PBS and blocked for 1 h at RT with MBB. 25 ?L of the phage culture supernatant was mixed with 25 ?L of MBB, added to the plate and incubated for 2 h at RT. The plate was washed 8 times with PBST before 50 ?L of anti-M13 antibody HRP conjugate (GE Healthcare) was added at a 1:7,000 dilution in MBB, and incubated for 1 h at RT. The plate was washed 8 times with PBST, and developed with 50 ?L 3,3,5,5-Tetramethylbenzidine (TMB) substrate (GeneTex). After 5-15 min, the reactions were stopped by adding 50 ?L of 2M H.sub.2SO.sub.4, and the signal was measured at an absorbance of 450 nm. Monoclonal clones with high signal intensity (Absorbance higher than 1) were sequenced by Sanger sequencing to identify unique VH domains binding to VEGF-121.

Example 4: Affinity Maturation Phage Display Library Construction

[0478] Anti-VEGF DotBody 13A6 was selected for affinity maturation, as its binding affinity is below 50 nM and it also blocked the interaction between VEGFA and the VEGF receptor 1 (VEGFR1). An affinity maturation phage display library was created by Kunkel mutagenesis, according to Bostrom J. et al. (14). by using the primers shown in Table 3. The library contained 1.1?10.sup.8 unique sequences, as estimated by serial dilutions upon library electroporation into TG1 cells, plating onto 2YT agar supplemented with 100 ?g/mL Carbenicillin, and subsequent sequencing of plasmids from 30 colonies.

TABLE-US-00007 TABLE3 DNAsequencesofprimersemployedtogenerate anaffinitymaturationphagedisplaylibrary ofanti-VEGFDotBody13A6.Thenumbersinthe sequencerepresentfrequencyofbasesA,T,C andGintheoligonucleotidesequence. Frequencycode5=70%A,10%G,10%C, 10%T;6=70%G,10%A,10%C,10%T;7= 70%C,10%A,10%G,10%T;8=70%T,10%A, 10%G,10%C. Primer name Primersequence aVEGF- TGTGCAATTTCTGGCTTC878788678678577657ATAGA 13A6 CTGGGTGCGTCAG CDR1 aVEGF- GAATGGGTTGCAAGGATT88877687867866887857765 13A6 7TATGCCGATAGCGTCAAG CDR2 aVEGF- ACTGCCGTCTATTATTGT66856587865787885787885 13A6 7558688888788757558776577857577678788657T CDR3 ACCGGGGTCAAGGAACA

Example 5: 13A6-Based Affinity Maturation Phage Display Selections

[0479] Neutravidin was immobilized onto Maxisorp Immuno Tubes (Thermo Scientific) at 10 ?g in 1 mL PBS overnight at 4? C. The tubes were washed twice in PBS, and blocked for 1 h at room temperature (RT) in MBB. Biotinylated VEGF-121 (Acro Biosystems) was added at different concentrations depending on the panning round (refer to Table 4). The protein was incubated for 1 h at RT, and non-bound protein removed by two washes with PBS. Negative selection tubes were prepared as described for biotinylated VEGF-121, but PBS was added in place of biotinylated VEGF-121.

[0480] The remaining selection procedures are similar to the Phage display selections from na?ve synthetic DotBody phage display libraries, with certain changes as summarised in Table 4.

TABLE-US-00008 TABLE 4 Affinity maturation selection conditions of 13A6-based phage display libraries against biotinylated VEGF-121. Changes Details Number of 5 rounds with increasing levels of stringency selection rounds Adaptor protein A biotin-binding protein was immobilized on Maxisorp plates as follows: Round 1: 20 ?g Neutravidin in 2 mL Round 2: 10 ?g Streptavidin in 2 mL Round 3: 10 ?g Neutravidin in 2 mL Round 4: 10 ?g Streptavidin in 2 mL Round 5: 10 ?g Neutravidin in 2 mL Amount of Decreasing biotinylated VEGF-121 concentration was incubated on the biotinylated VEGF- adaptor protein-coated tubes in the various selection round: 121 immobilized Round 1 to 2: 300 nM Round 3 to 5: 15 nM Amount of phages The library was diluted in 1 mL MBB at different final concentrations in the added to VEGF- various selection round: 121 Round 1: 1 ? 10.sup.13 pfu/mL of affinity matured phage display library Round 2: 1 ? 10.sup.12 pfu/mL of purified phages from previous round Rounds 3 to 5: 5 ? 10.sup.11 pfu/mL of purified phages from previous round Incubation time of The phages were incubated in the negative selection tube for 1 h at RT, then phages with transferred to the tube containing VEGF-121, and incubated at RT for VEGF-121 different durations in the various selection round: Round 1: 2 h Round 2: 1 h Round 3: 30 min Round 4: 10 min Round 5: 3 min Washing of non- Non-bound phages were removed following different wash regiments in the bound phages various selection round: Round 1: 3 times with MBB, 3 times with PBST, 2 times with PBS Round 2: 4 times with MBB, 4 times with PBST, 2 times with PBS Round 3: 10 times with MBB, 10 times with PBST, 2 times with PBS Rounds 4 and 5: 15 times with MBB, 15 times with PBST, 2 times with PBS

[0481] 13A6-based affinity maturation phage display selections gave rise to 16A2.1, 16C2.1 and 16A6.1.

Example 6: 16A2.1-Based Affinity Maturation Phage Display Library Construction

[0482] Anti-VEGF DotBody 16A2.1 was selected for affinity maturation, as its binding affinity is below 5 nM and it also blocked the interaction between VEGFA and the VEGF receptor 1 (VEGFR1). An affinity maturation phage display library was created by Kunkel mutagenesis, according to Bostrom J. et al. (14), by using the primers stated in Table 5. As CDR3 contained a potential N-glycosylation site (sequence NST), three primers were designed for this CDR, one in which the parental sequence was left intact, and two others in which it was changed as follows before performing the randomized primer design (changes underlined):

TABLE-US-00009 16A2.1(AST): SEVQLVESGGGLVQPGGSLRLSCAISGFALAETDIDWVRQAPGKGLEWVA RIFSSGGNTDYADSVKGRFTISADTSKNTVYLQMNSLRAEDTAVYYCGRS DSIAYNVILPASTYTARDYRGQGTLVTVSS 16A2.1(NSA): SEVQLVESGGGLVQPGGSLRLSCAISGFALAETDIDWVRQAPGKGLEWVA RIFSSGGNTDYADSVKGRFTISADTSKNTVYLQMNSLRAEDTAVYYCGRS DSIAYNVILPNSAYTARDYRGQGTLVTVSS

[0483] The library obtained contained 0.6?10.sup.8 unique sequences, as estimated by serial dilutions upon library electroporation into TG1 cells, plating onto 2YT agar supplemented with 100 ?g/mL Carbenicillin, and subsequent sequencing of plasmids from several colonies to determine mutation rates.

TABLE-US-00010 TABLE5 DNAsequencesofprimersemployedtogenerate anaffinitymaturationphagedisplaylibrary ofanti-VEGFDotBody16A2.1.Thenumbersin thesequencerepresentfrequencyofbasesA, T,CandGintheoligonucleotidesequence. Frequencycode5=70%A,10%G,10%C,10% T;6=70%G,10%A,10%C,10%T;7=70%C, 10%A,10%G,10%T;8=70%T,10%A,10%G, 10%C. Primer name Primersequence aVEGF- TGTGCAATTTCTGGCTTC678788678655577657ATAGA 16A2.1 CTGGGTGCGTCAG CDR1 aVEGF- GAATGGGTTGCAAGGATT88887887866866855857765 16A2.1 7TATGCCGATAGCGTCAAG CDR2 aVEGF- ACTGCCGTCTATTATTGT66856587865787858867885 16A2.1 7558688588788776558878577857577678565657T CDR3 ACCGGGGTCAAGGAACA aVEGF- ACTGCCGTCTATTATTGT66856587865787858867885 16A2.1 7558688588788776558878678857577678565657T (AST) ACCGGGGTCAAGGAACA CDR3 aVEGF- ACTGCCGTCTATTATTGT66856587865787858867885 16A2.1 7558688588788776678878577857577678565657T (NSA) ACCGGGGTCAAGGAACA CDR3

Example 7: 16C2.1-Based Affinity Maturation Phage Display Library Construction

[0484] Anti-VEGF DotBody 16C2.1 was selected for affinity maturation, as its binding affinity is below 5 nM and it also blocked the interaction between VEGFA and the VEGF receptor 1 (VEGFR1). An affinity maturation phage display library was created by Kunkel mutagenesis, according to Bostrom J. et al. (14), by using the primers stated in Table 6. The library obtained contained 1.1?10.sup.8 unique sequences, as estimated by serial dilutions upon library electroporation into TG1 cells, plating onto 2YT agar supplemented with 100 ?g/mL Carbenicillin, and subsequent sequencing of plasmids from several colonies to determine mutation rates:

TABLE-US-00011 TABLE6 DNAsequencesofprimersemployedtogenerate anaffinitymaturationphagedisplaylibrary ofanti-VEGFDotBody16C2.1.Thenumbersin thesequencerepresentfrequencyofbasesA, T,CandGintheoligonucleotidesequence. Frequencycode5=70%A,10%G,10%C,10% T;6=70%G,10%A,10%C,10%T;7=70%C, 10%A,10%G,10%T;8=70%T,10%A,10%G, 10%C. Primer name Primersequence aVEGF- TGTGCAATTTCTGGCTTC878788678678577657ATAGA 16C2.1 CTGGGTGCGTCAG CDR1 aVEGF- GAATGGGTTGCAAGGATT88887877687865788857765 16C2.1 7TATGCCGATAGCGTCAAG CDR2 aVEGF- ACTGCCGTCTATTATTGT66856577665757785767885 16C2.1 7558588888788657577776577857558678788757T CDR3 ACCGGGGTCAAGGAACA

Example 8: 16A2.1-Based and 16C2.1-Based Affinity Maturation Phage Display Selections, with Heat Challenge

[0485] 16A2.1-based and 16C2.1-based phage display libraries were panned separately, following identical procedures.

[0486] Neutravidin was immobilized onto Maxisorp Immuno Tubes (Thermo Scientific) at 10 ?g in 1 mL PBS overnight at 4? C. The tubes were washed twice in PBS, and blocked for 1 h at room temperature (RT) in MBB. Biotinylated VEGF-121 (Acro Biosystems) was added at different concentrations depending on the panning round (refer to Table 7). The protein was incubated for 1 h at RT, and non-bound protein removed by two washes with PBS. Negative selection tubes were prepared as described for biotinylated VEGF-121, but PBS was added in place of biotinylated VEGF-121.

[0487] The remaining selection procedures are similar to the Phage display selections from na?ve synthetic DotBody phage display libraries, with certain changes as summarised in Table 7.

TABLE-US-00012 TABLE 7 Affinity maturation selection conditions of 16A2.1-based and 16C2.1- based phage display libraries against biotinylated VEGF-121. Changes Details Number of 4 rounds with increasing levels of stringency selection rounds Adaptor protein A biotin-binding protein was immobilized on Maxisorp plates as follows: Round 1: 20 ?g Neutravidin in 2 mL Round 2: 10 ?g Neutravidin in 2 mL Round 3: 10 ?g Neutravidin in 2 mL Round 4: 10 ?g Neutravidin in 2 mL Amount of Decreasing biotinylated VEGF-121 concentration was incubated on the biotinylated VEGF- adaptor protein-coated tubes in the various selection round: 121 immobilized Round 1: 75 nM Round 2: 24 nM Round 3: 7.5 nM Round 4: 7.5 nM Amount of phages The library was diluted in 1 mL MBB at different final concentrations in the added to VEGF-121 various selection round: Round 1: 2 ? 10.sup.12 pfu/mL of affinity matured phage display library Round 2: 5 ? 10.sup.11 pfu/mL of purified phages from previous round Rounds 3: 5 ? 10.sup.11 pfu/mL of purified phages from previous round Rounds 4: 5 ? 10.sup.11 pfu/mL of purified phages from previous round Heat challenge The phages were incubated at different temperatures, spun-down at 15,000g for 5 min, before proceeding with selection. The temperatures are shown below: Round 1: no heat treatment Round 2: no heat treatment Round 3: 30 min at 55? C. Round 4: 30 min at 70? C. Incubation time The phages were incubated in the negative selection tube for 1 h at RT, then of phages with transferred to the tube containing VEGF-121, and incubated at RT for VEGF-121 different durations in the various selection round: Round 1: 1 h Round 2: 30 min Round 3: 30 min Round 4: 30 min Washing of non- Non-bound phages were removed following different wash regiments in the bound phages various selection round: Round 1: 3 times with MBB, 3 times with PBST Round 2: 10 times with MBB, 10 times with PBST, 2 times with PBS Round 3: 15 times with MBB, 15 times with PBST, 2 times with PBS Round 4: 15 times with MBB, 15 times with PBST, 2 times with PBS

[0488] 16A2.1-based affinity maturation phage display selections with heat challenge gave rise to 20A2.1 and 20A3.1.

[0489] 16C2.1-based affinity maturation phage display selections with heat challenge gave rise to 21A1.1, 21A8.1, 21D9.1 and 21E6.1.

Example 9: 16C2.1-Based Affinity Maturation Phage Display Selections, without Heat Challenge

[0490] The 1602.1-based phage display library was also panned without heat challenge, as follows.

[0491] Neutravidin was immobilized onto Maxisorp Immuno Tubes (Thermo Scientific) at 10 ?g in 1 mL PBS overnight at 4? C. The tubes were washed twice in PBS, and blocked for 1 h at room temperature (RT) in MBB. Biotinylated VEGF-165 (Acro Biosystems) was added at different concentrations depending on the panning round (refer to Table 8). The protein was incubated for 1 h at RT, and non-bound protein removed by two washes with PBS. Negative selection tubes were prepared as described for biotinylated VEGF-165, but PBS was added in place of biotinylated VEGF-165.

[0492] The remaining selection procedures are similar to the Phage display selections from na?ve synthetic DotBody phage display libraries, with certain changes as summarised in Table 8.

TABLE-US-00013 TABLE 8 Affinity maturation selection conditions of 16C2.1-based phage display libraries against biotinylated VEGF-165. Changes Details Number of 4 rounds with increasing levels of stringency selection rounds Adaptor protein A biotin-binding protein was immobilized on Maxisorp plates as follows: Round 1: 20 ?g Neutravidin in 2 mL Round 2: 10 ?g Streptavidin in 2 mL Round 3: 10 ?g Neutravidin in 2 mL Round 4: 10 ?g Neutravidin in 2 mL Amount of Decreasing biotinylated VEGF-165 concentration was incubated on the biotinylated VEGF- adaptor protein-coated tubes in the various selection round: 165 immobilized Round 1: 223 nM Round 2: 223 nM Round 3: 11 nM Round 4: 11 nM Amount of phages The library was diluted in 1 mL MBB at different final concentrations in the added to VEGF- various selection round: 165 Round 1: 1 ? 10.sup.13 pfu/mL of affinity matured phage display library Round 2: 1 ? 10.sup.12 pfu/mL of purified phages from previous round Rounds 3: 1 ? 10.sup.12 pfu/mL of purified phages from previous round Rounds 4: 5 ? 10.sup.11 pfu/mL of purified phages from previous round Incubation The phages were incubated in the negative selection tube for 1 h at RT, then conditions of transferred to the tube containing VEGF-165, and incubated at RT for phages with different durations in the various selection round, with ranibizumab added for VEGF-165 additional stringency in rounds 3 and 4: Round 1: 1 h Round 2: 30 min Round 3: 30 min, in the presence of 5 nM ranibizumab Round 4: 30 min, in the presence of 5 nM ranibizumab Washing of non- Non-bound phages were removed following different wash regiments in the bound phages various selection round: Round 1: 3 times with MBB, 3 times with PBST Round 2: 10 times with MBB, 10 times with PBST, 2 times with PBS Round 3: 15 times with MBB, 15 times with PBST, 2 times with PBS Round 4: 15 times with MBB, 15 times with PBST, 2 times with PBS

Example 10: Protein Production and Characterization of Binding Kinetics by Biolayer Interferometry

[0493] VEGFA-binding clones were cloned into a pET-based expression vector with a ATG codon in 5 of the open reading frame and a sequence coding for a hexahistidine tag in 3. They were produced recombinantly in E. coli and purified by immobilized-metal affinity chromatography, followed by desalting into PBS. The bivalent molecule 16A2.1x2 was also produced in the same way.

[0494] Binding characterization was performed using BLI (Satorius) at RT with a 1000 rpm flow-rate. Biotinylated human VEGF-165 (Acro Biosystem) was immobilized onto Streptavidin-coated tips at a concentration of 3 ?g/mL for 60 sec in BLI buffer (0.1% BSA and 0.01% Tween-20 in PBS). After a 30 sec baseline, anti-VEGFA DotBodies were associated at 8 different concentrations, including a blank reference, in BLI buffer for 60 sec, followed by a 400 sec dissociation in BLI buffer. The background buffer signal was subtracted using the 0 nM concentration reference, and the kinetics of binding were calculated using a global fit following a 1:1 binding model, using BLI Analysis Software. The bivalent molecule 16A2.1x2 was also characterized as described here, but with a 600 sec dissociation. To characterize the binding of all the clones against murine VEGFA, the same procedure was employed, but the immobilized target was replaced with biotinylated murine VEGF-164 (Acro Biosystem).

[0495] The sensorgrams are shown in FIGS. 2 and 3, and the binding data are shown in the following table:

TABLE-US-00014 Target K.sub.on K.sub.diss Binding Affinity antigen Molecule (M.sup.?1s.sup.?1) (s.sup.?1) (K.sub.D) Human 13A6 1.22 ? 10.sup.5 4.75 ? 10.sup.3 38.9 nM VEGF165 16A2.1 3.94 ? 10.sup.5 9.86 ? 10.sup.?4 2.5 nM 16A6.1 9.24 ? 10.sup.4 1.36 ? 10.sup.?3 14.7 nM 16A2.1x2 4.26 ? 10.sup.5 1.28 ? 10.sup.?4 0.301 nM 20A2.1 9.77 ? 10.sup.4 5.50 ? 10.sup.?4 5.54 nM 20A3.1 2.60 ? 10.sup.5 7.25 ? 10.sup.?4 2.78 nM 21A1.1 9.15 ? 10.sup.4 1.03 ? 10.sup.?3 11.3 nM 21A8.1 5.80 ? 10.sup.4 1.18 ? 10.sup.?3 20.0 nM 21D9.1 4.82 ? 10.sup.4 9.90 ? 10.sup.?4 20.5 nM 21E6.1 6.96 ? 10.sup.4 4.42 ? 10.sup.?4 6.3 nM 23D5.1 2.86 ? 10.sup.5 8.02 ? 10.sup.?4 2.8 nM Mouse 13A6 4.19 ? 10.sup.4 1.04 ? 10.sup.?2 248 nM VEGF164 16A2.1 1.16 ? 10.sup.5 1.93 ? 10.sup.?3 16.6 nM 16A6.1 1.22 ? 10.sup.5 2.25 ? 10.sup.?3 18.4 nM 20A2.1 1.32 ? 10.sup.5 1.35 ? 10.sup.?3 10.2 nM 20A3.1 1.02 ? 10.sup.5 8.89 ? 10.sup.?4 8.7 nM 21A1.1 1.67 ? 10.sup.5 2.31 ? 10.sup.?3 13.8 nM 21A8.1 6.34 ? 10.sup.4 2.92 ? 10.sup.?3 46.1 nM 21D9.1 5.80 ? 10.sup.4 2.43 ? 10.sup.?3 41.7 nM 21E6.1 8.28 ? 10.sup.4 1.17 ? 10.sup.?3 14.2 nM 23D5.1 2.05 ? 10.sup.5 1.69 ? 10.sup.?3 8.34 nM

Example 11: Competitive ELISA

[0496] To perform the competitive ELISA, the VEGFR1 was immobilized at a concentration of 2 ?g/mL onto a Maxisorp 96-well plate (Thermo Scientific) overnight at 4? C., washed three times with PBS and blocked for 1 h at RT with ELISA Block Buffer (EBB: 0.2% BSA in PBST). Human VEGF-121 at a concentration 0.5 nM was mixed with purified anti-VEGFA DotBodies at different concentrations following a 1:3 serial dilution starting at 500 nM and ending at 0.008 nM, with a 0 nM control. Ranibizumab was also mixed with human VEGF-121 using a 1:3 serial dilution starting at 30 nM and ending at 0.0005 nM, with a 0 nM control. The samples were incubated for 2 h at RT, and 50 ?L transferred onto the VEGFR1-coated plate. After 2 h incubation, the plate was washed 3 times with PBST. 50 ?L of streptavidin-HRP conjugate (Thermo Scientific) was added at a 1:5,000 dilution in EBB, and incubated for 1 h at RT. The plate was washed 5 times with PBST, and developed with 50 ?L 3,3,5,5-Tetramethylbenzidine (TMB) substrate (GeneTex). After 5-15 min, the reactions were stopped by adding 50 ?L of 2M H.sub.2SO.sub.4, and signal was measured at an absorbance of 450 nm.

[0497] The data was plotted using Graphpad Prism 9 software, and IC.sub.50 determined using a [inhibitor] vs. responsevariable slope (four parameters) non-linear regression curve.

[0498] The results are shown in FIGS. 4 and 5.

[0499] The IC.sub.50 values determined for the different molecules for inhibition of interaction between human VEGF-121 and human VEGFR1 based on the data in FIG. 4 were as follows: [0500] 13A6=5.9 ?M [0501] 16A2.1=12.2 nM [0502] 16A6.1=41.4 nM [0503] Ranibizumab=6.8 nM.

[0504] The IC.sub.50 values determined for the different molecules for inhibition of interaction between human VEGF-121 and human VEGFR1 based on the data in FIG. 5 were as follows: [0505] 16A2.1=3.0 nM [0506] 20A2.1=1.6 nM [0507] 20A3.1=0.9 nM [0508] 21A1.1=0.9 nM [0509] 21A8.1=1.8 nM [0510] 21D9.1=1.0 nM [0511] 21E6.1=7.5 nM [0512] 23D5.1=1.4 nM [0513] Ranibizumab=7.4 nM

Example 12: Evaluation of Thermostability by Analysis of Binding to VEGFA after Heat Challenge

[0514] VEGFA-binding DotBodies were incubated for 1 h at room temperature, 60? C., 70? C. or 80? C. in BLI buffer, at a concentration of 250 nM (heat challenge). After the heat challenge, the samples were centrifuged at 15,000 g for 5 min and the supernatant was employed to perform characterization of binding to human VEGF165 by BLI, as described in Example 10 above.

[0515] The results are shown in FIG. 6.

Example 13: Conclusions

[0516] The inventors have produced stabilized VH domain antibodies, which bind specifically to human VEGFA, and which cross-react with murine VEGFA.

[0517] Among the antigen-binding molecules produce, clone 13A6 has an affinity of 38.9 nM for human VEGFA and 248 nM for murine VEGFA. Clone 13A6 blocks the interaction between VEGFA and VEGF Receptor 1 (VEGFR1) with an IC.sub.50 of 5.9 ?M as measured by competitive ELISA.

[0518] 13A6 was subjected to affinity maturation by phage display to further improve its binding affinity against human and murine VEGFA. A new phage display library was generated, in which each CDR position was mutated with a ratio of approximately 50% of the residue present in 13A6's wild-type sequence, and 50% of any other amino acid. Affinity maturation selections against human VEGFA were performed with increasing levels of stringency, by lowering phage concentration, antigen concentration and binding time, while increasing the number of washes. This procedure led to the identification of several affinity-improved DotBodies.

[0519] Among these, clones, 16A2.1 and 16A6.1 have affinities against human VEGFA of 2.5 nM and 14.7 nM, respectively. Their affinities for murine VEGFA are 16.6 nM and 18.4 nM, respectively. Their abilities to block the VEGFA-VEGFR1 interaction were significantly improved, with IC50s estimated at 12.2 nM for 16A2.1 and 41.4 nM for 16A6.1.

[0520] To prove the modularity of the DotBodies generated against VEGFA, clone 16A2.1 was produced as a bivalent molecule comprising of two copies of 16A2.1 sequence connected by a flexible (GGGGS)x6-GGGG linker. The molecule could be produced recombinantly in E. coli, and had an improved affinity for VEGFA of 301 ?M. As VEGFA exists as a homodimer (11), the improved binding affinity suggests that the bivalent 16A2.1x2 molecule may be binding both VEGF monomers concurrently.

[0521] DotBodies 16A2.1 and 16C2.1 were selected for further activity improvements. Additionally, 16A2.1 contained a potential glycosylation site (NST) in CDR3, which needed to be removed while maintaining binding. Two new phage display libraries were generated, in which each CDR position was mutated with a ratio of approximately 50% of the residue present in 16A2.1 and 16C2.1 sequences, respectively, and 50% of any other amino-acid. For 16A2.1, the NST sequence in CDR3 was replaced by either AST or NSA when performing the affinity maturation library design, with the goal of removing either Asn (N) or Ser (S)/Thr (T) in the typical Nx(T/S)N-glycosylation motif (where x is any amino acid, except proline) (12,13). Binding and stability-based selections against human VEGFA were performed with heat challenges at increasing temperatures, while lowering the antigen concentration and increasing the number of washes, to identify the most stable, high affinity anti-VEGF DotBodies. An additional selection without heat challenge was performed for the 16C2.1-based library. The heat-challenged libraries led to the identification of clones 20A2.1, 20A3.1, 21A1.1, 21A8.1, 21D9.1, 21E9.1, while the non-heat-challenged selection identified DotBody 23D5.1 as the most enriched sequence. Importantly, DotBodies 20A2.1 and 20A3.1 did not contain the potential N-glycosylation site present in their parental clone 16A2.1.

[0522] All DotBodies identified retained binding to human VEGFA with affinities ranging from 2.8 nM to 20 nM, and to mouse VEGFA with affinities ranging from 8.3 nM to 46.1 nM. Their ability to block VEGF-VEGFR interaction was measured by competitive ELISA, with IC50s ranging from 0.9 nM to 7.5 nM. Ranibizumab was used as a control, with a measured IC50 of 7.4 nM. Parental DotBody 16A2.1 had an IC50=3 nM, and the newly identified DotBodies 20A2.1 and 20A3.1 showed improved blockade activity (IC50=1.6 nM and IC50=0.9 nM, respectively). The following DotBodies also displayed high blockade activity: 21A1.1 (IC50=0.9 nM), 21D9.1 (IC50=1.0 nM) and 23D5.1 (IC50=1.4 nM).

[0523] All the DotBodies identified through the heat-challenge selection retained binding to VEGFA after incubation at temperatures ranging between room-temperature and 80? C. 23D5.1, which was identified in selections without heat challenge, lost most of its binding activity at 60? C. or above.

[0524] In conclusion, through a series of tailored phage display library designs and selection strategies, the inventors produced a broad range of anti-VEGF DotBodies with mid- to low-nanomolar affinities, binding both human VEGFA and murine VEGFA. These DotBodies are able to block the VEGF-VEGFR interactions with IC50s in the low-micromolar to sub-nanomolar range. The majority of DotBodies identified, including five potent blockers16A2.1, 20A2.1, 20A3.1 and 21D9.1have high thermostability, retaining binding to human VEGFA after incubation at temperatures ranging from room temperature to 80? C. These DotBodies are modular, and can used to create multi-valent and/or multi-specific molecules by linking several DotBodies in tandem (e.g. by introducing linker sequences between the DotBodies).

REFERENCES

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