Cystine Knot Scaffold Platform

Abstract

The present invention provides non-naturally occurring cystine knot peptides (CKPs) that bind VEGF-A. In addition, methods of using non-naturally occurring CKPs that bind VEGF-A are provided, including diagnostic and therapeutic compositions and methods. Non-natural CKPs that bind low-density lipoprotein receptor-related protein 6 (LRP6) are also provided.

Description

[0001] This application is a divisional application of international application number PCT / US2016 / 052012, international application date September 15, 2016, Chinese national phase application number 201680052527.6, Chinese national phase application date March 9, 2018, and invention name “Cystine knot scaffold platform”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of priority to U.S. Provisional Application Serial No. 62 / 219,063, filed September 15, 2015, the contents of which are incorporated herein by reference in their entirety.

[0004] Submission of Sequence Listings in ASCII Text Files

[0005] The contents of the following submission in an ASCII text file are incorporated herein by reference in their entirety: Computer Readable Form (CRF) of a Sequence Listing (File Name: 146392026840SEQLIST.txt, Record Date: September 15, 2016, Size: 183KB). Background of the Invention

[0006] The design and engineering of new proteins from alternative protein scaffolds has become an emerging field in the last decade with a wide range of applications from structural biology and imaging tools to therapeutic agents currently in clinical trials (HK Binz et al., Nat Biotechnol 23, 1257-1268, 2005; HK Binz and A Pluckthun, Curr Opin Biotechnol 16, 459-469, 2005; SS Sidhu and S Koide, Curr Opin Struct Biol 17, 481-487, 2007; A Skerra, Curr Opin Biotechnol 18, 295-304, 2007; C Gronwall and SS Tahl, J Biotechnol 140, 254-269, 2009; T Wurch et al., Trends Biotechnol 30, 575-582, 2012; S Banta et al., Annu Rev Biomed Eng 15,93-113,2013).

[0007] Desirable physical properties of potential alternative scaffold molecules include high thermal stability and reversibility of thermal folding and unfolding. Several methods have been applied to increase the apparent thermal stability of proteins and enzymes, including rational design based on comparison with highly similar thermostable sequences, design of stable disulfide bonds, mutations that increase α-helical propensity, engineering of salt bridges, changes in the surface charge of proteins, directed evolution, and composition of consensus sequences (Lehmann and Wyss, Cur Open Biotechnology 12, 371-375, 2001).

[0008] Cysteine ​​knot peptides come from a wide range of sources and exhibit different pharmacological activities. They are about 30-50 amino acids long and contain six conserved cysteine ​​residues that form three disulfide bonds. One of the disulfide bonds penetrates a large loop formed by the other two disulfide bonds and their interconnected backbones, resulting in a characteristic knot topology with multiple loops exposed on the surface. Loops are defined as amino acid regions flanking the six conserved cysteine ​​residues and are highly variable in nature. In addition, the unique arrangement of disulfide bonds makes cystine knot peptides highly stable to thermal, proteolytic and chemical degradation.

[0009] Thus, there is a need to develop small, stable, artificial antibody-like molecules for a variety of therapeutic and diagnostic applications, such as ocular diseases and disorders. The present invention satisfies this need and other needs. SUMMARY OF THE INVENTION

[0010] In certain embodiments, provided herein are non-naturally occurring cystine knot peptides (CKPs) that bind vascular endothelial growth factor A (VEGF-A), wherein the CKP comprises a cystine scaffold structure:

[0011] Z1C1L1C2L2C3L3C4L4C5L5C6Z2

[0012] in:

[0013] Z1 and Z2 are any amino acids;

[0014] L1 is loop 1 and has a structure selected from the group consisting of X1X2X3X4X5X6 (SEQ ID NO: 2), X1X2X3X4X5X6X7 (SEQ ID NO: 3), X1X2X3X4X5X6X7X8 (SEQ ID NO: 4), X1X2X3X4X5X6X7X8X9 (SEQ ID NO: 5), and X1X2X3X4X5X6X7X8X9 (SEQ ID NO: 6). 10 (SEQ ID NO: 6), wherein X1-X 10 Each of is any amino acid;

[0015] L2 is loop 2 and has the structure: X1X2X3X4X5 (SEQ ID NO: 7), wherein each of X1-X5 is any amino acid or unnatural amino acid;

[0016] L3 is loop 3 and has the structure: X1X2X3, wherein each of X1–X3 is any amino acid or unnatural amino acid;

[0017] L4 is loop 4 and has the structure: X1, wherein X1 is any amino acid or unnatural amino acid;

[0018] L5 is loop 5 and has the structure: X1X2X3X4X5 (SEQ ID NO: 7), wherein each of X1-X5 is any amino acid or unnatural amino acid;

[0019] wherein the unnatural amino acid is selected from the group consisting of L-propargylglycine-PEG6-, L-sulfotyrosine, L-norleucine, L-1-naphthylalanine, L-2-naphthylalanine, L-2-chlorotryptophan, L-3-fluorotyrosine, and L-4-fluorophenylalanine; and

[0020] Wherein the CKP binds VEGF-A with an affinity of 500 pM or better.

[0021] In certain embodiments according to (or as applied to) any of the above embodiments, with reference to the wild-type Ecballium elaterium trypsin inhibitor EETI-II protein having the amino acid sequence shown in SEQ ID NO:1, the non-naturally occurring (CKP) that binds VEGF-A has altered disulfide bond connectivity; wherein the altered disulfide bond connectivity is C1-C4, C2-C3 and C5-C6.

[0022] In certain embodiments according to (or as applied to) any of the embodiments above, the unnatural amino acid is selected from the group consisting of L-propargylglycine-PEG6-, L-sulfotyrosine, L-norleucine, L-1-naphthylalanine, L-2-naphthylalanine, L-2-chlorotryptophan, L-3-fluorotyrosine, L-4-fluorophenylalanine, γ-benzyl-L-proline, γ-(4-fluoro-benzyl)-L-proline, 4-OH-L-proline, 4-fluoro-L-proline, 4-[4-(trifluoromethyl)-benzyl]-L-proline, 3,4-difluoro-L-phenylalanine, 3,4-dichloro-L-phenylalanine, 4-chloro-L-phenylalanine, 3-F,4-Cl-L-phenylalanine, 2-pyridone (NH para)-L-alanine, pyridone (NH meta)-L-alanine, 3-(1-N-methylindole)-L-alanine, 3-(1-N-ethylindole)-L-alanine, 3-(1-N-isopropylindole)-L-alanine, 3-(5-aza-indole)-L-alanine, 4-methyl-L-phenylalanine, 2-naphthyl-L-alanine, L-4,4'- biphenylalanine, 3-(3-quinolyl)-L-alanine, 3-(2-quinolyl)-L-alanine, 3-(2-quinoxalinyl)-L-alanine, 4-methyl-2-pyridyl-alanine, 4-ethyl-2-pyridyl-L-alanine, benzothiazole-L-alanine, benzothiophene-L-alanine, 3-isoquinolyl-L-alanine, tert-butyl-L-alanine (also known as L-neopentylglycine), 3-cyclobutyl-L-alanine, cyclopentyl-L-alanine, 5,5,5-trifluoro-L-leucine, tert-butyl-L-glycine (also known as L-tert-leucine), L-cyclopentylglycine, L-cyclobutylglycine, 3,4-hydroxy-L-phenylalanine, 3,4-fluoro-L-phenylalanine, 3-fluoro, 4-OH-L-phenylalanine, 2-chloro-L-tyrosine, 2-methyl-L-tyrosine, 2-ethyl-L-tyrosine, 4-(naphthalene-1-ol)-L-alanine, D-serine, L-β-homoserine, L-β-alanine, N-α-methylglycine, glycine amide, glycerol glycine ester, ethylene glycol glycine ester, oxetane-3-yl glycine ester and glycine morpholine amide.

[0023] In certain embodiments according to (or as applied to) any of the above embodiments, Z1 and / or Z2 are more than one amino acid, or non-natural amino acid. In certain embodiments, Z2 is two amino acids. In certain embodiments, Z2 is three amino acids.

[0024] In certain embodiments according to (or as applied to) any of the embodiments above, Z1 and / or Z2 is G.

[0025] In certain embodiments according to (or as applied to) any of the above embodiments, in L1, X3 is not I; X5 is not M; and / or X6 is not R. In certain embodiments according to (or as applied to) any of the above embodiments, in L1: X1 is an amino acid selected from P, Q, R, T, V, D, N, K, L, and X; X2 is an amino acid selected from T, D, L, V, I, R, P, N, and X; X3 is an amino acid selected from T, P, M, L, S, F, R, and X; X4 is an amino acid selected from R, T, Q, D, W, L, E, S, K, and X; X5 is an amino acid selected from F, P, V, E, K, L, I, and X; X6 is an amino acid selected from The invention relates to an amino acid selected from the group consisting of: L-propargylglycine-PEG6-, L-sulfotyrosine, L-norleucine, L-1-naphthylalanine, L-2-naphthylalanine, L-2-chlorotryptophan, L-3-fluorotyrosine and L-4-fluorophenylalanine. In certain embodiments according to (or as applied to) any of the above embodiments, in L1: X9 is an amino acid selected from L, I, V, D, E and X, wherein X is a non-natural amino acid and is selected from the group consisting of: L-propargylglycine-PEG6-, L-sulfotyrosine, L-norleucine, L-1-naphthylalanine, L-2-naphthylalanine, L-2-chlorotryptophan, L-3-fluorotyrosine and L-4-fluorophenylalanine. In certain embodiments according to (or as applied to) any of the above embodiments, in L1: X 10 is an amino acid selected from the group consisting of Y, T, M, N, F and X, wherein X is an unnatural amino acid and is selected from the group consisting of L-propargylglycine-PEG6-, L-sulfotyrosine, L-norleucine, L-1-naphthylalanine, L-2-naphthylalanine, L-2-chlorotryptophan, L-3-fluorotyrosine and L-4-fluorophenylalanine.

[0026] In certain embodiments according to (or as applied to) any of the embodiments above, X is an unnatural amino acid and is selected from the group consisting of L-propargylglycine-PEG6-, L-sulfotyrosine, L-norleucine, L-1-naphthylalanine, L-2-naphthylalanine, L-2-chlorotryptophan, L-3-fluorotyrosine, L-4-fluorophenylalanine, γ-benzyl-L-proline, γ-(4-fluoro-benzyl)-L-proline, 4-OH-L-proline, 4-fluoro-L-proline, 4-[4-(trifluoromethyl)-benzyl]-L-proline, Acid, 3,4-difluoro-L-phenylalanine, 3,4-dichloro-L-phenylalanine, 4-chloro-L-phenylalanine, 3-F,4-Cl-L-phenylalanine, 2-pyridone (NH para)-L-alanine, pyridone (NH meta)-L-alanine, 3-(1-N-methylindole)-L-alanine, 3-(1-N-ethylindole)-L-alanine, 3-(1-N-isopropylindole)-L-alanine, 3-(5-aza-indole)-L-alanine, 4-methyl-L-phenylalanine, 2-naphthyl-L-alanine, L-4,4' -biphenylalanine, 3-(3-quinolyl)-L-alanine, 3-(2-quinolyl)-L-alanine, 3-(2-quinoxalinyl)-L-alanine, 4-methyl-2-pyridyl-alanine, 4-ethyl-2-pyridyl-L-alanine, benzothiazole-L-alanine, benzothiophene-L-alanine, 3-isoquinolyl-L-alanine, tert-butyl-L-alanine (also known as L-neopentylglycine), 3-cyclobutyl-L-alanine, cyclopentyl-L-alanine, 5,5,5-trifluoro-L-leucine, tert-butyl-L-glycine (also known as L-tert-leucine), L-cyclopentylglycine, L-cyclobutylglycine, 3,4-hydroxy-L-phenylalanine, 3,4-fluoro-L-phenylalanine, 3-fluoro,4-OH-L-phenylalanine, 2-chloro-L-tyrosine, 2-methyl-L-tyrosine, 2-ethyl-L-tyrosine, 4-(naphthalene-1-ol)-L-alanine, D-serine, L-β-homoserine, L-β-alanine, N-α-methylglycine, glycine amide, glycerol glycine ester, glycol glycine ester, oxetane-3-yl glycine ester and glycine morpholine amide.

[0027] In certain embodiments according to (or as applied to) any of the above embodiments, in L5, each of X1-X5 is any amino acid, except that X2 is not proline (P). In certain embodiments according to (or as applied to) any of the above embodiments, in L5, each of X1-X5 is any amino acid, except that X4 is not glycine (G). In certain embodiments according to (or as applied to) any of the above embodiments, in L5: X1 is an amino acid selected from G, Q, H, R, L and Q; X2 is an amino acid selected from P, M, W, Y, F, L and H; X3 is an amino acid selected from N, F, H and Y; X4 is an amino acid selected from G, Q, D, N, K, H, E and S; and / or X5 is an amino acid selected from F, S and T.

[0028] In certain embodiments according to (or as applied to) any of the above embodiments, L1 has the structure X1X2X3X4X5X6X7X8 (SEQ ID NO: 4), wherein: X1 is an amino acid selected from P, Q and R; X2 is an amino acid selected from T, L and D; X3 is an amino acid selected from T, M and L; X4 is an amino acid selected from R, Q and D; X5 is an amino acid selected from F, P and V; X6 is an amino acid selected from K and F; X7 is an amino acid selected from Q and W; and X8 is an amino acid selected from Y, G and D. In certain embodiments according to (or as applied to) any of the above embodiments, L1 has the structure X1X2X3X4X5X6X7X8 X9 X 10 (SEQ ID NO:6), wherein: X1 is an amino acid selected from Q, R, T and V; X2 is an amino acid selected from T and D; X3 is P; X4 is an amino acid selected from T and W; X5 is an amino acid selected from F, E, P and K; X6 is an amino acid selected from N and P; X7 is an amino acid selected from W and H; X8 is an amino acid selected from A, D, E and W; X9 is an amino acid selected from L and I; and X 10 is an amino acid selected from the group consisting of Y, T, M and N.

[0029] In certain embodiments according to (or as applied to) any of the above embodiments, in L5: X1 is an amino acid selected from G, H and Q; X2 is an amino acid selected from P, M, W and Y; X3 is an amino acid selected from N and Y; X4 is an amino acid selected from G, Q and S; and X5 is an amino acid selected from F and S.

[0030] In certain embodiments according to (or as applied to) any of the above embodiments, L1 has the structure X1X2X3X4X5X6X7X8 (SEQ ID NO:4), wherein: X1 is an amino acid selected from D, Q, N and K; X2 is an amino acid selected from V, I, R, L and P; X3 is an amino acid selected from L, S, M, T and F; X4 is an amino acid selected from Q, L and E; X5 is P; X6 is an amino acid selected from F, L and Y; X7 is W; and X8 is G.

[0031] In certain embodiments according to (or as applied to) any of the above embodiments, in L5: X3 is Y; X5 is S; and X1, X2, and X4 are each any amino acid, except that X1 is not G, X2 is not P, X4 is not G, and / or X5 is not F. In certain embodiments according to (or as applied to) any of the above embodiments, in L5: X1 is an amino acid selected from H, L, R, and Q; X2 is an amino acid selected from W, F, and Y; X3 is Y; X4 is an amino acid selected from Q, N, K, H, and E; and X5 is S.

[0032] In certain embodiments according to (or as applied to) any of the above embodiments, L1 has the structure X1X2X3X4X5X6X7X8X9X 10 (SEQ ID NO:6), wherein: X1 is an amino acid selected from K, Q, L and R; X2 is an amino acid selected from N and D; X3 is an amino acid selected from P and L; X4 is an amino acid selected from L, T, S and K; X5 is an amino acid selected from F, V, I and L; X6 is an amino acid selected from N and D; X7 is W; X8 is an amino acid selected from A and S; X9 is an amino acid selected from L, V, E and D; and X 10 is an amino acid selected from Y and F.

[0033] In certain embodiments according to (or as applied to) any of the embodiments above, in L5: X1 is Q; X2 is an amino acid selected from L, F, M and H; X3 is an amino acid selected from F, Y and H; X4 is an amino acid selected from D, Q, N and K; and X5 is an amino acid selected from S and T.

[0034] In certain embodiments according to (or as applied to) any of the embodiments above, in L2, X1 is K, X2 is Q, X3 is D, X4 is S, and X5 is D.

[0035] In certain embodiments according to (or as applied to) any of the embodiments above, L1 has the structure X1X2X3X4X5X6X7X8 (SEQ ID NO: 4), wherein: X5 is P; X7 is W; X8 is G; and wherein X1, X2, X3, X4 and X6 are each any amino acid, except that X1 is not P, X2 is not R, X3 is not I, and / or X6 is not R. In certain embodiments according to (or as applied to) any of the embodiments above, L1 has the structure X1X2X3X4X5X6X7X8 (SEQ ID NO: 4), wherein: X1 is an amino acid selected from N and D; X2 is an amino acid selected from I and V; X3 is an amino acid selected from M and L; X4 is an amino acid selected from L, Q, D and K; X5 is P; X6 is an amino acid selected from F, Y, T, L and M; X7 is W; and X8 is G.

[0036] In certain embodiments according to (or as applied to) any of the above embodiments, in L5: X1 is an amino acid selected from Q, H, L and R; X2 is an amino acid selected from Y and W; X3 is Y; X4 is an amino acid selected from Q and N; and X5 is S. In certain embodiments according to (or as applied to) any of the above embodiments, in L5: X3 is Y; X5 is S; and X1, X2 and X4 are each any amino acid, except: X1 is not G, X2 is not P, and / or X4 is not G.

[0037] In certain embodiments according to (or as applied to) any of the embodiments above, in L2: X1 is an amino acid selected from G or E; X2 is an amino acid selected from Q, L, P, R, E and M; X3 is an amino acid selected from S, D and N; X4 is an amino acid selected from F, Y, L, M and I; and / or X5 is an amino acid selected from E, D, Q, L and S,

[0038] In certain embodiments according to (or as applied to) any of the embodiments above, in L3, X1 is L, X2 is A, and X3 is G.

[0039] In certain embodiments according to (or as applied to) any of the embodiments above, in L4, X1 is V or F.

[0040] In certain embodiments according to (or as applied to) any of the embodiments above, in L5, each of X1-X5 is any amino acid, except that X2 is not proline (P).

[0041] In certain embodiments according to (or as applied to) any of the embodiments above, in L5, each of X1-X5 is any amino acid, except that X4 is not glycine (G).

[0042] In certain embodiments according to (or as applied to) any of the above embodiments, in L5: X1 is any amino acid except G; X2 is any amino acid except P; X3 is any amino acid except N; X4 is any amino acid except G; and / or X5 is any amino acid except F.

[0043] In certain embodiments according to (or as applied to) any of the above embodiments, L1 has the structure XXXXXXXXXXXX (SEQ ID NO:4), wherein X1 is an amino acid selected from N, D and X; X2 is an amino acid selected from I, V and X; X3 is M or X; X4 is an amino acid selected from L, Q and X; X5 is P or X; X6 is F, Y or X; X7 is W or X; and X8 is G or X, wherein X is an unnatural amino acid selected from the group consisting of L-propargylglycine-PEG6-, L-sulfotyrosine, L-norleucine, L-1-naphthylalanine, L-2-naphthylalanine, L-2-chlorotryptophan, L-3-fluorotyrosine and L-4-fluorophenylalanine.

[0044] In certain embodiments according to (or as applied to) any of the embodiments above, X is an unnatural amino acid and is selected from the group consisting of L-propargylglycine-PEG6-, L-sulfotyrosine, L-norleucine, L-1-naphthylalanine, L-2-naphthylalanine, L-2-chlorotryptophan, L-3-fluorotyrosine, L-4-fluorophenylalanine, γ-benzyl-L-proline, γ-(4-fluoro-benzyl)-L-proline, 4-OH-L-proline, 4-fluoro-L-proline, 4-[4-(trifluoromethyl)-benzyl]-L-proline, Acid, 3,4-difluoro-L-phenylalanine, 3,4-dichloro-L-phenylalanine, 4-chloro-L-phenylalanine, 3-F,4-Cl-L-phenylalanine, 2-pyridone (NH para)-L-alanine, pyridone (NH meta)-L-alanine, 3-(1-N-methylindole)-L-alanine, 3-(1-N-ethylindole)-L-alanine, 3-(1-N-isopropylindole)-L-alanine, 3-(5-aza-indole)-L-alanine, 4-methyl-L-phenylalanine, 2-naphthyl-L-alanine, L-4,4' -biphenylalanine, 3-(3-quinolyl)-L-alanine, 3-(2-quinolyl)-L-alanine, 3-(2-quinoxalinyl)-L-alanine, 4-methyl-2-pyridyl-alanine, 4-ethyl-2-pyridyl-L-alanine, benzothiazole-L-alanine, benzothiophene-L-alanine, 3-isoquinolyl-L-alanine, tert-butyl-L-alanine (also known as L-neopentylglycine), 3-cyclobutyl-L-alanine, cyclopentyl-L-alanine, 5,5,5-trifluoro-L-leucine, tert-butyl-L-glycine (also known as L-tert-leucine), L-cyclopentylglycine, L-cyclobutylglycine, 3,4-hydroxy-L-phenylalanine, 3,4-fluoro-L-phenylalanine, 3-fluoro,4-OH-L-phenylalanine, 2-chloro-L-tyrosine, 2-methyl-L-tyrosine, 2-ethyl-L-tyrosine, 4-(naphthalene-1-ol)-L-alanine, D-serine, L-β-homoserine, L-β-alanine, N-α-methylglycine, glycine amide, glycerol glycine ester, glycol glycine ester, oxetane-3-yl glycine ester and glycine morpholine amide.

[0045] In certain embodiments according to (or as applied to) any of the above embodiments, in L3, each of X1-X3 is any amino acid or unnatural amino acid, except that X1 is not leucine (L), X2 is not alanine (A), and X3 is not glycine (G), wherein the unnatural amino acid is selected from the group consisting of: L-propargylglycine-PEG6-, L-sulfotyrosine, L-norleucine, L-1-naphthylalanine, L-2-naphthylalanine, L-2-chlorotryptophan, L-3-fluorotyrosine and L-4-fluorophenylalanine. In certain embodiments according to (or as applied to) any of the above embodiments, in L3: X1 is an amino acid selected from M, F, LV and X; X2 is an amino acid selected from S, N, Q, I, Y, E, V, T and X; X3 is an amino acid selected from D, Q, T, N, E, R and X, wherein X is an unnatural amino acid selected from the group consisting of: L-propargylglycine-PEG6-, L-sulfotyrosine, L-norleucine, L-1-naphthylalanine, L-2-naphthylalanine, L-2-chlorotryptophan, L-3-fluorotyrosine and L-4-fluorophenylalanine.

[0046] In certain embodiments according to (or as applied to) any of the above embodiments, in L4, Xi is any amino acid other than V or a non-natural amino acid selected from the group consisting of: L-propargylglycine-PEG6-, L-sulfotyrosine, L-norleucine, L-1-naphthylalanine, L-2-naphthylalanine, L-2-chlorotryptophan, L-3-fluorotyrosine, and L-4-fluorophenylalanine. In certain embodiments according to (or as applied to) any of the above embodiments, in L4, Xi is I, L, or X, wherein X is a non-natural amino acid selected from the group consisting of: L-propargylglycine-PEG6-, L-sulfotyrosine, L-norleucine, L-1-naphthylalanine, L-2-naphthylalanine, L-2-chlorotryptophan, L-3-fluorotyrosine, and L-4-fluorophenylalanine.

[0047] In certain embodiments according to (or as applied to) any of the above embodiments, in L5: X3 is Y or X; X5 is S or X; and X1, X2 and X4 are each any amino acid or X, except that X1 is not G, X2 is not P, and / or X4 is not G, wherein X is a non-natural amino acid selected from the group consisting of: L-propargylglycine-PEG6-, L-sulfotyrosine, L-norleucine, L-1-naphthylalanine, L-2-naphthylalanine, L-2-chlorotryptophan, L-3-fluorotyrosine and L-4-fluorophenylalanine. In certain embodiments according to (or as applied to) any of the above embodiments, in L5, each of X1-X5 is any amino acid, except that X2 is not proline (P). In certain embodiments according to (or as applied to) any of the above embodiments, in L5, each of X1-X5 is any amino acid, except that X4 is not glycine (G). In certain embodiments according to (or as applied to) any of the above embodiments, in L5: X1 is an amino acid selected from Q, H and X; X2 is an amino acid selected from Y, W and X; X3 is Y or X; X4 is an amino acid selected from Q, N or X; X5 is S or X, wherein X is an unnatural amino acid selected from the group consisting of: L-propargylglycine-PEG6-, L-sulfotyrosine, L-norleucine, L-1-naphthylalanine, L-2-naphthylalanine, L-2-chlorotryptophan, L-3-fluorotyrosine and L-4-fluorophenylalanine.

[0048] In certain embodiments according to (or as applied to) any of the embodiments above, X is an unnatural amino acid and is selected from the group consisting of L-propargylglycine-PEG6-, L-sulfotyrosine, L-norleucine, L-1-naphthylalanine, L-2-naphthylalanine, L-2-chlorotryptophan, L-3-fluorotyrosine, L-4-fluorophenylalanine, γ-benzyl-L-proline, γ-(4-fluoro-benzyl)-L-proline, 4-OH-L-proline, 4-fluoro-L-proline, 4-[4-(trifluoromethyl)-benzyl]-L-proline, , 3,4-difluoro-L-phenylalanine, 3,4-dichloro-L-phenylalanine, 4-chloro-L-phenylalanine, 3-F,4-Cl-L-phenylalanine, 2-pyridone (NH para)-L-alanine, pyridone (NH meta)-L-alanine, 3-(1-N-methylindole)-L-alanine, 3-(1-N-ethylindole)-L-alanine, 3-(1-N-isopropylindole)-L-alanine, 3-(5-aza-indole)-L-alanine, 4-methyl-L-phenylalanine, 2-naphthyl-L-alanine, L-4,4' -biphenylalanine, 3-(3-quinolyl)-L-alanine, 3-(2-quinolyl)-L-alanine, 3-(2-quinoxalinyl)-L-alanine, 4-methyl-2-pyridyl-alanine, 4-ethyl-2-pyridyl-L-alanine, benzothiazole-L-alanine, benzothiophene-L-alanine, 3-isoquinolyl-L-alanine, tert-butyl-L-alanine (also known as L-neopentylglycine), 3-cyclobutyl-L-alanine, cyclopentyl-L-alanine, 5,5,5-trifluoro-L-leucine, tert-butyl-L-glycine (also known as L-tert-leucine), L-cyclopentylglycine, L-cyclobutylglycine, 3,4-hydroxy-L-phenylalanine, 3,4-fluoro-L-phenylalanine, 3-fluoro,4-OH-L-phenylalanine, 2-chloro-L-tyrosine, 2-methyl-L-tyrosine, 2-ethyl-L-tyrosine, 4-(naphthalene-1-ol)-L-alanine, D-serine, L-β-homoserine, L-β-alanine, N-α-methylglycine, glycine amide, glycerol glycine ester, glycol glycine ester, oxetane-3-yl glycine ester and glycine morpholine amide.

[0049] In certain embodiments according to (or as applied to) any of the embodiments above, in L2: X1 is G or X; X2 is R, P or X; X3 is D or X; X4 is F, I or X; and X5 is E, D or X, wherein X is an unnatural amino acid selected from the group consisting of: L-propargylglycine-PEG6-, L-sulfotyrosine, L-norleucine, L-1-naphthylalanine, L-2-naphthylalanine, L-2-chlorotryptophan, L-3-fluorotyrosine, and L-4-fluorophenylalanine.

[0050] In certain embodiments according to (or as applied to) any of the embodiments above, the non-naturally occurring cystine knot peptide (CKP) that binds to vascular endothelial growth factor A (VEGF-A) comprises the amino acid sequence GCNIMLPFWGCGRDFECLQQCICQYYQSCG (SEQ ID NO: 103). In certain embodiments according to (or as applied to) any of the embodiments above, the non-naturally occurring cystine knot peptide (CKP) that binds to vascular endothelial growth factor A (VEGF-A) comprises the amino acid sequence GCNIMLPFWGCGRDFECVERCICQYYQSCG (SEQ ID NO: 104). In certain embodiments according to (or as applied to) any of the embodiments above, the non-naturally occurring cystine knot peptide (CKP) that binds to vascular endothelial growth factor A (VEGF-A) comprises the amino acid sequence GCNIMLPFWGCGRDFECMSDCICQYYQSCG (SEQ ID NO: 105). In certain embodiments according to (or as applied to) any of the embodiments above, the non-naturally occurring cystine knot peptide (CKP) that binds to vascular endothelial growth factor A (VEGF-A) comprises the amino acid sequence GCNIMLPFWGCGRDFECMNQCICQYYQSCG (SEQ ID NO: 106). In certain embodiments according to (or as applied to) any of the embodiments above, the non-naturally occurring cystine knot peptide (CKP) that binds to vascular endothelial growth factor A (VEGF-A) comprises the amino acid sequence GCNIMLPFWGCGRDFECMQTCICQYYQSCG (SEQ ID NO: 107). In certain embodiments according to (or as applied to) any of the embodiments above, the non-naturally occurring cystine knot peptide (CKP) that binds to vascular endothelial growth factor A (VEGF-A) comprises the amino acid sequence GCNIMLPFWGCGRDFECVYQCICQYYQSCG (SEQ ID NO: 108). In certain embodiments according to (or as applied to) any of the embodiments above, the non-naturally occurring cystine knot peptide (CKP) that binds to vascular endothelial growth factor A (VEGF-A) comprises the amino acid sequence GCNIMLPFWGCGRDFECFINCICQYYQSCG (SEQ ID NO: 109). In certain embodiments according to (or as applied to) any of the embodiments above, the non-naturally occurring cystine knot peptide (CKP) that binds to vascular endothelial growth factor A (VEGF-A) comprises the amino acid sequence GCNIMLPFWGCGRDFECVSQCICQYYQSCG (SEQ ID NO: 110).In certain embodiments according to (or as applied to) any of the embodiments above, the non-naturally occurring cystine knot peptide (CKP) that binds to vascular endothelial growth factor A (VEGF-A) comprises the amino acid sequence GCNIMLPFWGCGRDFECVTECICQYYQSCG (SEQ ID NO: 111). In certain embodiments according to (or as applied to) any of the embodiments above, the non-naturally occurring cystine knot peptide (CKP) that binds to vascular endothelial growth factor A (VEGF-A) comprises the amino acid sequence GCNIMLPFWGCGRDFECFYECICQYYQSCG (SEQ ID NO: 112). In certain embodiments according to (or as applied to) any of the embodiments above, the non-naturally occurring cystine knot peptide (CKP) that binds to vascular endothelial growth factor A (VEGF-A) comprises the amino acid sequence GCNIMLPFWGCGRDFECMEQCICQYYQSCG (SEQ ID NO: 113). In certain embodiments according to (or as applied to) any of the embodiments above, the non-naturally occurring cystine knot peptide (CKP) that binds to vascular endothelial growth factor A (VEGF-A) comprises the amino acid sequence GCNIMLPFWGCGRDFECVYRCICQYYQSCG (SEQ ID NO: 114). In certain embodiments according to (or as applied to) any of the embodiments above, the non-naturally occurring cystine knot peptide (CKP) that binds to vascular endothelial growth factor A (VEGF-A) comprises the amino acid sequence GCDVMQPYWGCGPDIDCFVRCLCHWYNSCG (SEQ ID NO: 139). In certain embodiments according to (or as applied to) any of the embodiments above, the non-naturally occurring cystine knot peptide (CKP) that binds to vascular endothelial growth factor A (VEGF-A) comprises the amino acid sequence GCDVMQPYWGCGPDIDCLSNCICHWYNSCG (SEQ ID NO: 140). In certain embodiments according to (or as applied to) any of the embodiments above, the non-naturally occurring cystine knot peptide (CKP) that binds to vascular endothelial growth factor A (VEGF-A) comprises the amino acid sequence GCNIMLPYWGCGRDFECMEQCICQYYQSCG (SEQ ID NO: 142). In certain embodiments according to (or as applied to) any of the embodiments above, the non-naturally occurring cystine knot peptide (CKP) that binds to vascular endothelial growth factor A (VEGF-A) comprises the amino acid sequence GCNIXLPFWGCGRDFECMSDCICQYYQSCG (SEQ ID NO: 144), wherein X is norleucine (Nle).In certain embodiments according to (or as applied to) any of the embodiments above, the non-naturally occurring cystine knot peptide (CKP) that binds to vascular endothelial growth factor A (VEGF-A) comprises the amino acid sequence GCNIXLPFWGCGRDFECVSQCICQYYQSCG (SEQ ID NO: 145), wherein X is norleucine (Nle). In certain embodiments according to (or as applied to) any of the embodiments above, the non-naturally occurring cystine knot peptide (CKP) that binds to vascular endothelial growth factor A (VEGF-A) comprises the amino acid sequence GCNIXLPYWGCGRDFECMEQCICQYYQSCG (SEQ ID NO: 146), wherein X is norleucine (Nle).

[0051] In certain embodiments according to (or as applied to) any of the embodiments above, the non-naturally occurring cystine knot peptide (CKP) that binds vascular endothelial growth factor A (VEGF-A) comprises the amino acid sequence GCDVXQPYWGCGPDIDCLSNCICHWYNSCG (SEQ ID NO: 224), wherein X is norleucine.

[0052] In certain embodiments, a non-naturally occurring cystine knot peptide (CKP) is provided that comprises an amino acid selected from the group consisting of:

[0053] GCNIMLPFWGCGRDFECMEQCICQYYQSCG(SEQ ID NO:113);

[0054] GCNIMLPFWGCGRDFECVYRCICQYYQSCG (SEQ ID NO: 114);

[0055] GCDVMQPYWGCGPDIDCFVRCLCHWYNSCG(SEQ ID NO:139);

[0056] GCDVMQPYWGCGPDIDCLSNCICHWYNSCG(SEQ ID NO:140);

[0057] GCNIMLPYWGCGRDFECMEQCICQYYQSCG(SEQ ID NO:142);

[0058] GCNIXLPFWGCGRDFECMSDCICQYYQSCG (SEQ ID NO: 144), wherein X is norleucine (Nle); GCNIXLPFWGCGRDFECVSQCICQYYQSCG (SEQ ID NO: 145), wherein X is norleucine (Nle); GCNIXLPYWGCGRDFECMEQCICQYYQSCG (SEQ ID NO: 146), wherein X is norleucine (Nle); and GCDVXQPYWGCGPDIDCLSNCICHWYNSCG (SEQ ID NO: 224), wherein X is norleucine. In certain embodiments according to (or as applied to) any of the embodiments above, the CKP comprises the amino acid sequence shown in GCNIMLPFWGCGRDFECMEQCICQYYQSCG (SEQ ID NO: 113). In certain embodiments according to (or as applied to) any of the above embodiments, the CKP comprises the amino acid sequence shown in GCNIMLPFWGCGRDFECVYRCICQYYQSCG (SEQ ID NO: 114). In certain embodiments according to (or as applied to) any of the above embodiments, the CKP comprises the amino acid sequence shown in GCDVMQPYWGCGPDIDCFVRCLCHWYNSCG (SEQ ID NO: 139). In certain embodiments according to (or as applied to) any of the above embodiments, the CKP comprises the amino acid sequence shown in GCDVMQPYWGCGPDIDCLSNCICHWYNSCG (SEQ ID NO: 140). In certain embodiments according to (or as applied to) any of the above embodiments, the CKP comprises the amino acid sequence shown in GCNIMLPYWGCGRDFECMEQCICQYYQSCG (SEQ ID NO: 142). In certain embodiments according to (or as applied to) any of the above embodiments, the CKP comprises the amino acid sequence shown in GCNIXLPFWGCGRDFECMSDCICQYYQSCG (SEQ ID NO: 144), wherein X is norleucine (Nle). In certain embodiments according to (or as applied to) any of the above embodiments, the CKP comprises the amino acid sequence shown in GCNIXLPFWGCGRDFECVSQCICQYYQSCG (SEQ ID NO: 145), wherein X is norleucine (Nle). In certain embodiments according to (or as applied to) any of the above embodiments, the CKP comprises the amino acid sequence shown in GCNIXLPYWGCGRDFECMEQCICQYYQSCG (SEQ ID NO: 146), wherein X is norleucine (Nle).In certain embodiments according to (or as applied to) any of the above embodiments, the CKP comprises the amino acid sequence shown in GCDVXQPYWGCGPDIDCLSNCICHWYNSCG (SEQ ID NO: 224), wherein X is norleucine. In certain embodiments according to (or as applied to) any of the above embodiments, the CKP binds to VEGF-A.

[0059] In certain embodiments, a non-naturally occurring cystine knot peptide (CKP) is provided that comprises an amino acid selected from the group consisting of:

[0060] GCDVX1QPYWGCGPDI-D / E-CLS-N / K / X2-CICHWYNSCG(SEQ ID NO:534), GCDVX1QPYWGCGPDI-N / K / X2-CLS-D / E-CICHWYNSCG(SEQ ID NO:535), GCNIX1LPYWGCGRDF-D / E-CME-N / K / X2-CICQYYQSCG (SEQ ID NO:538),

[0061] GCNIX1LPYWGCGRDF-N / K / X2-CME-D / E-CICQYYQSCG (SEQ ID NO: 539), GCNIX1LPFWGCGRDF-D / E-CVS-N / K / X2-CICQYYQSCG (SEQ ID NO: 540) and GCNIX1LPFWGCGRDF-N / K / X2-CVS-D / E-CICQYYQSCG (SEQ ID NO: 541), wherein X1 is norleucine and X2 is ornithine. In certain embodiments according to (or as applied to) any of the embodiments above, the CKP comprises the amino acid sequence shown in GCDVX1QPYWGCGPDI-D / E-CLS-N / K / X2-CICHWYNSCG (SEQ ID NO: 534), wherein X1 is norleucine and X2 is ornithine. In certain embodiments according to (or as applied to) any of the above embodiments, the CKP comprises the amino acid sequence shown in GCDVXQPYWGCGPDIDCLSKCICHWYNSCG (SEQ ID NO: 536), wherein X is norleucine. In certain embodiments according to (or as applied to) any of the above embodiments, the CKP comprises the amino acid sequence shown in GCDVX1QPYWGCGPDIDCLSX2CICHWYNSCG (SEQ ID NO: 537), wherein X1 is norleucine and X2 is ornithine. In certain embodiments according to (or as applied to) any of the above embodiments, the CKP comprises the amino acid sequence shown in GCDVX1QPYWGCGPDI-N / K / X2-CLS-D / E-CICHWYNSCG (SEQ ID NO: 535), wherein X1 is norleucine and X2 is ornithine. In certain embodiments according to (or as applied to) any of the above embodiments, the CKP comprises the amino acid sequence shown in GCNIX1LPYWGCGRDF-D / E-CME-N / K / X2-CICQYYQSCG (SEQ ID NO: 538), wherein X1 is norleucine and X2 is ornithine. In certain embodiments according to (or as applied to) any of the above embodiments, the CKP comprises the amino acid sequence shown in GCNIX1LPYWGCGRDFECMEKCICQYYQSCG (SEQ ID NO: 543), wherein X is norleucine. In certain embodiments according to (or as applied to) any of the above embodiments, the CKP comprises the amino acid sequence shown in GCNIX1LPYWGCGRDFECMEX2CICQYYQSCG (SEQ ID NO: 544), wherein X1 is norleucine and X2 is ornithine.In certain embodiments according to (or as applied to) any of the above embodiments, the CKP comprises the amino acid sequence shown in GCNIX1LPYWGCGRDF-N / K / X2-CME-D / E-CICQYYQSCG (SEQ ID NO: 539), wherein X1 is norleucine and X2 is ornithine. In certain embodiments according to (or as applied to) any of the above embodiments, the CKP comprises the amino acid sequence shown in GCNIX1LPFWGCGRDF-D / E-CVS-N / K / X2-CICQYYQSCG (SEQ ID NO: 540), wherein X1 is norleucine and X2 is ornithine. In certain embodiments according to (or as applied to) any of the above embodiments, the CKP comprises the amino acid sequence shown in GCNIXLPFWGCGRDFECVSKCICQYYQSCG (SEQ ID NO: 545), wherein X is norleucine. In certain embodiments according to (or as applied to) any of the above embodiments, the CKP comprises the amino acid sequence shown in GCNIX1LPFWGCGRDFECVSX2CICQYYQSCG (SEQ ID NO: 546), wherein X1 is norleucine and X2 is ornithine. In certain embodiments according to (or as applied to) any of the above embodiments, the CKP comprises the amino acid sequence shown in GCNIX1LPFWGCGRDF-N / K / X2-CVS-D / E-CICQYYQSCG (SEQ ID NO: 541), wherein X1 is norleucine and X2 is ornithine. In certain embodiments according to (or as applied to) any of the above embodiments, the CKP binds to VEGF-A.

[0062] In certain embodiments according to (or as applied to) any of the embodiments above, a non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A is provided, wherein the CKP comprises a cystine scaffold structure:

[0063] Z1C1L1C2L2C3L3C4L4C5L5C6Z2

[0064] in:

[0065] Z1 and Z2 are any amino acids;

[0066] L1 is Ring 1 and has a structure selected from the group consisting of: X1X2X3X4X5X6X7X8, X1X2X3X4X5X6X7X8X9, and X1X2X3X4X5X6X7X8X9X 10 , where X1-X 10 Each of is any amino acid;

[0067] L2 is loop 2 and has the structure: X1X2X3X4X5, where each of X1–X5 is any amino acid;

[0068] L3 is loop 3 and has the structure: X1X2X3, where each of X1–X3 is any amino acid;

[0069] L4 is loop 4 and has the structure: X1, wherein X1 is any amino acid;

[0070] L5 is loop 5 and has the structure: X1X2X3X4X5, wherein each of X1–X5 is any amino acid;

[0071] Wherein with reference to the wild-type EETI-II protein having the amino acid sequence shown in SEQ ID NO:1, the CKP has altered disulfide bonds; wherein the altered disulfide bonds are C1-C4, C2-C3 and C5-C6; and wherein the CKP has an alpha helix content percentage of at least 20%.

[0072] In certain embodiments according to (or as applied to) any of the above embodiments, Z1 and Z2 are any amino acid, more than one amino acid, or an unnatural amino acid. In certain embodiments according to (or as applied to) any of the above embodiments, X1-X2 in L1 is any amino acid, more than one amino acid, or an unnatural amino acid. 10In certain embodiments according to (or as applied to) any of the above embodiments, each of X1-X5 in L2 is any amino acid or unnatural amino acid. In certain embodiments according to (or as applied to) any of the above embodiments, each of X1-X3 in L3 is any amino acid or unnatural amino acid. In certain embodiments according to (or as applied to) any of the above embodiments, X1 in L4 is any amino acid or unnatural amino acid. In certain embodiments according to (or as applied to) any of the above embodiments, each of X1-X5 in L5 is any amino acid or unnatural amino acid. In certain embodiments, the unnatural amino acid is selected from the group consisting of: L-propargylglycine-PEG6-, L-sulfotyrosine, L-norleucine, L-1-naphthylalanine, L-2-naphthylalanine, L-2-chlorotryptophan, L-3-fluorotyrosine, L-4-fluorophenylalanine, γ-benzyl-L-proline, γ-(4-fluoro-benzyl)-L-proline, 4-OH-L-proline, 4-fluoro-L-proline, 4-[4-(trifluoromethyl)-benzyl]-L-proline, 3,4-difluoro-L-phenylalanine, Acid, 3,4-dichloro-L-phenylalanine, 4-chloro-L-phenylalanine, 3-F,4-Cl-L-phenylalanine, 2-pyridone (NH para)-L-alanine, pyridone (NH meta)-L-alanine, 3-(1-N-methylindole)-L-alanine, 3-(1-N-ethylindole)-L-alanine, 3-(1-N-isopropylindole)-L-alanine, 3-(5-aza-indole)-L-alanine, 4-methyl-L-phenylalanine, 2-naphthyl-L-alanine, L-4,4'-biphenylalanine, 3 -(3-quinolyl)-L-alanine, 3-(2-quinolyl)-L-alanine, 3-(2-quinoxalinyl)-L-alanine, 4-methyl-2-pyridyl-alanine, 4-ethyl-2-pyridyl-L-alanine, benzothiazole-L-alanine, benzothiophene-L-alanine, 3-isoquinolyl-L-alanine, tert-butyl-L-alanine (also known as L-neopentylglycine), 3-cyclobutyl-L-alanine, cyclopentyl-L-alanine, 5,5,5-trifluoro-L-leucine, tert-butyl-L-glycine (also known as L-tert- -leucine), L-cyclopentylglycine, L-cyclobutylglycine, 3,4-hydroxy-L-phenylalanine, 3,4-fluoro-L-phenylalanine, 3-fluoro,4-OH-L-phenylalanine, 2-chloro-L-tyrosine, 2-methyl-L-tyrosine, 2-ethyl-L-tyrosine, 4-(naphthalene-1-ol)-L-alanine, D-serine, L-β-homoserine, L-β-alanine, N-α-methylglycine, glycine amide, glycerol glycine ester, ethylene glycol glycine ester, oxetane-3-yl glycine ester and glycine morpholine amide.

[0073] In certain embodiments according to (or as applied to) any of the embodiments above, the non-naturally occurring (CKP) that binds VEGF-A binds to VEGF-A with an affinity of 500 pM or less. In certain embodiments according to (or as applied to) any of the embodiments above, the binding affinity is determined via surface plasmon resonance.

[0074] In certain embodiments according to (or as applied to) any of the above embodiments, Z1 and / or Z2 are more than one amino acid, or non-natural amino acids. In certain embodiments, Z2 is two amino acids. In certain embodiments, Z2 is three amino acids. In certain embodiments according to (or as applied to) any of the above embodiments, in L5, each of X1-X5 is any amino acid, except that X2 is not proline (P). In certain embodiments according to (or as applied to) any of the above embodiments, in L5, each of X1-X5 is any amino acid, except that X4 is not glycine (G).

[0075] In certain embodiments according to (or as applied to) any of the embodiments above, the C-terminal carboxyl group of the non-naturally occurring (CKP) that binds VEGF-A is modified (such as capped). In certain embodiments according to (or as applied to) any of the embodiments above, the N-terminal amino group of the non-naturally occurring (CKP) that binds VEGF-A is modified (such as capped). In certain embodiments according to (or as applied to) any of the embodiments above, the C-terminal carboxyl group of the non-naturally occurring (CKP) that binds VEGF-A is capped and the N-terminal amino group of the non-naturally occurring (CKP) that binds VEGF-A is modified (such as capped).

[0076] In certain embodiments according to (or as applied to) any of the embodiments above, the C-terminal carboxyl group of the non-naturally occurring (CKP) that binds VEGF-A is amidated. In certain embodiments according to (or as applied to) any of the embodiments above, the N-terminal amino group of the non-naturally occurring (CKP) that binds VEGF-A is acetylated. In certain embodiments according to (or as applied to) any of the embodiments above, the C-terminal carboxyl group of the non-naturally occurring (CKP) that binds VEGF-A is amidated and the N-terminal amino group of the non-naturally occurring (CKP) that binds VEGF-A is acetylated.

[0077] In certain embodiments according to (or as applied to) any of the above embodiments, the non-naturally occurring (CKP) that binds to VEGF-A inhibits VEGF-A activity. In certain embodiments according to (or as applied to) any of the above embodiments, the CKP has an IC of between about 0.5 nM and about 1.0 nM. 50 Inhibition of VEGF-A activity. In certain embodiments according to (or as applied to) any of the embodiments above, the non-naturally occurring EETI-II scaffold protein binds human VEGF-A, mouse VEGF-A, and rat VEGF-A.

[0078] In certain embodiments according to (or as applied to) any of the above embodiments, the non-naturally occurring CKP competes with the antibody G6.31 for binding to VEGF-A. In certain embodiments according to (or as applied to) any of the above embodiments, a non-naturally occurring CKP is provided that competes with the non-naturally occurring (CKP) that binds to VEGF-A in any of the above embodiments for binding to VEGF-A.

[0079] In certain embodiments according to (or as applied to) any of the above embodiments, the non-naturally occurring CKP binds to an epitope on VEGF-A, wherein the epitope includes at least one of the amino acid residues selected from the group consisting of: V14, V15, F17, D19, Y21, Q22, Y25, I46, K48, N62, D63, L66, M81, I83, K84, P85, H86, G88, Q89, I91, C104, R105 and P106.

[0080] In certain embodiments according to (or as applied to) any of the embodiments above, the residue is selected from the group consisting of: K48, N62 and D63. In certain embodiments according to (or as applied to) any of the embodiments above, the residue is selected from the group consisting of: Y21, Y25 and P106. In certain embodiments according to (or as applied to) any of the embodiments above, the residue is selected from the group consisting of: H86 and Q89. In certain embodiments according to (or as applied to) any of the embodiments above, the residue is selected from the group consisting of: M81, D19 and Q22. In certain embodiments according to (or as applied to) any of the embodiments above, the residue is selected from the group consisting of: F17, M81 and I91. In certain embodiments according to (or as applied to) any of the embodiments above, the residue is selected from the group consisting of: V14, F17, D19, Q22, M81 and I91. In certain embodiments according to (or as applied to) any of the embodiments above, the residue is selected from the group consisting of: Y25.

[0081] In certain embodiments according to (or as applied to) any of the above embodiments, the non-naturally occurring CKP that binds VEGF-A is conjugated to a therapeutic agent. In certain embodiments according to (or as applied to) any of the above embodiments, the non-naturally occurring CKP that binds VEGF-A is conjugated to a label. In certain embodiments according to (or as applied to) any of the above embodiments, the label is selected from the group consisting of a radioisotope, a fluorescent dye, and an enzyme.

[0082] In certain embodiments according to (or as applied to) any of the above embodiments, an isolated nucleic acid is provided, encoding the non-naturally occurring (CKP) that binds VEGF-A of any of the above embodiments. An expression vector is also provided, encoding the nucleic acid molecule of any of the above embodiments. A cell is also provided, comprising a cell of the expression vector of any of the above embodiments. A method of producing the non-naturally occurring (CKP) that binds VEGF-A of any of the above embodiments is also provided, the method comprising: culturing the cell of any of the above embodiments, and recovering the non-naturally occurring (CKP) that binds VEGF-A from the cell culture.

[0083] Also provided is a method of producing a non-naturally occurring (CKP) that binds VEGF-A of any one of the above embodiments, the method comprising chemically synthesizing the non-naturally occurring (CKP) that binds VEGF-A.

[0084] Provided herein is a composition comprising the non-naturally occurring (CKP) that binds VEGF-A of any of the above embodiments and a pharmaceutically acceptable carrier. In certain embodiments according to (or as applied to) any of the above embodiments, the composition comprises one or more additional compounds. In certain embodiments according to (or as applied to) any of the above embodiments, the additional compound binds a second biomolecule selected from the group consisting of: interleukin-6 (IL-6); interleukin-6 receptor (IL-6R); PDGF; angiopoietin; angiopoietin 2; Tie2; S1P; integrins αvβ3, αvβ5 and α5β1; betacellulin; apelin / APJ; erythropoietin; complement factor D; TNFα; HtrA1; VEGF receptor; ST-2 receptor; and proteins genetically associated with risk of age-related macular degeneration (AMD), such as complement pathway component C2, factor B, factor H, CFHR3, C3b, C5, C5a, C3a, HtrA1, ARMS2, TIMP3, HLA, interleukin-8 (IL-8), CX3CR1, TLR3, TLR4, CETP, LIPC, COL10A1 and TNFRSF10A. In certain embodiments according to (or as applied to) any of the embodiments above, the additional compound is a non-naturally occurring CKP. In certain embodiments according to (or as applied to) any of the embodiments above, the additional compound is an antibody or antigen-binding fragment thereof.

[0085] Provided herein is a method for treating an eye disease characterized by angiogenesis and / or vascular permeability or leakage in a subject, the method comprising administering to the subject an effective amount of a non-naturally occurring (CKP) in conjunction with VEGF-A in any of the above embodiments. In certain embodiments according to (or as applied to) any of the above embodiments, the method further comprises administering one or more additional compounds. In certain embodiments according to (or as applied to) any of the above embodiments, the non-naturally occurring CKP in conjunction with VEGF-A is administered simultaneously with one or more additional compounds. In certain embodiments according to (or as applied to) any of the above embodiments, the non-naturally occurring CKP in conjunction with VEGF-A is administered before or after one or more additional compounds. In certain embodiments according to (or as applied to) any of the above embodiments, the additional compound binds a second biomolecule selected from the group consisting of: interleukin-6 (IL-6); interleukin-6 receptor (IL-6R); PDGF; angiopoietin; angiopoietin 2; Tie2; S1P; integrins αvβ3, αvβ5 and α5β1; betacellulin; apelin / APJ; erythropoietin; complement factor D; TNFα; HtrA1; VEGF receptor; ST-2 receptor; and proteins genetically associated with risk of age-related macular degeneration (AMD), such as complement pathway component C2, factor B, factor H, CFHR3, C3b, C5, C5a, C3a, HtrA1, ARMS2, TIMP3, HLA, interleukin-8 (IL-8), CX3CR1, TLR3, TLR4, CETP, LIPC, COL10A1 and TNFRSF10A. In certain embodiments according to (or as applied to) any of the above embodiments, the additional compound is a non-naturally occurring CKP. In certain embodiments according to (or as applied to) any of the above embodiments, the additional compound is an antibody or an antigen-binding fragment thereof. In certain embodiments according to (or as applied to) any of the above embodiments, the eye disease is an intraocular neovascular disease selected from the group consisting of proliferative retinopathy, choroidal neovascularization (CNV), age-related macular degeneration (AMD), diabetic and other ischemic-related retinopathy, diabetic macular edema, pathological myopia, von Hippel-Lindau disease, ocular histoplasmosis, retinal vein occlusion (RVO) (including central retinal vein occlusion (CRVO) and branch retinal vein occlusion (BRVO)), corneal neovascularization, retinal neovascularization and retinopathy of prematurity (ROP).

[0086] In certain embodiments according to (or as applied to) any of the embodiments above, the non-naturally occurring CKP that binds VEGF-A or the composition is administered to a subject via an implantable device. In certain embodiments according to (or as applied to) any of the embodiments above, the implantable device is selected from the group consisting of an ocular insert, a slow release depot, an ocular plug / reservoir, a non-biodegradable ocular implant, or a biodegradable ocular implant.

[0087] In certain embodiments according to (or as applied to) any of the above embodiments, a composition comprising a non-naturally occurring (CKP) binding to VEGF-A of any of the above embodiments is provided for use in treating an eye disease characterized by angiogenesis and / or vascular permeability or leakage in a subject. In certain embodiments according to (or as applied to) any of the above embodiments, the eye disease is an intraocular neovascular disease selected from the group consisting of proliferative retinopathy, choroidal neovascularization (CNV), age-related macular degeneration (AMD), diabetic and other ischemic-related retinopathy, diabetic macular edema, pathological myopia, von Hippel-Lindau disease, ocular histoplasmosis, retinal vein occlusion (RVO) (including central retinal vein occlusion (CRVO) and branch retinal vein occlusion (BRVO)), corneal neovascularization, retinal neovascularization, and retinopathy of prematurity (ROP). In certain embodiments according to (or as applied to) any of the above embodiments, the composition is administered to the subject via an implantable device. In certain embodiments according to (or as applied to) any of the embodiments above, the implantable device is selected from the group consisting of an ocular insert, a slow release depot, an ocular plug / reservoir, a non-biodegradable ocular implant, or a biodegradable ocular implant.

[0088] In certain embodiments according to (or as applied to) any of the above embodiments, a composition comprising a non-naturally occurring (CKP) binding to VEGF-A of any of the above embodiments is provided for use in treating an eye disease characterized by angiogenesis and / or vascular permeability or leakage in a subject. In certain embodiments according to (or as applied to) any of the above embodiments, the eye disease is an intraocular neovascular disease selected from the group consisting of proliferative retinopathy, choroidal neovascularization (CNV), age-related macular degeneration (AMD), diabetic and other ischemic-related retinopathy, diabetic macular edema, pathological myopia, von Hippel-Lindau disease, ocular histoplasmosis, retinal vein occlusion (RVO) (including central retinal vein occlusion (CRVO) and branch retinal vein occlusion (BRVO)), corneal neovascularization, retinal neovascularization, and retinopathy of prematurity (ROP). In certain embodiments according to (or as applied to) any of the above embodiments, the drug is administered to the subject via an implantable device. In certain embodiments according to (or as applied to) any of the embodiments above, the implantable device is selected from the group consisting of an ocular insert, a slow release depot, an ocular plug / reservoir, a non-biodegradable ocular implant, or a biodegradable ocular implant.

[0089] In certain embodiments according to (or as applied to) any of the embodiments above, the non-naturally occurring (CKP) that binds VEGF-A is formulated for long-acting delivery.

[0090] Provided herein is a formulation comprising the non-naturally occurring (CKP) that binds VEGF-A of any of the above embodiments and PLGA. In certain embodiments according to (or as applied to) any of the above embodiments, the PLGA is a PLGA rod.

[0091] Also provided herein are non-naturally occurring cystine knot peptides (CKPs) that bind to human low-density lipoprotein receptor-related protein 6 (LRP6), wherein the CKP comprises a cystine scaffold structure:

[0092] Z1C1L1C2L2C3L3C4L4C5L5C6Z2;

[0093] in:

[0094] Z1 and Z2 are any amino acids;

[0095] L1 is Ring 1 and has a structure selected from the group consisting of X1X2X3X4X5X6, X1X2X3X4X5X6X7, X1X2X3X4X5X6X7X8, X1X2X3X4X5X6X7X8X9, and X1X2X3X4X5X6X7X8X9X 10 , where X1-X 10 Each of is any amino acid;

[0096] L2 is loop 2 and has the structure: X1X2X3X4X5, where each of X1–X5 is any amino acid;

[0097] L3 is loop 3 and has the structure: X1X2X3, where each of X1–X3 is any amino acid;

[0098] L4 is loop 4 and has the structure: X1, wherein X1 is any amino acid; and

[0099] L5 is loop 5 and has the structure: X1X2X3X4X5, wherein each of X1-X5 is any amino acid.

[0100] In certain embodiments according to (or as applied to) any of the above embodiments, Z1 and / or Z2 are more than one amino acid, or non-natural amino acid. In certain embodiments, Z2 is two amino acids. In certain embodiments, Z2 is three amino acids.

[0101] In certain embodiments according to (or as applied to) any of the embodiments above, Z1 and / or Z2 is G.

[0102] In certain embodiments according to (or as applied to) any of the above embodiments, in L1: X1 is an amino acid selected from R, V, M, A, G, N, S, and E; X2 is an amino acid selected from T, N, S, G, R, and A; X3 is an amino acid selected from N, R, H, V, K, S, G, I, and Y; X4 is an amino acid selected from R, V, N, I, K, S, and T; X5 is an amino acid selected from V, R, K, I, T, S, L, and N; and X6 is an amino acid selected from K, G, A, I, R, N, S, and V. In certain embodiments according to (or as applied to) any of the above embodiments, in L1: X7 is an amino acid selected from G, R, K, E, P, and T. In certain embodiments according to (or as applied to) any of the above embodiments, in L1: X8 is an amino acid selected from G, R, K, Q, A, and S. In certain embodiments according to (or as applied to) any of the above embodiments, in L1: X9 is an amino acid selected from R or G. In certain embodiments according to (or as applied to) any of the above embodiments, in L1: X 10 is an amino acid selected from the group consisting of E, W and G.

[0103] In certain embodiments according to (or as applied to) any of the embodiments above, in L5: X1 is an amino acid selected from G, S, N, Y, A and R; X2 is an amino acid selected from P, G, S, V, E, R, F and D; X3 is an amino acid selected from N, G, S, E, P, K, H and R; X4 is an amino acid selected from G, R, H, S, Q, V and D; and X5 is an amino acid selected from F, D, N, R, G, Y, S and T.

[0104] In certain embodiments according to (or as applied to) any of the above embodiments, in L2, X1 is K, X2 is Q, X3 is D, X4 is S, and X5 is D. In certain embodiments according to (or as applied to) any of the above embodiments, in L3, X1 is L, X2 is A, and X3 is G. In certain embodiments according to (or as applied to) any of the above embodiments, in L4, X1 is X. BRIEF DESCRIPTION OF THE DRAWINGS

[0105] Figure 1 Depicting the structure of the EETI-II cystine knot protein.

[0106] Figure 2A Shown are the results of experiments conducted to determine whether EGF_CKP9.54.90 disrupts the interactions between VEGF-A(8-109) and KDR; between VEGF-A(8-109) and Flt-1; between VEGF-A 165 and KDR; between VEGF-B and Flt-1; between VEGF-C and Flt-4; between VEGF-D and Flt-4; and between PIGF-2 and FLT-1.

[0107] Figure 2B Shown are the results of experiments conducted to determine whether EGF_CKP9.54.90 disrupts the interactions between VEGF-A(8-109) and KDR; between VEGF-A(8-109) and Flt-1; between VEGF-A 165 and KDR; between EGF and EGFR; between PDGF and PDGFR; between NGF and NGFR; and between IGF and IGFR.

[0108] Figure 3 Shown are the results of experiments performed to determine whether VEGF_CKP9.54.90, VEGF_CKP9.54, and VEGF_CKP9.63.12 inhibit trypsin protease activity.

[0109] Figure 4Shown are the results of experiments performed to determine whether VEGF_CKP9.54.90 and VEGF_CKP9.63.12 are resistant to trypsin digestion.

[0110] Figure 5 The structure of VEGF_CKP9.54.90, the structure of wild-type EETI-II are depicted, and a schematic diagram comparing the disulfide bonding pattern of CKP variants that bind VEGF to that of wild-type EETI-II is provided.

[0111] Figure 6 Depicted is the co-crystal structure of VEGF_CKP9.54.90 in complex with VEGF-A.

[0112] Figure 7 Depicted is a space filling model showing the binding interface of VEGF_CKP9.54.90, antibody G6.31 and domain 2 of Flt-2 on VEGF-A.

[0113] Figure 8 Depicted is a ribbon diagram model showing the binding interface of VEGF_CKP9.54.90, antibody G6.31 and domain 2 of Flt-2 on VEGF-A.

[0114] Figure 9 The contact residues on VEGF-A at the interaction surface between VEGF-A and VEGF_CKP9.54.90 are shown.

[0115] Figure 10 The binding interface of bevacizumab Fab, Z-domain and receptor-blocking peptide v108 on VEGF-A is shown.

[0116] Figure 11 Provides the results of experiments performed to determine the effects of amino acid substitution mutations in VEGF-A on the binding of VEGF_CKP9.54.90 to VEGF-A and the binding of VEGF_CKP9.63.12 to VEGFA. The results of the experiments are displayed relative to two different y axes.

[0117] Figure 12 Presented are the results of experiments performed to determine the effect of VEGF_CKP9.54.90 on CNV in rat eyes.

[0118] Figure 13 Provided to determine the IC of CKP variants that bind VEGF 50 The results of experiments conducted for the value.

[0119] Figure 14The structures of VEGF_CKP9.54 and VEGF_CKP9.63 are depicted. Figure 14 Also shown is a portion of the co-crystal structure of VEGF_CKP9.63 in complex with VEGF-A, which shows that the residue at position 8 within loop 1 of VEGF_CKP9.63 is able to form a hydrogen bond with the side chain of Gln22 of VEGF-A.

[0120] Figure 15 Shown are the results of phage competition ELISA experiments performed to evaluate the binding affinity of clones 9.54-28, 9.54, 9.54.1-2, 9.54.1-36, 9.54.1-42, 9.54.1-63, 9.54.1-90 and 9.54.1 to hVEGF(8-109).

[0121] Figure 16A Shown are the results of phage competition ELISA experiments performed to evaluate the binding affinity of clones 9.63.44-1 to 9.63.44-7.

[0122] Figure 16B Shown are the results of phage competition ELISA experiments performed to evaluate the binding affinity of clones 9.63.44-8 to 9.63.44-14. DETAILED DESCRIPTION OF THE INVENTION

[0123] A non-naturally occurring cystine knot peptide (CKP) is provided that specifically binds to human VEGF-A. The non-naturally occurring CKP exhibits one or more of the following properties: inhibits VEGF-A activity and IC 50 Between less than about 0.5 nM and less than about 1.0 nM; binds to human VEGF-A, mouse VEGF-A and rat VEGF-A; is resistant to trypsin digestion; disulfide bonds are C1-C4, C2-C3 and C5-C6; at least about at least about 15% to at least about 50% alpha helical content; binds to an epitope on VEGF-A that is different from the epitope bound by antibody G6.31, binds to an epitope on VEGF-A that is different from the epitope bound by bevacizumab and / or binds to an epitope on VEGF-A that is different from the epitope bound by Flt-1.

[0124] Also provided are chimeric molecules and conjugates comprising non-naturally occurring cystine knot peptides that bind to VEGF-A, nucleic acids encoding non-naturally occurring CKPs that bind to VEGF-A, and compositions (such as pharmaceutical compositions). Also provided are methods of using non-naturally occurring CKPs that bind to VEGF-A for treating eye diseases and / or conditions (such as ocular vascular proliferative diseases and / or conditions) caused by abnormal (such as excessive) angiogenesis and / or abnormal vascular permeability. Also provided are uses of non-naturally occurring CKPs that bind to VEGF-A in the preparation of drugs for treating eye diseases or conditions.

[0125] In a related aspect, non-naturally occurring CKPs that bind to human low-density lipoprotein receptor-related protein 6 (LRP6) are also provided.

[0126] Unless otherwise indicated, the practice of the present disclosure employs standard methods and conventional techniques in the fields of cell biology, toxicology, molecular biology, biochemistry, cell culture, immunology, oncology, recombinant DNA, and related fields, which are within the skill of the art. Such technical descriptions are in the literature and thus available to those skilled in the art. See, e.g., Alberts, B. et al., “Molecular Biology of the Cell,” 5th ed., Garland Science, New York, NY, 2008; Voet, D. et al., “Fundamentals of Biochemistry: Life at the Molecular Level,” 3rd ed., John Wiley & Sons, Hoboken, NJ, 2008; Sambrook, J. et al., “Molecular Cloning: A Laboratory Manual,” 3rd ed., Cold Spring Harbor Laboratory Press, 2001; Ausubel, F. et al., “Current Protocols in Molecular Biology,” John Wiley & Sons, New York, 1987 and periodically updated; Freshney, RI, “Culture of Animal Cells: A Manual of Basic Technique,” ​​4th ed., John Wiley & Sons, Somerset, NJ, 2000; and the series “Methods in Enzymology,” Academic Press, San Diego, CA.

[0127] definition

[0128] As used herein, "non-naturally occurring" means, for example, a polypeptide comprising an amino acid sequence not found in nature, or, for example, a nucleic acid comprising a nucleotide sequence not found in nature. The "non-naturally occurring cystine knot peptide" or "non-naturally occurring CKP" (or nucleic acids encoding them) provided herein do not have the amino acid sequence of the wild-type EETI-II protein, i.e., GCPRILMRCKQDSDCLAGCVCGPNGFCG (SEQ ID NO: 1), wherein loop 1 (L1) is the amino acid sequence PRILMR (SEQ ID NO: 92), loop 2 (L2) is the amino acid sequence KQDSD (SEQ ID NO: 93), loop 3 (L3) is the amino acid sequence LAG, loop 4 (L4) is the amino acid V, and loop 5 (L5) is the amino acid sequence GPNGF (SEQ ID NO: 15). The non-naturally occurring CKP provided herein can be produced by genetic engineering methods or by chemical synthesis methods. Therefore, the non-natural CKPs described herein can be recombinant, i.e., produced by cells or nucleic acids or vectors that have been modified by introducing heterologous nucleic acids or by changing natural nucleic acids into a form that is non-natural to the cell, or the cells are derived from cells so modified. Alternatively, the non-natural CKPs described herein can be produced by chemical peptide synthesis.

[0129] As used herein, the term "cystine knot peptide" or "CKP" refers to a peptide of 26-50 amino acids in length containing six conserved cysteine ​​residues that form three disulfide bonds. One of the disulfide bonds penetrates a macrocycle formed by the other two disulfide bonds and their interconnected backbones, thereby producing a characteristic knot topology with multiple loops exposed on the surface. Loops are defined as amino acid regions flanking the six conserved cysteine ​​residues and are highly variable in nature.

[0130] As used herein, "amino acid change" refers to the addition, deletion or substitution of at least one amino acid in, for example, a peptide sequence (such as in a WT EETI-II peptide sequence to produce a non-naturally occurring CKP, or in a non-naturally occurring CKP to produce another non-naturally occurring CKP).

[0131] An "isolated" non-naturally occurring CKP or composition is a CKP or composition that has been identified and separated and / or recovered from a component of its natural environment. The contaminant components of its natural environment are substances that will interfere with the diagnostic or therapeutic use of the non-naturally occurring CKP, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In a preferred embodiment, the non-naturally occurring CKP or composition will be purified (1) to greater than 95% by weight of the non-naturally occurring CKP, as determined by the Lowry method, and most preferably to greater than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence, such as by using a spinning cup sequencer, or (3) to homogeneity, by SDS-PAGE using Coomassie blue or preferably silver dye under reducing or non-reducing conditions. An isolated non-naturally occurring CKP includes an in situ CKP within a recombinant cell, since at least one component of the natural environment of the CKP will be absent. The isolated non-naturally occurring CKP will be prepared by at least one purification step.

[0132] "Percent (%) amino acid sequence identity" or "homology" relative to the polypeptide sequences identified herein is defined as the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the polypeptide being compared, after comparing the sequences with any conservative substitutions as part of the sequence identity. Alignment for determining the percentage of amino acid sequence identity can be achieved in a variety of ways within the capabilities of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for measuring the alignment, including any algorithm required to achieve maximum alignment within the full-length sequence being compared. However, for the purposes of this article, the amino acid sequence identity % values ​​are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and the source code has been submitted to the US Copyright Office, Washington DC, 20559 (US Copyright Office Washington DC 20559) with user documentation, where it is registered with US Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California. The ALIGN-2 program should be compiled for use on a UNIX operating system (preferably Digital UNIX V4.0D). All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0133] As used herein, the term "epitope" refers to a protein determinant that can be specifically bound by a non-natural CKP provided herein. An epitope can include about 3-10 amino acids in a three-dimensional conformation, which is unique to the epitope. These amino acids can be linear (i.e., continuous in the amino acid sequence) or they can be located at different parts of the protein (i.e., discontinuous in the amino acid sequence) in the protein. The method for determining the three-dimensional conformation of the amino acid in the protein or at the interface of two proteins is known in the art, and includes, for example, X-ray crystallography and 2-dimensional nuclear magnetic resonance.

[0134] The terms "disulfide bonding pattern (DBP)", "disulfide linkage" and "disulfide bonding pattern" refer to the cysteine ​​linkage pattern relative to the WT EETI-II protein. The WT EETI-II protein contains six conserved cysteine ​​residues (numbered 1-6) that form three disulfide bonds with linkages C1-C4, C2-C5 and C3-C6. The disulfide bonding pattern is topologically invariant, meaning that the disulfide bonds can only be altered by unwinding one or more disulfide bonds (such as using redox conditions).

[0135] "Subject," "patient," or "individual" for therapeutic purposes refers to any animal classified as a mammal, including humans, domesticated and farm animals, and zoo, sport or pet animals, such as dogs, horses, cats, cows, etc. Preferably, the mammal is a human.

[0136] An "effective amount" of a non-naturally occurring CKP (or a composition comprising such a non-naturally occurring CKP) as disclosed herein is an amount sufficient to achieve a particular stated purpose. An "effective amount" can be determined empirically and by known methods related to the stated purpose.

[0137] The term "therapeutically effective amount" refers to an amount of a non-naturally occurring CKP or composition as disclosed herein that is effective to "treat" a disease or condition in a mammal, such as a human patient. In the case of an ocular disease or condition, such as an ocular vascular proliferative disease or an ocular condition characterized by excessive angiogenesis, a therapeutically effective amount of a non-naturally occurring CKP that binds VEGF-A as described herein (or a composition comprising such a non-naturally occurring CKP that binds VEGF-A) refers to an amount that reduces, stops, or prevents at least one symptom of the ocular disease, such as a symptom or condition of the ocular disease described in further detail elsewhere herein. For example, an effective amount will be considered to be an amount sufficient to reduce or prevent symptoms of an ocular disease or ocular condition, such as an ocular vascular proliferative disease or an ocular condition characterized by excessive angiogenesis, such as complete or partial elimination and / or maintenance of an ocular disease as measured by optical coherence tomography (OCT) or improvement and / or maintenance of best corrected visual acuity (such as greater than 5 letters as assessed by the EDTRS eye chart), or a decrease in the size of neovascularization or neovascular permeability as assessed by fluorescein fundus angiography. An effective amount as used herein will also include an amount sufficient to prevent or delay the development of, for example, macular edema, enhanced permeability (such as retinal vascular permeability), the size of CNV lesions, and vision loss. An effective amount as used herein will also include an amount sufficient to prevent or delay the development of symptoms of an ocular disease, alter the course of a symptomatic disease (such as, but not limited to, slowing the progression of symptoms of an ocular disease), or reverse a symptom of a disease.

[0138] As used herein, "treatment" is a scheme to obtain beneficial or desired results (including clinical results). For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms caused by an eye disease, reducing the scope of an eye disease, stabilizing an eye disease (e.g., preventing or delaying disease deterioration), preventing or delaying the spread of a disease (such as, around eye tissue), preventing or delaying the recurrence of an eye disease, delaying or slowing the progression of an eye disease, improving the disease state, providing relief or elimination (partial or complete) of an eye disease, reducing the dosage of one or more other drugs required for treating an eye disease, delaying the progression of an eye disease, increasing or improving the quality of life, and / or preventing or delaying vision loss. "Treatment" also encompasses the reduction of the pathological consequences of an eye disease (such as, for example, vision loss). The methods provided herein contemplate any one or more of these aspects of treatment.

[0139] A "disorder" is any condition that would benefit from treatment using a non-naturally occurring CKP that binds VEGF-A as described herein. VEGF-A-related disorders to be treated herein include ocular diseases and disorders (such as ocular vascular proliferative diseases or ocular disorders characterized by excessive angiogenesis), as described elsewhere herein.

[0140] As used herein, by "pharmaceutically acceptable" or "pharmacologically compatible" is meant a substance that is not biologically or otherwise undesirable, e.g., the substance can be incorporated into a pharmaceutical composition administered to a patient without causing any significant undesirable biological effect or interacting in a deleterious manner with any other component of the composition in which it is present. Pharmaceutically acceptable carriers or excipients have preferably met the required standards of toxicology and manufacturing testing and / or are included on the Inactive Ingredient Guide prepared by the US Food and Drug Administration.

[0141] The term "detection" is intended to include determining the presence or absence of a substance, or quantifying the amount of a substance (such as a target ligand). Thus, the term refers to the use of the substances, compositions and methods provided herein for qualitative and quantitative determinations. In general, the specific technique used for detection is not critical to the practice of the present invention.

[0142] For example, "detection" according to the present invention may include: observing the presence or absence of a target ligand (including, but not limited to, a human low-density lipoprotein receptor-related protein 6 (LRP6) polypeptide or a human vascular endothelial growth factor A (VEGF-A) polypeptide); changes in the level of a target ligand; and / or changes in the biological function / activity of a target ligand. In certain embodiments, "detection" may include detecting the level of a target ligand (e.g., the level of a polypeptide of human LRP6 or human VEGF). Detection may include quantifying a change (increase or decrease) in any value between 10% and 90% or in any value between 30% and 60% or in any value exceeding 100% when compared to a control. Detection may include quantifying a change in any value between 2-fold and 10-fold (including endpoints) or more (e.g., 100-fold).

[0143] As used herein, the term "label" refers to a detectable compound or composition that is directly or indirectly conjugated to a non-naturally occurring CKP. The label itself can be detectable per se (e.g., a radioisotope label or a fluorescent label), or, in the case of an enzymatic label, can catalyze a chemical change in a detectable substrate compound or composition.

[0144] With respect to the binding of a non-naturally occurring CKP to a target ligand, the term "specifically binds to a particular target ligand" or "specifically binds to a particular target ligand" or "is specific for a particular target ligand" means binding that is measurably different from non-specific interactions. Specific binding can be measured, for example, by determining the binding of a molecule compared to the binding of a control molecule, which is typically a molecule of similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target, for example, an excess of unlabeled target. In this case, if the binding of the labeled target to the probe is competitively inhibited by an excess of unlabeled target, specific binding is indicated. In certain embodiments, the extent of binding of the non-naturally occurring CKP to a "non-target" ligand will be less than about 10% of the binding of the non-naturally occurring CKP to its target ligand (such as LRP6 or VEGF-A), as determined by, for example, fluorescence activated cell sorting (FACS) analysis or radioimmunoprecipitation (RIA). In certain embodiments, the non-naturally occurring CKP of the present disclosure specifically binds to a target ligand (such as human low-density lipoprotein receptor-related protein 6 (LRP6) or human vascular endothelial growth factor A (VEGF-A)) with a dissociation constant (Kd) of equal to or less than 100 nM, optionally less than 10 nM, optionally less than 1 nM, optionally less than 0.5 nM, optionally less than 0.1 nM, optionally less than 0.01 nM, or optionally less than 0.005 nM; measured at a temperature of about 4°C, 25°C, 37°C, or 45°C.

[0145] References herein to "about" a value or parameter refer to the usual error ranges for the respective values ​​that are readily known to those skilled in the art. References herein to "about" a value or parameter include (and describe) aspects relating to the value or parameter itself. For example, a description referring to "about X" includes a description of "X".

[0146] It should be understood that aspects and embodiments of the invention described herein include "comprising," "consisting of," and "consisting essentially of" aspects and embodiments.

[0147] All references cited herein, including patent applications and publications, are hereby incorporated by reference in their entirety.

[0148] Non-naturally occurring cystine knot peptide (CKP) that binds to human vascular endothelial growth factor A (VEGF-A)

[0149] In certain embodiments, provided herein are non-naturally occurring cystine knot peptides (CKPs) that bind vascular endothelial growth factor A (VEGF-A), wherein the CKP comprises the following cystine scaffold structure (ie, scaffold structure I):

[0150] Z1C1L1C2L2C3L3C4L4C5L5C6Z2 (I)

[0151] in:

[0152] Z1 and Z2 are any amino acids;

[0153] L1 is Ring 1 and has a structure selected from the group consisting of: X1X2X3X4X5X6X7X8, X1X2X3X4X5X6X7X8X9, and X1X2X3X4X5X6X7X8X9X 10 , where X1-X 10 Each of is any amino acid;

[0154] L2 is Ring 2 and has the structure: X1X2X3X4X5, wherein each of X1-X5 is any amino acid or an unnatural amino acid selected from the group consisting of L-propargylglycine-PEG6-, L-sulfotyrosine, L-norleucine, L-1-naphthylalanine, L-2-naphthylalanine, L-2-chlorotryptophan, L-3-fluorotyrosine, L-4-fluorophenylalanine, γ-benzyl-L-proline, γ-(4-fluoro-benzyl)-L-proline, 4-OH-L-proline, 4-fluoro-L-proline, 4-[4-(trifluoromethyl)-] ... )-benzyl]-L-proline, 3,4-difluoro-L-phenylalanine, 3,4-dichloro-L-phenylalanine, 4-chloro-L-phenylalanine, 3-F,4-Cl-L-phenylalanine, 2-pyridone (NH para)-L-alanine, pyridone (NH meta)-L-alanine, 3-(1-N-methylindole)-L-alanine, 3-(1-N-ethylindole)-L-alanine, 3-(1-N-isopropylindole)-L-alanine, 3-(5-aza-indole)-L-alanine, 4-methyl-L-phenylalanine, 2-naphthyl-L-alanine , L-4,4'-biphenylalanine, 3-(3-quinolyl)-L-alanine, 3-(2-quinolyl)-L-alanine, 3-(2-quinoxalinyl)-L-alanine, 4-methyl-2-pyridyl-alanine, 4-ethyl-2-pyridyl-L-alanine, benzothiazole-L-alanine, benzothiophene-L-alanine, 3-isoquinolyl-L-alanine, tert-butyl-L-alanine (also known as L-neopentylglycine), 3-cyclobutyl-L-alanine, cyclopentyl-L-alanine, 5,5,5-trifluoro-L-leucine, tert-butyl-L-glycine acid (also known as L-tert-leucine), L-cyclopentylglycine, L-cyclobutylglycine, 3,4-hydroxy-L-phenylalanine, 3,4-fluoro-L-phenylalanine, 3-fluoro,4-OH-L-phenylalanine, 2-chloro-L-tyrosine, 2-methyl-L-tyrosine, 2-ethyl-L-tyrosine, 4-(naphthalene-1-ol)-L-alanine, D-serine, L-β-homoserine, L-β-alanine, N-α-methylglycine, glycine amide, glycerol glycine ester, ethylene glycol glycine ester, oxetan-3-yl glycine ester, and glycine morpholine amide.

[0155] L3 is Ring 3 and has the structure: X1X2X3, wherein each of X1-X3 is any amino acid or an unnatural amino acid selected from the group consisting of L-propargylglycine-PEG6-, L-sulfotyrosine, L-norleucine, L-1-naphthylalanine, L-2-naphthylalanine, L-2-chlorotryptophan, L-3-fluorotyrosine, L-4-fluorophenylalanine, γ-benzyl-L-proline, γ-(4-fluoro-benzyl)-L-proline, 4-OH-L-proline, 4-fluoro-L-proline, 4-[4-(trifluoromethyl)-benzyl] ... yl]-L-proline, 3,4-difluoro-L-phenylalanine, 3,4-dichloro-L-phenylalanine, 4-chloro-L-phenylalanine, 3-F,4-Cl-L-phenylalanine, 2-pyridone (NH para)-L-alanine, pyridone (NH meta)-L-alanine, 3-(1-N-methylindole)-L-alanine, 3-(1-N-ethylindole)-L-alanine, 3-(1-N-isopropylindole)-L-alanine, 3-(5-aza-indole)-L-alanine, 4-methyl-L-phenylalanine, 2-naphthyl-L-alanine, L -4,4'-biphenylalanine, 3-(3-quinolyl)-L-alanine, 3-(2-quinolyl)-L-alanine, 3-(2-quinoxalinyl)-L-alanine, 4-methyl-2-pyridyl-alanine, 4-ethyl-2-pyridyl-L-alanine, benzothiazole-L-alanine, benzothiophene-L-alanine, 3-isoquinolyl-L-alanine, tert-butyl-L-alanine (also known as L-neopentylglycine), 3-cyclobutyl-L-alanine, cyclopentyl-L-alanine, 5,5,5-trifluoro-L-leucine, tert-butyl-L-glycine (also known as L-tert-leucine), L-cyclopentylglycine, L-cyclobutylglycine, 3,4-hydroxy-L-phenylalanine, 3,4-fluoro-L-phenylalanine, 3-fluoro,4-OH-L-phenylalanine, 2-chloro-L-tyrosine, 2-methyl-L-tyrosine, 2-ethyl-L-tyrosine, 4-(naphthalene-1-ol)-L-alanine, D-serine, L-β-homoserine, L-β-alanine, N-α-methylglycine, glycine amide, glycerol glycine ester, glycol glycine ester, oxetan-3-yl glycine ester, and glycine morpholine amide;

[0156] L4 is Ring 4 and has the structure: X1, wherein X1 is any amino acid or an unnatural amino acid selected from the group consisting of L-propargylglycine-PEG6-, L-sulfotyrosine, L-norleucine, L-1-naphthylalanine, L-2-naphthylalanine, L-2-chlorotryptophan, L-3-fluorotyrosine, L-4-fluorophenylalanine, γ-benzyl-L-proline, γ-(4-fluoro-benzyl)-L-proline, 4-OH-L-proline, 4-fluoro-L-proline, 4-[4-(trifluoromethyl)-benzyl]-L-proline, 3,4-difluoro-L-phenylalanine, 3,4-dichloro-L-phenylalanine, 4-chloro-L-phenylalanine, 3-F,4-Cl-L-phenylalanine, 2-pyridone (NH para)-L-alanine, pyridone (NH meta)-L-alanine, 3-(1-N-methylindole)-L-alanine, 3-(1-N-ethylindole)-L-alanine, 3-(1-N-isopropylindole)-L-alanine, 3-(5-aza-indole)-L-alanine, 4-methyl-L-phenylalanine, 2-naphthyl-L-alanine, L-4,4'- biphenylalanine, 3-(3-quinolyl)-L-alanine, 3-(2-quinolyl)-L-alanine, 3-(2-quinoxalinyl)-L-alanine, 4-methyl-2-pyridyl-alanine, 4-ethyl-2-pyridyl-L-alanine, benzothiazole-L-alanine, benzothiophene-L-alanine, 3-isoquinolyl-L-alanine, tert-butyl-L-alanine (also known as L-neopentylglycine), 3-cyclobutyl-L-alanine, cyclopentyl-L-alanine, 5,5,5-trifluoro-L-leucine, tert-butyl-L-glycine (also known as (L-tert-leucine), L-cyclopentylglycine, L-cyclobutylglycine, 3,4-hydroxy-L-phenylalanine, 3,4-fluoro-L-phenylalanine, 3-fluoro, 4-OH-L-phenylalanine, 2-chloro-L-tyrosine, 2-methyl-L-tyrosine, 2-ethyl-L-tyrosine, 4-(naphthalene-1-ol)-L-alanine, D-serine, L-β-homoserine, L-β-alanine, N-α-methylglycine, glycine amide, glycerol glycine ester, ethylene glycol glycine ester, oxetane-3-yl glycine ester and glycine morpholine amide;

[0157] L5 is Ring 5 and has the structure: X1X2X3X4X5, wherein each of X1-X5 is any amino acid or an unnatural amino acid selected from the group consisting of L-propargylglycine-PEG6-, L-sulfotyrosine, L-norleucine, L-1-naphthylalanine, L-2-naphthylalanine, L-2-chlorotryptophan, L-3-fluorotyrosine, L-4-fluorophenylalanine, γ-benzyl-L-proline, γ-(4-fluoro-benzyl)-L-proline, 4-OH-L-proline, 4-fluoro-L-proline, 4-[4-(trifluoromethyl)-L-proline] -benzyl]-L-proline, 3,4-difluoro-L-phenylalanine, 3,4-dichloro-L-phenylalanine, 4-chloro-L-phenylalanine, 3-F,4-Cl-L-phenylalanine, 2-pyridone (NH para)-L-alanine, pyridone (NH meta)-L-alanine, 3-(1-N-methylindole)-L-alanine, 3-(1-N-ethylindole)-L-alanine, 3-(1-N-isopropylindole)-L-alanine, 3-(5-aza-indole)-L-alanine, 4-methyl-L-phenylalanine, 2-naphthyl-L-alanine, L-4,4'-biphenylalanine, 3-(3-quinolyl)-L-alanine, 3-(2-quinolyl)-L-alanine, 3-(2-quinoxalinyl)-L-alanine, 4-methyl-2-pyridyl-alanine, 4-ethyl-2-pyridyl-L-alanine, benzothiazole-L-alanine, benzothiophene-L-alanine, 3-isoquinolyl-L-alanine, tert-butyl-L-alanine (also known as L-neopentylglycine), 3-cyclobutyl-L-alanine, cyclopentyl-L-alanine, 5,5,5-trifluoro-L-leucine, tert-butyl-L-glycine (also known as L-tert-leucine), L-cyclopentylglycine, L-cyclobutylglycine, 3,4-hydroxy-L-phenylalanine, 3,4-fluoro-L-phenylalanine, 3-fluoro,4-OH-L-phenylalanine, 2-chloro-L-tyrosine, 2-methyl-L-tyrosine, 2-ethyl-L-tyrosine, 4-(naphthalen-1-ol)-L-alanine, D-serine, L-β-homoserine, L-β-alanine, N-α-methylglycine, glycine amide, glycerol glycine ester, ethylene glycol glycine ester, oxetan-3-yl glycine ester, and glycine morpholine amide; and

[0158] Wherein the CKP binds VEGF-A with an affinity of 500 pM or less.

[0159] In certain embodiments, the C-terminus of the non-naturally occurring cystine knot peptide (CKP) that binds to VEGF-A is modified (such as capped). In certain embodiments, the N-terminus of the non-naturally occurring cystine knot peptide (CKP) that binds to VEGF-A is modified (such as capped). In certain embodiments, both the C- and N-termini of the non-naturally occurring cystine knot peptide (CKP) that binds to VEGF-A are modified (such as capped). In certain embodiments, the C-terminal carboxyl of the non-naturally occurring cystine knot peptide (CKP) that binds to VEGF-A is amidated. In certain embodiments, the N-terminal amine of the non-naturally occurring cystine knot peptide (CKP) that binds to VEGF-A is acetylated. In certain embodiments, the C-terminal carboxyl of the non-naturally occurring cystine knot peptide (CKP) that binds to VEGF-A is amidated and the N-terminal amine of the non-naturally occurring cystine knot peptide (CKP) that binds to VEGF-A is acetylated.

[0160] In certain embodiments, the non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A has altered disulfide bond connections - with reference to the wild-type EETI-II protein having the amino acid sequence shown in SEQ ID NO: 1; wherein the altered disulfide bond connections are C1-C4, C2-C3, and C5-C6.

[0161] In certain embodiments, Z1 and / or Z2 of the non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A is G. In certain embodiments, Z1 and / or Z2 comprise more than one amino acid. In certain embodiments, Z1 and / or Z2 comprise 4 amino acids. In certain embodiments, Z1 and / or Z2 comprise 5 amino acids. In certain embodiments, Z1 and / or Z2 are non-natural amino acids. In certain embodiments, the non-natural amino acid is N-acetylglycine or glycinamide. In certain embodiments, the non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A comprises L1, wherein X3 is not I; wherein X5 is not M; and / or wherein X6 is not R. In certain embodiments, the non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A comprises L1, wherein X1 is an amino acid selected from P, Q, R, T, V, D, N, K, L, and X; wherein X2 is an amino acid selected from T, D, L, V, I, R, P, N, and X; wherein X3 is an amino acid selected from T, P, M, L, S, F, R, and X; wherein X4 is an amino acid selected from R, T, Q, D, W, L, E, S, K, and X; wherein wherein X5 is an amino acid selected from F, P, V, E, K, L, I and X; wherein X6 is an amino acid selected from K, N, F, P, L, Y, T, D, M and X; wherein X7 is an amino acid selected from Q, W, H and / or X; and / or wherein X8 is an amino acid selected from Y, A, G, D, E, W, S and X, wherein X is an unnatural amino acid and is selected from the group consisting of: L-propargylglycine-PEG6-, L-sulfotyrosine, L-norleucine , L-1-naphthylalanine, L-2-naphthylalanine, L-2-chlorotryptophan, L-3-fluorotyrosine, L-4-fluorophenylalanine, γ-benzyl-L-proline, γ-(4-fluoro-benzyl)-L-proline, 4-OH-L-proline, 4-fluoro-L-proline, 4-[4-(trifluoromethyl)-benzyl]-L-proline, 3,4-difluoro-L-phenylalanine, 3,4-dichloro-L-phenylalanine, 4-chloro-L-phenylalanine Alanine, 3-F,4-Cl-L-phenylalanine, 2-pyridone (NH para)-L-alanine, pyridone (NH meta)-L-alanine, 3-(1-N-methylindole)-L-alanine, 3-(1-N-ethylindole)-L-alanine, 3-(1-N-isopropylindole)-L-alanine, 3-(5-aza-indole)-L-alanine, 4-methyl-L-phenylalanine, 2-naphthyl-L-alanine, L-4,4'-biphenylalanine, 3-(3-quinolyl)-L-alanine, 3-(2-quinolyl)-L-alanine, 3-(2-quinoxalinyl)-L-alanine, 4-methyl-2-pyridyl-alanine, 4-ethyl-2-pyridyl-L-alanine, benzothiazole-L-alanine, benzothiophene-L-alanine, 3-isoquinolyl-L-alanine, tert-butyl-L-alanine (also known as L-neopentylglycine), 3-cyclobutyl-L-alanine, cyclopentyl-L-alanine, 5,5,5-trifluoro-L-leucine, tert-butyl-L-glycine ( also known as L-tert-leucine), L-cyclopentylglycine, L-cyclobutylglycine, 3,4-hydroxy-L-phenylalanine, 3,4-fluoro-L-phenylalanine, 3-fluoro,4-OH-L-phenylalanine, 2-chloro-L-tyrosine, 2-methyl-L-tyrosine, 2-ethyl-L-tyrosine, 4-(naphthalene-1-ol)-L-alanine, D-serine, L-β-homoserine, L-β-alanine, N-α-methylglycine, glycine amide, glycerol glycine ester, ethylene glycol glycine ester, oxetane-3-yl glycine ester, and glycine morpholine amide. In certain embodiments, the non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A comprises L1, wherein X9 is an amino acid selected from L, I, V, D, E, and X, wherein X is a non-natural amino acid and is selected from the group consisting of: L-propargylglycine-PEG6-, L-sulfotyrosine, L-norleucine, L-1-naphthylalanine, L-2-naphthylalanine, L-2-chlorotryptophan, L-3-fluorotyrosine, 4-(trifluoromethyl)-benzyl)-L-proline, 3,4-difluoro-L-phenylalanine, 3,4-dichloro-L-phenylalanine, 4-chloro-L-phenylalanine, 3-F,4-Cl-L-phenylalanine, 2-pyridone (NH para) -L-alanine, pyridone (NH meta)-L-alanine, 3-(1-N-methylindole)-L-alanine, 3-(1-N-ethylindole)-L-alanine, 3-(1-N-isopropylindole)-L-alanine, 3-(5-aza-indole)-L-alanine, 4-methyl-L-phenylalanine, 2-naphthyl-L-alanine, L-4,4'-biphenylalanine, 3-(3-quinolyl)-L-alanine acid, 3-(2-quinolyl)-L-alanine, 3-(2-quinoxalinyl)-L-alanine, 4-methyl-2-pyridyl-alanine, 4-ethyl-2-pyridyl-L-alanine, benzothiazole-L-alanine, benzothiophene-L-alanine, 3-isoquinolyl-L-alanine, tert-butyl-L-alanine (also known as L-neopentylglycine), 3-cyclobutyl-L-alanine, cyclopentyl-L-alanine, 5,5,5-trifluoro-L-leucine, tert-butyl-L-glycine (also known as L-tert-leucine), L-cyclopentylglycine, L-cyclobutylglycine, 3,4-hydroxy-L-phenylalanine, 3,4-fluoro-L-phenylalanine, 3-fluoro, 4-OH-L-phenylalanine, 2-chloro-L-tyrosine, 2-methyl-L-tyrosine, 2-ethyl-L-tyrosine, 4-(naphthalene-1-ol)-L-alanine, D-serine, L-β-homoserine, L-β-alanine, N-α-methylglycine, glycineamide, glycerol glycine ester, glycol glycine ester, oxetane-3-yl glycine ester and glycine morpholine amide. In certain embodiments, the non-naturally occurring cystine knot peptide (CKP) that binds to VEGF-A comprises L1, wherein X, 10is an amino acid selected from the group consisting of Y, T, M, N, F and X, wherein X is an unnatural amino acid and is selected from the group consisting of L-propargylglycine-PEG6-, L-sulfotyrosine, L-norleucine, L-1-naphthylalanine, L-2-naphthylalanine, L-2-chlorotryptophan, L-3-fluorotyrosine, L-4-fluorophenylalanine, γ-benzyl-L-proline, γ-(4-fluoro-benzyl)-L-proline, 4-OH-L-proline, 4-fluoro-L-proline, 4-[4-(trifluoromethyl)-benzyl]-L-proline, 3,4 -Difluoro-L-phenylalanine, 3,4-dichloro-L-phenylalanine, 4-chloro-L-phenylalanine, 3-F,4-Cl-L-phenylalanine, 2-pyridone (NH para)-L-alanine, pyridone (NH meta)-L-alanine, 3-(1-N-methylindole)-L-alanine, 3-(1-N-ethylindole)-L-alanine, 3-(1-N-isopropylindole)-L-alanine, 3-(5-aza-indole)-L-alanine, 4-methyl-L-phenylalanine, 2-naphthyl-L-alanine, L-4,4'-biphenyl 4-Methyl-2-pyridyl-alanine, 4-ethyl-2-pyridyl-alanine, benzothiazole-L-alanine, benzothiophene-L-alanine, 3-isoquinolyl-L-alanine, tert-butyl-L-alanine (also known as L-neopentylglycine), 3-cyclobutyl-L-alanine, cyclopentyl-L-alanine, 5,5,5-trifluoro-L-leucine, tert-butyl-L-glycine (also known as L-tert-leucine), L-cyclopentylglycine, L-cyclobutylglycine, 3,4-hydroxy-L-phenylalanine, 3,4-fluoro-L-phenylalanine, 3-fluoro,4-OH-L-phenylalanine, 2-chloro-L-tyrosine, 2-methyl-L-tyrosine, 2-ethyl-L-tyrosine, 4-(naphthalene-1-ol)-L-alanine, D-serine, L-β-homoserine, L-β-alanine, N-α-methylglycine, glycine amide, glycerol glycine ester, ethylene glycol glycine ester, oxetane-3-yl glycine ester and glycine morpholine amide.

[0162] In certain embodiments, the non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A comprises L5, wherein each of X1-X5 is any amino acid or a non-natural amino acid selected from the group consisting of L-propargylglycine-PEG6-, L-sulfotyrosine, L-norleucine, L-1-naphthylalanine, L-2-naphthylalanine, L-2-chlorotryptophan, L-3-fluorotyrosine, L-4-fluorophenylalanine, γ-benzyl-L-proline, γ-(4-fluoro-benzyl)-L-proline, 4-OH-L-proline, 4-fluoro-L-proline, 4- [4-(Trifluoromethyl)-benzyl]-L-proline, 3,4-difluoro-L-phenylalanine, 3,4-dichloro-L-phenylalanine, 4-chloro-L-phenylalanine, 3-F,4-Cl-L-phenylalanine, 2-pyridone (NH para)-L-alanine, pyridone (NH meta)-L-alanine, 3-(1-N-methylindole)-L-alanine, 3-(1-N-ethylindole)-L-alanine, 3-(1-N-isopropylindole)-L-alanine, 3-(5-aza-indole)-L-alanine, 4-methyl-L-phenylalanine, 2-naphthyl-L-alanine , L-4,4'-biphenylalanine, 3-(3-quinolyl)-L-alanine, 3-(2-quinolyl)-L-alanine, 3-(2-quinoxalinyl)-L-alanine, 4-methyl-2-pyridyl-alanine, 4-ethyl-2-pyridyl-L-alanine, benzothiazole-L-alanine, benzothiophene-L-alanine, 3-isoquinolyl-L-alanine, tert-butyl-L-alanine (also known as L-neopentylglycine), 3-cyclobutyl-L-alanine, cyclopentyl-L-alanine, 5,5,5-trifluoro-L-leucine, tert-butyl-L-glycine (also known as L- tert-leucine), L-cyclopentylglycine, L-cyclobutylglycine, 3,4-hydroxy-L-phenylalanine, 3,4-fluoro-L-phenylalanine, 3-fluoro,4-OH-L-phenylalanine, 2-chloro-L-tyrosine, 2-methyl-L-tyrosine, 2-ethyl-L-tyrosine, 4-(naphthalen-1-ol)-L-alanine, D-serine, L-β-homoserine, L-β-alanine, N-α-methylglycine, glycineamide, glycerol glycine ester, glycol glycine ester, oxetane-3-ylglycine ester and glycine morpholineamide; except that X2 is not proline (P).

[0163] In certain embodiments, the non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A comprises L5, wherein each of X1-X5 is any amino acid or a non-natural amino acid selected from the group consisting of L-propargylglycine-PEG6-, L-sulfotyrosine, L-norleucine, L-1-naphthylalanine, L-2-naphthylalanine, L-2-chlorotryptophan, L-3-fluorotyrosine, L-4-fluorophenylalanine, γ-benzyl-L-proline, γ-(4-fluoro-benzyl)-L-proline, 4-OH-L-proline, 4-fluoro-L-proline, 4- [4-(Trifluoromethyl)-benzyl]-L-proline, 3,4-difluoro-L-phenylalanine, 3,4-dichloro-L-phenylalanine, 4-chloro-L-phenylalanine, 3-F,4-Cl-L-phenylalanine, 2-pyridone (NH para)-L-alanine, pyridone (NH meta)-L-alanine, 3-(1-N-methylindole)-L-alanine, 3-(1-N-ethylindole)-L-alanine, 3-(1-N-isopropylindole)-L-alanine, 3-(5-aza-indole)-L-alanine, 4-methyl-L-phenylalanine, 2-naphthyl-L-alanine , L-4,4'-biphenylalanine, 3-(3-quinolyl)-L-alanine, 3-(2-quinolyl)-L-alanine, 3-(2-quinoxalinyl)-L-alanine, 4-methyl-2-pyridyl-alanine, 4-ethyl-2-pyridyl-L-alanine, benzothiazole-L-alanine, benzothiophene-L-alanine, 3-isoquinolyl-L-alanine, tert-butyl-L-alanine (also known as L-neopentylglycine), 3-cyclobutyl-L-alanine, cyclopentyl-L-alanine, 5,5,5-trifluoro-L-leucine, tert-butyl-L-glycine (also known as L- tert-leucine), L-cyclopentylglycine, L-cyclobutylglycine, 3,4-hydroxy-L-phenylalanine, 3,4-fluoro-L-phenylalanine, 3-fluoro, 4-OH-L-phenylalanine, 2-chloro-L-tyrosine, 2-methyl-L-tyrosine, 2-ethyl-L-tyrosine, 4-(naphthalen-1-ol)-L-alanine, D-serine, L-β-homoserine, L-β-alanine, N-α-methylglycine, glycine amide, glycerol glycine ester, ethylene glycol glycine ester, oxetane-3-yl glycine ester and glycine morpholine amide; except that X4 is not glycine (G). In certain embodiments, the non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A comprises L5, wherein X1 is an amino acid selected from G, Q, H, R, L and Q; wherein X2 is an amino acid selected from P, M, W, Y, F, L and H; wherein X3 is an amino acid selected from N, F, H and Y; wherein X4 is an amino acid selected from G, Q, D, N, K, H, E and S; and / or wherein X5 is an amino acid selected from F, S and T.In certain embodiments, the non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A comprises L2, wherein X1 is K, X2 is Q, X3 is D, X4 is S, and X5 is D. In certain embodiments, the non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A comprises L3, wherein X1 is L, X2 is A, and X3 is G. In certain embodiments, the non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A comprises L4, wherein X1 is V or F.

[0164] In certain embodiments, the non-naturally occurring cystine knot peptide (CKP) that binds to VEGF-A comprises: L1 comprising the structure X1X2X3X4X5X6X7X8, wherein: X1 is an amino acid selected from P, Q, and R; X2 is an amino acid selected from T, L, and D; X3 is an amino acid selected from T, M, and L; X4 is an amino acid selected from R, Q, and D; X5 is an amino acid selected from F, P, and V; X6 is an amino acid selected from K and F; X7 is an amino acid selected from Q and W; and X8 is an amino acid selected from Y, G, and D. In certain embodiments, the non-naturally occurring cystine knot peptide (CKP) that binds to VEGF-A comprises: L1 comprising the structure X1X2X3X4X5X6X7X8 X9 X 10 L1, wherein X1 is an amino acid selected from Q, R, T and V; X2 is an amino acid selected from T and D; X3 is P; X4 is an amino acid selected from T and W; X5 is an amino acid selected from F, E, P and K; X6 is an amino acid selected from N and P; X7 is an amino acid selected from W and H; X8 is an amino acid selected from A, D, E and W; X9 is an amino acid selected from L and I; and X 10 is an amino acid selected from the group consisting of Y, T, M, and N. In certain embodiments, a non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A comprises L5, wherein X1 is an amino acid selected from the group consisting of G, H, and Q; X2 is an amino acid selected from the group consisting of P, M, W, and Y; X3 is an amino acid selected from the group consisting of N and Y; X4 is an amino acid selected from the group consisting of G, Q, and S; and X5 is an amino acid selected from the group consisting of F and S. In certain embodiments, a non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A comprises L2, wherein X1 is K, X2 is Q, X3 is D, X4 is S, and X5 is D. In certain embodiments, a non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A comprises L3, wherein X1 is L, X2 is A, and X3 is G. In certain embodiments, a non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A comprises L4, wherein X1 is V or F.

[0165] In certain embodiments, a non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A comprises L1 having the structure X1X2X3X4X5X6X7X8, wherein: X1 is an amino acid selected from D, Q, N, and K; X2 is an amino acid selected from V, I, R, L, and P; X3 is an amino acid selected from L, S, M, T, and F; X4 is an amino acid selected from Q, L, and E; X5 is P; X6 is an amino acid selected from F, L, and Y; X7 is W; and X8 is G. In certain embodiments, a non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A comprises L5, wherein X3 is Y; X5 is S; and wherein X1, X2, and X4 are each any amino acid, except that X1 is not G, X2 is not P, X4 is not G, and / or X5 is not F. In certain embodiments, the non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A comprises L5, wherein X1 is an amino acid selected from H, L, R, and Q; X2 is an amino acid selected from W, F, and Y; X3 is Y; X4 is an amino acid selected from Q, N, K, H, and E; and X5 is S. In certain embodiments, the non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A comprises L2, wherein X1 is K, X2 is Q, X3 is D, X4 is S, and X5 is D. In certain embodiments, the non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A comprises L3, wherein X1 is L, X2 is A, and X3 is G. In certain embodiments, the non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A comprises L4, wherein X1 is V or F.

[0166] In certain embodiments, the non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A comprises: comprising the structure X1X2X3X4X5X6X7X8 X9 X 10 L1, wherein X1 is an amino acid selected from K, Q, L and R; X2 is an amino acid selected from N and D; X3 is an amino acid selected from P and L; X4 is an amino acid selected from L, T, S and K; X5 is an amino acid selected from F, V, I and L; X6 is an amino acid selected from N and D; X7 is W; X8 is an amino acid selected from A and S; X9 is an amino acid selected from L, V, E and D; and X 10is an amino acid selected from the group consisting of Y and F. In certain embodiments, the non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A comprises L5, wherein X1 is Q; X2 is an amino acid selected from the group consisting of L, F, M, and H; X3 is an amino acid selected from the group consisting of F, Y, and H; X4 is an amino acid selected from the group consisting of D, Q, N, and K; and X5 is an amino acid selected from the group consisting of S and T. In certain embodiments, the non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A comprises L2, wherein X1 is K, X2 is Q, X3 is D, X4 is S, and X5 is D. In certain embodiments, the non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A comprises L3, wherein X1 is L, X2 is A, and X3 is G. In certain embodiments, the non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A comprises L4, wherein X1 is V or F.

[0167] In certain embodiments, a non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A comprises: L1 comprising the structure X1X2X3X4X5X6X7X8, wherein: X5 is P; X7 is W; X8 is G; and wherein X1, X2, X3, X4, and X6 are each any amino acid, except that X1 is not P, X2 is not R, X3 is not I, and / or X6 is not R. In certain embodiments, a non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A comprises: L1 comprising the structure X1X2X3X4X5X6X7X8, wherein X1 is an amino acid selected from N and D; X2 is an amino acid selected from I and V; X3 is an amino acid selected from M and L; X4 is an amino acid selected from L, Q, D, and K; X5 is P; X6 is an amino acid selected from F, Y, T, L, and M; X7 is W; and X8 is G. In certain embodiments, the non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A comprises L5, wherein X1 is an amino acid selected from Q, H, L, and R; X2 is an amino acid selected from Y and W; X3 is Y; X4 is an amino acid selected from Q and N; and X5 is S. In certain embodiments, the non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A comprises L5, wherein X3 is Y; X5 is S; and wherein each of X1, X2, and X4 is any amino acid, except that X1 is not G, X2 is not P, and / or X4 is not G. In certain embodiments, the non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A comprises L2, wherein X1 is an amino acid selected from G or E; X2 is an amino acid selected from Q, L, P, R, E, and M; X3 is an amino acid selected from S, D, and N; X4 is an amino acid selected from F, Y, L, M, and I; and / or X5 is an amino acid selected from E, D, Q, L, and S. In certain embodiments, the non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A comprises L3, wherein X1 is L, X2 is A, and X3 is G. In certain embodiments, the non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A comprises L4, wherein X1 is V or F.

[0168] In certain embodiments, the non-naturally occurring cystine knot peptide (CKP) that binds to VEGF-A comprises L5, wherein each of X1-X5 is any amino acid, except that X2 is not proline (P). In certain embodiments, the non-naturally occurring cystine knot peptide (CKP) that binds to VEGF-A comprises L5, wherein each of X1-X5 is any amino acid, except that X4 is not glycine (G). In certain embodiments, the non-naturally occurring cystine knot peptide (CKP) that binds to VEGF-A comprises L5, wherein X1 is any amino acid except G; X2 is any amino acid except P; X3 is any amino acid except N; X4 is any amino acid except G; and / or X5 is any amino acid except F.

[0169] In certain embodiments, the non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A comprises: L1 comprising the structure X1X2X3X4X5X6X7X8, wherein X1 is an amino acid selected from N, D, and X; X2 is an amino acid selected from I, V, and X; X3 is M or X; X4 is an amino acid selected from L, Q, and X; X5 is P or X; X6 is F, Y, or X; X7 is W or X; and X8 is G or X, wherein X is a non-natural amino acid selected from the group consisting of L-propargylglycine-PEG6-, L-sulfotyrosine, L -norleucine, L-1-naphthylalanine, L-2-naphthylalanine, L-2-chlorotryptophan, L-3-fluorotyrosine, L-4-fluorophenylalanine, γ-benzyl-L-proline, γ-(4-fluoro-benzyl)-L-proline, 4-OH-L-proline, 4-fluoro-L-proline, 4-[4-(trifluoromethyl)-benzyl]-L-proline, 3,4-difluoro-L-phenylalanine, 3,4-dichloro-L-phenylalanine, 4-chloro-L-phenylalanine, 3-F,4-Cl-L-phenylalanine, 2-pyridone (NH para )-L-alanine, pyridone (NH meta)-L-alanine, 3-(1-N-methylindole)-L-alanine, 3-(1-N-ethylindole)-L-alanine, 3-(1-N-isopropylindole)-L-alanine, 3-(5-aza-indole)-L-alanine, 4-methyl-L-phenylalanine, 2-naphthyl-L-alanine, L-4,4'-biphenylalanine, 3-(3-quinolyl)-L-alanine, 3-(2-quinolyl)-L-alanine, 3-(2-quinoxalinyl)-L-alanine, 4-methyl-2 -pyridyl-alanine, 4-ethyl-2-pyridyl-L-alanine, benzothiazole-L-alanine, benzothiophene-L-alanine, 3-isoquinolyl-L-alanine, tert-butyl-L-alanine (also known as L-neopentylglycine), 3-cyclobutyl-L-alanine, cyclopentyl-L-alanine, 5,5,5-trifluoro-L-leucine, tert-butyl-L-glycine (also known as L-tert-leucine), L-cyclopentylglycine, L-cyclobutylglycine, 3,4-hydroxy-L-phenylalanine, 3,4-fluoro-L-phenylalanine, 3-fluoro,4-OH-L-phenylalanine, 2-chloro-L-tyrosine, 2-methyl-L-tyrosine, 2-ethyl-L-tyrosine, 4-(naphthalene-1-ol)-L-alanine, D-serine, L-β-homoserine, L-β-alanine, N-α-methylglycine, glycine amide, glycerol glycine ester, ethylene glycol glycine ester, oxetane-3-yl glycine ester and glycine morpholine amide. In certain embodiments, the non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A comprises L3, wherein each of X1-X3 is any amino acid or a non-natural amino acid selected from the group consisting of: L-propargylglycine-PEG6-, L-sulfotyrosine, L-norleucine, L-1-naphthylalanine, L-2-naphthylalanine, L-2-chlorotryptophan, L-3-fluorotyrosine, L-4-fluorophenylalanine, γ-benzyl-L-proline, γ- (4-Fluoro-benzyl)-L-proline, 4-OH-L-proline, 4-Fluoro-L-proline, 4-[4-(trifluoromethyl)-benzyl]-L-proline, 3,4-difluoro-L-phenylalanine, 3,4-dichloro-L-phenylalanine, 4-chloro-L-phenylalanine, 3-F,4-Cl-L-phenylalanine, 2-pyridone (NH para)-L-alanine, pyridone (NH meta)-L-alanine, 3-(1-N-methylindole)-L-alanine, 3-( 1-N-ethylindole)-L-alanine, 3-(1-N-isopropylindole)-L-alanine, 3-(5-aza-indole)-L-alanine, 4-methyl-L-phenylalanine, 2-naphthyl-L-alanine, L-4,4'-biphenylalanine, 3-(3-quinolyl)-L-alanine, 3-(2-quinolyl)-L-alanine, 3-(2-quinoxalinyl)-L-alanine, 4-methyl-2-pyridyl-alanine, 4-ethyl-2-pyridyl-L-alanine alanine, benzothiazole-L-alanine, benzothiophene-L-alanine, 3-isoquinolyl-L-alanine, tert-butyl-L-alanine (also known as L-neopentylglycine), 3-cyclobutyl-L-alanine, cyclopentyl-L-alanine, 5,5,5-trifluoro-L-leucine, tert-butyl-L-glycine (also known as L-tert-leucine), L-cyclopentylglycine, L-cyclobutylglycine, 3,4-hydroxy-L-phenylalanine, 3,4-fluoro-L-phenylalanine, 3-fluoro,4-OH-L-phenylalanine, 2-chloro-L-tyrosine, 2-methyl-L-tyrosine, 2-ethyl-L-tyrosine, 4-(naphthalene-1-ol)-L-alanine, D-serine, L-β-homoserine, L-β-alanine, N-α-methylglycine, glycinamide, glycerol glycinate, ethylene glycol glycinate, oxetane-3-yl glycinate, and glycine morpholine amide, except that X1 is not leucine (L), X2 is not alanine (A), and X3 is not glycine (G), wherein the unnatural amino acid is selected from the group consisting of: L-alkyne Propylglycine-PEG6-, L-sulfotyrosine, L-norleucine, L-1-naphthylalanine, L-2-naphthylalanine, L-2-chlorotryptophan, L-3-fluorotyrosine, L-4-fluorophenylalanine, γ-benzyl-L-proline, γ-(4-fluoro-benzyl)-L-proline, 4-OH-L-proline, 4-fluoro-L-proline, 4-[4-(trifluoromethyl)-benzyl]-L-proline, 3,4-difluoro-L-phenylalanine, 3,4-dichloro-L-phenylalanine, 4-chloro-L-phenylalanine, 3-F,4-Cl-L-phenyl Alanine, 2-pyridone (NH para)-L-alanine, pyridone (NH meta)-L-alanine, 3-(1-N-methylindole)-L-alanine, 3-(1-N-ethylindole)-L-alanine, 3-(1-N-isopropylindole)-L-alanine, 3-(5-aza-indole)-L-alanine, 4-methyl-L-phenylalanine, 2-naphthyl-L-alanine, L-4,4'-biphenylalanine, 3-(3-quinolyl)-L-alanine, 3-(2-quinolyl)-L-alanine, 3-(2-quinoxalinyl)-L-alanine , 4-methyl-2-pyridyl-alanine, 4-ethyl-2-pyridyl-L-alanine, benzothiazole-L-alanine, benzothiophene-L-alanine, 3-isoquinolyl-L-alanine, tert-butyl-L-alanine (also known as L-neopentylglycine), 3-cyclobutyl-L-alanine, cyclopentyl-L-alanine, 5,5,5-trifluoro-L-leucine, tert-butyl-L-glycine (also known as L-tert-leucine), L-cyclopentylglycine, L-cyclobutylglycine, 3,4-hydroxy-L-phenylalanine, 3,4-fluoro-L-phenylalanine, 3-fluoro,4-OH-L-phenylalanine, 2-chloro-L-tyrosine, 2-methyl-L-tyrosine, 2-ethyl-L-tyrosine, 4-(naphthalene-1-ol)-L-alanine, D-serine, L-β-homoserine, L-β-alanine, N-α-methylglycine, glycineamide, glycerol glycine ester, ethylene glycol glycine ester, oxetane-3-yl glycine ester, and glycine morpholineamide. In certain embodiments, the non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A comprises L3, wherein X1 is an amino acid selected from M, F, LV, and X; X2 is an amino acid selected from S, N, Q, I, Y, E, V, T, and X; and X3 is an amino acid selected from D, Q, T, N, E, R, and X, wherein X is a non-natural amino acid selected from the group consisting of L-propargylglycine-PEG6-, L-sulfotyrosine, L-norleucine, L-1-naphthylalanine, L-2-naphthylalanine, L -2-Chlorotryptophan, L-3-fluorotyrosine, L-4-fluorophenylalanine, γ-benzyl-L-proline, γ-(4-fluoro-benzyl)-L-proline, 4-OH-L-proline, 4-fluoro-L-proline, 4-[4-(trifluoromethyl)-benzyl]-L-proline, 3,4-difluoro-L-phenylalanine, 3,4-dichloro-L-phenylalanine, 4-chloro-L-phenylalanine, 3-F,4-Cl-L-phenylalanine, 2-pyridone (NH para)-L-alanine, pyridone (NH meta)- L-alanine, 3-(1-N-methylindole)-L-alanine, 3-(1-N-ethylindole)-L-alanine, 3-(1-N-isopropylindole)-L-alanine, 3-(5-aza-indole)-L-alanine, 4-methyl-L-phenylalanine, 2-naphthyl-L-alanine, L-4,4'-biphenylalanine, 3-(3-quinolyl)-L-alanine, 3-(2-quinolyl)-L-alanine, 3-(2-quinoxalinyl)-L-alanine, 4-methyl-2-pyridyl-alanine, 4-ethyl-2-pyridyl-L-alanine, benzothiazole-L-alanine, benzothiophene-L-alanine, 3-isoquinolyl-L-alanine, tert-butyl-L-alanine (also known as L-neopentylglycine), 3-cyclobutyl-L-alanine, cyclopentyl-L-alanine, 5,5,5-trifluoro-L-leucine, tert-butyl-L-glycine (also known as L-tert-leucine), L-cyclopentylglycine, L-cyclobutylglycine, 3,4-hydroxy-L-phenylalanine, 3,4-fluoro-L-phenylalanine, 3-fluoro,4-OH-L-phenylalanine, 2-chloro-L-tyrosine, 2-methyl-L-tyrosine, 2-ethyl-L-tyrosine, 4-(naphthalene-1-ol)-L-alanine, D-serine, L-β-homoserine, L-β-alanine, N-α-methylglycine, glycineamide, glycerol glycine ester, ethylene glycol glycine ester, oxetane-3-yl glycine ester, and glycine morpholineamide. In certain embodiments, the non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A comprises L4, wherein X1 is any amino acid except V, or a non-natural amino acid selected from the group consisting of: L-propargylglycine-PEG6-, L-sulfotyrosine, L-norleucine, L-1-naphthylalanine, L-2-naphthylalanine, L-2-chlorotryptophan, L-3-fluorotyrosine, L-4-fluorophenylalanine, γ-benzyl-L-proline, γ-(4 -Fluoro-benzyl)-L-proline, 4-OH-L-proline, 4-fluoro-L-proline, 4-[4-(trifluoromethyl)-benzyl]-L-proline, 3,4-difluoro-L-phenylalanine, 3,4-dichloro-L-phenylalanine, 4-chloro-L-phenylalanine, 3-F,4-Cl-L-phenylalanine, 2-pyridone (NH para)-L-alanine, pyridone (NH meta)-L-alanine, 3-(1-N-methylindole)-L-alanine, 3-(1 -N-ethylindole)-L-alanine, 3-(1-N-isopropylindole)-L-alanine, 3-(5-aza-indole)-L-alanine, 4-methyl-L-phenylalanine, 2-naphthyl-L-alanine, L-4,4'-biphenylalanine, 3-(3-quinolyl)-L-alanine, 3-(2-quinolyl)-L-alanine, 3-(2-quinoxalinyl)-L-alanine, 4-methyl-2-pyridyl-alanine, 4-ethyl-2-pyridyl-L-alanine alanine, benzothiazole-L-alanine, benzothiophene-L-alanine, 3-isoquinolyl-L-alanine, tert-butyl-L-alanine (also known as L-neopentylglycine), 3-cyclobutyl-L-alanine, cyclopentyl-L-alanine, 5,5,5-trifluoro-L-leucine, tert-butyl-L-glycine (also known as L-tert-leucine), L-cyclopentylglycine, L-cyclobutylglycine, 3,4-hydroxy-L-phenylalanine, 3,4-fluoro-L-phenylalanine, 3-fluoro,4-OH-L-phenylalanine, 2-chloro-L-tyrosine, 2-methyl-L-tyrosine, 2-ethyl-L-tyrosine, 4-(naphthalene-1-ol)-L-alanine, D-serine, L-β-homoserine, L-β-alanine, N-α-methylglycine, glycineamide, glycerol glycine ester, ethylene glycol glycine ester, oxetane-3-yl glycine ester, and glycine morpholineamide. In certain embodiments, the non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A comprises L4, wherein X1 is I, L, or X, wherein X is a non-natural amino acid selected from the group consisting of L-propargylglycine-PEG6-, L-sulfotyrosine, L-norleucine, L-1-naphthylalanine, L-2-naphthylalanine, L-2-chlorotryptophan, L-3-fluorotyrosine, L-4-fluorophenylalanine, γ-benzyl-L-proline, γ-(4- 4-(4-(trifluoromethyl)-benzyl)-L-proline, 4-(4-(trifluoromethyl)-benzyl)-L-proline, 3,4-difluoro-L-phenylalanine, 3,4-dichloro-L-phenylalanine, 4-chloro-L-phenylalanine, 3-F,4-Cl-L-phenylalanine, 2-pyridone (NH para)-L-alanine, pyridone (NH meta)-L-alanine, 3-(1-N-methylindole)-L-alanine, 3-(1- N-ethylindole)-L-alanine, 3-(1-N-isopropylindole)-L-alanine, 3-(5-aza-indole)-L-alanine, 4-methyl-L-phenylalanine, 2-naphthyl-L-alanine, L-4,4'-biphenylalanine, 3-(3-quinolyl)-L-alanine, 3-(2-quinolyl)-L-alanine, 3-(2-quinoxalinyl)-L-alanine, 4-methyl-2-pyridyl-alanine, 4-ethyl-2-pyridyl-L-alanine acid, benzothiazole-L-alanine, benzothiophene-L-alanine, 3-isoquinolyl-L-alanine, tert-butyl-L-alanine (also known as L-neopentylglycine), 3-cyclobutyl-L-alanine, cyclopentyl-L-alanine, 5,5,5-trifluoro-L-leucine, tert-butyl-L-glycine (also known as L-tert-leucine), L-cyclopentylglycine, L-cyclobutylglycine, 3,4-hydroxy-L-phenylalanine, 3,4-fluoro-L-phenylalanine, 3-fluoro,4-OH-L-phenylalanine, 2-chloro-L-tyrosine, 2-methyl-L-tyrosine, 2-ethyl-L-tyrosine, 4-(naphthalene-1-ol)-L-alanine, D-serine, L-β-homoserine, L-β-alanine, N-α-methylglycine, glycineamide, glycerol glycine ester, ethylene glycol glycine ester, oxetane-3-yl glycine ester, and glycine morpholineamide. In certain embodiments, the non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A comprises L5, wherein X3 is Y or a non-natural amino acid selected from the group consisting of L-propargylglycine-PEG6-, L-sulfotyrosine, L-norleucine, L-1-naphthylalanine, L-2-naphthylalanine, L-2-chlorotryptophan, L-3-fluorotyrosine, L-4-fluorophenylalanine, γ-benzyl-L-proline, γ-(4-fluoro-benzyl)- L-proline, 4-OH-L-proline, 4-fluoro-L-proline, 4-[4-(trifluoromethyl)-benzyl]-L-proline, 3,4-difluoro-L-phenylalanine, 3,4-dichloro-L-phenylalanine, 4-chloro-L-phenylalanine, 3-F,4-Cl-L-phenylalanine, 2-pyridone (NH para)-L-alanine, pyridone (NH meta)-L-alanine, 3-(1-N-methylindole)-L-alanine, 3-(1-N-ethyl indole)-L-alanine, 3-(1-N-isopropylindole)-L-alanine, 3-(5-aza-indole)-L-alanine, 4-methyl-L-phenylalanine, 2-naphthyl-L-alanine, L-4,4'-biphenylalanine, 3-(3-quinolyl)-L-alanine, 3-(2-quinolyl)-L-alanine, 3-(2-quinoxalinyl)-L-alanine, 4-methyl-2-pyridyl-alanine, 4-ethyl-2-pyridyl-L-alanine, benzothiazole-L-alanine, benzothiophene-L-alanine, 3-isoquinolyl-L-alanine, tert-butyl-L-alanine (also known as L-neopentylglycine), 3-cyclobutyl-L-alanine, cyclopentyl-L-alanine, 5,5,5-trifluoro-L-leucine, tert-butyl-L-glycine (also known as L-tert-leucine), L-cyclopentylglycine, L-cyclobutylglycine, 3,4-hydroxy-L-phenylalanine, 3,4-fluoro-L-phenylalanine, 3-fluoro,4-OH-L-phenylalanine, 2-chloro-L-tyrosine, 2-methyl-L-tyrosine, 2-ethyl-L-tyrosine, 4-(naphthalene-1-ol)-L-alanine, D-serine, L-β-homoserine, L-β-alanine, N-α-methylglycine, glycinamide, glycerol glycine ester, ethylene glycol glycine ester, oxetane-3-yl glycine ester and glycine morpholine amide; wherein X5 is S or an unnatural amino acid selected from the group consisting of: L-propargylglycine-PEG6-, L-sulfotyrosine, L-norleucine, L-1-naphthylalanine, L-2-naphthylalanine, L-2-chlorotryptophan, L-3-fluorotyrosine, L-4-fluorophenylalanine, γ-benzyl-L-proline, γ-(4-fluoro-benzyl)-L-proline, 4-OH-L-proline, 4-fluoro-L-proline, 4-[4-(trifluoromethyl)-benzyl]-L-proline, 3,4-difluoro-L-phenylalanine, 3,4-dichloro-L-phenylalanine, 4-chloro-L-phenylalanine, 3-F,4-Cl-L-phenylalanine, 2-pyridone (NH para)-L- Alanine, pyridone (NH meta)-L-alanine, 3-(1-N-methylindole)-L-alanine, 3-(1-N-ethylindole)-L-alanine, 3-(1-N-isopropylindole)-L-alanine, 3-(5-aza-indole)-L-alanine, 4-methyl-L-phenylalanine, 2-naphthyl-L-alanine, L-4,4'-biphenylalanine, 3-(3-quinolyl)-L-alanine, 3-(2-quinolyl)-L-alanine, 3-(2-quinoxalinyl)-L-alanine, 4-methyl-2-pyridinyl 4-Hydroxy-L-phenylalanine, 4-ethyl-2-pyridyl-L-alanine, benzothiazole-L-alanine, benzothiophene-L-alanine, 3-isoquinolyl-L-alanine, tert-butyl-L-alanine (also known as L-neopentylglycine), 3-cyclobutyl-L-alanine, cyclopentyl-L-alanine, 5,5,5-trifluoro-L-leucine, tert-butyl-L-glycine (also known as L-tert-leucine), L-cyclopentylglycine, L-cyclobutylglycine, 3,4-hydroxy-L-phenylalanine, 3,4-fluoro-L-phenylalanine, 3-fluoro,4-OH-L-phenylalanine, 2-chloro-L-tyrosine, 2-methyl-L-tyrosine, 2-ethyl-L-tyrosine, 4-(naphthalene-1-ol)-L-alanine, D-serine, L-β-homoserine, L-β-alanine, N-α-methylglycine, glycine amide, glycerol glycine ester, ethylene glycol glycine ester, oxetane-3-yl glycine ester and glycine morpholine amide; and wherein X1, X2 and X4 are each any amino acid or a non-natural amino acid selected from the group consisting of: L-propargylglycine-PEG6-, L-sulfon ... Tyrosine, L-norleucine, L-1-naphthylalanine, L-2-naphthylalanine, L-2-chlorotryptophan, L-3-fluorotyrosine, L-4-fluorophenylalanine, γ-benzyl-L-proline, γ-(4-fluoro-benzyl)-L-proline, 4-OH-L-proline, 4-fluoro-L-proline, 4-[4-(trifluoromethyl)-benzyl]-L-proline, 3,4-difluoro-L-phenylalanine, 3,4-dichloro-L-phenylalanine, 4-chloro-L-phenylalanine, 3-F,4-Cl-L-phenylalanine, 2-pyridone (N H para)-L-alanine, pyridone (NH meta)-L-alanine, 3-(1-N-methylindole)-L-alanine, 3-(1-N-ethylindole)-L-alanine, 3-(1-N-isopropylindole)-L-alanine, 3-(5-aza-indole)-L-alanine, 4-methyl-L-phenylalanine, 2-naphthyl-L-alanine, L-4,4'-biphenylalanine, 3-(3-quinolyl)-L-alanine, 3-(2-quinolyl)-L-alanine, 3-(2-quinoxalinyl)-L-alanine, 4-methyl -2-pyridyl-alanine, 4-ethyl-2-pyridyl-L-alanine, benzothiazole-L-alanine, benzothiophene-L-alanine, 3-isoquinolyl-L-alanine, tert-butyl-L-alanine (also known as L-neopentylglycine), 3-cyclobutyl-L-alanine, cyclopentyl-L-alanine, 5,5,5-trifluoro-L-leucine, tert-butyl-L-glycine (also known as L-tert-leucine), L-cyclopentylglycine, L-cyclobutylglycine, 3,4-hydroxy-L-phenylalanine, 3,4-fluoro-L-phenylalanine, 3-fluoro,4-OH-L-phenylalanine, 2-chloro-L-tyrosine, 2-methyl-L-tyrosine, 2-ethyl-L-tyrosine, 4-(naphthalene-1-ol)-L-alanine, D-serine, L-β-homoserine, L-β-alanine, N-α-methylglycine, glycine amide, glycerol glycine ester, ethylene glycol glycine ester, oxetane-3-yl glycine ester and glycine morpholine amide, except that X1 is not G, X2 is not P, and / or X4 is not G. In certain embodiments, the non-naturally occurring cystine knot peptide (CKP) that binds to VEGF-A comprises L5, wherein each of X1-X5 is any amino acid, except that X2 is not proline (P). In certain embodiments, the non-naturally occurring cystine knot peptide (CKP) that binds to VEGF-A comprises L5, wherein each of X1-X5 is any amino acid, except that X4 is not glycine (G). In certain embodiments, the non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A comprises L5, wherein X1 is an amino acid selected from Q, H, and X; X2 is an amino acid selected from Y, W, and X; X3 is Y or X; X4 is an amino acid selected from Q, N, or X; X5 is S or X, wherein X is a non-natural amino acid selected from the group consisting of L-propargylglycine-PEG6-, L-sulfotyrosine, L-norleucine, L-1-naphthylalanine, L-2-naphthylalanine, L- 2-Chlorotryptophan, L-3-fluorotyrosine, L-4-fluorophenylalanine, γ-benzyl-L-proline, γ-(4-fluoro-benzyl)-L-proline, 4-OH-L-proline, 4-fluoro-L-proline, 4-[4-(trifluoromethyl)-benzyl]-L-proline, 3,4-difluoro-L-phenylalanine, 3,4-dichloro-L-phenylalanine, 4-chloro-L-phenylalanine, 3-F,4-Cl-L-phenylalanine, 2-pyridone (NH para)-L-alanine, pyridone Indole (NH meta)-L-alanine, 3-(1-N-methylindole)-L-alanine, 3-(1-N-ethylindole)-L-alanine, 3-(1-N-isopropylindole)-L-alanine, 3-(5-aza-indole)-L-alanine, 4-methyl-L-phenylalanine, 2-naphthyl-L-alanine, L-4,4'-biphenylalanine, 3-(3-quinolyl)-L-alanine, 3-(2-quinolyl)-L-alanine, 3-(2-quinoxalinyl)-L- alanine, 4-methyl-2-pyridyl-alanine, 4-ethyl-2-pyridyl-L-alanine, benzothiazole-L-alanine, benzothiophene-L-alanine, 3-isoquinolyl-L-alanine, tert-butyl-L-alanine (also known as L-neopentylglycine), 3-cyclobutyl-L-alanine, cyclopentyl-L-alanine, 5,5,5-trifluoro-L-leucine, tert-butyl-L-glycine (also known as L-tert-leucine), L-cyclopentylglycine, L-cyclobutylglycine, 3,4-Hydroxy-L-phenylalanine, 3,4-fluoro-L-phenylalanine, 3-fluoro,4-OH-L-phenylalanine, 2-chloro-L-tyrosine, 2-methyl-L-tyrosine, 2-ethyl-L-tyrosine, 4-(naphthalene-1-ol)-L-alanine, D-serine, L-β-homoserine, L-β-alanine, N-α-methylglycine, glycineamide, glycerol glycine ester, ethylene glycol glycine ester, oxetane-3-yl glycine ester and glycine morpholineamide. In certain embodiments, the non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A comprises L2, wherein X1 is G or X; X2 is R, P or X; X3 is D or X; X4 is F, I or X; and X5 is E, D or X, wherein X is a non-natural amino acid selected from the group consisting of L-propargylglycine-PEG6-, L-sulfotyrosine, L-norleucine, L-1-naphthylalanine, L-2-naphthylalanine, L-2-chlorotryptophan, L-3-fluorotyrosine, L -4-Fluorophenylalanine, γ-benzyl-L-proline, γ-(4-fluoro-benzyl)-L-proline, 4-OH-L-proline, 4-fluoro-L-proline, 4-[4-(trifluoromethyl)-benzyl]-L-proline, 3,4-difluoro-L-phenylalanine, 3,4-dichloro-L-phenylalanine, 4-chloro-L-phenylalanine, 3-F,4-Cl-L-phenylalanine, 2-pyridone (NH para)-L-alanine, pyridone (NH meta)-L-alanine, 3-(1-N- methylindole)-L-alanine, 3-(1-N-ethylindole)-L-alanine, 3-(1-N-isopropylindole)-L-alanine, 3-(5-aza-indole)-L-alanine, 4-methyl-L-phenylalanine, 2-naphthyl-L-alanine, L-4,4'-biphenylalanine, 3-(3-quinolyl)-L-alanine, 3-(2-quinolyl)-L-alanine, 3-(2-quinoxalinyl)-L-alanine, 4-methyl-2-pyridyl-alanine, 4-ethyl-2 -pyridyl-L-alanine, benzothiazole-L-alanine, benzothiophene-L-alanine, 3-isoquinolyl-L-alanine, tert-butyl-L-alanine (also known as L-neopentylglycine), 3-cyclobutyl-L-alanine, cyclopentyl-L-alanine, 5,5,5-trifluoro-L-leucine, tert-butyl-L-glycine (also known as L-tert-leucine), L-cyclopentylglycine, L-cyclobutylglycine, 3,4-hydroxy-L-phenylalanine, 3,4-fluoro-L-phenylalanine, 3-fluoro,4-OH-L-phenylalanine, 2-chloro-L-tyrosine, 2-methyl-L-tyrosine, 2-ethyl-L-tyrosine, 4-(naphthalene-1-ol)-L-alanine, D-serine, L-β-homoserine, L-β-alanine, N-α-methylglycine, glycineamide, glycerol glycine ester, ethylene glycol glycine ester, oxetane-3-yl glycine ester and glycine morpholine amide.

[0170] In certain embodiments, the C-terminus of the non-naturally occurring cystine knot peptide (CKP) that binds to VEGF-A is modified (such as capped). In certain embodiments, the N-terminus of the non-naturally occurring cystine knot peptide (CKP) that binds to VEGF-A is modified (such as capped). In certain embodiments, the C- and N-termini of the non-naturally occurring cystine knot peptide (CKP) that binds to VEGF-A are modified (such as capped). In certain embodiments, the C-terminal carboxyl of the non-naturally occurring cystine knot peptide (CKP) that binds to VEGF-A is amidated. In certain embodiments, the N-terminal amine of the non-naturally occurring cystine knot peptide (CKP) that binds to VEGF-A is acetylated. In certain embodiments, the C-terminal carboxyl of the non-naturally occurring cystine knot peptide (CKP) that binds to VEGF-A is amidated and the N-terminal amine of the non-naturally occurring cystine knot peptide (CKP) that binds to VEGF-A is acetylated.

[0171] Also provided herein are non-naturally occurring cystine knot peptides (CKPs) that bind VEGF-A, wherein the CKP comprises a cystine scaffold structure (i.e., Scaffold Structure I) provided below:

[0172] Z1C1L1C2L2C3L3C4L4C5L5C6Z2 (I)

[0173] in:

[0174] Z1 and Z2 are any amino acids;

[0175] L1 is Ring 1 and has a structure selected from the group consisting of: X1X2X3X4X5X6X7X8, X1X2X3X4X5X6X7X8X9, and X1X2X3X4X5X6X7X8X9X 10 , where X1-X 10 Each of is any amino acid;

[0176] L2 is loop 2 and has the structure: X1X2X3X4X5, where each of X1–X5 is any amino acid;

[0177] L3 is loop 3 and has the structure: X1X2X3, where each of X1–X3 is any amino acid;

[0178] L4 is loop 4 and has the structure: X1, wherein X1 is any amino acid;

[0179] L5 is loop 5 and has the structure: X1X2X3X4X5, wherein each of X1-X5 is any amino acid; wherein,

[0180] With reference to the wild-type EETI-II protein having the amino acid sequence shown in SEQ ID NO: 1, the CKP has altered disulfide bonds; wherein the altered disulfide bonds are C1-C4, C2-C3 and C5-C6; and wherein the CKP has an alpha helical content percentage of at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45% or at least about 50%, including any ranges between these values.

[0181] In certain embodiments, the non-naturally occurring CKP binds VEGF-A with an affinity of 500 pM or less.

[0182] In certain embodiments, the binding affinity of a non-naturally occurring CKP for VEGF-A is determined via, for example, surface plasmon resonance or other assays described in detail in the Examples below.

[0183] In certain embodiments, Z1 and / or Z2 of the non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A is G. In certain embodiments, Z1 and / or Z2 comprises more than one amino acid. In certain embodiments, Z1 and / or Z2 comprises 4 amino acids. In certain embodiments, Z1 and / or Z2 comprises 5 amino acids. In certain embodiments, Z1 and / or Z2 is a non-natural amino acid. In certain embodiments, the non-natural amino acid is N-acetylglycine or glycinamide.

[0184] In certain embodiments, the C-terminus of the non-naturally occurring cystine knot peptide (CKP) that binds to VEGF-A is modified (such as capped). In certain embodiments, the N-terminus of the non-naturally occurring cystine knot peptide (CKP) that binds to VEGF-A is modified (such as capped). In certain embodiments, the C- and N-termini of the non-naturally occurring cystine knot peptide (CKP) that binds to VEGF-A are modified (such as capped). In certain embodiments, the C-terminal carboxyl of the non-naturally occurring cystine knot peptide (CKP) that binds to VEGF-A is amidated. In certain embodiments, the N-terminal amine of the non-naturally occurring cystine knot peptide (CKP) that binds to VEGF-A is acetylated. In certain embodiments, the C-terminal carboxyl of the non-naturally occurring cystine knot peptide (CKP) that binds to VEGF-A is amidated and the N-terminal amine of the non-naturally occurring cystine knot peptide (CKP) that binds to VEGF-A is acetylated.

[0185] In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises L5, wherein each of X1-X5 is any amino acid, except that X2 is not proline (P). In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises L5, wherein each of X1-X5 is any amino acid, except that X4 is not glycine (G). In certain embodiments, the non-naturally occurring CKP that binds VEGF-A has an IC of about 0.5 nM to about 1.0 nM. 50 Inhibit VEGF-A activity. In certain embodiments, the degree of inhibition is measured via cell IC 50 The assay was determined as described in further detail in the Examples below.

[0186] In certain embodiments, the non-naturally occurring CKP that binds to VEGF-A comprises: L1 comprising the following amino acid sequence: HMMYDY (SEQ ID NO: 231) or K / P / Q / RK / T / L / DW / T / M / LQ / R / DW / F / P / VW / K / FY / Q / WM / Y / G / D (SEQ ID NO: 115) or E / G / P / Q / R / T / VT / E / A / DD / T / I / PW / V / Q / T / WY / F / N / E / P / KP / E / W / N / PH / Q / K / W / HQ / F / E / A / D / WI / L / HD / W / P / Y / T / M / N (SEQ ID NO: 232), with reference to the above Scaffold Structure I. In certain embodiments, the non-naturally occurring CKP that binds to VEGF-A further comprises: L2 comprising the amino acid sequence KQDSD (SEQ ID NO: 93). In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L3 comprising the amino acid sequence LAG. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L4 comprising V or F. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L5 comprising the amino acid sequence G / E / Y / Q / HP / M / W / YN / Y / WG / D / T / Q / R / SF / A / E / S (SEQ ID NO: 20) or SWWPSL (SEQ ID NO: 237).

[0187] In certain embodiments, the non-naturally occurring CKP that binds to VEGF-A comprises: L1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-14 and 225-230, with reference to Scaffold Structure I above. In certain embodiments, the non-naturally occurring CKP that binds to VEGF-A further comprises: L2 comprising the amino acid sequence KQDSD (SEQ ID NO: 93). In certain embodiments, the non-naturally occurring CKP that binds to VEGF-A further comprises: L3 comprising the amino acid sequence LAG. In certain embodiments, the non-naturally occurring CKP that binds to VEGF-A comprises: L4 comprising V or F. In certain embodiments, the non-naturally occurring CKP that binds to VEGF-A further comprises: L5 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 15-18 and 233-238). The amino acid sequences of SEQ ID NOs 8-18, 225-230, and 233-238 are provided in Table 1 below.

[0188] Table 1

[0189]

[0190]

[0191] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence of any one of SEQ ID NOs: 21-17 and 239-244. The amino acid sequences of SEQ ID NOs: 21-17 and 239-244 are provided in Table 2 below.

[0192] Table 2

[0193] GCETDWYPHQIDCKQDSDCLAGCVCGPNGFCG (SEQ ID NO:239) GCGETVFEQFLWCKQDSDCLAGCVCGPNGFCG (SEQ ID NO:240) GCHMMYDYCKQDSDCLAGCVCEMYDACG (SEQ ID NO:241) GCKKWQWWYMCKQDSDCLAGCVCYPWTECG (SEQ ID NO:242) GCPAIQNWKEHPCKQDSDCLAGCVCSWWPSLCG (SEQ ID NO:243) GCPTTRFKQYCKQDSDCLAGCVCGPNGFCG (SEQ ID NO:21) GCQDPTFNWALYCKQDSDCLAGCVCQMYQSCG (SEQ ID NO:22) GCQLMHPFWGCKQDSDCLAGCVCHWYRSCG (SEQ ID NO:244) GCQLMQPFWGCKQDSDCLAGCVCHWYQSCG (SEQ ID NO:23) GCRDLDVKWDCKQDSDCLAGCFCQYYSSCG (SEQ ID NO:24) GCRTPWEPHDITCKQDSDCLAGCVCGPNGFCG (SEQ ID NO:25) GCTTPWPPHEIMCKQDSDCLAGCVCGPNGFCG (SEQ ID NO:26) GCVTPWKPHWINCKQDSDCLAGCVCGPNGFCG (SEQ ID NO:27)

[0194] In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises: L1 comprising the amino acid sequence Q / H / E / N / DL / V / R / P / IM / F / LQ / E / R / LPF / A / L / SWG (SEQ ID NO: 358), with reference to the above Scaffold Structure I. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L2 comprising the amino acid sequence KQDSD (SEQ ID NO: 93). In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L3 comprising the amino acid sequence LAG. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L4 comprising amino acid V. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L5 comprising the amino acid sequence HWYQS (SEQ ID NO: 17).

[0195] In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises: L1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 33, 36, and 245-253, with reference to Scaffold Structure I. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L2 comprising the amino acid sequence KQDSD (SEQ ID NO: 93). In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L3 comprising the amino acid sequence LAG. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L4 comprising amino acid V. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L5 comprising the amino acid sequence HWYQS (SEQ ID NO: 17). The amino acid sequences of SEQ ID NOs 33, 36, and 245-253 are provided in Table 3 below.

[0196] Table 3

[0197] HLFEPLWG (SEQ ID NO:245) QVMRPFWG (SEQ ID NO:246) QVMQPAWG (SEQ ID NO:247) HRLQPLWG (SEQ ID NO:248) ELLQPSWG (SEQ ID NO:249) NPMLPFWG (SEQ ID NO:368) NVLLPLWG (SEQ ID NO:250) DIMQPLWG (SEQ ID NO:36) DLMQPLWG (SEQ ID NO:251) NPMLPLWG (SEQ ID NO:252) QVLQPSWG (SEQ ID NO:253)

[0198] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence of any one of SEQ ID NOs: 265-275. The amino acid sequences of SEQ ID NOs: 265-275 are provided in Table 4 below.

[0199] Table 4

[0200]

[0201]

[0202] In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L1 comprising the amino acid sequence QLMQPFWG (SEQ ID NO: 10), with reference to Scaffold Structure I. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L2 comprising the amino acid sequence KQDSD (SEQ ID NO: 93). In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L3 comprising the amino acid sequence LAG. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L4 comprising amino acid V. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L5 comprising the amino acid sequence R / HWYN / Q / HS (SEQ ID NO: 359).

[0203] In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L1 comprising the amino acid sequence QLMQPFWG (SEQ ID NO: 10), with reference to the above Scaffold Structure I. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L2 comprising the amino acid sequence KQDSD (SEQ ID NO: 93). In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L3 comprising the amino acid sequence LAG. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L4 comprising amino acid V. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L5 comprising an amino acid sequence selected from the group consisting of HWYQS (SEQ ID NO: 17), RWYHS (SEQ ID NO: 43), and RWYNS (SEQ ID NO: 133).

[0204] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 23, 276, and 278. SEQ ID NOs 23, 276, and 278 are provided in Table 5 below.

[0205] Table 5

[0206] GCQLMQPFWGCKQDSDCLAGCVCRWYNSCG (SEQ ID NO:276) GCQLMQPFWGCKQDSDCLAGCVCHWYQSCG (SEQ ID NO:23) GCQLMQPFWGCKQDSDCLAGCVCRWYHSCG(SEQ ID NO:278)

[0207] In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises: L1 comprising the amino acid sequence Q / D / K / N / A / R / HL / V / I / R / P / V / -M / L / S / T / FQ / E / L / HPF / L / M / Y / SWG (SEQ ID NO: 40), with reference to Scaffold Structure I. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L2 comprising the amino acid sequence KQDSD (SEQ ID NO: 93). In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L3 comprising the amino acid sequence LAG. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L4 comprising amino acid V. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L5 comprising the amino acid sequence H / L / R / Q / -W / F / YYQ / N / K / H / D / ES (SEQ ID NO: 360).

[0208] In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises: L1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 28-39 and 254-261, with reference to Scaffold Structure I. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L2 comprising the amino acid sequence KQDSD (SEQ ID NO: 93). In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L3 comprising the amino acid sequence LAG. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L4 comprising amino acid V. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L5 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 41-46, 133, 262-264, and 567. The amino acid sequences of SEQ ID NOs: 17, 28-39, 41-46, 133, 254-264, and 567 are provided in Table 6 below.

[0209] Table 6

[0210]

[0211]

[0212] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 47-59 and 279-286. The amino acid sequences of SEQ ID NOs: 47-59 and 279-286 are provided in Table 7 below.

[0213] Table 7

[0214]

[0215]

[0216] In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises: L1 comprising the amino acid sequence Q / D / K / W / E / L / RD / NP / R / L / TT / S / L / KF / V / L / IN / DWA / S / GL / V / E / T / Q / DF / Y (SEQ ID NO: 70), with reference to Scaffold Structure I. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L2 comprising the amino acid sequence KQDSD (SEQ ID NO: 93). In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L3 comprising the amino acid sequence LAG. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L4 comprising amino acid V. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L5 comprising the amino acid sequence Q / RM / L / F / HY / F / HD / Q / N / KS / T (SEQ ID NO: 80).

[0217] In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises: L1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 60-69 and 287-291. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L2 comprising the amino acid sequence KQDSD (SEQ ID NO: 93). In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L3 comprising the amino acid sequence LAG. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L4 comprising amino acid V. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L5 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 71-79, 274, and 292. The amino acid sequences of SEQ ID NOs: 16, 60-69, 71-79, 274, and 287-292 are provided in Table 8 below.

[0218] Table 8

[0219]

[0220] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 81-91 and 293-297. The amino acid sequences of SEQ ID NOs: 81-91 and 293-297 are provided in Table 9 below.

[0221] Table 9

[0222]

[0223]

[0224] In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises: L1 comprising the amino acid sequence NIMLPFWG (SEQ ID NO: 33), with reference to Scaffold Structure I. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L2 comprising the amino acid sequence K / G / Q / S / NQ / L / P / A / V / T / R / W / K / G / YD / S / E / NS / F / Y / L / F / Q / MD / E / N / A / L / F / H / Q (SEQ ID NO: 98). In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L3 comprising the amino acid sequence LAG. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L4 comprising amino acid V. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L5 comprising the amino acid sequence QYYQS (SEQ ID NO: 45).

[0225] In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises: L1 comprising the amino acid sequence NIMLPFWG (SEQ ID NO: 33), with reference to Scaffold Structure I. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 94-97 and 298-309. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L3 comprising the amino acid sequence LAG. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L4 comprising amino acid V. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L5 comprising the amino acid sequence QYYQS (SEQ ID NO: 45). The amino acid sequences of SEQ ID NOs: 94-97 and 298-309 are provided in Table 10 below.

[0226] Table 10

[0227]

[0228]

[0229] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 99-102 and 310-321. The amino acid sequences of SEQ ID NOs: 99-102 and 310-321 are provided in Table 11 below.

[0230] Table 11

[0231] GCNIMLPFWGCGQSFECLAGCVCQYYQSCG(SEQ ID NO:99) GCNIMLPFWGCGLDYDCLAGCVCQYYQSCG(SEQ ID NO:100) GCNIMLPFWGCGPELNCLAGCVCQYYQSCG(SEQ ID NO:310) GCNIMLPFWGCQADYACLAGCVCQYYQSCG(SEQ ID NO:311) GCNIMLPFWGCGVDYLCLAGCVCQYYQSCG(SEQ ID NO:312) GCNIMLPFWGCGTNFLCLAGCVCQYYQSCG(SEQ ID NO:313) GCNIMLPFWGCSRDFDCLAGCVCQYYQSCG(SEQ ID NO:314) GCNIMLPFWGCNRDFLCLAGCVCQYYQSCG(SEQ ID NO:315) GCNIMLPFWGCGWDQFCLAGCVCQYYQSCG(SEQ ID NO:316) GCNIMLPFWGCGKDFHCLAGCVCQYYQSCG(SEQ ID NO:317) GCNIMLPFWGCGPDLQCLAGCVCQYYQSCG(SEQ ID NO:101) GCNIMLPFWGCSGDFACLAGCVCQYYQSCG(SEQ ID NO:318) GCNIMLPFWGCGKELNCLAGCVCQYYQSCG(SEQ ID NO:319) GCNIMLPFWGCGWSMDCLAGCVCQYYQSCG(SEQ ID NO:320) GCNIMLPFWGCGYDLQCLAGCVCQYYQSCG(SEQ ID NO:321) GCNIMLPFWGCGRDFECLAGCVCQYYQSCG(SEQ ID NO:102)

[0232] In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises: L1 comprising the amino acid sequence NIM / LL / S / T / Q / N / E / DPF / Y / S-WG (SEQ ID NO: 454), with reference to Scaffold Structure I. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises: L1 comprising the amino acid sequence NIMLPFWG (SEQ ID NO: 33), with reference to Scaffold Structure I. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L2 comprising the amino acid sequence GRDFE (SEQ ID NO: 97). In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L3 comprising the amino acid sequence L / V / M / FA / Q / E / S / N / Y / I / TG / Q / R / D / T / N / E. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L4 comprising amino acid V or I. In certain embodiments, the non-naturally occurring VEGF-A binding CKP further comprises: L5 comprising the amino acid sequence QYYQS (SEQ ID NO: 45).

[0233] In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises: an L1 comprising an amino acid sequence selected from the group consisting of NIMLPFWG (SEQ ID NO:33), NILLPFWG (SEQ ID NO:396), NILLPYWG (SEQ ID NO:397), NIMSPFWG (SEQ ID NO:398), NIMTPFWG (SEQ ID NO:399), NIMQPFWG (SEQ ID NO:400), NIMNPFWG (SEQ ID NO:401), NIMEPFWG (SEQ ID NO:402), NIMDPFWG (SEQ ID NO:403), NIMLPSWG (SEQ ID NO:414), and NIMLPYWG (SEQ ID NO:141), with reference to Scaffold Structure I. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises: an L1 comprising the amino acid sequence NIMLPFWG (SEQ ID NO:33), with reference to Scaffold Structure V or I. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L2 comprising the amino acid sequence GRDFE (SEQ ID NO: 97). In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L3 comprising an amino acid sequence selected from the group consisting of: LQQ, VER, MSD, MNQ, MQT, VYQ, FIN, VSQ, VTE, FYE, MEQ and VYR. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L4 comprising amino acid I. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L5 comprising the amino acid sequence QYYQS (SEQ ID NO: 45).

[0234] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 103-114. The amino acid sequences of SEQ ID NOs: 103-114 are provided in Table 12 below.

[0235] Table 12

[0236]

[0237]

[0238] In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises: L1 comprising the amino acid sequence DVMQPYWG (SEQ ID NO: 35), with reference to Scaffold Structure I. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L2 comprising the amino acid sequence K / G / D / A / EQ / E / R / V / P / D / M / G / N / L / A / FD / N / Y / SS / F / L / I / M / Y / V / N / ED / L / Q / S / E / T / L / A / N (SEQ ID NO: 121). In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L3 comprising the amino acid sequence LAG. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L4 comprising amino acid V. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L5 comprising the amino acid sequence HWYNS (SEQ ID NO: 46).

[0239] In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises: L1 comprising the amino acid sequence DVMQPYWG (SEQ ID NO: 35), with reference to Scaffold Structure I. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 117-120, 211, and 322-339. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L3 comprising the amino acid sequence LAG. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L4 comprising amino acid V. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L5 comprising the amino acid sequence HWYNS (SEQ ID NO: 46). The amino acid sequences of SEQ ID NOs: 117-120, 211, and 322-339 are provided in Table 13 below.

[0240] Table 13

[0241]

[0242] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 122-126 and 340-357. The amino acid sequences of SEQ ID NOs: 122-126 and 340-357 are provided in Table 14 below.

[0243] Table 14

[0244] GCDVMQPYWGCGENFLCLAGCVCHWYNSCG(SEQ ID NO:122) GCDVMQPYWGCGRDLQCLAGCVCHWYNSCG(SEQ ID NO:340) GCDVMQPYWGCGVDLSCLAGCVCHWYNSCG(SEQ ID NO:341) GCDVMQPYWGCGPDIDCLAGCVCHWYNSCG(SEQ ID NO:123) GCDVMQPYWGCGDDLECLAGCVCHWYNSCG(SEQ ID NO:342) GCDVMQPYWGCGVDMTCLAGCVCHWYNSCG(SEQ ID NO:343) GCDVMQPYWGCGMDIECLAGCVCHWYNSCG(SEQ ID NO:344) GCDVMQPYWGCDGDYQCLAGCVCHWYNSCG(SEQ ID NO:345) GCDVMQPYWGCGNDVSCLAGCVCHWYNSCG(SEQ ID NO:346) GCDVMQPYWGCGRDMDCLAGCVCHWYNSCG(SEQ ID NO:124) GCDVMQPYWGCAGDELCLAGCVCHWYNSCG(SEQ ID NO:347) GCDVMQPYWGCGLDEECLAGCVCHWYNSCG(SEQ ID NO:348) GCDVMQPYWGCDGDFDCLAGCVCHWYNSCG(SEQ ID NO:349) GCDVMQPYWGCAGDFECLAGCVCHWYNSCG(SEQ ID NO:350) GCDVMQPYWGCEMDFDCLAGCVCHWYNSCG(SEQ ID NO:125) GCDVMQPYWGCGNSFECLAGCVCHWYNSCG(SEQ ID NO:351) GCDVMQPYWGCGQDLTCLAGCVCHWYNSCG(SEQ ID NO:352) GCDVMQPYWGCGENLACLAGCVCHWYNSCG(SEQ ID NO:353) GCDVMQPYWGCGQDYNCLAGCVCHWYNSCG(SEQ ID NO:354) GCDVMQPYWGCGADLSCLAGCVCHWYNSCG(SEQ ID NO:355) GCDVMQPYWGCGFDMDCLAGCVCHWYNSCG(SEQ ID NO:356) GCDVMQPYWGCGESLSCLAGCVCHWYNSCG(SEQ ID NO:126) GCDVMQPYWGCDLNYECLAGCVCHWYNSCG(SEQ ID NO:357)

[0245] In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises: L1 comprising the amino acid sequence DVM / LQ / K / DPY / M / T / LWG (SEQ ID NO: 130), with reference to Scaffold Structure I. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises:

[0246] L2 comprising the amino acid sequence KQDSD (SEQ ID NO: 93). In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L3 comprising the amino acid sequence LAG. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L4 comprising amino acid V. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L5 comprising the amino acid sequence H / L / Q / RWYNS (SEQ ID NO: 134).

[0247] In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises: L1 comprising an amino acid sequence selected from SEQ ID NOs: 127-129, with reference to Scaffold Structure I. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L2 comprising the amino acid sequence KQDSD (SEQ ID NO: 93). In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L3 comprising the amino acid sequence LAG. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L4 comprising amino acid V. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L5 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 131-133. The amino acid sequences of SEQ ID NOs: 127-129 and 131-133 are provided in Table 15 below:

[0248] Table 15

[0249] DVMKPMWG(SEQ ID NO:127) QWYNS(SEQ ID NO:131) DVLDPTWG(SEQ ID NO:128) LWYNS(SEQ ID NO:132) DVLQPLWG(SEQ ID NO:129) RWYNS(SEQ ID NO:133)

[0250] In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 135-137. The amino acid sequences of SEQ ID NOs: 135-127 are provided in Table 16 below:

[0251] Table 16

[0252] GCDVMKPMWGCKQDSDCLAGCVCQWYNSCG(SEQ ID NO:135) GCDVLDPTWGCKQDSDCLAGCVCLWYNSCG(SEQ ID NO:136) GCDVLQPLWGCKQDSDCLAGCVCRWYNSCG(SEQ ID NO:137)

[0253] In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises: L1 comprising the amino acid sequence DVMQPYWG (SEQ ID NO: 35), with reference to Scaffold Structure I. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L2 comprising the amino acid sequence GPDID (SEQ ID NO: 118). In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L3 comprising the amino acid sequence L / FA / V / SG / R / N. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L4 comprising an amino acid selected from V, I and L. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L5 comprising the amino acid sequence HWYNS (SEQ ID NO: 46).

[0254] In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises: L1 comprising the amino acid sequence DVMQPYWG (SEQ ID NO: 35), with reference to Scaffold Structure I. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L2 comprising the amino acid sequence GPDID (SEQ ID NO: 118). In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L3 comprising the amino acid sequences FVR and LSN. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L4 comprising an amino acid selected from V, I and L. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L5 comprising the amino acid sequence HWYNS (SEQ ID NO: 46).

[0255] In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCDVMQPYWGCGPDIDCFVRCLCHWYNSCG (SEQ ID NO: 139). In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCDVMQPYWGCGPDIDCLSNCICHWYNSCG (SEQ ID NO: 140).

[0256] In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises L1, L2, L3, L4 and / or L5 of any one of the non-naturally occurring CKPs that bind VEGF-A disclosed herein. Thus, in certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises: L1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-14, 28-39, 60-69, 127-129, 141, 225-230, 245-261, 287-291, 396-403 and 414, with reference to Scaffold Structure I. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 93-97, 117-120, 211, 298-309 and 322-339. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L3 comprising an amino acid sequence selected from the group consisting of: LAG, LQQ, VER, MSD, MNQ, MQT, VYQ, FIN, VSQ, VTE, FYE, MEQ and VYR, FVR and LSN. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L4 comprising amino acids V, F, I or L. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A further comprises: L5 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 15-18, 41-46, 71-79, 131-133, 233-238, 262-264 and 292. In certain embodiments, the C-terminus of the non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A is modified (such as capped). In certain embodiments, the N-terminus of the non-naturally occurring cystine knot peptide (CKP) that binds to VEGF-A is modified (such as capped). In certain embodiments, the C- and N-termini of the non-naturally occurring cystine knot peptide (CKP) that binds to VEGF-A are modified (such as capped). In certain embodiments, the C-terminal carboxyl group of the non-naturally occurring cystine knot peptide (CKP) that binds to VEGF-A is amidated. In certain embodiments, the N-terminal amine of the non-naturally occurring cystine knot peptide (CKP) that binds to VEGF-A is acetylated. In certain embodiments, the C-terminal carboxyl group of the non-naturally occurring cystine knot peptide (CKP) that binds to VEGF-A is amidated and the N-terminal amine of the non-naturally occurring cystine knot peptide (CKP) that binds to VEGF-A is acetylated.

[0257] In certain embodiments, at least one amino acid is deleted from the CKPs provided herein that bind VEGF-A. In certain embodiments, at least one amino acid is deleted from the N-terminus. In certain embodiments, at least one amino acid is deleted from the C-terminus. In certain embodiments, at least one amino acid is deleted from the N-terminus and the C-terminus. In certain embodiments, at least one internal amino acid is deleted. In certain embodiments, the non-naturally occurring CKPs that bind VEGF-A comprise the amino acid sequence CNIMLPYWGCGRDFECLAGCVCQYYQSC (SEQ ID NO: 217). In certain embodiments, the C-terminus of the non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A is modified (such as capped). In certain embodiments, the N-terminus of the non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A is modified (such as capped). In certain embodiments, the C- and N-termini of the non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A are both modified (such as capped). In certain embodiments, the C-terminal carboxyl group of the non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A is amidated. In certain embodiments, the N-terminal amine of the non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A is acetylated. In certain embodiments, the C-terminal carboxyl group of the non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A is amidated and the N-terminal amine of the non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A is acetylated.

[0258] In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises at least one amino acid addition. In certain embodiments, at least one amino acid is added to the N-terminus. In certain embodiments, at least one amino acid is added to the C-terminus. In certain embodiments, at least one amino acid is added to the N-terminus and the C-terminus.

[0259] In certain embodiments, two amino acids are added to the N-terminus of the non-naturally occurring CKPs that bind to VEGF-A provided herein. In certain embodiments, two amino acids are added to the N-terminus of the non-naturally occurring CKPs that bind to VEGF-A, and the CKP is shown in GCNIMLPFWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 102). In certain embodiments, the two amino acids added to the N-terminus of SEQ ID NO: 102 are F / I / G / T / V / LH / A / S / R. In certain embodiments, the two amino acids added to the N-terminus of SEQ ID NO: 102 are selected from the group consisting of: FH, IA, GS, TR, VH and LS. In certain embodiments, the non-naturally occurring CKPs that bind to VEGF-A comprise the amino acid sequence shown in GCNIMLPFWGCGRDFECLAGCVCQYYQSCGFH (SEQ ID NO: 379). In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence shown in GCNIMLPFWGCGRDFECLAGCVCQYYQSCGIA (SEQ ID NO: 380). In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence shown in GCNIMLPFWGCGRDFECLAGCVCQYYQSCGGS (SEQ ID NO: 381). In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence shown in GCNIMLPFWGCGRDFECLAGCVCQYYQSCGTR (SEQ ID NO: 382). In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence shown in GCNIMLPFWGCGRDFECLAGCVCQYYQSCGVH (SEQ ID NO: 383). In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence shown in GCNIMLPFWGCGRDFECLAGCVCQYYQSCGLS (SEQ ID NO: 384).

[0260] In certain embodiments, two amino acids are added to the N-terminus of a non-naturally occurring CKP that binds VEGF-A, the CKP being shown in GCDVLQPYWGCGPDIDCLSNCICHWYNSCG (SEQ ID NO: 386). In certain embodiments, the two amino acids added to the N-terminus of SEQ ID NO: 102 are R / W / P / D / Q / E / ST / K / E / F / Q / L / S. In certain embodiments, the two amino acids added to the N-terminus of SEQ ID NO: 102 are selected from the group consisting of RT, WK, PL, DE, QF, EQ, PT, RL, and SL. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence shown in GCDVLQPYWGCGPDIDCLSNCICHWYNSCGRT (SEQ ID NO: 387). In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence shown in GCDVLQPYWGCGPDIDCLSNCICHWYNSCGWK (SEQ ID NO: 388). In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence shown in GCDVLQPYWGCGPDIDCLSNCICHWYNSCGPL (SEQ ID NO: 389). In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence shown in GCDVLQPYWGCGPDIDCLSNCICHWYNSCGDE (SEQ ID NO: 390). In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence shown in GCDVLQPYWGCGPDIDCLSNCICHWYNSCGQF (SEQ ID NO: 391). In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence shown in GCDVLQPYWGCGPDIDCLSNCICHWYNSCGEQ (SEQ ID NO: 392). In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence shown in GCDVLQPYWGCGPDIDCLSNCICHWYNSCGPT (SEQ ID NO: 393). In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence shown in GCDVLQPYWGCGPDIDCLSNCICHWYNSCGRL (SEQ ID NO: 394).In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence set forth in GCDVLQPYWGCGPDIDCLSNCICHWYNSCGSL (SEQ ID NO: 395).

[0261] In certain embodiments, three amino acids are added to the N-terminus of the non-naturally occurring CKPs that bind to VEGF-A provided herein. In certain embodiments, two amino acids are added to the N-terminus of the non-naturally occurring CKPs that bind to VEGF-A, and the CKP is shown in GCNIMLPFWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 102). In certain embodiments, the three amino acids added to the N-terminus of SEQ ID NO: 102 are P / N / T / D / E / Y / WL / Y / F / H / D / PI / Q / V / K / S / Y / H. In certain embodiments, the three amino acids added to the N-terminus of SEQ ID NO: 102 are selected from the group consisting of: PLI, NYQ, PLQ, TFQ, DLV, EHK, YLS, WDY, WPH, and PHQ. In certain embodiments, the non-naturally occurring CKPs that bind to VEGF-A comprise the amino acid sequence shown in GCNIMLPFWGCGRDFECLAGCVCQYYQSCGPLI (SEQ ID NO: 369). In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence shown in GCNIMLPFWGCGRDFECLAGCVCQYYQSCGNYQ (SEQ ID NO: 370). In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence shown in GCNIMLPFWGCGRDFECLAGCVCQYYQSCGPLQ (SEQ ID NO: 371). In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence shown in GCNIMLPFWGCGRDFECLAGCVCQYYQSCGTFQ (SEQ ID NO: 372). In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence shown in GCNIMLPFWGCGRDFECLAGCVCQYYQSCGDLV (SEQ ID NO: 373). In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence shown in GCNIMLPFWGCGRDFECLAGCVCQYYQSCGEHK (SEQ ID NO: 374). In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence set forth in GCNIMLPFWGCGRDFECLAGCVCQYYQSCGYLS (SEQ ID NO: 375).In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence set forth in GCNIMLPFWGCGRDFECLAGCVCQYYQSCGWDY (SEQ ID NO: 376). In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence set forth in GCNIMLPFWGCGRDFECLAGCVCQYYQSCGWPH (SEQ ID NO: 377). In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence set forth in GCNIMLPFWGCGRDFECLAGCVCQYYQSCGPHQ (SEQ ID NO: 378).

[0262] In certain embodiments, the C-terminus of the non-naturally occurring cystine knot peptide (CKP) that binds to VEGF-A is modified (such as capped). In certain embodiments, the N-terminus of the non-naturally occurring cystine knot peptide (CKP) that binds to VEGF-A is modified (such as capped). In certain embodiments, both the C- and N-termini of the non-naturally occurring cystine knot peptide (CKP) that binds to VEGF-A are modified (such as capped). In certain embodiments, the C-terminal carboxyl of the non-naturally occurring cystine knot peptide (CKP) that binds to VEGF-A is amidated. In certain embodiments, the N-terminal amine of the non-naturally occurring cystine knot peptide (CKP) that binds to VEGF-A is acetylated. In certain embodiments, the C-terminal carboxyl of the non-naturally occurring cystine knot peptide (CKP) that binds to VEGF-A is amidated and the N-terminal amine of the non-naturally occurring cystine knot peptide (CKP) that binds to VEGF-A is acetylated.

[0263] In certain embodiments, the non-naturally occurring CKP that binds VEGF-A is a variant of the non-naturally occurring CKP that binds VEGF-A described herein. In certain embodiments, such variants comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 amino acid substitutions in one or more of the sequences shown in the following sequences: SEQ ID NO: 8-14, 28-39, 60-69, 127-129, 141, 225-230, 245-261, 287-291, 396-403, and 414; SEQ ID NO: 414; NO:93-97, 117-120, 211, 298-309 and 322-339; amino acid sequences LAG, LQQ, VER, MSD, MNQ, MQT, VYQ, FIN, VSQ, VTE, FYE, MEQ, and VYR, FVR and LSN; and / or 15-18, 41-46, 71-79, 131-133, 233-238, 262-264 and 292. In certain embodiments, the amino acid substitution is a conservative amino acid substitution. In certain embodiments, the amino acid substitution does not substantially reduce the ability of the non-natural CKP binding to VEGF-A to bind to human VEGF-A. For example, conservative changes that do not substantially reduce VEGF-A binding affinity can be made (e.g., conservative substitutions as provided herein). Using the methods described in the following examples, the binding affinity of the variants of the non-natural CKP binding to VEGF-A can be evaluated.

[0264] Conservative substitutions are shown below in Table 17 under the heading "Conservative Substitutions". More substantial changes are provided in Table 17 under the heading "Exemplary Substitutions" and are further described below based on amino acid side chain classes. Amino acid substitutions can be introduced into variants of non-naturally occurring CKPs that bind VEGF-A and the products screened for the desired activity (e.g., retained / improved VEGF-A binding).

[0265] Table 17: Conservative substitutions

[0266]

[0267]

[0268] Non-conservative substitutions will entail exchanging a member of one of these classes for a member of another class.

[0269] Exemplary substitution variants are non-naturally occurring CKPs in conjunction with VEGF-A of affinity maturation, which can be conveniently produced, for example, using affinity maturation techniques (such as those described herein) based on phage display. In short, one or more residues in L1, L2, L3, L4 and / or L5 are changed (i.e., added, deleted or replaced), and the CKP in conjunction with VEGF-A of the variant is displayed on phage and screened for VEGF-A binding affinity. In certain embodiments of affinity maturation, by any of a variety of methods (e.g., error-prone PCR, loop shuffling or oligonucleotide directed mutagenesis), diversity is introduced into the variable gene selected for maturation. A secondary library is then established. The library is subsequently screened to identify any non-naturally occurring CKP variants with desired affinity to VEGF-A. In certain embodiments, introducing diversity relates to a scheme for a ring, wherein several residues in L1, L2, L3, L4 and / or L5 (e.g., about 5, about 4-6 or about 6-10 residues at a time) are randomized. The L1, L2, L3, L4 and / or L5 residues involved in binding to the target ligand can be identified, for example, using alanine scanning mutagenesis or modeling.

[0270] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNIMLPYWGCGQSFECLAGCVCQYYQSCG (SEQ ID NO: 215).

[0271] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNIMLPYWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 216).

[0272] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNIMLPYWGCGRDFECLAKCVCQYYQSCG (SEQ ID NO: 542).

[0273] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNIMLPYWGCGRDFECMSDCICQYYQSCG (SEQ ID NO: 363).

[0274] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNIMLPYWGCGRDFECMSDCICQYYQSCG (SEQ ID NO: 364).

[0275] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNIMLPYWGCGRDFECMNQCICQYYQSCG (SEQ ID NO: 222).

[0276] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNIMLPYWGCGRDFECFYECICQYYQSCG (SEQ ID NO: 223).

[0277] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNIMLPYWGCGRDFECMEQCICQYYQSCG (SEQ ID NO: 142).

[0278] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNILLPFWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 405).

[0279] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNILLPYWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 406).

[0280] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNIMSPFWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 407).

[0281] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNIMTPFWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 408).

[0282] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNIMQPFWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 409).

[0283] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNIMNPFWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 410).

[0284] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNIMEPFWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 411).

[0285] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNIMDPFWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 412).

[0286] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNIMLPSWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 415).

[0287] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNIMLPFWGCGRDFECLSGCVCQYYQSCG (SEQ ID NO: 421).

[0288] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNIMLPFWGCGRDFECLTGCVCQYYQSCG (SEQ ID NO: 422).

[0289] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNIMLPFWGCGRDFECLEGCVCQYYQSCG (SEQ ID NO: 423).

[0290] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNIMLPYWGCGRDFECLAGCLCQYYQSCG (SEQ ID NO: 424).

[0291] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNIMLPYWGCGRDFECLAGCICQYYQSCG (SEQ ID NO: 425).

[0292] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNIMLPYWGCGRDFECLAGCVCQYYQSCS (SEQ ID NO: 431).

[0293] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNILLPYWGCGRDFECMEQCICQYYQSCG (SEQ ID NO: 435).

[0294] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCDVLQPYWGCGPDIDCLSNCICHWYNSCG (SEQ ID NO: 386).

[0295] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNILLPFWGCGRDFECVSQCICQYYQSCG (SEQ ID NO: 547).

[0296] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNILQPFWGCGRDFECVSQCICQYYQSCG (SEQ ID NO: 548).

[0297] In certain embodiments, one or more amino acids in the sequence of the non-naturally occurring CKP in combination with VEGF-A provided herein are replaced by non-natural amino acids. In certain embodiments, the one or more amino acids are replaced by the same non-natural amino acids. In certain embodiments, the one or more amino acids are each replaced by different non-natural amino acids. In certain embodiments, the non-naturally occurring CKP in combination with VEGF-A comprises non-natural amino acids at any amino acid position in L1, L2, L3, L4 and / or L5, with reference to Scaffold Structure I.

[0298] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNIMLPYWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 216), wherein the N-terminal glycine is capped with a C(═O)-oxetan-3-yl group.

[0299] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNIMLPYWGCGRDFECLAXCVCQYYQSCG (SEQ ID NO: 568, wherein X is ornithine.

[0300] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence XCNIMLPYWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 361), wherein X is N-acetylglycine.

[0301] In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIMLPXWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 362), wherein X is sulfotyrosine. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIMLPXWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 362), wherein X is 3,4-difluoro-L-phenylalanine. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIMLPXWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 362), wherein X is 3,4-dichloro-L-phenylalanine. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIMLPXWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 362), wherein X is 4-chloro-L-phenylalanine. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIMLPXWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 362), wherein X is 3-F, 4-Cl-L-phenylalanine. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIMLPXWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 362), wherein X is 2-pyridone (NH para)-L-alanine. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIMLPXWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 362), wherein X is pyridone (NH meta)-L-alanine.

[0302] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNIXLPFWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 218), wherein X is norleucine.

[0303] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNIMLPFXGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 219), wherein X is 1-naphthylalanine.

[0304] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNIMLPFXGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 219), wherein X is 2-naphthylalanine.

[0305] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence XCNIMLPFWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 221), wherein X is PEG6-propargylglycine.

[0306] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNIXLPYWGCGRDFECMSDCICQYYQSCG (SEQ ID NO: 365), wherein X is norleucine.

[0307] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNIXLPFWGCGRDFECMSDCICQYYQSCG (SEQ ID NO: 144), wherein X is norleucine.

[0308] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNIXLPFWGCGRDFECVSQCICQYYQSCG (SEQ ID NO: 145), wherein X is norleucine.

[0309] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNIX1LPFWGCGRDF-D / E-CVS-N / K / X 2-CICQYYQSCG (SEQ ID NO: 540), wherein X1 is norleucine and X2 is ornithine. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIXLPFWGCGRDFECVSKCICQYYQSCG (SEQ ID NO: 545), wherein X is norleucine. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIX1LPFWGCGRDFECVSX2CICQYYQSCG (SEQ ID NO: 546), wherein X1 is norleucine and X2 is ornithine. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIX1LPFWGCGRDF-N / K / X2-CVS-D / E-CICQYYQSCG (SEQ ID NO: 541), wherein X1 is norleucine and X2 is ornithine.

[0310] In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIXLPFWGCGRDFKCVS-D / E-CICQYYQSCG (SEQ ID NO: 561, wherein X is norleucine. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence CNIXLPFWGCGRDFKCVSDCICQYYQSCG (SEQ ID NO: 562, wherein X is norleucine. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence CNIXLPFWGCGRDFKCVSECICQYYQSCG (SEQ ID NO: 563, wherein X is norleucine. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIX1LPFWGCGRDFX2CVS-D / E-CICQYYQSCG (SEQ ID NO: 564, where X1 is norleucine and X2 is ornithine. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIX1LPFWGCGRDFX2CVSDCICQYYQSCG (SEQ ID NO: 565, where X1 is norleucine and X2 is ornithine. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIX1LPFWGCGRDFX2CVSECICQYYQSCG (SEQ ID NO: 566, where X1 is norleucine and X2 is ornithine.

[0311] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNIXLPYWGCGRDFECMEQCICQYYQSCG (SEQ ID NO: 146), wherein X is norleucine.

[0312] In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIX1LPYWGCGRDF-D / E-CME-N / K / X2-CICQYYQSCG (SEQ ID NO: 538), wherein X1 is norleucine and X2 is ornithine. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIX1LPYWGCGRDFECMEKCICQYYQSCG (SEQ ID NO: 543), wherein X is norleucine. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIX1LPYWGCGRDFECMEX2CICQYYQSCG (SEQ ID NO: 544), wherein X1 is norleucine and X2 is ornithine.

[0313] In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIX1LPYWGCGRDF-N / K / X2-CME-D / E-CICQYYQSCG (SEQ ID NO: 539), wherein X1 is norleucine and X2 is ornithine. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIXLPYWGCGRDFKCME-D / E-CICQYYQSCG (SEQ ID NO: 555), wherein X is norleucine. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIXLPYWGCGRDFKCMEDCICQYYQSCG (SEQ ID NO: 556), wherein X is norleucine. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIXLPYWGCGRDFKCMEECICQYYQSCG (SEQ ID NO: 557), wherein X is norleucine. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIX1LPYWGCGRDFX2CME-D / E-CICQYYQSCG (SEQ ID NO: 558), wherein X1 is norleucine and X2 is ornithine. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIX1LPYWGCGRDFX2CMEDCICQYYQSCG (SEQ ID NO: 559), wherein X1 is norleucine and X2 is ornithine. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIX1LPYWGCGRDFX2CMEECICQYYQSCG (SEQ ID NO: 560), wherein X1 is norleucine and X2 is ornithine.

[0314] In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCDVX1QPYWGCGPDI-D / E-CLS-N / K / X2-CICHWYNSCG (SEQ ID NO: 534), wherein X1 is norleucine and X2 is ornithine. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCDVXQPYWGCGPDIDCLSKCICHWYNSCG (SEQ ID NO: 536), wherein X is norleucine. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCDVX1QPYWGCGPDIDCLSX2CICHWYNSCG (SEQ ID NO: 537), wherein X1 is norleucine and X2 is ornithine. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCDVX1QPYWGCGPDI-N / K / X2-CLS-D / E-CICHWYNSCG (SEQ ID NO: 535), wherein X1 is norleucine and X2 is ornithine. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCDVXQPYWGCGPDIDCLSNCICHWYNSCG (SEQ ID NO: 224), wherein X is norleucine. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCDVXQPYWGCGPDIKCLS-D / E-CICHWYNSCG (SEQ ID NO: 549), wherein X is norleucine. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCDVXQPYWGCGPDIKCLSDCICHWYNSCG (SEQ ID NO: 550), wherein X is norleucine. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCDVXQPYWGCGPDIKCLSECICHWYNSCG (SEQ ID NO: 551), wherein X is norleucine. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCDVX1QPYWGCGPDIX2CLS-D / E-CICHWYNSCG (SEQ ID NO: 552), wherein X1 is norleucine and X2 is ornithine. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCDVX1QPYWGCGPDIX2CLSDCICHWYNSCG (SEQ ID NO: 553), wherein X1 is norleucine and X2 is ornithine.In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCDVX1QPYWGCGPDIX2CLSECICHWYNSCG (SEQ ID NO: 554), wherein X1 is norleucine and X2 is ornithine.

[0315] In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIMLXFWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 413), wherein X is γ-benzyl-L-proline. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIMLXFWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 413), wherein X is γ-(4-fluoro-benzyl)-L-proline. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIMLXFWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 413), wherein X is 4-OH-L-proline. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIMLXFWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 413), wherein X is 4-fluoro-L-proline. In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNIMLXFWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 413), wherein X is 4-[4-(trifluoromethyl)benzyl]-L-proline.

[0316] In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIMLPYXGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 417), wherein X is N-methylindole. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIMLPYXGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 417), wherein X is N-ethylindole. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIMLPYXGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 417), wherein X is N-isopropylindole. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIMLPYXGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 417), wherein X is 5-aza-indole.

[0317] In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIMLPYWGCGRDXECLAGCVCQYYQSCG (SED ID NO: 419), wherein X is 4-methyl-L-phenylalanine. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIMLPYWGCGRDXECLAGCVCQYYQSCG (SED ID NO: 419), wherein X is 2-naphthyl-L-alanine. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIMLPYWGCGRDXECLAGCVCQYYQSCG (SED ID NO: 419), wherein X is 2-quinolyl-alanine. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIMLPYWGCGRDXECLAGCVCQYYQSCG (SED ID NO: 419), wherein X is 4,4'-biphenyl-L-alanine. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIMLPYWGCGRDXECLAGCVCQYYQSCG (SED ID NO: 419), wherein X is 3-(3-quinolinyl)-L-alanine. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIMLPYWGCGRDXECLAGCVCQYYQSCG (SED ID NO: 419), wherein X is 3-(2-quinolinyl)-L-alanine. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIMLPYWGCGRDXECLAGCVCQYYQSCG (SED ID NO: 419), wherein X is 3-(2-quinoxalinyl)-L-alanine. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIMLPYWGCGRDXECLAGCVCQYYQSCG (SED ID NO: 419), wherein X is 4-methyl-2-pyridyl-alanine. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIMLPYWGCGRDXECLAGCVCQYYQSCG (SED ID NO: 419), wherein X is 4-ethyl-2-pyridyl-L-alanine. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIMLPYWGCGRDXECLAGCVCQYYQSCG (SED ID NO: 419), wherein X is benzothiazole-L-alanine.In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIMLPYWGCGRDXECLAGCVCQYYQSCG (SED ID NO: 419), wherein X is benzothiophene-L-alanine. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIMLPYWGCGRDXECLAGCVCQYYQSCG (SED ID NO: 419), wherein X is 3-(3-isoquinolyl)-L-alanine.

[0318] In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIMLPYWGCGRDFECXAGCVCQYYQSCG (SEQ ID NO: 420), wherein X is tert-butyl-L-alanine (also known as L-neopentylglycine). In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIMLPYWGCGRDFECXAGCVCQYYQSCG (SEQ ID NO: 420), wherein X is 3-cyclobutyl-L-alanine. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIMLPYWGCGRDFECXAGCVCQYYQSCG (SEQ ID NO: 420), wherein X is 3-cyclopentyl-L-alanine. In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNIMLPYWGCGRDFECXAGCVCQYYQSCG (SEQ ID NO: 420), wherein X is 5,5,5-trifluoro-L-leucine.

[0319] In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIMLPYWGCGRDFECLAGCXCQYYQSCG (SEQ ID NO: 426), wherein X is L-tert-leucine. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIMLPYWGCGRDFECLAGCXCQYYQSCG (SEQ ID NO: 426), wherein X is tert-butyl-L-alanine (also known as L-neopentylglycine). In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIMLPYWGCGRDFECLAGCXCQYYQSCG (SEQ ID NO: 426), wherein X is L-cyclopentylglycine. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIMLPYWGCGRDFECLAGCXCQYYQSCG (SEQ ID NO: 426), wherein X is 3-cyclopentyl-L-alanine. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIMLPYWGCGRDFECLAGCXCQYYQSCG (SEQ ID NO: 426), wherein X is L-cyclobutyl-L-glycine. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIMLPYWGCGRDFECLAGCXCQYYQSCG (SEQ ID NO: 426), wherein X is 3-cyclobutyl-L-alanine. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIMLPYWGCGRDFECLAGCXCQYYQSCG (SEQ ID NO: 426), wherein X is 5,5,5-trifluoro-L-leucine.

[0320] In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIMLPYWGCGRDFECLAGCVCQXYQSCG (SEQ ID NO: 428), wherein X is 2-pyridone. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIMLPYWGCGRDFECLAGCVCQXYQSCG (SEQ ID NO: 428), wherein X is 3,4-hydroxy-L-phenylalanine. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIMLPYWGCGRDFECLAGCVCQXYQSCG (SEQ ID NO: 428), wherein X is 3,4-fluorophenylalanine. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIMLPYWGCGRDFECLAGCVCQXYQSCG (SEQ ID NO: 428), wherein X is 3-fluoro, 4-OH-L-phenylalanine.

[0321] In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIMLPYWGCGRDFECLAGCVCQYXQSCG (SEQ ID NO: 430), wherein X is 2-chloro-L-tyrosine. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIMLPYWGCGRDFECLAGCVCQYXQSCG (SEQ ID NO: 430), wherein X is 2-methyl-L-tyrosine. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIMLPYWGCGRDFECLAGCVCQYXQSCG (SEQ ID NO: 430), wherein X is 2-ethyl-L-tyrosine or 4-(naphthalene-1-ol-)-L-alanine.

[0322] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNIMLPYWGCGRDFECLAGCVCQYYQSCX (SEQ ID NO: 432), wherein X is D-serine, L-β-homoserine, L-β-alanine, N-α-methylglycine, glycine whose carboxyl terminus is converted to a glycerol ester, glycine whose carboxyl terminus is converted to an ethylene glycol ester, glycine whose carboxyl terminus is converted to an oxetane ester, or glycine morpholineamide.

[0323] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNIXQPYWGCGRDFECMEQCICQYYQSCG (SEQ ID NO: 436), wherein X is norleucine.

[0324] In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIX1LPYWGCGRDFECX2EQCICQYYQSCG (SEQ ID NO: 437). In certain embodiments, X1 and X2 are the same non-natural amino acids. In certain embodiments, X1 and X2 are different non-natural amino acids. In certain embodiments, X1 and X2 are norleucine. In certain embodiments, X1 is norleucine and X2 is 3-cyclobutyl-L-alanine.

[0325] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNIXLPYWGCGRDFECLEQCICQYYQSCG (SEQ ID NO: 438), wherein X is norleucine.

[0326] In certain embodiments, the non-naturally occurring CKP that binds to VEGF-A comprises the amino acid sequence GCNIX1LPYWGCGRDFECX2EQCX3CQYYQSCG (SEQ ID NO: 439). In certain embodiments, X1, X2 and / or X3 are the same non-natural amino acids. In certain embodiments, X1, X2 and / or X3 are not the same non-natural amino acids. In certain embodiments, X1 is norleucine, X2 is 3-cyclobutyl-L-alanine, and X3 is cyclobutyl-L-glycine. In certain embodiments, X1 is norleucine, X2 is 3-cyclobutyl-L-alanine, and X3 is 3-cyclobutyl-L-alanine. In certain embodiments, X1 is norleucine, X2 is 3-cyclobutyl-L-alanine, and X3 is norleucine.

[0327] In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence CNIX1QPYWGCGRDFECX2EQCX3CQYYQSCG (SEQ ID NO: 440). In certain embodiments, X1, X2 and / or X3 are the same non-natural amino acids. In certain embodiments, X1, X2 and / or X3 are not the same non-natural amino acids. In certain embodiments, X1 is norleucine, X2 is 3-cyclobutyl-L-alanine, and X3 is cyclobutyl-L-glycine. In certain embodiments, X1 is norleucine, X2 is 3-cyclobutyl-L-alanine, and X3 is 3-cyclobutyl-L-alanine.

[0328] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNILLPYWGCGRDFECXEQCICQYYQSCG (SEQ ID NO: 441), wherein X is 3-cyclobutyl-L-alanine or tert-butyl-L-alanine (also known as L-neopentylglycine).

[0329] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNILLPYWGCGRDFECMEQCXCQYYQSCG (SEQ ID NO: 442), wherein X is cyclobutyl-L-glycine or 3-cyclobutyl-L-alanine.

[0330] In certain embodiments, the non-naturally occurring CKP that binds to VEGF-A comprises the amino acid sequence X1CNIX2LPYWGCGRDFECMEQCICQYYQSCX3 (SEQ ID NO: 443). In certain embodiments, X1, X2 and / or X3 are the same non-natural amino acids. In certain embodiments, X1, X2 and / or X3 are not the same non-natural amino acids. In certain embodiments, X1 is N-acetylglycine, X2 is norleucine, and X3 is glycinamide.

[0331] In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence X1CNILLPYWGCGRDFECMEQCICQYYQSCX2 (SEQ ID NO: 444). In certain embodiments, X1 and X2 are the same non-natural amino acids. In certain embodiments, X1 and X2 are different non-natural amino acids. In certain embodiments, X1 is N-acetylglycine and X2 is glycinamide.

[0332] In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence X1CNILQPYWGCGRDFECMEQCICQYYQSCX2 (SEQ ID NO: 445). In certain embodiments, X1 and X2 are the same non-natural amino acids. In certain embodiments, X1 and X2 are different non-natural amino acids. In certain embodiments, X1 is N-acetylglycine and X2 is glycinamide.

[0333] In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence X1CNILQPYWGCGRDFECLEQCICQYYQSCX2 (SEQ ID NO: 446). In certain embodiments, X1 and X2 are the same non-natural amino acids. In certain embodiments, X1 and X2 are different non-natural amino acids. In certain embodiments, X1 is N-acetylglycine and X2 is glycinamide.

[0334] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCDVLQPYWGCGPDIDCXSNCICHWYNSCG (SEQ ID NO: 447), wherein X is 3-cyclobutyl-L-alanine or tert-butyl-L-alanine (L-neopentylglycine).

[0335] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCDVLQPYWGCGPDIDCLSNCXCHWYNSCG (SEQ ID NO: 448), wherein X is cyclobutyl-L-glycine.

[0336] In certain embodiments, the non-naturally occurring CKP that binds to VEGF-A comprises the amino acid sequence GCDVX1QPYWGCGPDIDCX2SNC2X3CHWYNSCG (SEQ ID NO: 449). In certain embodiments, X1, X2 and / or X3 are the same non-natural amino acids. In certain embodiments, X1, X2 and / or X3 are not the same non-natural amino acids. In certain embodiments, X1 is norleucine, X2 is 3-cyclobutyl-L-alanine, and X3 is cyclobutyl-L-glycine. In certain embodiments, X1 is norleucine, X2 is 3-cyclobutyl-L-alanine, and X3 is 3-cyclobutyl-L-alanine.

[0337] In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCDVLQPYWGCGPDIDCX1SNC2X2CHWYNSCG (SEQ ID NO: 450). In certain embodiments, X1 and X2 are the same non-natural amino acids. In certain embodiments, X1 and X2 are different non-natural amino acids. In certain embodiments, X1 is 3-cyclobutyl-L-alanine and X2 is cyclobutyl-L-glycine. In certain embodiments, X1 is 3-cyclobutyl-L-alanine and X2 is 3-cyclobutyl-L-alanine.

[0338] In certain embodiments, the non-naturally occurring CKP that binds to VEGF-A comprises the amino acid sequence X1CDVLQPYWGCGPDIDCX2SNC2X3CHWYNSCX4 (SEQ ID NO: 451). In certain embodiments, X1, X2, X3 and / or X4 are the same non-natural amino acids. In certain embodiments, X1, X2, X3 and / or X4 are not the same non-natural amino acids. In certain embodiments, X1 is N-acetylglycine, X2 is 3-cyclobutyl-L-alanine, X3 is cyclobutyl-L-glycine, and X4 is glycinamide. In certain embodiments, X1 is acetylglycine, X2 is cyclobutyl-L-alanine, X3 is cyclobutyl-L-alanine, and X4 is glycinamide.

[0339] In certain embodiments, the non-naturally occurring CKP that binds to VEGF-A comprises the amino acid sequence X1CDVX2QPYWGCGPDIDCLSNCICHWYNSCX3 (SEQ ID NO: 452). In certain embodiments, X1, X2 and / or X3 are the same non-natural amino acids. In certain embodiments, X1, X2 and / or X3 are not the same non-natural amino acids. In certain embodiments, X1 is N-acetylglycine, X2 is norleucine, and X3 is glycinamide.

[0340] In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence X1CDVLQPYWGCGPDIDCLSNCICHWYNSCX2 (SEQ ID NO: 453). In certain embodiments, X1 and X2 are the same non-natural amino acids. In certain embodiments, X1 and X2 are different non-natural amino acids. In certain embodiments, X1 is N-acetylglycine and X2 is glycinamide.

[0341] In certain embodiments, the C-terminus of the non-naturally occurring cystine knot peptide (CKP) that binds to VEGF-A is modified (such as capped). In certain embodiments, the N-terminus of the non-naturally occurring cystine knot peptide (CKP) that binds to VEGF-A is modified (such as capped). In certain embodiments, the C- and N-termini of the non-naturally occurring cystine knot peptide (CKP) that binds to VEGF-A are modified (such as capped). In certain embodiments, the C-terminal carboxyl of the non-naturally occurring cystine knot peptide (CKP) that binds to VEGF-A is amidated. In certain embodiments, the N-terminal amine of the non-naturally occurring cystine knot peptide (CKP) that binds to VEGF-A is acetylated. In certain embodiments, the C-terminal carboxyl of the non-naturally occurring cystine knot peptide (CKP) that binds to VEGF-A is amidated and the N-terminal amine of the non-naturally occurring cystine knot peptide (CKP) that binds to VEGF-A is acetylated.

[0342] Structural characteristics

[0343] In certain embodiments, the structure of the non-naturally occurring CKP that binds VEGF-A provided herein has a disulfide bond connection different from that of the WT EETI-II protein, i.e., different from the C1-C4, C2-C5 and C3-C6 disulfide bond pattern characteristics of the WT EETI-II. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A provided herein has a disulfide bond connection of C1-C4, C2-C3 and C5-C6. The method for determining the disulfide bond connection of the CKP that binds VEGF-A, for example, non-naturally occurring CKP that binds VEGF-A includes, for example, by solving and analyzing the co-crystal structure of the CKP that binds VEGF-A and VEGF-A complex, after partial reduction alkylation, via mass spectrometry, or after proteolytic digestion, via mass spectrometry, such as Sampoli et al. (2000) Proteins Struct Funct Gen. 40, 168-174, etc., for structural calculation.

[0344] In certain embodiments, the non-naturally occurring CKP that binds VEGF-A has an alpha helical content of at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50%, including any ranges between these values. In certain embodiments, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, or at least 18 amino acids of the non-naturally occurring CKP that binds VEGF-A form an alpha helix. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A has disulfide bonds of C1-C4, C2-C3, and C5-C6 and an alpha helical content of at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50%, including any ranges between these values. In certain embodiments, the alpha helical content of the non-naturally occurring CKP that binds VEGF-A is determined by, for example, circular dichroism (CD), optical rotation dispersion (ORD), nuclear magnetic resonance (NMR) by solving and analyzing the co-crystal structure of the non-naturally occurring CKP that binds VEGF-A in complex with VEGF-A after partial reductive alkylation or by mass spectrometry after proteolytic digestion.

[0345] In certain embodiments, the non-naturally occurring CKPs that bind VEGF-A provided herein compete for VEGF-A binding with a second non-naturally occurring CKP that binds VEGF-A, wherein the second non-naturally occurring CKP that binds VEGF-A comprises: L1 comprising the amino acid sequence NIMLPFWG (SEQ ID NO: 33); L2 comprising the amino acid sequence KQDSD (SEQ ID NO: 93); L3 comprising the amino acid sequence LAG; L4 comprising amino acid V, and L5 comprising the amino acid sequence QYYQS (SEQ ID NO: 45), with reference to Scaffold Structure 1. In certain embodiments, the non-naturally occurring CKPs that bind VEGF-A provided herein compete for VEGF-A binding with a second non-naturally occurring CKP that binds VEGF-A, wherein the second non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIMLPFWGCKQDSDCLAGCVCQYYQSCG (SEQ ID NO: 52).

[0346] In certain embodiments, the non-naturally occurring VEGF-A binding CKPs provided herein bind to the same epitope on VEGF-A that is bound by a second non-naturally occurring VEGF-A binding CKP, wherein the second non-naturally occurring VEGF-A binding CKP comprises: L1 comprising the amino acid sequence NIMLPFWG (SEQ ID NO:33); L2 comprising the amino acid sequence KQDSD (SEQ ID NO:93); L3 comprising the amino acid sequence LAG; L4 comprising amino acid V, and L5 comprising the amino acid sequence QYYQS (SEQ ID NO:45), with reference to Scaffold Structure 1. In certain embodiments, the non-naturally occurring VEGF-A binding CKPs provided herein bind to the same epitope on VEGF-A that is bound by a second non-naturally occurring VEGF-A binding CKP, wherein the second non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNIMLPFWGCKQDSDCLAGCVCQYYQSCG (SEQ ID NO:52).

[0347] In certain embodiments, the non-naturally occurring CKPs that bind VEGF-A provided herein compete for binding to VEGF-A with a second non-naturally occurring CKP that binds VEGF-A, wherein the second CKP that binds VEGF-A comprises: L1 comprising the amino acid sequence NIMLPFWG (SEQ ID NO: 33); L2 comprising the amino acid sequence GRDFE (SEQ ID NO: 97); L3 comprising the amino acid sequence LAG; L4 comprising amino acid V, and L5 comprising the amino acid sequence QYYQS (SEQ ID NO: 45), with reference to Scaffold Structure 1. In certain embodiments, the non-naturally occurring CKPs that bind VEGF-A provided herein compete for binding to VEGF-A with a second non-naturally occurring CKP that binds VEGF-A, wherein the second non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIMLPFWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 102).

[0348] In certain embodiments, the non-naturally occurring VEGF-A binding CKPs provided herein bind to the same epitope on VEGF-A that is bound by a second non-naturally occurring VEGF-A binding CKP, wherein the second non-naturally occurring VEGF-A binding CKP comprises: L1 comprising the amino acid sequence NIMLPFWG (SEQ ID NO:33); L2 comprising the amino acid sequence GRDFE (SEQ ID NO:97); L3 comprising the amino acid sequence LAG; L4 comprising amino acid V, and L5 comprising the amino acid sequence QYYQS (SEQ ID NO:45), with reference to Scaffold Structure 1. In certain embodiments, the non-naturally occurring VEGF-A binding CKPs provided herein bind to the same epitope on VEGF-A that is bound by a second non-naturally occurring VEGF-A binding CKP, wherein the second non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNIMLPFWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO:102).

[0349] In certain embodiments, the non-naturally occurring CKP that binds VEGF-A provided herein competes for binding to VEGF-A with a second non-naturally occurring CKP that binds VEGF-A, wherein the second non-naturally occurring CKP that binds VEGF-A comprises: L1 comprising the amino acid sequence DVMQPYWG (SEQ ID NO: 35); L2 comprising the amino acid sequence KQDSD (SEQ ID NO: 93); L3 comprising the amino acid sequence LAG; L4 comprising amino acid V, and L5 comprising the amino acid sequence HWYNS (SEQ ID NO: 46), with reference to Scaffold Structure 1. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A provided herein competes for binding to VEGF-A with a second non-naturally occurring CKP that binds VEGF-A, wherein the second non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCDVMQPYWGCKQDSDCLAGCVCHWYNSCG (SEQ ID NO: 55).

[0350] In certain embodiments, the non-naturally occurring VEGF-A binding CKPs provided herein bind to the same epitope on VEGF-A that is bound by a second non-naturally occurring VEGF-A binding CKP, wherein the second non-naturally occurring VEGF-A binding CKP comprises: L1 comprising the amino acid sequence DVMQPYWG (SEQ ID NO: 35); L2 comprising the amino acid sequence KQDSD (SEQ ID NO: 93); L3 comprising the amino acid sequence LAG; L4 comprising amino acid V, and L5 comprising the amino acid sequence HWYNS (SEQ ID NO: 46), with reference to Scaffold Structure 1. In certain embodiments, the non-naturally occurring VEGF-A binding CKPs provided herein bind to the same epitope on VEGF-A that is bound by a second non-naturally occurring VEGF-A binding CKP, wherein the second non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCDVMQPYWGCKQDSDCLAGCVCHWYNSCG (SEQ ID NO: 55).

[0351] In certain embodiments, the non-naturally occurring CKP proteins that bind to VEGF-A provided herein compete with a second non-naturally occurring CKP that binds to VEGF-A for binding to VEGF-A, wherein the second CKP that binds to VEGF-A comprises: L1 comprising the amino acid sequence DVMQPYWG (SEQ ID NO: 35); L2 comprising the amino acid sequence GPDID (SEQ ID NO: 118); L3 comprising the amino acid sequence LAG; L4 comprising amino acid V, and L5 comprising the amino acid sequence HWYNS (SEQ ID NO: 46), with reference to Scaffold Structure 1. In certain embodiments, the non-naturally occurring CKP that binds to VEGF-A provided herein competes with a second non-naturally occurring CKP that binds to VEGF-A for binding to VEGF-A, wherein the second non-naturally occurring CKP that binds to VEGF-A comprises the amino acid sequence GCDVMQPYWGCGPDIDCLAGCVCHWYNSCG (SEQ ID NO: 123).

[0352] In certain embodiments, the non-naturally occurring VEGF-A binding CKPs provided herein bind to the same epitope on VEGF-A that is bound by a second non-naturally occurring VEGF-A binding CKP, wherein the second non-naturally occurring VEGF-A binding CKP comprises: L1 comprising the amino acid sequence DVMQPYWG (SEQ ID NO: 35); L2 comprising the amino acid sequence GPDID (SEQ ID NO: 118); L3 comprising the amino acid sequence LAG; L4 comprising amino acid V, and L5 comprising the amino acid sequence HWYNS (SEQ ID NO: 46), with reference to Scaffold Structure 1. In certain embodiments, the non-naturally occurring VEGF-A binding CKPs provided herein bind to the same epitope on VEGF-A that is bound by a second non-naturally occurring VEGF-A binding CKP, wherein the second non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCDVMQPYWGCGPDIDCLAGCVCHWYNSCG (SEQ ID NO: 123).

[0353] In certain embodiments, the non-naturally occurring CKPs that bind to VEGF-A provided herein bind to an epitope of VEGF-A that comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more than ten amino acids selected from the group consisting of: V14, V15, F17, D19, Y21, Q22, Y25, I46, K48, N62, D63, L66, M81, I83, K84, P85, H86, Q87, G88, Q89, I91, C104, R105, and P106. In certain embodiments, the non-naturally occurring CKPs that bind to VEGF-A provided herein bind to an epitope of VEGF-A that comprises K48, N62, and D63. In certain embodiments, the non-naturally occurring CKPs that bind to VEGF-A provided herein bind to an epitope of VEGF-A, the epitope comprising H86. In certain embodiments, the non-naturally occurring CKPs that bind to VEGF-A provided herein bind to an epitope of VEGF-A, the epitope comprising Y21, Y25 and P106. In certain embodiments, the non-naturally occurring CKPs that bind to VEGF-A provided herein bind to an epitope of VEGF-A, the epitope comprising M81, D19 and Q22. In certain embodiments, the non-naturally occurring CKPs that bind to VEGF-A provided herein bind to an epitope of VEGF-A, the epitope comprising F17, M81 and I91. In certain embodiments, the non-naturally occurring CKPs that bind to VEGF-A provided herein bind to an epitope of VEGF-A, the epitope comprising V14, F17, D19, Q22, M81 and I91. In certain embodiments, the non-naturally occurring VEGF-A binding CKP provided herein binds to an epitope of VEGF-A comprising Q22 and Y25.

[0354] In certain embodiments, the non-naturally occurring CKPs that bind to VEGF-A provided herein bind to an epitope on VEGF-A that overlaps with the epitope of VEGF-A that is bound by the anti-VEGF-A antibody G6.31 (Fuh et al. (2006) J. Biol. Chem. 281, 6625-6631). In certain embodiments, the non-naturally occurring CKPs that bind to VEGF-A provided herein bind to an epitope on VEGF-A that overlaps with the epitope of VEGF-A that is bound by Flt-1. In certain embodiments, the non-naturally occurring CKPs that bind to VEGF-A provided herein bind to an epitope on VEGF-A that overlaps with the epitope of VEGF-A that is bound by bevacizumab.

[0355] Features

[0356] In certain embodiments, a non-naturally occurring CKP that "specifically binds" VEGF-A (such as human VEGF-A, mouse VEGF-A, and / or rat VEGF-A) has a binding affinity of no more than about 1×10 -7 M, preferably not more than about 1×10 -8 and most preferably no more than about 1×10 -9 M), but its binding affinity for a homologue of VEGF-A or other growth factor is at least about 50 times weaker, or at least about 500 times weaker, or at least about 1000 times weaker than its binding affinity for VEGF-A.

[0357] In certain embodiments, the degree of binding of the non-naturally occurring CKPs that bind to VEGF-A provided herein to, for example, non-target proteins (e.g., homologs of VEGFA, such as VEGF-B, VEGF-C, and VEGF-D) or other growth factors (such as P1GF, EGF, NGF, IGF, and PDGF) is less than about 10% of the binding of the non-naturally occurring CKPs that bind to VEGF-A to VEGF-A, as determined by methods known in the art such as ELISA, fluorescence activated cell sorting (FACS) analysis, or radioimmunoprecipitation (RIA). Specific binding can be measured, for example, by determining the binding of a molecule compared to the binding of a control molecule, which is generally a molecule of a similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule, which is similar to the target, for example, an excess of unlabeled target. In this case, if the binding of the labeled target to the probe is competitively inhibited by an excess of unlabeled target, specific binding is indicated. Additional methods for evaluating the binding of non-naturally occurring CKPs that "specifically bind" VEGF-A are described in the Examples.

[0358] As used herein, the terms "specifically bind to" a particular polypeptide or an epitope on a particular polypeptide target, "specifically bind to" a particular polypeptide or an epitope on a particular polypeptide target, or "have specificity for" a particular polypeptide or an epitope on a particular polypeptide target can be exhibited, for example, by a molecule that has at least about 10 -4 M, or at least about 10 -5 M, or at least about 10 -6 M, or at least about 10 -7 M, or at least about 10 -8 M, or at least about 10 -9 M, or at least about 10 -10 M, or at least about 10 -11 M, or at least about 10 -12In one embodiment, the term "specifically binds" refers to a molecule wherein the molecule binds to a specific polypeptide or an epitope on a specific polypeptide without substantially binding to any other polypeptide or polypeptide epitope.

[0359] In certain embodiments, the non-naturally occurring CKP that binds VEGF-A binds VEGF-A with a Kd of about 1 pM to about 500 nM. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A binds VEGF-A with a Kd of about 1 pM to about 50 pM, about 50 pM to about 250 pM, about 250 pM to about 500 pM, about 500 pM to 750 pM, about 750 pM to about 1 nM, about 1 nM to about 25 nM, about 25 nM to about 50 nM, 50 nM to about 100 nM, about 100 nM to about 250 nM, or about 250 nM to about 500 nM.

[0360] In certain embodiments, the non-naturally occurring CKP that binds to VEGF-A binds to human VEGF-A, mouse VEGF-A and / or rat VEGF-A. In certain embodiments, the non-naturally occurring CKP that binds to VEGF-A that binds to human VEGF-A, mouse VEGF-A and rat VEGF-A comprises: L1 comprising the amino acid sequence NIMLPFWG (SEQ ID NO: 33); L2 comprising the amino acid sequence KQDSD (SEQ ID NO: 93); L3 comprising the amino acid sequence LAG; L4 comprising amino acid V, and L5 comprising the amino acid sequence QYYQS (SEQ ID NO: 45), with reference to Scaffold Structure 1. In certain embodiments, the non-naturally occurring CKP that binds to VEGF-A that binds to human VEGF-A, mouse VEGF-A and rat VEGF-A comprises the amino acid sequence GCNIMLPFWGCKQDSDCLAGCVCQYYQSCG (SEQ ID NO: 52).

[0361] In certain embodiments, the non-naturally occurring CKP that binds VEGF-A binds human VEGF-A, mouse VEGF-A, and rat VEGF-A, comprising: L1 comprising the amino acid sequence NIMLPFWG (SEQ ID NO: 33); L2 comprising the amino acid sequence GRDFE (SEQ ID NO: 97); L3 comprising the amino acid sequence LAG; L4 comprising amino acid V, and L5 comprising the amino acid sequence QYYQS (SEQ ID NO: 45), with reference to Scaffold Structure 1. In certain embodiments, the non-naturally occurring CKP that binds human VEGF-A, mouse VEGF-A, and rat VEGF-A comprises the amino acid sequence GCNIMLPFWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 102).

[0362] In certain embodiments, the non-naturally occurring CKP that binds to human VEGF-A, mouse VEGF-A, and rat VEGF-A comprises: L1 comprising the amino acid sequence DVMQPYWG (SEQ ID NO: 35); L2 comprising the amino acid sequence KQDSD (SEQ ID NO: 93); L3 comprising the amino acid sequence LAG; L4 comprising amino acid V, and L5 comprising the amino acid sequence HWYNS (SEQ ID NO: 46), with reference to Scaffold Structure 1. In certain embodiments, the non-naturally occurring CKP that binds to human VEGF-A, mouse VEGF-A, and rat VEGF-A comprises the amino acid sequence GCDVMQPYWGCKQDSDCLAGCVCHWYNSCG (SEQ ID NO: 55).

[0363] In certain embodiments, the non-naturally occurring VEGF-A binding CKP that binds human VEGF-A, mouse VEGF-A, and rat VEGF-A comprises: L1 comprising the amino acid sequence DVMQPYWG (SEQ ID NO: 35); L2 comprising the amino acid sequence GPDID (SEQ ID NO: 118); L3 comprising the amino acid sequence LAG; L4 comprising amino acid V, and L5 comprising the amino acid sequence HWYNS (SEQ ID NO: 46), with reference to Scaffold Structure 1. In certain embodiments, the non-naturally occurring VEGF-A binding CKP that binds human VEGF-A, mouse VEGF-A, and rat VEGF-A comprises the amino acid sequence GCDVMQPYWGCGPDIDCLAGCVCHWYNSCG (SEQ ID NO: 123).

[0364] In certain embodiments, the non-naturally occurring CKPs described herein that bind VEGF-A have an IC of less than about 0.5 nM, less than about 0.6 nM, less than about 0.7 nM, less than about 0.8 nM, less than about 0.9 nM, or less than about 1.0 nM. 50 values, including any ranges between those values.

[0365] In certain embodiments, the non-naturally occurring CKP that binds VEGF-A does not inhibit trypsin protease activity, such as in a peptide substrate cleavage assay (eg, the peptide substrate cleavage assay described in the Examples).

[0366] In certain embodiments, the non-naturally occurring CKP that binds VEGF-A is resistant to trypsin digestion. In certain embodiments, after incubation with trypsin at 37°C for 24 hours, about 30% or less, about 25% or less, or about 20% or less of the non-naturally occurring CKP that binds VEGF-A is cleaved at Arg13 within loop 2.

[0367] Also contemplated are nucleic acid molecules encoding the non-naturally occurring CKPs that bind to VEGF-A described herein, expression vectors comprising nucleic acid molecules encoding the non-naturally occurring CKPs that bind to VEGF-A, and cells comprising the nucleic acid molecules. Also provided herein are methods of producing the non-naturally occurring CKPs that bind to VEGF-A described herein by culturing the cells, expressing the non-naturally occurring CKPs that bind to VEGF-A, and recovering the non-naturally occurring CKPs that bind to VEGF-A from the cell culture.

[0368] In certain embodiments, a non-naturally occurring CKP that binds VEGF-A is produced via in vitro translation, as described elsewhere herein.

[0369] In certain embodiments, a non-naturally occurring CKP that binds VEGF-A is produced via chemical peptide synthesis, for example by grafting chemically synthesized L1, L2, L3, L4 and / or L5 peptides onto a scaffold framework (such as scaffold structure I), or by chemically synthesizing the entire non-naturally occurring CKP that binds VEGF-A.

[0370] Non-naturally occurring cystine knot peptide (CKP) that binds to human low-density lipoprotein receptor (LDL)-related protein 6 (LRP6)

[0371] The LDL receptor is a transmembrane cell surface protein involved in receptor-mediated endocytosis of lipoproteins and protein ligands. Human LDL receptor-related protein 6 (LRP6) (Accession Nos.: NM_002336 (mRNA) and NP_002327 (protein); UniProtKB: O75581) acts as a receptor, or with Frizzled (a co-receptor for Wnt), and thereby transmits the canonical Wnt / β-catenin signaling cascade (Katoh et al. (2007) Clin Cancer Res 13: 4042-4045). Through its interaction with the Wnt / β-catenin signaling cascade, LRP6 plays a role in the regulation of cell differentiation, proliferation, and migration, and in the development of many cancer types (Li et al. (2004) Oncogene 23:9129-9135; Tung et al. (2012) PLoS ONE 7(5):e36565.doi:10.1371 / journal.pone.0036565; Liu et al. (2010) Proc Natl Acad Sci USA 107:5136-5141).

[0372] Wnt signaling is involved in many biological pathways. In terms of disease, it is related to cancer and metastatic disease, osteoporosis and other bone metabolism and diseases, neuronal and neurodegenerative diseases, rheumatoid arthritis and other inflammatory diseases. This inhibition of Wnt signaling by blocking LRP6 may have a wide range of therapeutic benefits. Bone loss is a serious medical problem, not only in the process of postmenopausal osteoporosis, but also in rheumatoid arthritis. Bone degradation occurs in multiple myeloma and in bone metastasis. Therefore, therapeutic strategies aimed at strengthening bones, fracture prevention or restoring damaged bones are of great importance (Kawai et al. (2011) Nat. Rev. Drug Discov. 10, 141–156; Mason and Williams (2010) J. Osteoporosis, vol. 2010, article ID 460120, 9 pages; doi: 10.4061 / 2010 / 460120). Due to their role in inhibiting new bone formation, the Wnt pathway inhibitors DKK1 and SOST are considered very promising therapeutic targets; antibodies with the function of neutralizing SOST have shown significant preclinical activity (Ominsky et al. (2010) J. Bone Miner. Res. 25, 948-959) and are now in human clinical trials (Padhi et al. (2011) J. Bone Miner. Res. 26, 19-26).

[0373] Misregulated Wnt signaling has been implicated in diseases ranging from osteoporosis to cancer (Clevers (2006) Cell 127:469-80; MacDonald et al. 2009. Dev Cell 17:9-26; Nusse (2008) Cell Res 18:523-7; Polakis (2007) Curr Opin Genet Dev 17:45-51). This list has been expanded to include metabolic disorders (Mani et al. (2007) Science 315:1278-82) and neurodegeneration (Caricasole et al. (2004) J Neurosci 24:6021-7; De Ferrari et al. (2007) Proc Natl Acad Sci USA 104:9434-9). A particularly clear link exists between mutations in the protein adenomatous polyposis coli (APC), which prevents efficient regulation of β-catenin levels, and colorectal cancer (Polakis (2007) Curr Opin Genet Dev 17:45-51). Particular attention should also be paid to the strong genetic relationship between LRP5 and bone homeostasis. Loss-of-function mutations in LRP5 cause the autosomal recessive disorder osteoporosis-pseudoglioma syndrome (OPPG), which is characterized by low bone mass, ocular defects, and a tendency to fractures (Gong et al. (2001) Cell 107:513-23).

[0374] Provided herein are non-naturally occurring CKPs that bind to human low-density lipoprotein receptor-related protein 6 (LRP6), wherein the non-natural CKP comprises the following cystine scaffold structure (i.e., scaffold structure I):

[0375] Z1C1L1C2L2C3L3C4L4C5L5C6Z2 (I)

[0376] in:

[0377] Z1 and Z2 are any amino acids;

[0378] L1 is Ring 1 and has a structure selected from the group consisting of X1X2X3X4X5X6, X1X2X3X4X5X6X7, X1X2X3X4X5X6X7X8, X1X2X3X4X5X6X7X8X9, and X1X2X3X4X5X6X7X8X9X 10 , where X1-X 10 Each of is any amino acid;

[0379] L2 is loop 2 and has the structure: X1X2X3X4X5, where each of X1–X5 is any amino acid;

[0380] L3 is loop 3 and has the structure: X1X2X3, where each of X1–X3 is any amino acid;

[0381] L4 is loop 4 and has the structure: X1, wherein X1 is any amino acid; and

[0382] L5 is loop 5 and has the structure: X1X2X3X4X5, wherein each of X1-X5 is any amino acid.

[0383] In certain embodiments, Z1 and / or Z2 of the non-naturally occurring cystine knot peptide (CKP) that binds LRP6 is G. In certain embodiments, Z1 and / or Z2 comprises more than one amino acid. In certain embodiments, Z1 and / or Z2 comprises 4 amino acids. In certain embodiments, Z1 and / or Z2 comprises 5 amino acids.

[0384] In certain embodiments, the non-naturally occurring CKP that binds LRP6 comprises L1, wherein X1 is an amino acid selected from R, V, M, A, G, N, S, and E; wherein X2 is an amino acid selected from T, N, S, G, R, and A; wherein X3 is an amino acid selected from N, R, H, V, K, S, G, I, and Y; wherein X4 is an amino acid selected from R, V, N, I, K, S, and T; wherein X5 is an amino acid selected from V, R, K, I, T, S, L, and N; and wherein X6 is an amino acid selected from K, G, A, I, R, N, S, and V. In certain embodiments, the non-naturally occurring CKP that binds LRP6 comprises L1, wherein X7 is an amino acid selected from G, R, K, E, P, and T. In certain embodiments, the non-naturally occurring CKP that binds LRP6 comprises L1, wherein X8 is an amino acid selected from G, R, K, Q, A, and S. In certain embodiments, the non-naturally occurring CKP that binds LRP6 comprises L1, wherein X9 is an amino acid selected from R or G. In certain embodiments, the non-naturally occurring CKP that binds LRP6 comprises L1, wherein X9 is an amino acid selected from R or G. 10is an amino acid selected from E, W, and G. In certain embodiments, the non-naturally occurring CKP that binds to LRP6 comprises L5, wherein X1 is an amino acid selected from G, S, N, Y, A, and R; wherein X2 is an amino acid selected from P, G, S, V, E, R, F, and D; wherein X3 is an amino acid selected from N, G, S, E, P, K, H, and R; wherein X4 is an amino acid selected from G, R, H, S, Q, V, and D; and wherein X5 is an amino acid selected from F, D, N, R, G, Y, S, and T. In certain embodiments, the non-naturally occurring CKP that binds to LRP6 comprises L2, wherein X1 is K, X2 is Q, X3 is D, X4 is S, and X5 is D. In certain embodiments, the non-naturally occurring CKP that binds to LRP6 comprises L3, wherein X1 is L, X2 is A, and X3 is G. In certain embodiments, the non-naturally occurring CKP that binds to LRP6 comprises L4, wherein X1 is V.

[0385] In certain embodiments, the non-naturally occurring CKP that binds LRP6 competitively inhibits the binding of a competing molecule to human LRP6. In certain embodiments, the competing molecule is an anti-LRP6 antibody. In certain embodiments, the competing molecule is a second non-naturally occurring CKP that binds LRP6.

[0386] Non-naturally occurring CKPs that bind to overlapping or similar regions on the target can be identified by competitive inhibition / binding assays. Such assays are well known in the art and are described in, for example, SJ Mather (ed.) 1996. Current Directions in Radiopharmaceutical Research and Development, 169-179, Kluwer Academic Publishers; Zettner (1973) Clin. Chem. 19, 699-705; Gao (2012) Analytical Methods 4, 3718-3723.

[0387] In certain embodiments, the non-naturally occurring LRP6-binding CKP binds to the same human LRP6 epitope that is bound by a second non-naturally occurring LRP6-binding CKP comprising: an amino acid sequence comprising V / R / N / S / E / GN / S / G / RR / V / K / S / N / I / YV / N / I / R / S / TR / K / I / NG / I / R / K / S / A (SEQ ID NO: 185) or A / R / M / V / G / SN / T / S / AR / N / HV / R / KK / V / IR / K / A / N / S / VT / G / R / K / PS / G / R / A (SEQ ID NO: 186) or R / A / QS / AG / S / N / IN / KT / S / L / RI / R / VR / E / KK / Q / A / RR / G / QE / W / G / R (SEQ ID NO: 187). NO:187); L1 comprising the amino acid sequence KQDSD (SEQ ID NO:93); L3 comprising the amino acid sequence LAG; L4 comprising amino acid V; and L5 comprising the amino acid sequence G / S / N / Y / A / RP / G / S / V / E / R / F / DN / G / S / E / P / K / H / RG / R / H / S / Q / V / DF / D / N / R / G / Y / S / T (SEQ ID NO:188), with reference to Scaffold Structure I.

[0388] In certain embodiments, the non-naturally occurring CKP that binds LRP6 comprises: L1 comprising the amino acid sequence V / R / N / S / E / GN / S / G / RR / V / K / S / N / I / YV / N / I / R / S / TR / K / I / NG / I / R / K / S / A (SEQ ID NO: 185) or A / R / M / V / G / SN / T / S / AR / N / HV / R / KK / V / IR / K / A / N / S / VT / G / R / K / PS / G / R / A (SEQ ID NO: 186) or R / ASG / S / NN / KT / S / LI / RR / EK / Q / AR / GE / W / G (SEQ ID NO: 187). In certain embodiments, the non-naturally occurring CKP that binds LRP6 further comprises: L2 comprising the amino acid sequence KQDSD (SEQ ID NO: 93). In certain embodiments, the non-naturally occurring CKP that binds LRP6 further comprises: L3 comprising the amino acid sequence LAG. In certain embodiments, the non-naturally occurring CKP that binds LRP6 further comprises: L4 comprising the amino acid V. In certain embodiments, the non-naturally occurring CKP that binds LRP6 further comprises: L5 comprising the amino acid sequence G / S / N / Y / A / RP / G / S / V / E / R / F / DN / G / S / E / P / K / H / RG / R / H / S / Q / V / DF / D / N / R / G / Y / S / T (SEQ ID NO: 188).

[0389] In certain embodiments, the non-naturally occurring CKP that binds to LRP6 comprises L1 and / or L5 of any one of the non-naturally occurring CKPs that bind to LRP6 disclosed herein. In certain embodiments, the non-naturally occurring CKP that binds to LRP6 comprises: L1 comprising the amino acid sequence shown in any one of SEQ ID NOs: 147-168 and 367, with reference to Scaffold Structure I. In certain embodiments, the non-naturally occurring CKP that binds to LRP6 further comprises: L2 comprising the amino acid sequence shown in SEQ ID NO: 93. In certain embodiments, the non-naturally occurring CKP that binds to LRP6 further comprises: L3 comprising the amino acid sequence LAG. In certain embodiments, the non-naturally occurring CKP that binds to LRP6 further comprises: L4 comprising amino acid V. In certain embodiments, the non-naturally occurring CKP that binds to LRP6 further comprises: L5 comprising the amino acid sequence shown in any one of SEQ ID NOs: 19 and 169-184.

[0390] The amino acid sequences of L1 and L5 described above are provided in Table 18 below:

[0391] Table 18

[0392]

[0393]

[0394] In certain embodiments, the non-naturally occurring LRP6-binding CKP comprises: L1 comprising the amino acid sequence RTNRVKGG (SEQ ID NO: 147); L2 comprising the amino acid sequence KQDSD (SEQ ID NO: 93); L3 comprising the amino acid sequence LAG; L4 comprising amino acid V; and L5 comprising the amino acid sequence GPNGF (SEQ ID NO: 19), according to reference scaffold structure I.

[0395] In certain embodiments, the non-naturally occurring LRP6-binding CKP comprises: L1 comprising the amino acid sequence VNRVRG (SEQ ID NO: 148); L2 comprising the amino acid sequence KQDSD (SEQ ID NO: 93); L3 comprising the amino acid sequence LAG; L4 comprising amino acid V; and L5 comprising the amino acid sequence SGGRD (SEQ ID NO: 169), according to reference scaffold structure I.

[0396] In certain embodiments, the non-naturally occurring LRP6-binding CKP comprises: L1 comprising the amino acid sequence MNHVKARR (SEQ ID NO: 149); L2 comprising the amino acid sequence KQDSD (SEQ ID NO: 93); L3 comprising the amino acid sequence LAG; L4 comprising amino acid V; and L5 comprising the amino acid sequence GPNGF (SEQ ID NO: 19), with reference to Scaffold Structure I.

[0397] In certain embodiments, the non-naturally occurring LRP6-binding CKP comprises: L1 comprising the amino acid sequence RSVNKI (SEQ ID NO: 150); L2 comprising the amino acid sequence KQDSD (SEQ ID NO: 93); L3 comprising the amino acid sequence LAG; L4 comprising amino acid V; and L5 comprising the amino acid sequence GSSRN (SEQ ID NO: 170), with reference to Scaffold Structure I.

[0398] In certain embodiments, the non-naturally occurring LRP6-binding CKP comprises: L1 comprising the amino acid sequence VNKIKG (SEQ ID NO: 151); L2 comprising the amino acid sequence KQDSD (SEQ ID NO: 93); L3 comprising the amino acid sequence LAG; L4 comprising amino acid V; and L5 comprising the amino acid sequence GVEGR (SEQ ID NO: 29), with reference to Scaffold Structure I.

[0399] In certain embodiments, the non-naturally occurring LRP6-binding CKP comprises: L1 comprising the amino acid sequence RNSIKR (SEQ ID NO: 152); L2 comprising the amino acid sequence KQDSD (SEQ ID NO: 93); L3 comprising the amino acid sequence LAG; L4 comprising amino acid V; and L5 comprising the amino acid sequence SVGHG (SEQ ID NO: 172), with reference to Scaffold Structure I.

[0400] In certain embodiments, the non-naturally occurring LRP6-binding CKP comprises: L1 comprising the amino acid sequence VSNRVNKG (SEQ ID NO: 153); L2 comprising the amino acid sequence KQDSD (SEQ ID NO: 93); L3 comprising the amino acid sequence LAG; L4 comprising amino acid V; and L5 comprising the amino acid sequence GPNGF (SEQ ID NO: 19), with reference to Scaffold Structure I.

[0401] In certain embodiments, the non-naturally occurring LRP6-binding CKP comprises: L1 comprising the amino acid sequence RGNIIK (SEQ ID NO: 154); L2 comprising the amino acid sequence KQDSD (SEQ ID NO: 93); L3 comprising the amino acid sequence LAG; L4 comprising amino acid V; and L5 comprising the amino acid sequence NESRG (SEQ ID NO: 173), with reference to Scaffold Structure I.

[0402] In certain embodiments, the non-naturally occurring LRP6-binding CKP comprises: L1 comprising the amino acid sequence RSGNTIRKRE (SEQ ID NO: 155); L2 comprising the amino acid sequence KQDSD (SEQ ID NO: 93); L3 comprising the amino acid sequence LAG; L4 comprising amino acid V; and L5 comprising the amino acid sequence GGPGG (SEQ ID NO: 174), with reference to Scaffold Structure I.

[0403] In certain embodiments, the non-naturally occurring LRP6-binding CKP comprises: L1 comprising the amino acid sequence ASSNSIRQGW (SEQ ID NO: 156); L2 comprising the amino acid sequence KQDSD (SEQ ID NO: 93); L3 comprising the amino acid sequence LAG; L4 comprising amino acid V; and L5 comprising the amino acid sequence GPKSN (SEQ ID NO: 175), with reference to Scaffold Structure I.

[0404] In certain embodiments, the non-naturally occurring LRP6-binding CKP comprises: L1 comprising the amino acid sequence RSNRIR (SEQ ID NO: 157); L2 comprising the amino acid sequence KQDSD (SEQ ID NO: 93); L3 comprising the amino acid sequence LAG; L4 comprising amino acid V; and L5 comprising the amino acid sequence YGHGD (SEQ ID NO: 176), with reference to Scaffold Structure I.

[0405] In certain embodiments, the non-naturally occurring LRP6-binding CKP comprises: L1 comprising the amino acid sequence RSNKLREARG (SEQ ID NO: 158); L2 comprising the amino acid sequence KQDSD (SEQ ID NO: 93); L3 comprising the amino acid sequence LAG; L4 comprising amino acid V; and L5 comprising the amino acid sequence GSRQD (SEQ ID NO: 177), with reference to Scaffold Structure I.

[0406] In certain embodiments, the non-naturally occurring LRP6-binding CKP comprises: L1 comprising the amino acid sequence VNSVKR (SEQ ID NO: 159); L2 comprising the amino acid sequence KQDSD (SEQ ID NO: 93); L3 comprising the amino acid sequence LAG; L4 comprising amino acid V; and L5 comprising the amino acid sequence SRGVN (SEQ ID NO: 178), with reference to Scaffold Structure I.

[0407] In certain embodiments, the non-naturally occurring LRP6-binding CKP comprises: L1 comprising the amino acid sequence GSNKIRPR (SEQ ID NO: 160); L2 comprising the amino acid sequence KQDSD (SEQ ID NO: 93); L3 comprising the amino acid sequence LAG; L4 comprising amino acid V; and L5 comprising the amino acid sequence GPNDF (SEQ ID NO: 179), with reference to Scaffold Structure I.

[0408] In certain embodiments, the non-naturally occurring LRP6-binding CKP comprises: L1 comprising the amino acid sequence NRIRNS (SEQ ID NO: 161); L2 comprising the amino acid sequence KQDSD (SEQ ID NO: 93); L3 comprising the amino acid sequence LAG; L4 comprising amino acid V; and L5 comprising the amino acid sequence GRGDY (SEQ ID NO: 180), with reference to Scaffold Structure I.

[0409] In certain embodiments, the non-naturally occurring LRP6-binding CKP comprises: L1 comprising the amino acid sequence SRNSIK (SEQ ID NO: 162); L2 comprising the amino acid sequence KQDSD (SEQ ID NO: 93); L3 comprising the amino acid sequence LAG; L4 comprising amino acid V; and L5 comprising the amino acid sequence ASGSS (SEQ ID NO: 181), with reference to Scaffold Structure I.

[0410] In certain embodiments, the non-naturally occurring LRP6-binding CKP comprises: L1 comprising the amino acid sequence SNYVKR (SEQ ID NO: 163); L2 comprising the amino acid sequence KQDSD (SEQ ID NO: 93); L3 comprising the amino acid sequence LAG; L4 comprising amino acid V; and L5 comprising the amino acid sequence SPGGR (SEQ ID NO: 182), with reference to Scaffold Structure I.

[0411] In certain embodiments, the non-naturally occurring LRP6-binding CKP comprises: L1 comprising the amino acid sequence RANRVSGR (SEQ ID NO: 164); L2 comprising the amino acid sequence KQDSD (SEQ ID NO: 93); L3 comprising the amino acid sequence LAG; L4 comprising amino acid V; and L5 comprising the amino acid sequence GPNGF (SEQ ID NO: 19), with reference to Scaffold Structure I.

[0412] In certain embodiments, the non-naturally occurring LRP6-binding CKP comprises: L1 comprising the amino acid sequence SNRVKVRA (SEQ ID NO: 165); L2 comprising the amino acid sequence KQDSD (SEQ ID NO: 93); L3 comprising the amino acid sequence LAG; L4 comprising amino acid V; and L5 comprising the amino acid sequence GPNGF (SEQ ID NO: 19), with reference to Scaffold Structure I.

[0413] In certain embodiments, the non-naturally occurring LRP6-binding CKP comprises: L1 comprising the amino acid sequence ENRTKG (SEQ ID NO: 166); L2 comprising the amino acid sequence KQDSD (SEQ ID NO: 93); L3 comprising the amino acid sequence LAG; L4 comprising amino acid V; and L5 comprising the amino acid sequence GFRGT (SEQ ID NO: 183), according to reference scaffold structure I.

[0414] In certain embodiments, the non-naturally occurring LRP6-binding CKP comprises: L1 comprising the amino acid sequence GNKIRA (SEQ ID NO: 167); L2 comprising the amino acid sequence KQDSD (SEQ ID NO: 93); L3 comprising the amino acid sequence LAG; L4 comprising amino acid V; and L5 comprising the amino acid sequence RDRVG (SEQ ID NO: 184), with reference to Scaffold Structure I.

[0415] In certain embodiments, the non-naturally occurring LRP6-binding CKP comprises: L1 comprising the amino acid sequence ANRVKRTS (SEQ ID NO: 168); L2 comprising the amino acid sequence KQDSD (SEQ ID NO: 93); L3 comprising the amino acid sequence LAG; L4 comprising amino acid V; and L5 comprising the amino acid sequence GPNGF (SEQ ID NO: 19), with reference to Scaffold Structure I.

[0416] In certain embodiments, the non-naturally occurring LRP6-binding CKP comprises: L1 comprising the amino acid sequence QAINRVKRQR (SEQ ID NO: 367); L2 comprising the amino acid sequence KQDSD (SEQ ID NO: 93); L3 comprising the amino acid sequence LAG; L4 comprising amino acid V; and L5 comprising the amino acid sequence GPNGF (SEQ ID NO: 19), with reference to Scaffold Structure I.

[0417] In certain embodiments, the non-naturally occurring LRP6-binding CKP comprises an amino acid sequence set forth in any one of SEQ ID NOs: 189-210 and 366. SEQ ID NOs: 189-210 and 366 are provided below.

[0418] GCRTNRVKGGCKQDSDCLAGCVCGPNGFCG(SEQ ID NO:189)

[0419] GCVNRVRGCKQDSDCLAGCVCSGGRDCG(SEQ ID NO:190)

[0420] GCMNHVKARRCKQDSDCLAGCVCGPNGFCG(SEQ ID NO:191)

[0421] GCRSVNKICKQDSDCLAGCVCGSSRNCG(SEQ ID NO:192)

[0422] GCVNKIKGCKQDSDCLAGCVCGVEGRCG(SEQ ID NO:193)

[0423] GCRNSIKRCKQNSDCLAGCVCSVGHGCG(SEQ ID NO:194)

[0424] GCVSNRVNKGCKQDSDCLAGCVCGPNGFCG(SEQ ID NO:195)

[0425] GCRGNIIKCKQDSDCLAGCVCNESRGCG(SEQ ID NO:196)

[0426] GCRSGNTIRKRECKQDSDCLAGCVCGGPGGCG(SEQ ID NO:197)

[0427] GCASSNSIRQGWCKQDSDCLAGCVCGPKSNCG(SEQ ID NO:198)

[0428] GCRSNRIRCKQDSDCLAGCVCYGHGDCG(SEQ ID NO:199)

[0429] GCRSNKLREARGCKQDSDCLAGCVCGSRQDCG(SEQ ID NO:200)

[0430] GCVNSVKRCKQDSDCLAGCVCSRGVNCG(SEQ ID NO:201)

[0431] GCGSNKIRPRCKQDSDCLAGCVCGPNDFCG(SEQ ID NO:202)

[0432] GCNRIRNSCKQDSDCLAGCVCGRGDYCG(SEQ ID NO:203)

[0433] GCSRNSIKCKQDSDCLAGCVCASGSSCG(SEQ ID NO:204)

[0434] GCSNYVKRCKQDSDCLAGCVCSPGGRCG(SEQ ID NO:205)

[0435] GCRANRVSGRCKQDSDCLAGCVCGPNGFCG(SEQ ID NO:206)

[0436] GCSNRVKVRACKQDSDCLAGCVCGPNGFCG(SEQ ID NO:207)

[0437] GCENRTKGCKQDSDCLAGCVCGFRGTCG(SEQ ID NO:208)

[0438] GCGNKIRACKQDSDCLAGCCVCRDRVGCG(SEQ ID NO:209)

[0439] GCANRVKRTSCKQDSDCLAGCVCGPNGFCG(SEQ ID NO:210)

[0440] GCQAINRVKRQRCKQDSDCLAGCVCGPNGFCG(SEQ ID NO:366)

[0441] In certain embodiments, the non-natural CKP in combination with LRP6 is a variant of the non-natural CKP in combination with LRP6 described herein. In certain embodiments, this variant is included in one or more sequences shown in SEQ ID NO:19, 93, 147-168, 169-184 and 189-210 and / or at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 amino acid replacements in the amino acid sequence LAG. In certain embodiments, amino acid replacement is a conservative amino acid replacement. In certain embodiments, amino acid replacement does not substantially reduce the ability of the non-natural CKP in combination with LRP6 to combine with human LRP6. For example, conservative changes (e.g., conservative replacements as provided herein) that do not substantially reduce LRP6 binding affinity can be made. Using the method described in the following examples, the binding affinity of the variant of the non-natural CKP in combination with LRP6 can be evaluated.

[0442] Conservative substitutions are shown above in Table 17 under the heading "Conservative Substitutions". More substantial changes are provided in Table 17 under the heading "Exemplary Substitutions" and are further described below based on amino acid side chain classes. Amino acid substitutions can be introduced into variants of non-naturally occurring CKPs that bind to LRP6 and the products screened for the desired activity (e.g., retained / improved LRP6 binding).

[0443] Non-conservative substitutions will require exchanging a member of one of these categories for a member of another category. Exemplary substitution variants are non-naturally occurring CKPs that bind to LRP6 for affinity maturation, which can be conveniently produced, for example, using affinity maturation techniques based on phage display (such as those described herein). In short, one or more residues in L1, L2, L3, L4, and / or L5 are changed (i.e., added, deleted, or replaced), and the variant's CKPs that bind to LRP6 are displayed on phage and screened for LRP6 binding affinity. In certain embodiments of affinity maturation, diversity is introduced into the variable gene selected for maturation by any of a variety of methods (e.g., error-prone PCR, ring shuffling, or oligonucleotide directed mutagenesis). A secondary library is then established. The library is then screened to identify any non-naturally occurring CKP variants with a desired affinity for LRP6. In certain embodiments, the introduction of diversity involves a loop-directed approach in which several residues (e.g., about 5, about 4-6, or about 6-10 residues at a time) in L1, L2, L3, L4, and / or L5 are randomized. The L1, L2, L3, L4, and / or L5 residues involved in binding to a target ligand can be identified, for example, using alanine scanning mutagenesis or modeling.

[0444] In certain embodiments, the non-naturally occurring CKP "specifically binds" human LRP6 (i.e., has a binding affinity of no more than about 1×10 -7 M, preferably not more than about 1×10 -8 and most preferably no more than about 1×10 -9 M), but has a binding affinity for another LRP protein that is at least about 50 times weaker, or at least about 500 times weaker, or at least about 1000 times weaker than its binding affinity for LRP6.

[0445] In certain embodiments, the degree of binding of the non-naturally occurring CKP binding to LRP6 to a non-target protein (e.g., LRP6 homologs, such as LRP1, LRP1B, LRP2, LRP3, LRP4, LRP5, LRP8, LRP10, LRP11, and LRP12) is less than about 10% of the binding of the non-naturally occurring CKP binding to LRP6 to human LRP6, as determined by methods known in the art such as ELISA, fluorescence activated cell sorting (FACS) analysis, or radioimmunoprecipitation (RIA). Specific binding can be measured, for example, by determining the binding of a molecule compared to the binding of a control molecule, which is generally a molecule of a similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule, which is similar to a target, for example, an excess of unlabeled target. In this case, if the binding of the labeled target to the probe is competitively inhibited by an excess of unlabeled target, specific binding is indicated. As used herein, the terms "specifically bind to" a particular polypeptide or an epitope on a particular polypeptide target, "specifically bind to" a particular polypeptide or an epitope on a particular polypeptide target, or "have specificity for" a particular polypeptide or an epitope on a particular polypeptide target can be exhibited, for example, by a molecule that has at least about 10 -4 M, or at least about 10 -5 M, or at least about 10 -6 M, or at least about 10 -7 M, or at least about 10 -8 M, or at least about 10 -9 M, or at least about 10 -10 M, or at least about 10 -11 M, or at least about 10 -12 In one embodiment, the term "specifically binds" refers to a molecule wherein the molecule binds to a specific polypeptide or an epitope on a specific polypeptide without substantially binding to any other polypeptide or polypeptide epitope.

[0446] In certain embodiments, the non-naturally occurring CKP that binds LRP6 binds to human LRP6 with a Kd of about 1 pM to about 500 nM. In certain embodiments, the non-naturally occurring CKP that binds LRP6 that specifically binds to LRP6 binds to human LRP6 with a Kd of about 1 pM to about 50 pM, about 50 pM to about 250 pM, about 250 pM to about 500 pM, about 500 pM to 750 pM, about 750 pM to about 1 nM, about 1 nM to about 25 nM, about 25 nM to about 50 nM, 50 nM to about 100 nM, about 100 nM to about 250 nM, or about 250 nM to about 500 nM, including any ranges between these values.

[0447] In certain embodiments, the non-naturally occurring LRP6-binding CKP inhibits Wnt1 signaling, eg, as determined using the methods described in the Examples below.

[0448] Also contemplated are nucleic acid molecules encoding the non-naturally occurring CKP that binds to LRP6, expression vectors comprising nucleic acid molecules encoding the non-naturally occurring CKP that binds to LRP6, and cells comprising the nucleic acid molecules. Also provided herein are methods for producing a non-naturally occurring CKP that binds to LRP6 by culturing the cells, expressing the non-naturally occurring CKP that binds to LRP6, and recovering the non-naturally occurring CKP that binds to LRP6 from the cell culture.

[0449] In certain embodiments, a non-naturally occurring LRP6-binding CKP is produced via in vitro translation, as described elsewhere herein.

[0450] As described elsewhere herein, non-naturally occurring LRP6-binding CKPs are generated via chemical peptide synthesis, e.g., by grafting chemically synthesized L1, L2, L3, L4 and / or L5 peptides onto the EETI-II framework, or by chemically synthesizing the entire non-naturally occurring LRP6-binding CKP.

[0451] In certain embodiments, the non-naturally occurring LRP6-binding CKP is used as a therapeutic agent in the treatment of a disease or condition in which excessive LRP6 activity is implicated.

[0452] Production method

[0453] In certain embodiments, a non-naturally occurring CKP that binds VEGF-A or a non-naturally occurring CKP that binds LRP6 is produced by genetic engineering. A variety of methods for mutagenesis (together with appropriate screening or selection methods) have been previously described. Such mutagenesis methods include, but are not limited to, for example, error-prone PCR, loop shuffling or oligonucleotide-directed mutagenesis, random nucleotide insertion, or other methods prior to recombination. Other details about these methods are described in, for example, Abou-Nadler et al. (2010) Bioengineered Bugs 1, 337-340; Firth et al. (2005) Bioinformatics 21, 3314-3315; Cirino et al. (2003) Methods Mol Biol 231, 3-9; Pirakitikulr (2010) Protein Sci 19, 2336-2346; Steffens et al. (2007) J. Biomol Tech 18, 147-149; and other literature. Therefore, in certain embodiments, a non-naturally occurring CKP that binds to VEGF-A or a non-naturally occurring CKP that binds to LRP6 produced by genetic engineering techniques is provided.

[0454] In certain embodiments, a non-naturally occurring CKP that binds VEGF-A or a non-naturally occurring CKP that binds LRP6 is produced by in vitro translation. In short, in vitro translation requires cloning one or more protein coding sequences into a vector containing a promoter, producing mRNA by transcribing the cloned one or more sequences with RNA polymerase, and synthesizing the protein by translating the mRNA in vitro, for example, using a cell-free extract. The desired variant protein can be simply produced by changing the cloned protein coding sequence. Many mRNAs can be effectively translated in wheat germ extract or in rabbit reticulocyte lysates. Further details regarding in vitro translation are described in, for example, Hope et al. (1985) Cell 43, 177-188; Hope et al. (1986) Cell 46, 885-894; Hope et al. (1987) EMBO J. 6, 2781-2784; Hope et al. (1988) Nature 333, 635-640; and Melton et al. (1984) Nucl. Acids Res. 12, 7057-7070.

[0455] Therefore, a nucleic acid molecule encoding a non-naturally occurring CKP in combination with VEGF-A or a non-naturally occurring CKP in combination with LRP6 described herein is provided. An expression vector operably connected to a nucleic acid molecule is also provided, and the nucleic acid molecule encodes a non-naturally occurring CKP in combination with VEGF-A or a non-naturally occurring CKP in combination with LRP6. A host cell (including, for example, a prokaryotic host cell such as Escherichia coli (E. coli), a eukaryotic host cell such as a yeast cell, a mammalian cell, a CHO cell, etc.) is also provided, which comprises a nucleic acid encoding a non-naturally occurring CKP in combination with VEGF-A or a non-naturally occurring CKP in combination with LRP6.

[0456] In certain embodiments, a non-naturally occurring CKP that binds VEGF-A or a non-naturally occurring CKP that binds LRP6 is produced by in vitro translation. In short, in vitro translation requires cloning one or more protein coding sequences into a vector containing a promoter, producing mRNA by transcribing the cloned one or more sequences with RNA polymerase, and synthesizing the protein by translating the mRNA in vitro, for example, using a cell-free extract. The desired mutant protein can be simply produced by changing the cloned protein coding sequence. Many mRNAs can be effectively translated in wheat germ extract or in rabbit reticulocyte lysates. Further details regarding in vitro translation are described in, for example, Hope et al. (1985) Cell 43, 177-188; Hope et al. (1986) Cell 46, 885-894; Hope et al. (1987) EMBO J. 6, 2781-2784; Hope et al. (1988) Nature 333, 635-640; and Melton et al. (1984) Nucl. Acids Res. 12, 7057-7070.

[0457] In certain embodiments, a non-naturally occurring CKP that binds VEGF-A or a non-naturally occurring CKP that binds LRP6 is produced by chemistry. In certain embodiments, chemically synthesized L1, L2, L3, L4 and / or L5 peptides are grafted onto an EETI-II-based framework (such as Scaffold Structure I) to produce a non-naturally occurring CKP that binds VEGF-A or a non-naturally occurring CKP that binds LRP6. In certain embodiments, the entire non-naturally occurring CKP that binds VEGF-A or the entire non-naturally occurring CKP that binds LRP6 is chemically synthesized. Methods for solid phase and liquid phase peptide synthesis are well known in the art and are described in detail in, for example, Methods of Molecular Biology, 35, Peptide Synthesis Protocols, (MW Pennington and BM Dunn, eds.), Springer, 1994; Welsch et al. (2010) Curr Opin Chem Biol 14, 1-15; Methods of Enzymology, 289, Solid Phase Peptide Synthesis, (GB Fields, ed.), Academic Press, 1997; Chemical Approaches to the Synthesis of Peptides and Proteins, (P. Lloyd-Williams, F. Albericio and E. Giralt, eds.), CRC Press, 1997; Fmoc Solid Phase Peptide Synthesis, A Practical Approach, (WC Chan, PD White, eds.), Oxford University Press, 2000; Solid Phase Synthesis, A Practical Guide, (SF Kates, F Albericio, eds.), Marcel Dekker, 2000; P. Seneci, Solid-Phase Synthesis and Combinatorial Technologies, John Wiley & Sons, 2000; Synthesis of Peptides and Peptidomimetics (M. Goodman, editor-in-chief, A. Felix, L. Moroder, C. Tmiolo, editors), Thieme, 2002; NLBenoiton, Chemistry of Peptide Synthesis, CRC Press, 2005; Methods in Molecular Biology, 298, Peptide Synthesis and Applications, (edited by J. Howl) Humana Press, 2005; and Amino Acids, Peptides and Proteins in Organic Chemistry, Volume 3, Building Blocks, Catalysts and Coupling Chemistry, (ed. AB Hughs) Wiley-VCH, 2011. .

[0458] Chimeric molecules comprising non-naturally occurring EETI-II proteins

[0459] If advantageous, the non-naturally occurring CKPs described herein (such as non-naturally occurring CKPs that bind to VEGF-A or non-naturally occurring CKPs that bind to LRP6) can also be modified in a manner to form a chimeric molecule comprising a non-naturally occurring CKP fused (e.g., recombinantly fused) to another heterologous polypeptide or amino acid sequence. In certain embodiments, such chimeric molecules comprise a fusion of a non-naturally occurring CKP described herein (such as non-naturally occurring CKPs that bind to VEGF-A or non-naturally occurring CKPs that bind to LRP6) with an antibody to form, for example, a bivalent molecule or a bispecific molecule.

[0460] In certain embodiments, a chimeric molecule comprises a fusion of a non-naturally occurring CKP described herein (such as a non-naturally occurring CKP that binds VEGF-A or a non-naturally occurring CKP that binds LRP6) and a second portion (such as a protein transduction domain) that targets the chimeric molecule for delivery to a different tissue, or, for example, across the blood-brain barrier, using, for example, the protein transduction domain of the human immunodeficiency virus TAT ​​protein (Schwarze et al., 1999, Science 285:1569-72).

[0461] In certain embodiments, the non-natural CKP provided herein can be used as a bi- or multi-specific (for different target ligands or different epitopes on the same target ligand) in a polymeric form. For example, a dimeric bispecific non-natural CKP has a subunit specific for a first target protein or epitope and a second subunit specific for a second target protein or epitope. The non-natural CKP protein subunits can be connected in a variety of conformations that can increase titer and thereby increase the affinity of the combination with the target ligand.

[0462] In certain embodiments, chimeric molecules provided herein include two or more (such as three, four, five, six, seven, eight, nine, ten or more than ten) non-natural CKP proteins. In certain embodiments, nucleic acid can be engineered to encode two or more copies of a single non-natural CKP, which is transcribed and translated in series to produce a covalently attached polymer of the same subunit. In certain embodiments, nucleic acid can be engineered to encode two or more different non-natural CKPs, which are transcribed and translated in series to produce a covalently attached polymer of different subunits, which are combined with different epitopes of, for example, a single target ligand or, for example, different target ligands.

[0463] In another embodiment, such a chimeric molecule comprises a fusion of a non-naturally occurring CKP described herein (such as a non-naturally occurring CKP that binds VEGF-A or a non-naturally occurring CKP that binds LRP6) and a tag polypeptide, the tag polypeptide providing an epitope to which an anti-tag antibody can selectively bind. The epitope tag is typically located at the amino- or carboxyl-terminus of the non-naturally occurring CKP. Using antibodies to the tag polypeptide, the presence of such an epitope-tagged form of the non-naturally occurring CKP protein can be detected. Furthermore, the provision of the epitope tag allows the non-naturally occurring CKP to be easily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag. A variety of tag polypeptides and their respective antibodies are known in the art. Examples include: poly-histidine (poly-His) or poly-histidine-glycine (poly-His-Gly) tags; influenza HA tag polypeptide and its antibody 12CA5 (Field et al. (1988) Mol. Cell. Biol. 8, 2159-2165); c-myc tag and 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies against them (Evan et al. (1985) Mol. Cell. Biol. 5, 3610-3616]; and herpes simplex virus glycoprotein D (gD) tag and its antibody (Paborsky et al. (1990) Protein Eng., 3, 547-553). Other tag polypeptides include Flag-peptide (Hopp et al. (1988) BioTechnology, 6, 1204-1210); KT3 epitope peptide (Martin et al. (1992) Science, 255, 192-194]); α-tubulin epitope peptide (Skinner et al. (1991) J. Biol. Chem. 266, 15163-15166); and T7 gene 10 protein peptide tag (Lutz-Freyermuth et al. (1990) Proc. Natl. Acad. Sci. USA 87, 6393-6397].

[0464] In certain embodiments, the chimeric molecule may comprise a fusion of a non-naturally occurring CKP protein described herein (such as a non-naturally occurring CKP that binds VEGF-A or a non-naturally occurring CKP that binds LRP6) and an immunoglobulin or a specific region of an immunoglobulin. For a bivalent form of a chimeric molecule (e.g., an "immunoadhesin"), this fusion may be directed to the Fc region of an IgG molecule. The Ig fusion provided herein includes a polypeptide containing about or only residues 94-243, residues 33-53, or residues 33-52 of a person in at least one variable region within an Ig molecule. In a particularly preferred embodiment, the immunoglobulin fusion includes hinges, CH2 and CH3, or hinges, CH1, CH2, and CH3 regions of an IgG1 molecule. Regarding the production of immunoglobulin fusions, see also U.S. Patent No. 5,428,130, issued June 27, 1995. In certain embodiments, the non-naturally occurring CKP described herein (such as the non-naturally occurring CKP in combination with VEGF-A or the non-naturally occurring CKP in combination with LRP6) is, for example, fused at the N or C-terminus with the constant region (Fc) of IgG. In certain embodiments, the non-naturally occurring CKP / Fc fusion molecule activates the complement component of the immune response. In certain embodiments, the non-naturally occurring CKP / Fc fusion protein increases the therapeutic value of the non-naturally occurring CKP. In certain embodiments, the non-naturally occurring CKP protein described herein (such as the non-naturally occurring CKP in combination with VEGF-A or the non-naturally occurring CKP in combination with LRP6) is, for example, fused (such as recombinantly fused) at the N or C-terminus with a complement protein (such as Clq). Various publications describe methods for obtaining non-naturally occurring proteins whose half-life is altered by introducing an FcRn binding polypeptide into the molecule (WO 1997 / 43316, US 5869046, US 5747035, WO 1996 / 32478, WO 1991 / 14438), or by fusing the protein to an antibody whose FcRn binding affinity is retained but whose affinity for other Fc receptors has been greatly reduced (WO 1999 / 43713), or by fusing the FcRn binding domain of an antibody (WO 2000 / 09560, US 4703039). Specific techniques and methods for increasing the half-life of physiologically active molecules (e.g., non-naturally occurring CKPs) can also be found in US7083784. In certain embodiments, the non-naturally occurring CKP proteins provided herein (such as a non-naturally occurring CKP that binds to VEGF-A or a non-naturally occurring CKP that binds to LRP6) are fused to an Fc region from IgG comprising the following amino acid residue mutations (numbered according to the EU index in Kabat): M252Y / S254T / T256E or H433K / N434F / Y436H.

[0465] In certain embodiments, the non-naturally occurring CKP protein described herein (such as a non-naturally occurring CKP in combination with VEGF-A or a non-naturally occurring CKP in combination with LRP6) is fused to a molecule that increases or prolongs in vivo or serum half-life. In certain embodiments, the non-naturally occurring CKP described herein (such as a non-naturally occurring CKP in combination with VEGF-A or a non-naturally occurring CKP in combination with LRP6) is fused to albumin (such as human serum albumin (HSA)), polyethylene glycol (PEG), polysaccharides, immunoglobulin molecules (IgG), complement, hemoglobin, binding peptides, lipoproteins or other factors to increase its half-life in the bloodstream and / or its tissue penetration.

[0466] By the techniques of gene shuffling, motif shuffling, exon shuffling and / or codon shuffling (collectively referred to as "DNA shuffling"), additional chimeric molecules comprising a non-naturally occurring CKP that binds to VEGF-A or a non-naturally occurring CKP that binds to LRP6 can be generated. DNA shuffling can be used to alter the activity of a non-naturally occurring CKP (e.g., a non-naturally occurring CKP with higher affinity and lower dissociation rate). See generally, US 5605793, US 5811238, US 5830721, US 5834252, US 5837458, Patten et al. (1997) Curr. Opinion Biotechnol. 8, 724-33; Harayama (1998) Trends Biotechnol. 16, 76-82; Hansson et al., (1999) J. Mol. Biol. 287, 265-76; and Lorenzo and Blasco, (1998) Biotechniques 24, 308-313.

[0467] In certain embodiments, random mutagenesis is performed by error-prone PCR, random nucleotide insertion, or other methods prior to recombination to alter the non-naturally occurring CKPs that bind to VEGF-A or non-naturally occurring CKPs that bind to LRP6 provided herein. One or more portions of a polynucleotide encoding a scaffold that binds to a specific target can be recombined with one or more components, motifs, segments, parts, domains, fragments, etc. of one or more heterologous molecules.

[0468] Any of these fusions can be produced by standard techniques, for example, by expression of the fusion protein from a recombinant fusion gene constructed using publicly available gene sequences or by chemical peptide synthesis.

[0469] A conjugate comprising a non-naturally occurring CKP that binds VEGF-A or a non-naturally occurring CKP that binds LRP6.

[0470] Provided herein are immunoconjugates comprising a non-naturally occurring CKP described herein (such as a non-naturally occurring CKP that binds VEGF-A or a non-naturally occurring CKP that binds LRP6) conjugated to a cytotoxic agent such as a chemotherapeutic agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof), or a radioactive isotope (i.e., a radioconjugate).

[0471] Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII and PAP-S), Momordica charantia inhibitor, curcin, crotin, Saponaria officinalis toxin, scutellaria baicalensis ... officinalis inhibitors, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and tricothecenes. Other toxins include maytansine and maytansinoids, calicheamicin, and other cytotoxic agents. A variety of radionuclides can be used to produce radioconjugated non-naturally occurring CKPs. Examples include 212 Bi, 131 I. 131 In, 90 Y and 186 Re.

[0472] Conjugates of the non-naturally occurring CKPs described herein (such as a non-naturally occurring CKP that binds VEGF-A or a non-naturally occurring CKP that binds LRP6) with, for example, a cytotoxic agent are prepared using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazo compounds (such as bis(p-azidobenzoyl)hexanediamine), Derivatives (such as di-(p-diazo The ricin immunotoxin is prepared as described in Vitetta et al., Science, 238:1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionuclides to non-naturally occurring CKPs provided herein. See WO94 / 11026.

[0473] In another embodiment, the non-naturally occurring CKPs described herein (such as a non-naturally occurring CKP that binds VEGF-A or a non-naturally occurring CKP that binds LRP6) can be conjugated to a "receptor" (such as streptavidin (streptavidin)) for use in "pre-targeting" of the eye, wherein the non-naturally occurring EETI-II scaffold protein-receptor conjugate is administered to an ocular patient, followed by removal of unbound conjugate from the circulation using a clearing agent, and then administration of a "ligand" (e.g., avidin) conjugated to a cytotoxic agent (e.g., a radionuclide) or a therapeutic agent.

[0474] In certain embodiments, the non-naturally occurring CKPs provided herein (such as non-naturally occurring CKPs that bind VEGF-A or non-naturally occurring CKPs that bind LRP6) can be used as bi- or multi-specific (for different target ligands or different epitopes on the same target ligand) in a multimeric form. Connections can be covalent or non-covalent. For example, a dimeric bispecific non-naturally occurring CKP has a subunit specific for a first target protein or epitope and a second subunit specific for a second target protein or epitope. Non-naturally occurring CKP subunits can be connected in a variety of conformations that can increase potency and thereby increase the affinity of binding to a target ligand or bind to multiple target ligands.

[0475] In certain embodiments, the non-naturally occurring CKP provided herein is engineered to provide a reactive group for conjugation. In certain embodiments, the N-terminus and / or C-terminus may also be used to provide a reactive group for conjugation. In certain embodiments, the N-terminus is conjugated to a part (for example, but not limited to PEG), and the C-terminus is conjugated to another part (for example, but not limited to biotin), or vice versa.

[0476] Provide non-natural CKPs described herein (such as non-natural CKPs for binding VEGF-A or non-natural CKPs for binding LRP6) conjugated to one or more parts, including but not limited to peptides, polypeptides, proteins, fusion proteins, nucleic acid molecules, small molecules, mimetic agents, synthetic drugs, inorganic molecules and organic molecules. Also provided are the uses of non-natural CKPs described herein (such as non-natural CKPs for binding VEGF-A or non-natural CKPs for binding LRP6) chemically conjugated (including both covalent and non-covalent conjugation) to heterologous proteins or polypeptides (or fragments thereof, to polypeptides of at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100 amino acids). The fusion does not necessarily need to be direct, but can occur through the linker sequence described herein.

[0477] In certain embodiments, the non-naturally occurring CKP described herein (such as a non-naturally occurring CKP that binds VEGF-A or a non-naturally occurring CKP that binds LRP6) or an analog or derivative thereof can be conjugated to a diagnostic or detectable agent. Such non-naturally occurring CKP conjugates can be used to monitor or predict the development or progression of a disease as part of a clinical trial operation, such as determining the efficacy of a particular therapy. Such diagnosis and detection can be accomplished by coupling the non-naturally occurring CKP to a detectable substance, including but not limited to: various enzymes, such as but not limited to horseradish peroxidase, alkaline phosphatase, β-galactosidase or acetylcholinesterase; prosthetic groups, such as but not limited to streptavidin biotin and avidin / biotin; fluorescent substances, such as but not limited to umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; luminescent materials, such as but not limited to luminol; bioluminescent materials, such as but not limited to luciferase, luciferin and aequorin; radioactive substances, such as but not limited to iodine ( 131 I. 125 I. 123 I. 121 I), carbon ( 14 C) Sulfur ( 35 S), tritium (3 H), Indium ( 115 In, 113 In, 112 In, 111 In) and technetium ( 99 Tc), thallium ( 201 Ti、Ga( 68 Ga, 67 Ga), Palladium ( 103 Pd), Mo ( 99 Mo), xenon ( 133 Xe、Fluorine( 18 F), 153 Sm, 177 Lu, 159 Gd, 149 Pm, 140 La, 175 Yb, 166 Ho, 90 Y. 47 Sc, 186 Re, 188 Re, 142 Pr, 105 Rh, 97 Such as 68 Ge, 57 Co. 65 Zn, 85 Sr. 32 P. 153 Gd, 169 Yb, 51 Cr, 54 Mn, 75 Se, 113 Sn and 117 Tn; positron-emitting metals, non-radioactive paramagnetic metal ions, and molecules that are radiolabeled or conjugated to specific radioisotopes using various positron emission tomography techniques.

[0478] Also provided are non-naturally occurring CKPs conjugated to therapeutic moieties (such as non-naturally occurring CKPs that bind VEGF-A or non-naturally occurring CKPs that bind LRP6). In certain embodiments, the non-naturally occurring CKP can be conjugated to a therapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent, or a radioactive metal ion, e.g., an alpha-emitter. A cytotoxin or cytotoxic agent includes any agent that is harmful to cells.

[0479] In certain embodiments, a non-naturally occurring CKP described herein (such as a non-naturally occurring CKP that binds VEGF-A or a non-naturally occurring CKP that binds LRP6) is conjugated to a therapeutic moiety, such as a radioactive metal ion such as an α-emitter (such as213 Bi) or can be used to convert radio metal ions (including but not limited to, 131 In, 131 Lu, 131 Y. 131 Ho, 131Sm) conjugated to a macrocyclic chelator of a polypeptide. In certain embodiments, the macrocyclic chelator is 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA), which can be connected to a non-naturally occurring CKP via a linker molecule. Such linker molecules are generally known in the art and are described in, for example, Denardo et al. (1998) Clin Cancer Res. 4, 2483-90; Peterson et al. (1999) Bioconjug. Chem. 10, 553-557; and Zimmerman et al. (1999) Nucl. Med. Biol. 26, 943-50.

[0480] Techniques for conjugating therapeutic moieties to antibodies are well known and can be applied to the non-naturally occurring CKPs disclosed herein, see, e.g., Amon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy," Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56. (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery," Controlled Drug Delivery (2nd ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," Monoclonal Antibodies 84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibodies In Cancer Therapy", Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., 1982, Immunol. Rev. 62: 119-58. Similar protocols can be adapted for use with the non-naturally occurring CKPs provided herein.

[0481] The therapeutic moiety or drug conjugated to the non-naturally occurring CKP described herein (such as a non-naturally occurring CKP that binds VEGF-A or a non-naturally occurring CKP that binds LRP6) should be selected to achieve the desired preventive or therapeutic effect for a particular condition in a subject. When deciding which therapeutic moiety or drug to conjugate to a stent, a clinician or other medical staff should consider the following aspects: the nature of the disease, the severity of the disease, and the condition of the subject.

[0482] In certain embodiments, the non-naturally occurring CKPs described herein (such as non-naturally occurring CKPs that bind VEGF-A or non-naturally occurring CKPs that bind LRP6) can also be attached to a solid support, which is particularly useful for immunoassays or purification of target antigens. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

[0483] Covalent modification

[0484] Covalent modification of the non-naturally occurring CKPs described herein (such as non-naturally occurring CKPs that bind VEGF-A or non-naturally occurring CKPs that bind LRP6) is also contemplated. One class of covalent modifications includes reacting the targeted amino acid residues of the non-naturally occurring CKPs with an organic derivatization agent that can react with the selected side chains or N- or C-terminal residues of the non-naturally occurring CKPs. Derivatization using a bifunctional agent can be used, for example, to crosslink the non-naturally occurring CKPs with a water-insoluble support matrix or surface used in a method for purifying a target ligand, and vice versa. Commonly used cross-linking agents include, for example, 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters (e.g., esters formed with 4-azidosalicylic acid), homobifunctional imidates (including disuccinimidyl esters such as 3,3'-dithiobis(succinimidyl-propionate)), bifunctional maleimides such as di-N-maleimido-1,8-octane, and agents such as methyl-3-[(p-azidophenyl)-dithio]propioimidate.

[0485] Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of the hydroxyl groups of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine and histidine side chains (TECreighton, Proteins: Structure and Molecular Properties, WH Freeman & Co., San Francisco, pp. 79-86 (1983)), acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

[0486] Another type of covalent modification of the non-naturally occurring CKP includes linking the non-naturally occurring CKP to one of a variety of non-proteinaceous polymers (e.g., polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes) in the manner described in US 4640835, US 4496689, US 4301144, US 4670417, US 4791192, or US 4179337.

[0487] The term "polyethylene glycol" or "PEG" means a polyethylene glycol compound or a derivative thereof, with or without a coupling agent, coupling or activating moiety (e.g., with a thiol, triflate, tresylate, aziridine, oxirane, N-hydroxysuccinimide or maleimide moiety). The term "PEG" is intended to mean polyethylene glycol of a molecular weight of 500 to 150,000 Da, including analogs thereof, wherein, for example, the terminal OR-group has been replaced by a methoxy group (referred to as mPEG).

[0488] In certain embodiments, the non-naturally occurring CKP described herein (such as a non-naturally occurring CKP that binds VEGF-A or a non-naturally occurring CKP that binds LRP6) is derivatized with polyethylene glycol (PEG). PEG is a linear water-soluble polymer of ethylene oxide repeating units with two terminal hydroxyl groups. PEGs are classified according to their molecular weight, which is generally in the range of about 500 daltons to about 40,000 daltons. In a currently preferred embodiment, the PEG used has a molecular weight in the range of 5,000 daltons to about 20,000 daltons. The PEG coupled to the non-naturally occurring CKP provided herein can be branched or unbranched (e.g., Monfardini, C. et al. 1995 Bioconjugate Chem 6: 62-69). PEG is commercially available from Nektar Inc., Sigma Chemical Co. and other companies. Such PEGs include, but are not limited to, monomethoxypolyethylene glycol (MePEG-OH), monomethoxypolyethylene glycol-succinate (MePEG-S), monomethoxypolyethylene glycol-succinimidyl succinate (MePEG-S-NHS), monomethoxypolyethylene glycol-amine (MePEG-NH2), monomethoxypolyethylene glycol-trifluoroethanesulfonate (MePEG-TRES), and monomethoxypolyethylene glycol-imidazolyl-carbonyl (MePEG-IM).

[0489] In certain embodiments, the hydrophilic polymer (e.g., PEG) employed is capped at one end with a non-reactive group (such as a methoxy or ethoxy group). Thereafter, the molecule is activated by reacting with a suitable activating agent such as a cyanuric halide (e.g., cyanuric chloride, cyanuric bromide, or cyanuric fluoride), a diimidazole, an anhydride reagent (e.g., a dihalo succinic anhydride, such as dibromosuccinic anhydride), an acyl azide, p-diazo Benzyl ether, 3-(p-diazo The polymer is activated at the other end by reactions such as phenoxy)-2-hydroxypropyl ether). The activated polymer is then reacted with the non-natural CKP herein (such as the non-natural CKP in combination with VEGF-A or the non-natural CKP in combination with LRP6) to produce the non-natural CKP derivatized by the polymer. Alternatively, the functional groups in the non-natural CKP provided herein can be activated for reaction with the polymer, or known coupling methods can be used to connect two groups in a planned (concerted) coupling reaction. It is easy to understand that the non-natural CKP provided herein can be derivatized with PEG using many other reaction schemes known and used by those skilled in the art.

[0490] Liposomes

[0491] The non-naturally occurring CKP disclosed herein (such as non-naturally occurring CKP in combination with VEGF-A or non-naturally occurring CKP in combination with LRP6) can also be formulated into liposomes. By methods known in the art, such as Epstein et al., Proc Natl Acad Sci USA, 82: 3688 (1985); Hwang et al., Proc Natl Acad Sci USA, 77: 4030 (1980); and the method described in U.S. Patent Nos. 4,485,045 and 4,544,545, liposomes containing non-naturally occurring EETI-II scaffold proteins described herein are prepared. Liposomes with increased circulation time are disclosed in U.S. Patent No. 5,013,556.

[0492] Particularly useful liposomes can be produced by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). The liposomes are extruded through a filter of defined pore size to produce liposomes having a desired diameter. A second therapeutic agent is optionally contained in the liposomes. See Gabizon et al., J. National Cancer Inst., 81(19):1484(1989). Pharmaceutical compositions and preparations comprising a non-naturally occurring cystine knot peptide (CKP) that binds to human vascular endothelial growth factor A (VEGF-A).

[0493] In certain embodiments, provided herein are pharmaceutical compositions comprising non-naturally occurring CKPs in combination with VEGF-A and pharmaceutically acceptable excipients. In certain embodiments, the composition may also contain a buffer, a carrier, a stabilizer, a preservative and / or a bulking agent so that the composition is suitable for ocular administration to a patient to achieve the desired effect or result. In certain embodiments, the pharmaceutical composition comprises one or more penetration enhancers that allow non-naturally occurring CKPs in combination with VEGF-A to penetrate the cornea. Examples of such penetration enhancers include, for example, surfactants, bile acids, chelating agents, preservatives, cyclodextrins (i.e., cylindrical oligonucleotides with hydrophilic outer surfaces and lipophilic inner surfaces that form complexes with lipophilic drugs), etc. Such penetration enhancers increase chemical stability and bioavailability and reduce local irritation. In certain embodiments, provided herein are pharmaceutical compositions that additionally include agents that increase the absorption and distribution of non-naturally occurring CKPs in combination with VEGF-A in various ocular compartments. In certain embodiments, provided herein are pharmaceutical compositions that include cross-linked polyacrylic acid, which can improve ocular bioavailability by virtue of its mucoadhesive properties. In certain embodiments, the pharmaceutical compositions provided herein comprise a bioadhesive polymer.

[0494] In certain embodiments, the pharmaceutical compositions provided herein are formulated as in-situ gelling systems, such as liquids based on viscous polymers that exhibit sol-to-gel phase transfer on the surface of the eye due to changes in specific physicochemical parameters (particle strength, temperature, pH, or solvent exchange) when the composition contacts tears. In certain embodiments, the pharmaceutical compositions provided herein are formulated as eye sprays. In certain embodiments, the pharmaceutical compositions provided are formulated as liposomes. In certain embodiments, the pharmaceutical compositions provided herein are formulated as vesicles (niosomes) (i.e., vesicles based on nonionic surfactants containing, for example, cholesterol as an excipient). In certain embodiments, the pharmaceutical compositions provided herein are formulated as pharmacoplasts (i.e., vesicles formed by amphiphilic drugs). In certain embodiments, the pharmaceutical compositions provided herein are formulated as microemulsions. Additional details on various ocular drug formulations are provided in, for example, Gaikwad et al. (2013) IndoAmer J Pharm Res. 3, 3216-3232; Achouri et al. (2012) Drug Dev Indust Pharm. 39, 1599-1617; Lu (2010) Recent Pat Drug Deliv Formul. 4, 49-57; Baranowski et al. (2014) SciWorld J. doi.org / 10.1155 / 2014 / 861904; Lang (1995) Adv Drug Deliv Rev. 16, 39-43; Short (2008) Toxicologic Path. 36, 49-62; and elsewhere.

[0495] In certain embodiments, pharmaceutical compositions comprising a non-naturally occurring CKP that binds VEGF-A as described herein are stable at room temperature (such as at about 20-25° C.) for about 0.5 weeks, about 1.0 weeks, about 1.5 weeks, about 2.0 weeks, about 2.5 weeks, 3.5 weeks, about 4.0 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, or more than 6 months, including any ranges between these values. In certain embodiments, pharmaceutical compositions comprising a non-naturally occurring CKP that binds VEGF-A as described herein are stable under accelerated conditions (such as storage at about 37° C.) for about 0.5 weeks, about 1.0 weeks, about 1.5 weeks, about 2.0 weeks, about 2.5 weeks, 3.5 weeks, about 4.0 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, or more than 6 months, including any ranges between these values.

[0496] Treatment methods using non-naturally occurring cystine knot peptides (CKPs) that bind to vascular endothelial growth factor A (VEGF-A)

[0497] Vascular endothelial growth factor (VEGF-A), a dimeric glycoprotein of about 40 kDa, is a potent endothelial cell mitogen that stimulates proliferation, migration and tube formation, causing angiogenic growth of new blood vessels and increased vascular permeability. Hypoxic conditions in the retina and cornea induce the expression of vascular endothelial growth factor (VEGF-A), and abnormal (such as excessive or otherwise inappropriate) growth of leaky blood vessels contributes to the pathology of several debilitating eye diseases, including, for example, diabetic blindness, retinopathy, primary diabetic retinopathy, age-related macular degeneration (AMD), proliferative diabetic retinopathy (PDR), retinopathy of prematurity (ROP), choroidal neovascularization (CNV), diabetic macular edema, pathological myopia, von Rippel-Lindau disease, ocular histoplasmosis, retinal vein occlusion (RVO) (both branch retinal vein occlusion (BRVO) and central retinal vein occlusion (CRVO)), corneal neovascularization, retinal neovascularization and erythema. VEGF-A-induced angiogenesis is deleterious, and retinal, trabecular, or corneal neovascularization can ultimately lead to vision loss.

[0498] In certain embodiments, provided herein is a method for treating an ocular disease or condition in a subject, the method comprising administering to the subject an effective amount of a non-naturally occurring CKP described herein that binds VEGF-A or a composition (such as a pharmaceutical composition) comprising a non-naturally occurring CKP that binds VEGF-A as described herein. In certain embodiments, a composition (such as a pharmaceutical composition) comprising a non-naturally occurring CKP that binds VEGF-A as described herein is provided for use in treating an ocular disease or condition in a subject. In certain embodiments, a non-naturally occurring CKP that binds VEGF-A as described herein (or a CKP composition comprising such a non-naturally occurring CKP) is provided for use in the preparation of a medicament for treating an ocular disease and condition in a subject.

[0499] In certain embodiments, the subject to be treated is a mammal (e.g., a human, non-human primate, rat, mouse, cattle, horse, pig, sheep, goat, dog, cat, etc.). In certain embodiments, the subject is a human. In certain embodiments, the subject is a clinical patient, a clinical trial volunteer, an experimental animal, etc. In certain embodiments, the subject is suspected of having an eye disease or condition characterized by abnormal angiogenesis and / or abnormal vascular permeability (such as those described herein) or is at risk of having the eye disease or condition. In certain embodiments, the subject has been diagnosed as having an eye disease or condition characterized by abnormal angiogenesis and / or abnormal vascular permeability (such as those described herein).

[0500] In certain embodiments, the ocular disease or disorder is an ocular vascular proliferative disease, such as an ocular vascular proliferative disease selected from the group consisting of diabetic blindness, retinopathy, primary diabetic retinopathy, age-related macular degeneration (AMD), proliferative diabetic retinopathy (PDR), retinopathy of prematurity (ROP), choroidal neovascularization (CNV), diabetic macular edema, pathological myopia, von Rippel-Lindau disease, ocular histoplasmosis, retinal vein occlusion (RVO) (both branch retinal vein occlusion (BRVO) and central retinal vein occlusion (CRVO)), corneal neovascularization, retinal neovascularization and erythema. In certain embodiments, corneal neovascularization leads to eye infection, eye inflammation, eye trauma (including chemical burns) or loss of the limbal stem cell barrier. In certain embodiments, corneal neovascularization is caused by herpetic keratitis, trachoma or onchocerciasis.

[0501] In certain embodiments, an effective amount of a non-naturally occurring CKP that binds VEGF-A described herein (or a composition comprising such a non-naturally occurring CKP that binds VEGF-A described herein) is administered directly to the eye of a subject (such as intravitreally or topically), as described in further detail elsewhere herein.

[0502] In certain embodiments, the non-naturally occurring CKP in combination with VEGF-A described herein (or a composition comprising such a non-naturally occurring CKP) is administered in combination with a second agent. For patients whose eye diseases or conditions are triggered by inflammatory reactions, a combination therapy using an anti-inflammatory agent may be considered. For example, the combination of steroids with the non-naturally occurring CKP in combination with VEGF-A described herein (or a composition comprising such a non-natural CKP) to respectively reduce inflammation and prevent new blood vessels from forming in patients with, for example, corneal neovascularization may be particularly advantageous. Patients with eye diseases or conditions secondary to bacterial, viral, fungal or acanthamoebal infections may benefit from the administration of the non-naturally occurring CKP in combination with VEGF-A described herein (or a composition comprising such a non-naturally occurring CKP) and an antimicrobial agent and optionally an anti-inflammatory agent. Patients with corneal stromal vessels due to eye diseases or conditions are at risk of swelling due to immune rejection after corneal transplantation. Therefore, administration of the non-naturally occurring CKPs described herein that bind VEGF-A (or compositions comprising such non-naturally occurring CKPs) prior to (and optionally also after) corneal transplantation may be particularly beneficial to patients with corneal stromal vessels, since successful reduction of corneal vascularization will reduce the risk of transplant rejection. In certain embodiments, the non-naturally occurring CKPs described herein that bind VEGF-A (or compositions comprising such non-naturally occurring CKPs) are administered in combination with a second anti-angiogenic agent. In certain embodiments, the non-naturally occurring CKPs described herein that bind VEGF-A (or compositions comprising such non-naturally occurring CKPs) are administered in combination with a matrix metalloproteinase (MMP) inhibitor.

[0503] In certain embodiments, the non-naturally occurring CKP described herein that binds VEGF-A (or a composition comprising such a non-naturally occurring CKP) is administered in combination with a second therapy. In certain embodiments, the second therapy is laser photocoagulation therapy (LPT). LPT uses a laser to cause controlled damage to the retina, thereby producing a beneficial therapeutic effect. Short-pulsed lasers can seal leaking blood vessels, destroy abnormal blood vessels, seal retinal holes, or destroy abnormal tissues at the back of the eye. It is fast, non-invasive, and generally does not require anesthesia other than anesthetic eye drops. LPT technology and devices are readily available to ophthalmologists (see Lock et al. (2010) Med J Malaysia 65: 88-94). Other details about LPT can be found in, for example, WO 2014 / 033184.

[0504] In certain embodiments, the second therapy is photodynamic therapy (PDT). PDT uses photoactivated molecules to cause local damage to the neovascular endothelium, resulting in vascular occlusion. Light is delivered to the retina as a single circular spot via a fiber optic cable and a slit lamp using a suitable ophthalmic magnifier (laser treatment). Prior to laser treatment, a photoactivated compound is injected into the circulation, and damage is applied by photoactivation of the compound in areas plagued by neovascularization. A commonly used photoactivated compound is verteporfin. Verteporfin is mainly transported in plasma by lipoprotein. Once verteporfin is activated by light in the presence of oxygen, highly reactive short-lived singlet oxygen and reactive oxygen free radicals are produced, thus damaging the endothelium around the blood vessel. The known damaged endothelium releases procoagulant factors and vasoactive factors via lipoxygenase (leukotrienes) and cyclooxygenase (eicosanoids, such as thromboxane (thromboxane)) pathways, resulting in platelet aggregation, fibrin clot formation and vasoconstriction. Verteporfin appears to preferentially accumulate in neovascular tissue to some extent. The laser wavelength for the photoactivation of photoactivated compounds can vary depending on the specific photoactivated compounds used. Other details about PDT can be found in, for example, WO 2014 / 033184.

[0505] In certain embodiments, the second therapy is diathermy and cautery, wherein the vessel is occluded by applying a coagulation current via a monopolar diathermy unit or by thermal cautery using an electrolytic needle inserted into a feeder vessel at the limbus.

[0506] Application

[0507] In certain embodiments, a non-naturally occurring CKP that binds VEGF-A (or a composition comprising such a non-naturally occurring CKP) is administered, for example, by injection (e.g., subconjunctival injection, intracorneal injection, or intravitreal injection). The administration of an aqueous form typically has a typical volume of 20-150 μl (e.g., 40-60 μl or 50 μl). The injection can be via a 30-gauge × 1 / 2-inch (12.7 mm) needle. In certain embodiments, a non-naturally occurring CKP that binds VEGF-A (or a composition comprising such a non-naturally occurring CKP) is provided in a pre-filled sterile syringe ready for administration. In certain embodiments, the syringe has a low silicone content or is silicone-free. The syringe can be made of glass. The advantage of using a pre-filled syringe for delivery is that any contamination of the sterile antagonist solution before administration can be avoided. The pre-filled syringe also provides an easier-to-handle administration for ophthalmologists. For further details on intravitreal administration, see, for example, WO 2014 / 033184, Fagan et al. (2013) Clin Exp Ophthalmol. 41, 500-507; Avery et al. (2014) Retina. 34 Suppl 12, S1-S18; and Doshi et al. (2015) Seminar Ophthalmol. 26, 104-113.

[0508] In certain embodiments, a non-naturally occurring CKP that binds VEGF-A (or a composition comprising such a non-naturally occurring CKP) is administered topically (e.g., in the form of eye drops). Additional details on topical drug delivery to the eye are found, for example, in Loftsson et al. (2012) Acta Ophthalmologica. 90, 603-608; Patel et al. (2013) World J. Pharmacol. 2, 47-64; Freeman et al. (2009) Exp Rev Ophthalmol. 4, 59-64; and Boddu et al. (2014) Recent Patents on Drug Delivery and Formulation. 8, 27-36.

[0509] In certain embodiments, non-naturally occurring CKP in combination with VEGF-A (or a composition comprising such non-naturally occurring CKP) is continuously delivered to the eye using an intravitreal device. In certain embodiments, non-naturally occurring CKP in combination with VEGF-A (or a composition comprising such non-naturally occurring CKP) is administered via an eye insert (including, but not limited to, for example, Ocusert, Lactiser, soluble ocular drug insert (SODI), Minidisc, contact lens, membrane, filter paper strip, artificial tear insert, and collagen cover). See, for example, Gaikwad et al. (2013) Indo Amer J Pharm Res.3, 3216-3232). In certain embodiments, non-naturally occurring CKP in combination with VEGF-A (or a composition comprising such non-naturally occurring CKP) is administered as a slow release storage, an eye plug / reservoir, an eye implant (such as a sclera or vitreous implant). Various sclera and intravitreal delivery systems are known in the art. These delivery systems are generally non-biodegradable and can be active or passive. For example, WO 2010 / 088548 describes a delivery system with a rigid body that uses passive diffusion to deliver a therapeutic agent. WO 2002 / 100318 discloses a delivery system with a flexible body that allows active administration via a pressure differential. Alternatively, active delivery can be achieved by an implantable micropump. An example of an intravitreal delivery system that uses a micropump to deliver a therapeutic agent is the Ophthalmic MicroPump System sold by Replenish, Inc. TM, which can be programmed to deliver a set amount of therapeutic agent for a predetermined number of times. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A (or a composition comprising such a non-naturally occurring CKP) is placed in a small capsule-like container (e.g., a silicone elastomer cup). The container is usually implanted above the iris in the eye. The container includes a release opening. The release of the non-naturally occurring CKP that binds VEGF-A (or a composition comprising such a non-naturally occurring CKP) can be controlled by a membrane between the non-naturally occurring CKP that binds VEGF-A (or a composition comprising such a non-naturally occurring CKP) and the opening or by means of a micropump connected to the container. Alternatively, the non-naturally occurring CKP that binds VEGF-A (or a composition comprising such a non-naturally occurring CKP) can be deposited in a slow-release matrix that prevents the antagonist from rapidly diffusing out of the container. Preferably, the intravitreal device is designed to release the non-naturally occurring CKP that binds VEGF-A (or a composition comprising such a non-naturally occurring CKP) at a higher initial rate in the first month. The release rate is slowly reduced to a rate that is about 50% lower than the initial rate, for example, during the first month after implantation. The container may be of a size sufficient to accommodate a supply of a non-naturally occurring CKP that binds VEGF-A (or a composition comprising such a non-naturally occurring CKP) that lasts for about four to six months. Since a reduced dose of a non-naturally occurring CKP that binds VEGF-A (or a composition comprising such a non-naturally occurring CKP) may be sufficient to achieve effective treatment when administered continuously, the supply in the container may last for one year or longer, preferably about two years, and more preferably about three years. Because only a minor surgery is required to implant the delivery system and intravitreal injections are avoided, patient compliance issues with repeated intravitreal injections can be avoided. The intravitreal concentration of a non-naturally occurring CKP that binds VEGF-A (or a composition comprising such a non-naturally occurring CKP) is reduced, and therefore the risk of potential side effects caused by the non-naturally occurring CKP that binds VEGF-A (or a composition comprising such a non-naturally occurring CKP) entering the circulation is reduced.

[0510] In certain embodiments, non-naturally occurring CKPs (or compositions comprising such non-naturally occurring CKPs) that bind VEGF-A are administered by iontophoresis. Iontophoresis is a non-invasive technique in which a small electric current is applied to enhance the penetration of ionized drugs into tissues (see, e.g., Myles et al. (2005) Adv Drug Deliv Rev 57, 2063-79 and Eljarrat-Binstock et al. (2006) J Controlled Release 110, 479-89). Drugs are administered with electrodes carrying the same charge as the drug, and electrodes with opposite charges are placed elsewhere on the body to complete the circuit. The drug acts as a conductor for the current through the tissue.

[0511] Additional details on administering drugs to the eye are provided in, for example, Kuno et al. (2011) Polymers 3, 193-221; Short (2008) Toxicologic Path. 36, 49-62; Ghate et al. (2006) Expert Opin Drug Deliv 3, 275-87; Davis et al. (2004) Curr Opin Mol Therap 6, 195-205; Gaudana et al. (2010) AAPS J. 12, 348-360; and elsewhere.

[0512] Slow release / long-acting delivery formulations

[0513] In certain embodiments, a non-naturally occurring CKP that binds VEGF-A (or a composition comprising such a non-naturally occurring CKP) is provided as a slow release formulation.Slow release formulations are typically obtained by mixing the therapeutic agent with a biodegradable polymer or encapsulating it into microparticles.

[0514] The slow release formulation according to the present invention generally comprises a non-naturally occurring CKP in combination with VEGF-A (or a composition comprising such a non-naturally occurring CKP), a polymeric carrier and a release modifier for regulating the release rate of the non-naturally occurring CKP in combination with VEGF-A (or a composition comprising such a non-naturally occurring CKP) from the polymeric carrier. By changing the preparation conditions of the polymer-based delivery composition...

Claims

1. A non-naturally occurring peptide that binds to human low-density lipoprotein receptor-related protein 6 (LRP6), wherein the peptide is selected from the group consisting of the amino acid sequence shown in any one of SEQ ID NOs: 189-210.

2. A composition comprising the peptide according to claim 1 and a pharmaceutically acceptable carrier.

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