Cystine knot stent platform

Abstract

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

Description

[0001] This application is a divisional application of International Application No. PCT / US2016 / 052012, with an international filing date of September 15, 2016, China National Phase Application No. 202210278625.9, and a China National Phase filing date of March 9, 2018, with the invention title "Cystine Knot Scaffold Platform".

[0002] Cross - reference to related applications

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

[0004] Submission of a Sequence Listing in ASCII text file

[0005] The following submitted content in the ASCII text file is incorporated herein by reference in its entirety: the computer - readable form (CRF) of the sequence listing (filename: 146392026840SEQLIST.txt, date of record: September 15, 2016, size: 183 KB). Background of the Invention

[0007] The design and engineering of novel proteins from alternative protein scaffolds has emerged as a new 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; ASkerra, Curr Opin Biotechnol 18, 295 - 304, 2007; C Gronwall and S Stahl, 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).

[0008] 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 homologous thermostable sequences, design of stabilizing disulfide bonds, mutations to increase α-helical propensity, engineering of salt bridges, alteration of the surface charge of proteins, directed evolution, and composition of consensus sequences (Lehmann and Wyss, Curr Opin Biotechnol 12, 371-375, 2001).

[0009] Cystine-knot peptides are derived from a wide range of sources and exhibit diverse pharmacological activities. They are approximately 30-50 amino acids in length and contain six conserved cysteine residues that form three disulfide bonds. One of the disulfide bonds penetrates a large ring formed by the other two disulfide bonds and the backbone to which they are attached, resulting in a characteristic knotted 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 the disulfide bonds renders cystine-knot peptides highly stable to thermal degradation, proteolytic degradation, and chemical degradation.

[0010] Accordingly, there is a need to develop small, stable, artificial antibody-like molecules for a variety of therapeutic and diagnostic applications, such as eye diseases and disorders. The present invention meets this need and other needs. SUMMARY OF THE INVENTION

[0012] In certain embodiments, provided herein is a non-naturally occurring cystine-knot peptide (CKP) that binds vascular endothelial growth factor A (VEGF-A), wherein the CKP comprises a cystine scaffold structure:

[0013] Z1C1L1C2L2C3L3C4L4C5L5C6Z2

[0014] Wherein:

[0015] Z1 and Z2 are any amino acids;

[0016] 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 X1X2X3X4X5X6X7X8X9X 10 (SEQ ID NO:6), wherein each of X1-X 10 is any amino acid;

[0017] L2 is loop 2 and has the structure: X1X2X3X4X5 (SEQ ID NO:7), where each of X1–X5 is any amino acid or non-natural amino acid;

[0018] L3 is loop 3 and has the structure: X1X2X3, where each of X1–X3 is any amino acid or non-natural amino acid;

[0019] L4 is loop 4 and has the structure: X1, where X1 is any amino acid or non-natural amino acid;

[0020] L5 is loop 5 and has the structure: X1X2X3X4X5 (SEQ ID NO:7), where each of X1–X5 is any amino acid or non-natural amino acid;

[0021] wherein the non-natural amino acids are selected from the group consisting of: L-propargylglycine-PEG6-, L-sulphotyrosine, L-norleucine, L-1-naphthylalanine, L-2-naphthylalanine, L-2-chlorotryptophan, L-3-fluorotyrosine and L-4-fluorophenylalanine; and

[0022] wherein the CKP binds to VEGF-A with an affinity of 500 pM or better.

[0023] 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 to VEGF-A has an altered disulphide connectivity; wherein the altered disulphide connectivity is C1-C4, C2-C3 and C5-C6.

[0024] In certain embodiments according to (or as applied to) any of the above embodiments, the unnatural amino acid is selected from the group consisting of: L-propargylglycine-PEG6-, L-sulfo 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 (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-L-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-(naphth-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.

[0025] In certain embodiments according to (or as applied to) any of the above embodiments, Z1 and / or Z2 is more than one amino acid, or an unnatural amino acid. In certain embodiments, Z2 is two amino acids. In certain embodiments, Z2 is three amino acids.

[0026] In certain embodiments according to (or as applied to) any of the above embodiments, Z1 and / or Z2 is G.

[0027] 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 K, N, F, P, L, Y, T, D, M, and X; X7 is an amino acid selected from Q, W, H, and / X; and / or X8 is an amino acid selected from Y, A, G, D, E, W, S, and X, where 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: X9 is an amino acid selected from L, I, V, D, E, and X, where 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 Y, T, M, N, F, and X, where 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.

[0028] In certain embodiments according to (or as applied to) any of the above embodiments, X is a non-natural amino acid and is selected from the group consisting of: L-propargylglycine-PEG6-, L-sulfo 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 (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-(naphth-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.

[0029] 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.

[0030] In certain embodiments according to (or as applied to) any of the above embodiments, L1 has the structure X1X2X3X4X5X6X7X8 (SEQ ID NO:4), where: 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), where: 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 Y, T, M, and N.

[0031] 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.

[0032] In certain embodiments according to (or as applied to) any of the above embodiments, L1 has the structure X1X2X3X4X5X6X7X8 (SEQ ID NO:4), where: 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.

[0033] 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.

[0034] 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), where: 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.

[0035] In certain embodiments according to (or as applied to) any of the above embodiments, 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.

[0036] 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.

[0037] In certain embodiments according to (or as applied to) any of the above embodiments, L1 has the structure X1X2X3X4X5X6X7X8 (SEQ ID NO:4), where: X5 is P; X7 is W; X8 is G; and where 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 above embodiments, L1 has the structure X1X2X3X4X5X6X7X8 (SEQ ID NO:4), where: 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.

[0038] 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 that: X1 is not G, X2 is not P, and / or X4 is not G.

[0039] In certain embodiments according to (or as applied to) any of the above embodiments, 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.

[0040] 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.

[0041] In certain embodiments according to (or as applied to) any of the above embodiments, in L4, X1 is V or F.

[0042] 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).

[0043] 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).

[0044] In certain embodiments according to (or as applied to) any of the above embodiments, in L5: X1 is any amino acid other than G; X2 is any amino acid other than P; X3 is any amino acid other than N; X4 is any amino acid other than G; and / or X5 is any amino acid other than F.

[0045] In certain embodiments according to (or as applied to) any of the above embodiments, L1 has the structure X1X2X3X4X5X6X7X8 (SEQ ID NO:4), where 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, where X is a non-natural amino acid selected from the group consisting of: L-propargylglycine-PEG6-, L-sulfo tyrosine, L-norleucine, L-1-naphthylalanine, L-2-naphthylalanine, L-2-chlorotryptophan, L-3-fluorotyrosine, and L-4-fluorophenylalanine.

[0046] In certain embodiments in accordance with (or as applied to) any of the above embodiments, X is a non-natural amino acid and is selected from the group consisting of: L-propargylglycine-PEG6-, L-sulfo 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 (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-azaindole)-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, glycylamide, glycerol glycine ester, ethylene glycol glycine ester, oxetan-3-yl glycine ester, and glycine morpholine amide.

[0047] 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 non-natural amino acid, except that X1 is not leucine (L), X2 is not alanine (A), and X3 is not glycine (G), wherein the non-natural amino acids are 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, L, V, 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 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.

[0048] In certain embodiments according to (or as applied to) any of the above embodiments, in L4, X1 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, 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, and L-4-fluorophenylalanine.

[0049] 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, where 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, where 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.

[0050] In certain embodiments according to (or as applied to) any of the above embodiments, 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, L-4-fluorophenylalanine, γ-benzyl-L-proline, γ-(4-fluoro-benzyl)-L-proline, 4-OH-L-proline, 4-fluor-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-L-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-(naphth-1-ol)-L-alanine, D-serine, L-β-homoserine, L-β-alanine, N-α-methylglycine, glycylamide, glycerol glycine ester, ethylene glycol glycine ester, oxetane-3-yl glycine ester, and glycine morpholine amide.

[0051] In certain embodiments according to (or as applied to) any of the above embodiments, 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, where 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.

[0052] In certain embodiments according to (or as applied to) any of the above embodiments, a non-naturally occurring cystine knot peptide (CKP) that binds 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 above embodiments, a non-naturally occurring cystine knot peptide (CKP) that binds 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 above embodiments, a non-naturally occurring cystine knot peptide (CKP) that binds 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 above embodiments, a non-naturally occurring cystine knot peptide (CKP) that binds 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 above embodiments, a non-naturally occurring cystine knot peptide (CKP) that binds 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 above embodiments, a non-naturally occurring cystine knot peptide (CKP) that binds 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 above embodiments, a non-naturally occurring cystine knot peptide (CKP) that binds 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 above embodiments, a non-naturally occurring cystine knot peptide (CKP) that binds 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 above embodiments, a non-naturally occurring cystine knot peptide (CKP) that binds 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 above embodiments, a non-naturally occurring cystine knot peptide (CKP) that binds 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 above embodiments, a non-naturally occurring cystine knot peptide (CKP) that binds 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 above embodiments, a non-naturally occurring cystine knot peptide (CKP) that binds 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 above embodiments, a non-naturally occurring cystine knot peptide (CKP) that binds 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 above embodiments, a non-naturally occurring cystine knot peptide (CKP) that binds 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 above embodiments, a non-naturally occurring cystine knot peptide (CKP) that binds 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 above embodiments, a non-naturally occurring cystine knot peptide (CKP) that binds vascular endothelial growth factor A (VEGF-A) comprises the amino acid sequence GCNIXLPFWGCGRDFECMSDCICQYYQSCG (SEQ ID NO: 144), where X is norleucine (Nle).In certain embodiments according to (or as applied to) any of the above embodiments, a non-naturally occurring cystine knot peptide (CKP) that binds vascular endothelial growth factor A (VEGF-A) comprises the amino acid sequence GCNIXLPFWGCGRDFECVSQCICQYYQSCG (SEQ ID NO: 145), where X is norleucine (Nle). In certain embodiments according to (or as applied to) any of the above embodiments, a non-naturally occurring cystine knot peptide (CKP) that binds vascular endothelial growth factor A (VEGF-A) comprises the amino acid sequence GCNIXLPYWGCGRDFECMEQCICQYYQSCG (SEQ ID NO: 146), where X is norleucine (Nle).

[0053] In certain embodiments according to (or as applied to) any of the above embodiments, a 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), where X is norleucine.

[0054] In certain embodiments, a non-naturally occurring cystine knot peptide (CKP) is provided that comprises an amino acid selected from the group consisting of:

[0055] GCNIMLPFWGCGRDFECMEQCICQYYQSCG (SEQ ID NO: 113);

[0056] GCNIMLPFWGCGRDFECVYRCICQYYQSCG (SEQ ID NO: 114);

[0057] GCDVMQPYWGCGPDIDCFVRCLCHWYNSCG (SEQ ID NO: 139);

[0058] GCDVMQPYWGCGPDIDCLSNCICHWYNSCG (SEQ ID NO: 140);

[0059] GCNIMLPYWGCGRDFECMEQCICQYYQSCG (SEQ ID NO: 142);

[0060] 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 above embodiments, the CKP comprises

[0061] 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

[0062] 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

[0063] 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

[0064] 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

[0065] 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

[0066] 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.

[0067] In certain embodiments, a non-naturally occurring cystine knot peptide (CKP) is provided that comprises amino acids selected from the group consisting of:

[0068] 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),

[0069] 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), where 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-D / E-CLS-N / K / X2-CICHWYNSCG (SEQ ID NO:534), where 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), where 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), where 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), where 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), where 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 GCNIXLPYWGCGRDFECMEKCICQYYQSCG (SEQ ID NO:543), where 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), where 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.

[0070] In certain embodiments according to (or as applied to) any of the above embodiments, there is provided a non-naturally occurring cystine knot peptide (CKP) that binds to VEGF-A, wherein the CKP comprises a cystine scaffold structure:

[0071] Z1C1L1C2L2C3L3C4L4C5L5C6Z2

[0072] Wherein:

[0073] Z1 and Z2 are any amino acids;

[0074] L1 is Loop 1 and has a structure selected from the group consisting of: X1X2X3X4X5X6X7X8, X1X2X3X4X5X6X7X8X9, and X1X2X3X4X5X6X7X8X9X 10 , wherein X1-X 10 each of which is any amino acid;

[0075] L2 is loop 2 and has the structure: X1X2X3X4X5, where each of X1–X5 is an arbitrary amino acid;

[0076] L3 is loop 3 and has the structure: X1X2X3, where each of X1–X3 is an arbitrary amino acid;

[0077] L4 is loop 4 and has the structure: X1, where X1 is an arbitrary amino acid;

[0078] L5 is loop 5 and has the structure: X1X2X3X4X5, where each of X1–X5 is an arbitrary amino acid;

[0079] Wherein, with reference to the wild-type Ecballium elaterium trypsin inhibitor EETI-II protein having the amino acid sequence shown in SEQ ID NO:1, said CKP has an altered disulfide bond connectivity; wherein the altered disulfide bond connectivity is C1-C4, C2-C3, and C5-C6; and wherein said CKP has an alpha helix content percentage of at least 20%.

[0080] In certain embodiments according to (or as applied to) any of the above embodiments, Z1 and Z2 are arbitrary amino acids, more than one amino acid, or non-natural amino acids. In certain embodiments according to (or as applied to) any of the above embodiments, X1-X in L1 10Each of them is an arbitrary 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 L2 is an arbitrary 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 an arbitrary amino acid or unnatural amino acid. In certain embodiments according to (or as applied to) any of the above embodiments, X1 in L4 is an arbitrary 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 an arbitrary amino acid or unnatural amino acid. In certain embodiments, the unnatural amino acids are 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, glycylamide, glycerol glycine ester, ethylene glycol glycine ester, oxetan-3-yl glycine ester, and glycine morpholine amide.

[0081] In certain embodiments according to (or as applied to) any of the above embodiments, the non-naturally occurring (CKP) that binds to 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 above embodiments, the binding affinity is determined by surface plasmon resonance.

[0082] 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).

[0083] In certain embodiments according to (or as applied to) any of the above embodiments, the C-terminal carboxyl group of the non-naturally occurring (CKP) that binds to VEGF-A is modified (such as capped). In certain embodiments according to (or as applied to) any of the above embodiments, the N-terminal amino group of the non-naturally occurring (CKP) that binds to VEGF-A is modified (such as capped). In certain embodiments according to (or as applied to) any of the above embodiments, the C-terminal carboxyl group of the non-naturally occurring (CKP) that binds to VEGF-A is capped and the N-terminal amino group of the non-naturally occurring (CKP) that binds to VEGF-A is modified (such as capped).

[0084] In certain embodiments according to (or as applied to) any of the above embodiments, the C-terminal carboxyl group of the non-naturally occurring (CKP) that binds to VEGF-A is amidated. In certain embodiments according to (or as applied to) any of the above embodiments, the N-terminal amino group of the non-naturally occurring (CKP) that binds to VEGF-A is acetylated. In certain embodiments according to (or as applied to) any of the above embodiments, the C-terminal carboxyl group of the non-naturally occurring (CKP) that binds to VEGF-A is amidated and the N-terminal amino group of the non-naturally occurring (CKP) that binds to VEGF-A is acetylated.

[0085] In certain embodiments according to (or as applied to) any of the above embodiments, a non-naturally occurring (CKP) that binds 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 between about 0.5 nM and about 1.0 nM 50 inhibits VEGF-A activity. In certain embodiments according to (or as applied to) any of the above embodiments, the non-naturally occurring EETI-II scaffold protein binds human VEGF-A, mouse VEGF-A, and rat VEGF-A.

[0086] 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 VEGF-A in any of the above embodiments for binding to VEGF-A.

[0087] In certain embodiments according to (or as applied to) any of the above embodiments, a non-naturally occurring CKP that binds an epitope on VEGF-A, the epitope comprising 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.

[0088] In certain embodiments according to (or as applied to) any of the above embodiments, the residues are selected from the group consisting of: K48, N62, and D63. In certain embodiments according to (or as applied to) any of the above embodiments, the residues are selected from the group consisting of: Y21, Y25, and P106. In certain embodiments according to (or as applied to) any of the above embodiments, the residues are selected from the group consisting of: H86 and Q89. In certain embodiments according to (or as applied to) any of the above embodiments, the residues are selected from the group consisting of: M81, D19, and Q22. In certain embodiments according to (or as applied to) any of the above embodiments, the residues are selected from the group consisting of: F17, M81, and I91. In certain embodiments according to (or as applied to) any of the above embodiments, the residues are 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 above embodiments, the residues are selected from the group consisting of: Y25.

[0089] In certain embodiments according to (or as applied to) any of the above embodiments, a 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, a 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.

[0090] In certain embodiments according to (or as applied to) any of the above embodiments, there is provided an isolated nucleic acid encoding a non-naturally occurring (CKP) that binds VEGF-A of any of the above embodiments. There is also provided an expression vector encoding the nucleic acid molecule of any of the above embodiments. There is also provided a cell comprising the expression vector of any of the above embodiments. There is also provided a method of producing a non-naturally occurring (CKP) that binds VEGF-A of any of the above embodiments, 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.

[0091] There is also provided a method of producing a non-naturally occurring (CKP) that binds VEGF-A of any of the above embodiments, the method comprising chemically synthesizing the non-naturally occurring (CKP) that binds VEGF-A.

[0092] Provided herein is a composition comprising a 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 the risk of age-related macular degeneration (AMD), such as complement pathway components 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 antigen-binding fragment thereof.

[0093] Provided herein is a method of treating an ocular disease in a subject characterized by angiogenesis and / or vascular permeability or leakage, the method comprising administering to the subject an effective amount of a non-naturally occurring (CKP) that binds VEGF-A of 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 that binds 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 that binds 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 the risk of age-related macular degeneration (AMD), such as complement pathway components 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 ocular 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 ischemia-related retinopathies, diabetic macular edema, pathologic 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).

[0094] In certain embodiments according to (or as applied to) any of the above embodiments, 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 above embodiments, the implantable device is selected from the group consisting of: an ocular insert, a sustained release depot, an ocular plug / reservoir, a non-biodegradable ocular implant, or a biodegradable ocular implant.

[0095] In certain embodiments according to (or as applied to) any of the above embodiments, there is provided a composition comprising the non-naturally occurring (CKP) that binds VEGF-A of any of the above embodiments for use in the treatment of an ocular disease in a subject characterized by angiogenesis and / or vascular permeability or leakage. In certain embodiments according to (or as applied to) any of the above embodiments, the ocular 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 ischemia-related retinopathies, diabetic macular edema, pathologic 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 a subject via an implantable device. In certain embodiments according to (or as applied to) any of the above embodiments, the implantable device is selected from the group consisting of: an ocular insert, a sustained release depot, an ocular plug / reservoir, a non-biodegradable ocular implant, or a biodegradable ocular implant.

[0096] In certain embodiments according to (or as applied to) any of the above embodiments, there is provided a composition comprising a non-naturally occurring (CKP) that binds VEGF-A of any of the above embodiments for use in treating an ocular 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 ocular 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 ischemia-related retinopathies, diabetic macular edema, pathologic 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 above embodiments, the implantable device is selected from the group consisting of: an ocular insert, a sustained release depot, an ocular plug / reservoir, a non-biodegradable ocular implant, or a biodegradable ocular implant.

[0097] In certain embodiments according to (or as applied to) any of the above embodiments, the non-naturally occurring (CKP) that binds VEGF-A is formulated for long-acting delivery.

[0098] There is provided herein a formulation comprising a 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.

[0099] There is also provided herein a non-naturally occurring cystine knot peptide (CKP) that binds to human low density lipoprotein receptor-related protein 6 (LRP6), wherein the CKP comprises a cystine scaffold structure:

[0100] Z1C1L1C2L2C3L3C4L4C5L5C6Z2;

[0101] wherein:

[0102] Z1 and Z2 are any amino acids;

[0103] L1 is loop 1 and has a structure selected from the group consisting of: X1X2X3X4X5X6, X1X2X3X4X5X6X7, X1X2X3X4X5X6X7X8, X1X2X3X4X5X6X7X8X9, and X1X2X3X4X5X6X7X8X9X 10 , where each of X1 - X 10 is any amino acid;

[0104] L2 is loop 2 and has a structure: X1X2X3X4X5, where each of X1–X5 is any amino acid;

[0105] L3 is loop 3 and has a structure: X1X2X3, where each of X1–X3 is any amino acid;

[0106] L4 is loop 4 and has a structure: X1, where X1 is any amino acid; and

[0107] L5 is loop 5 and has a structure: X1X2X3X4X5, where each of X1–X5 is any amino acid.

[0108] In certain embodiments according to (or as applied to) any of the above embodiments, Z1 and / or Z2 is more than one amino acid, or a non-natural amino acid. In certain embodiments, Z2 is two amino acids. In certain embodiments, Z2 is three amino acids.

[0109] In certain embodiments according to (or as applied to) any of the above embodiments, Z1 and / or Z2 is G.

[0110] 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 E, W, and G.

[0111] In certain embodiments according to (or as applied to) any of the above embodiments, 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.

[0112] 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

[0114] Figure 1 Depicts the structure of the EETI-II cystine knot protein.

[0115] Figure 2A Shows the results of experiments conducted to determine whether EGF_CKP9.54.90 disrupts the interaction between VEGF-A(8-109) and KDR; between VEGF-A(8-109) and Flt-1; between VEGF-A165 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.

[0116] Figure 2B Shows the results of experiments conducted to determine whether EGF_CKP9.54.90 disrupts the interaction 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.

[0117] Figure 3 Shows the results of experiments conducted to determine whether VEGF_CKP9.54.90, VEGF_CKP9.54, and VEGF_CKP9.63.12 inhibit trypsin protease activity.

[0118] Figure 4Show the results of experiments conducted to determine whether VEGF_CKP9.54.90 and VEGF_CKP9.63.12 are resistant to trypsin digestion.

[0119] Figure 5 Depict the structure of VEGF_CKP9.54.90, the structure of wild-type EETI-II, and provide a schematic diagram comparing the disulfide bond connectivity pattern of the CKP variant that binds VEGF with that of wild-type EETI-II.

[0120] Figure 6 Depict the co-crystal structure of VEGF_CKP9.54.90 complexed with VEGF-A.

[0121] Figure 7 Depict 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.

[0122] Figure 8 Depict 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.

[0123] Figure 9 Show the contact residues on VEGF-A at the interaction surface between VEGF-A and VEGF_CKP9.54.90.

[0124] Figure 10 Show the binding interface of bevacizumab Fab, Z-domain, and receptor-blocking peptide v108 on VEGF-A.

[0125] Figure 11 Provide the results of experiments conducted to determine the effect 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 shown relative to two different y-axes.

[0126] Figure 12 Provide the results of experiments conducted to determine the effect of VEGF_CKP9.54.90 on CNV in rat eyes.

[0127] Figure 13 Provide for determining the IC 50 value of the CKP variant that binds VEGF.

[0128] Figure 14Depict the structures of VEGF_CKP9.54 and VEGF_CKP9.63. Figure 14 Also shown is a portion of the co-crystal structure of VEGF_CKP9.63 complexed 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.

[0129] Figure 15 Show the results of phage competition ELISA experiments conducted to evaluate the binding affinities 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 for hVEGF(8-109).

[0130] Figure 16A Show the results of phage competition ELISA experiments conducted to evaluate the binding affinities of clones 9.63.44-1 to 9.63.44-7.

[0131] Figure 16B Show the results of phage competition ELISA experiments conducted to evaluate the binding affinities of clones 9.63.44-8 to 9.63.44-14. Detailed Description of the Invention

[0133] Provide non-naturally occurring cystine knot peptides (CKPs) that specifically bind to human VEGF-A. Such non-naturally occurring CKPs exhibit one or more of the following properties: inhibit VEGF-A activity and have an IC 50 between less than about 0.5 nM and less than about 1.0 nM; bind to human VEGF-A, mouse VEGF-A, and rat VEGF-A; are resistant to trypsin digestion; have disulfide bonds linked as C1-C4, C2-C3, and C5-C6; have an α-helix content of at least about at least about 15% to at least about 50%; bind to an epitope on VEGF-A that is different from the epitope bound by antibody G6.31, bind to an epitope on VEGF-A that is different from the epitope bound by bevacizumab, and / or bind to an epitope on VEGF-A that is different from the epitope bound by Flt-1.

[0134] Also provided are chimeric molecules and conjugates comprising non-naturally occurring cystine knot peptides that bind VEGF-A, nucleic acids encoding non-naturally occurring CKP that binds VEGF-A, and compositions such as pharmaceutical compositions. Also provided are methods of using non-naturally occurring CKP that binds VEGF-A for treating eye diseases and / or disorders (such as ocular angiogenic diseases and / or disorders) caused by abnormal (such as excessive) angiogenesis and / or abnormal vascular permeability. Also provided is the use of non-naturally occurring CKP that binds VEGF-A in the preparation of a medicament for treating an eye disease or disorder.

[0135] In related aspects, non-naturally occurring CKP that binds human low density lipoprotein receptor-related protein 6 (LRP6) is also provided.

[0136] 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 techniques are described in the literature and are thus available to those of skill in the art. See, for example, 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 periodic updates; Freshney, R. I., “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.

[0137] Definitions

[0138] As used herein, "non-naturally occurring" means, for example, a polypeptide that contains an amino acid sequence not found in nature, or a nucleic acid that contains 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), where 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. Thus, the non-naturally occurring CKP described herein can be recombinant, i.e., produced by a cell or nucleic acid or vector that has been modified by introducing a heterologous nucleic acid or altering a native nucleic acid into a form that is non-native to the cell, or the cell is derived from a cell so modified. Alternatively, the non-naturally occurring CKP described herein can be produced by chemical peptide synthesis.

[0139] As used herein, the term "cystine knot peptide" or "CKP" refers to a peptide that is 26-50 amino acids in length and contains six conserved cysteine residues that form three disulfide bonds. One of the disulfide bonds penetrates a large ring formed by the other two disulfide bonds and the backbone to which they are attached, thereby producing a characteristic knot topology with multiple loops exposed on the surface. Loops are defined as the amino acid regions flanking the six conserved cysteine residues and are inherently highly variable.

[0140] As used herein, "amino acid alteration" means the addition, deletion, or substitution of at least one amino acid in, for example, a peptide sequence (such as in the 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).

[0141] A "separated" non-naturally occurring CKP or composition is a CKP or composition that has been identified and separated and / or recovered from the components 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 greater than 99% by weight, (2) purified to an extent 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) purified to homogeneity, by SDS-PAGE using Coomassie blue or preferably silver stain under reducing or non-reducing conditions. Separated non-naturally occurring CKP includes in situ CKP in recombinant cells because at least one component of the natural environment of the CKP will be absent. Separated non-naturally occurring CKP will be prepared by at least one purification step.

[0142] "Percent (%) amino acid sequence identity" or "homology" relative to the polypeptide sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the polypeptide being compared, after aligning the sequences such that any conservative substitutions are considered part of the sequence identity. The alignment for determining the percent amino acid sequence identity can be achieved in a variety of ways within the skill in the art, e.g., 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 algorithms needed to achieve the maximum alignment over the full length of the sequences being compared. However, for the purposes herein, 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 U.S. Copyright Office, Washington, D.C., 20559, along with the user documentation, where it is registered under U.S. 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 are not changed.

[0143] As used herein, the term "epitope" refers to a protein determinant that can be specifically bound by a non-naturally occurring CKP provided herein. An epitope can comprise from about 3 to 10 amino acids in a three-dimensional conformation that is unique to the epitope. These amino acids can be linear within the protein (i.e., contiguous in the amino acid sequence) or they can be located in different portions of the protein (i.e., non-contiguous in the amino acid sequence). Methods for determining the three-dimensional conformation of amino acids within a protein or at the interface of two proteins are known in the art and include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance.

[0144] The terms "disulfide bonding pattern (DBP)", "disulfide linkage", and "disulfide 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 breaking one or more disulfide bonds (such as using redox conditions).

[0145] A "subject", "patient", or "individual" for therapeutic purposes refers to any animal classified as a mammal, including humans, domesticated and farm animals, and zoo, sports, or pet animals such as dogs, horses, cats, cattle, etc. Preferably, the mammal is a human.

[0146] 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 specifically stated purpose. The "effective amount" can be determined empirically and by known methods relevant to the purpose.

[0147] The term "therapeutically effective amount" refers to the amount of a non-naturally occurring CKP or composition as disclosed herein that is effective to "treat" a disease or disorder in a mammal, such as a human patient. In the case of an ocular disease or ocular disorder (such as an ocular vascular proliferative disease or an ocular disorder characterized by excessive angiogenesis), a therapeutically effective amount of a non-naturally occurring CKP that binds VEGF-A (or a composition comprising such a non-naturally occurring VEGF-A-binding CKP) as described herein refers to the amount that reduces, terminates, or prevents at least one symptom of the ocular disease (such as the symptoms or disorders of the ocular disease further described in detail elsewhere herein). For example, an effective amount will be considered to be an amount that is sufficient to reduce or prevent the symptoms of an ocular disease or ocular disorder (such as an ocular vascular proliferative disease or an ocular disorder characterized by excessive angiogenesis), such as the complete or partial elimination of the ocular disease and / or the maintenance or improvement of best corrected visual acuity (such as greater than 5 letters as evaluated by the EDTRS visual acuity chart), or the reduction in the size of neovascularization or neovascular permeability as evaluated by fluorescein fundus angiography. An effective amount as used herein will also include an amount sufficient to prevent or delay, for example, the development of 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 the symptomatic disease (such as, but not limited to, slowing the progression of the symptoms of the ocular disease), or reverse the symptoms of the disease.

[0148] As used herein, "treatment" is a regimen that achieves a beneficial or desired result, including a clinical result. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviation of one or more symptoms caused by an ocular disease, reduction in the scope of the ocular disease, stabilization of the ocular disease (such as preventing or delaying disease progression), prevention or delay of the spread of the disease (such as to the tissues surrounding the eye), prevention or delay of the recurrence of the ocular disease, delay or slowing of the progression of the ocular disease, improvement of the disease state, provision of alleviation or elimination (partial or complete) of the ocular disease, reduction in the dosage of one or more other medications required to treat the ocular disease, delay in the progression of the ocular disease, increase or improvement in quality of life, and / or prevention or delay of vision loss. "Treatment" also encompasses reduction in the pathological consequences (such as, for example, vision loss) of an ocular disease. The methods provided herein contemplate any one or more of these aspects of treatment.

[0149] "Disorder" refers to any condition that would benefit from treatment with the non-naturally occurring CKP in combination with VEGF-A described herein. VEGF-A-related disorders to be treated herein include ocular diseases and disorders (such as ocular angiogenic diseases or ocular disorders characterized by excessive angiogenesis), as described elsewhere herein.

[0150] As used herein, "pharmaceutically acceptable" or "pharmacologically compatible" means 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 effects or interacting in a harmful 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 toxicological and manufacturing tests and / or are included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration.

[0151] The term "detect" 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 determination. Generally, the specific techniques for detection are not critical to the practice of the invention.

[0152] For example, "detecting" according to the invention can 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); a change in the level of a target ligand; and / or a change in the biological function / activity of a target ligand. In certain embodiments, "detecting" can include detecting the level of a target ligand (e.g., the polypeptide level of human LRP6 or human VEGF). Detection can include quantifying a change (increase or decrease) of any value between 10% and 90% or any value between 30% and 60% or any value greater than 100% compared to a control. Detection can include quantifying a change of any value between 2-fold and 10-fold (including the endpoints) or more (e.g., 100-fold).

[0153] As used herein, the word "label" refers to a detectable compound or composition that is conjugated directly or indirectly 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 alteration of a detectable substrate compound or composition.

[0154] Regarding the binding of a non-naturally occurring CKP to a target ligand, the terms "specifically binds to a specific target ligand" or "binds specifically to a specific target ligand" or "is specific for a specific target ligand" mean binding that is measurably different from non-specific interactions. Specific binding can be measured, for example, by determining the binding of a molecule relative 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, such as an excess of unlabeled target. In this case, if the binding of a labeled target to a probe is competitively inhibited by an excess of unlabeled target, specific binding is indicated. In certain embodiments, the degree of binding of a 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 radioimmuno-precipitation (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.

[0155] References herein to "about" a value or parameter refer to the ordinary error range of each value that would be readily known to a person skilled in the art. References herein to "about" a value or parameter include (and describe) aspects that relate to the value or parameter itself. For example, a description of "about X" includes a description of "X".

[0156] It should be understood that aspects and embodiments of the inventions described herein include aspects and embodiments of "comprising", "consisting of", and "consisting essentially of".

[0157] All references cited herein, including patent applications and publications, are incorporated herein by reference in their entirety.

[0158] A non-naturally occurring cystine knot peptide (CKP) that binds to human vascular endothelial growth factor A (VEGF-A)

[0159] In certain embodiments, provided herein is a non-naturally occurring cystine knot peptide (CKP) that binds to vascular endothelial growth factor A (VEGF-A), wherein the CKP comprises the following cystine scaffold structure (i.e., scaffold structure I):

[0160] Z1C1L1C2L2C3L3C4L4C5L5C6Z2(I)

[0161] Wherein:

[0162] Z1 and Z2 are arbitrary amino acids;

[0163] L1 is Loop 1 and has a structure selected from the group consisting of: X1X2X3X4X5X6X7X8, X1X2X3X4X5X6X7X8X9, and X1X2X3X4X5X6X7X8X9X 10 , wherein each of X1-X 10 is an arbitrary amino acid;

[0164] L2 is Ring 2 and has the structure: X1X2X3X4X5, where each of X1–X5 is an arbitrary amino acid or a non-natural amino acid selected from the group consisting of: L-propargylglycine-PEG6-, L-sulphotyrosine, 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-(naphth-1-ol)-L-alanine, D-serine, L-β-homoserine, L-β-alanine, N-α-methylglycine, glycylamide, glycerol glycine ester, ethylene glycol glycine ester, oxetan-3-yl glycine ester, and glycine morpholine amide.

[0165] L3 is Ring 3 and has the structure: X1X2X3, where each of X1–X3 is an arbitrary amino acid or a non-natural amino acid selected from the group consisting of: L-propargylglycine-PEG6-, L-sulfo 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 (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, glycylamide, glycerol glycine ester, ethylene glycol glycine ester, oxetan-3-yl glycine ester, and glycine morpholine amide;

[0166] L4 is Ring 4 and has the structure: X1, where X1 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-fluor-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-(naphth-1-ol)-L-alanine, D-serine, L-β-homoserine, L-β-alanine, N-α-methylglycine, glycinamide, glycerol glycine ester, ethylene glycol glycine ester, oxetan-3-yl glycine ester, and glycine morpholine amide;

[0167] L5 is Ring 5 and has the structure: X1X2X3X4X5, where each of X1–X5 is an arbitrary 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, glycylamide, glycerol glycine ester, ethylene glycol glycine ester, oxetan-3-yl glycine ester, and glycine morpholine amide; and

[0168] wherein the CKP binds to VEGF-A with an affinity of 500 pM or less.

[0169] In certain embodiments, the C-terminus of a non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A is modified (such as capped). In certain embodiments, the N-terminus of a non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A is modified (such as capped). In certain embodiments, both the C- and N-termini of a non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A are modified (such as capped). In certain embodiments, the C-terminal carboxyl group of a non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A is amidated. In certain embodiments, the N-terminal amine of a non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A is acetylated. In certain embodiments, the C-terminal carboxyl group of a non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A is amidated and the N-terminal amine of a non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A is acetylated.

[0170] In certain embodiments, a non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A has an altered disulfide connectivity - with reference to the wild-type Ecballium elaterium trypsin inhibitor EETI-II protein having the amino acid sequence shown in SEQ ID NO:1; wherein the altered disulfide connectivity is C1-C4, C2-C3, and C5-C6.

[0171] In certain embodiments, Z1 and / or Z2 of a 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 are non-natural amino acids. In certain embodiments, the non-natural amino acid is N-acetylglycine or glycylamide. In certain embodiments, a non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A comprises L1, where X3 is not I; where X5 is not M; and / or where X6 is not R. In certain embodiments, a non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A comprises L1, where X1 is an amino acid selected from P, Q, R, T, V, D, N, K, L, and X; where X2 is an amino acid selected from T, D, L, V, I, R, P, N, and X; where X3 is an amino acid selected from T, P, M, L, S, F, R, and X; where X4 is an amino acid selected from R, T, Q, D, W, L, E, S, K, and X; where X5 is an amino acid selected from F, P, V, E, K, L, I, and X; where X6 is an amino acid selected from K, N, F, P, L, Y, T, D, M, and X; where X7 is an amino acid selected from Q, W, H, and / X; and / or where X8 is an amino acid selected from Y, A, G, D, E, W, S, and X, where X is a non-natural amino acid and is selected from the group consisting of: L-propargylglycine-PEG6-, L-sulphotyrosine, 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 glycinate, ethylene glycol glycinate, oxetan-3-yl glycinate, 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 the group consisting of 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, L-4-fluorophenylalanine, γ-benzyl-L-proline, γ-(4-fluoro-benzyl)-L-proline, 4-OH-L-proline, 4-fluor-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-dihydroxy-L-phenylalanine, 3,4-difluoro-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 glycinate, ethylene glycol glycinate, oxetan-3-yl glycinate, and glycine morpholine amide. In certain embodiments, the non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A comprises L1, wherein X, 10is an amino acid selected from Y, T, M, N, F, and X, where 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, 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, glycylamide, glycerol glycine ester, ethylene glycol glycine ester, oxetan-3-yl glycine ester, and glycine morpholine amide.

[0172] In certain embodiments, a non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A comprises L5, where each of X1–X5 is any amino acid or a non-natural amino acid selected from the group consisting of: L-propargylglycine-PEG6-, L-sulfo 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 (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-L-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; except that X2 is not proline (P).

[0173] 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-fluor-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-L-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-(naphth-1-ol)-L-alanine, D-serine, L-β-homoserine, L-β-alanine, N-α-methylglycine, glycylamide, 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, where 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, where 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, where X1 is V or F.

[0174] In certain embodiments, the non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A comprises: L1 comprising the structure X1X2X3X4X5X6X7X8, where: 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 VEGF-A comprises: L1 comprising the structure X1X2X3X4X5X6X7X8 X9 X 10 of L1, where 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 Y, T, M, and N. In certain embodiments, the non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A comprises L5, where 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. In certain embodiments, the non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A comprises L2, where 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, where 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, where X1 is V or F.

[0175] 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, a 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, 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.

[0176] In certain embodiments, a non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A comprises: L1 comprising the structure X1X2X3X4X5X6X7X8 X9 X 10 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 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 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. 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.

[0177] 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, a 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, 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, and / or X4 is not G. In certain embodiments, a 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, 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.

[0178] In certain embodiments, the non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A comprises L5, where 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 VEGF-A comprises L5, where 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, where X1 is any amino acid other than G; X2 is any amino acid other than P; X3 is any amino acid other than N; X4 is any amino acid other than G; and / or X5 is any amino acid other than F.

[0179] In certain embodiments, a non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A comprises: L1 comprising the structure X1X2X3X4X5X6X7X8, where 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, where X is a non-natural amino acid selected from the group consisting of: L-propargylglycine-PEG6-, L-sulfo 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 (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-azaindole)-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, glycinamide, glycerol glycinate, ethylene glycol glycinate, oxetan-3-yl glycinate, and glycine morpholine amide. In certain embodiments, a 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-fluor-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-azaindole)-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-L-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, 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, 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-azaindole)-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-L-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, glycinamide, glycerol glycinate, ethylene glycol glycinate, oxetan-3-yl glycinate, and glycine morpholinamide. 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 the group consisting of M, F, L, V, and X; X2 is an amino acid selected from the group consisting of S, N, Q, I, Y, E, V, T, and X; and X3 is an amino acid selected from the group consisting of D, Q, T, N, E, R, and X, where X is a non-natural amino acid selected from the group consisting of: L-propargylglycine-PEG6-, L-sulfo 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 (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-azaindole)-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-L-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, glycinamide, glycerol glycinate, ethylene glycol glycinate, oxetan-3-yl glycinate, and glycine morpholinamide. In certain embodiments, the non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A comprises L4, wherein X1 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, 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-azaindole)-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, glycinamide, glycerol glycinate, ethylene glycol glycinate, oxetan-3-yl glycinate, and glycine morpholinamide. In certain embodiments, the non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A comprises L4, wherein X1 is I, L, or X, where 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-azaindole)-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-L-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, glycinamide, glycerol glycinate, ethylene glycol glycinate, oxetan-3-yl glycinate, and glycine morpholinamide. In certain embodiments, a 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-sulfo 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-fluor-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-azaindole)-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-L-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, glycinamide, glycerol glycinate, ethylene glycol glycinate, oxetan-3-yl glycinate, and glycine morpholine amide; wherein X5 is S or a non-natural amino acid selected from the group consisting of: L-propargylglycine-PEG6-, L-sulfo 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 (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-azaindole)-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-L-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 glycinate, ethylene glycol glycinate, oxetan-3-yl glycinate, and glycine morpholine amide; and wherein X1, X2, and X4 are each an arbitrary amino acid or a non-natural amino acid selected from the group consisting of: L-propargylglycine-PEG6-, L-sulfo 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 (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-azaindole)-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-L-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, glycinamide, glycerol glycinate, ethylene glycol glycinate, oxetan-3-yl glycinate, and glycine morpholine amide, except that X1 is not G, X2 is not P, and / or X4 is not G. In certain embodiments, a non-naturally occurring cystine knot peptide (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, a non-naturally occurring cystine knot peptide (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, a 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, and X; X5 is S or X, wherein X is a non-natural amino acid selected from the group consisting of: L-propargylglycine-PEG6-, L-sulfo 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 (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-azaindole)-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-L-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, glycinamide, glycerol glycinate, ethylene glycol glycinate, oxetan-3-yl glycinate, and glycine morpholinamide. In certain embodiments, the non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A comprises L2, where 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, where 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-(naphthalen-1-ol)-L-alanine, D-serine, L-β-homoserine, L-β-alanine, N-α-methylglycine, glycinamide, glycerol glycinate, ethylene glycol glycinate, oxetan-3-yl glycinate, and glycine morpholine amide.,

[0180] In certain embodiments, the C-terminus of a non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A is modified (such as capped). In certain embodiments, the N-terminus of a 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 a non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A are modified (such as capped). In certain embodiments, the C-terminal carboxyl of a non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A is amidated. In certain embodiments, the N-terminal amine of a non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A is acetylated. In certain embodiments, the C-terminal carboxyl of a non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A is amidated and the N-terminal amine of a non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A is acetylated.

[0181] Also provided herein are non-naturally occurring cystine knot peptides (CKPs) that bind VEGF-A, wherein the CKP comprises a cystine scaffold structure provided below (i.e., scaffold structure I):

[0182] Z1C1L1C2L2C3L3C4L4C5L5C6Z2 (I)

[0183] Wherein:

[0184] Z1 and Z2 are any amino acids;

[0185] L1 is loop 1 and has a structure selected from the group consisting of: X1X2X3X4X5X6X7X8, X1X2X3X4X5X6X7X8X9, and X1X2X3X4X5X6X7X8X9X 10 , where each of X1-X 10 is any amino acid;

[0186] L2 is loop 2 and has the structure: X1X2X3X4X5, where each of X1–X5 is any amino acid;

[0187] L3 is loop 3 and has the structure: X1X2X3, where each of X1–X3 is any amino acid;

[0188] L4 is Loop 4 and has the structure: X1, where X1 is any amino acid;

[0189] L5 is Loop 5 and has the structure: X1X2X3X4X5, where each of X1–X5 is any amino acid; wherein,

[0190] 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 CKP has an altered disulfide bond connectivity; wherein the altered disulfide bond connectivity is C1-C4, C2-C3, and C5-C6; and wherein the CKP has an alpha helix 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 range between these values.

[0191] In certain embodiments, the non-naturally occurring CKP binds VEGF-A with an affinity of 500 pM or less.

[0192] In certain embodiments, the binding affinity of the non-naturally occurring CKP for VEGF-A is determined via, for example, surface plasmon resonance or other assays described in detail in the following examples.

[0193] 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.

[0194] In certain embodiments, the C-terminus of a non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A is modified (such as capped). In certain embodiments, the N-terminus of a 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 a non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A are modified (such as capped). In certain embodiments, the C-terminal carboxyl group of a non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A is amidated. In certain embodiments, the N-terminal amine of a non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A is acetylated. In certain embodiments, the C-terminal carboxyl group of a non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A is amidated and the N-terminal amine of a non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A is acetylated.

[0195] In certain embodiments, a 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, a non-naturally occurring CKP that binds VEGF-A comprises L5, and each of X1–X5 is any amino acid, except that X4 is not glycine (G). In certain embodiments, a non-naturally occurring CKP that binds VEGF-A has an IC 50 that inhibits VEGF-A activity. In certain embodiments, the degree of inhibition is determined via cell IC 50 assay as further described in detail in the following examples.

[0196] In certain embodiments, a non-naturally occurring CKP that binds VEGF-A comprises: L1 having the following amino acid sequence: HMMYDY (SEQ ID NO:231) or

[0197] K / P / Q / R-K / T / L / D-W / T / M / L-Q / R / D-W / F / P / V-W / K / F-Y / Q / W-M / Y / G / D (SEQ ID NO:115) or

[0198] E / G / P / Q / R / T / V-T / E / A / D-D / T / I / P-W / V / Q / T / W-Y / F / N / E / P / K-P / E / W / N / P-H / Q / K / W / H-Q / F / E / A / D / W-I / L / H-D / W / P / Y / T / M / N (SEQ ID NO:232), with reference to the above-mentioned 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 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 / H-P / M / W / Y-N / Y / W-G / D / T / Q / R / S-F / A / E / S (SEQ ID NO:20) or SWWPSL (SEQ ID NO:237).

[0199] 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 NO:8-14 and 225-230, with reference to the above-mentioned 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 comprises: L4 comprising V or F. 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 and 233-238). The amino acid sequences of SEQ ID NO 8-18, 225-230 and 233-238 are provided in Table 1 below.

[0200] Table 1

[0201]

[0202]

[0203] In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises any one of the amino acid sequences of SEQ ID NO:21-17 and 239-244. The amino acid sequences of SEQ ID NO:21-17 and 239-244 are provided in Table 2 below.

[0204] Table 2

[0205]

[0206] In certain embodiments, a non-naturally occurring VEGF-A-binding CKP comprises: L1 comprising the amino acid sequence Q / H / E / N / D-L / V / R / P / I-M / F / L-Q / E / R / L-P-F / A / L / S-W-G (SEQ ID NO: 358), with reference to scaffold structure I above. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP further comprises: L2 comprising the amino acid sequence KQDSD (SEQ ID NO: 93). In certain embodiments, the non-naturally occurring VEGF-A-binding CKP further comprises: L3 comprising the amino acid sequence LAG. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP further comprises: L4 comprising the amino acid V. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP further comprises: L5 comprising the amino acid sequence HWYQS (SEQ ID NO: 17).

[0207] In certain embodiments, a non-naturally occurring VEGF-A-binding CKP 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 VEGF-A-binding CKP further comprises: L2 comprising the amino acid sequence KQDSD (SEQ ID NO: 93). In certain embodiments, the non-naturally occurring VEGF-A-binding CKP further comprises: L3 comprising the amino acid sequence LAG. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP further comprises: L4 comprising the amino acid V. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP 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.

[0208] Table 3

[0209] 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)

[0210] In certain embodiments, a non-naturally occurring VEGF-A-binding CKP comprises any one of the amino acid sequences of SEQ ID NOs: 265-275. The amino acid sequences of SEQ ID NOs: 265-275 are provided in Table 4 below.

[0211] Table 4

[0212]

[0213]

[0214] In certain embodiments, the non-naturally occurring VEGF-A-binding CKP 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 VEGF-A-binding CKP further comprises: L2 comprising the amino acid sequence KQDSD (SEQ ID NO:93). In certain embodiments, the non-naturally occurring VEGF-A-binding CKP further comprises: L3 comprising the amino acid sequence LAG. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP further comprises: L4 comprising the amino acid V. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises: L5 comprising the amino acid sequence R / H-W-Y-N / Q / H-S (SEQ ID NO:359).

[0215] In certain embodiments, the non-naturally occurring VEGF-A-binding CKP further comprises: L1 comprising the amino acid sequence QLMQPFWG (SEQ ID NO:10), with reference to the above-described scaffold structure I. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP further comprises: L2 comprising the amino acid sequence KQDSD (SEQ ID NO:93). In certain embodiments, the non-naturally occurring VEGF-A-binding CKP further comprises: L3 comprising the amino acid sequence LAG. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP further comprises: L4 comprising the amino acid V. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP 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).

[0216] In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises an amino acid sequence selected from the group consisting of SEQ ID NO:23, 276, and 278. SEQ ID NOs 23, 276, and 278 are provided in Table 5 below.

[0217] Table 5

[0218] GCQLMQPFWGCKQDSDCLAGCVCRWYNSCG (SEQ ID NO:276) GCQLMQPFWGCKQDSDCLAGCVCHWYQSCG (SEQ ID NO:23) GCQLMQPFWGCKQDSDCLAGCVCRWYHSCG (SEQ ID NO:278)

[0219] In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises: comprising the amino acid sequence

[0220] L1 of Q / D / K / N / A / R / H-L / V / I / R / P / V / -M / L / S / T / F-Q / E / L / H-P-F / L / M / Y / S-W-G (SEQ ID NO:40), with reference to scaffold structure I. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP further comprises: L2 comprising the amino acid sequence KQDSD (SEQ ID NO:93). In certain embodiments, the non-naturally occurring VEGF-A-binding CKP further comprises: L3 comprising the amino acid sequence LAG. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP further comprises: L4 comprising the amino acid V. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP further comprises: comprising the amino acid sequence

[0221] L5 of H / L / R / Q / -W / F / Y-Y-Q / N / K / H / D / E-S (SEQ ID NO:360).

[0222] In certain embodiments, the non-naturally occurring VEGF-A-binding CKP 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 VEGF-A-binding CKP further comprises: L2 comprising the amino acid sequence KQDSD (SEQ ID NO:93). In certain embodiments, the non-naturally occurring VEGF-A-binding CKP further comprises: L3 comprising the amino acid sequence LAG. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP further comprises: L4 comprising the amino acid V. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP 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.

[0223] Table 6

[0224]

[0225]

[0226] 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.

[0227] Table 7

[0228]

[0229]

[0230] In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises: L1 comprising the amino acid sequence

[0231] Q / D / K / W / E / L / R-D / N-P / R / L / T-T / S / L / K-F / V / L / I-N / D-W-A / S / G-L / V / E / T / Q / D-F / Y (SEQ ID NO:70), with reference to scaffold structure I. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP further comprises: L2 comprising the amino acid sequence KQDSD (SEQ ID NO:93). In certain embodiments, the non-naturally occurring VEGF-A-binding CKP further comprises: L3 comprising the amino acid sequence LAG. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP further comprises: L4 comprising the amino acid V. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP further comprises: comprising the amino acid sequence

[0232] Q / R-M / L / F / H-Y / F / H-D / Q / N / K-S / T (SEQ ID NO:80) of L5.

[0233] In certain embodiments, the non-naturally occurring VEGF-A-binding CKP 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 VEGF-A-binding CKP further comprises: L2 comprising the amino acid sequence KQDSD (SEQ ID NO:93). In certain embodiments, the non-naturally occurring VEGF-A-binding CKP further comprises: L3 comprising the amino acid sequence LAG. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP further comprises: L4 comprising the amino acid V. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP 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.

[0234] Table 8

[0235]

[0236] 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.

[0237] Table 9

[0238]

[0239]

[0240] In certain embodiments, the non-naturally occurring VEGF-A-binding CKP 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 VEGF-A-binding CKP further comprises: L2 comprising the amino acid sequence

[0241] K / G / Q / S / N-Q / L / P / A / V / T / R / W / K / G / Y-D / S / E / N-S / F / Y / L / F / Q / M-D / E / N / A / L / F / H / Q (SEQ ID NO: 98). In certain embodiments, the non-naturally occurring VEGF-A-binding CKP further comprises: L3 comprising the amino acid sequence LAG. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP further comprises: L4 comprising the amino acid V. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP further comprises: L5 comprising the amino acid sequence QYYQS (SEQ ID NO: 45).

[0242] In certain embodiments, the non-naturally occurring VEGF-A-binding CKP 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 VEGF-A-binding CKP 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 VEGF-A-binding CKP further comprises: L3 comprising the amino acid sequence LAG. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP further comprises: L4 comprising the amino acid V. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP 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.

[0243] Table 10

[0244]

[0245]

[0246] 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.

[0247] Table 11

[0248] 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)

[0249] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises: L1 comprising the amino acid sequence N-I-M / L-L / S / T / Q / N / E / D-P-F / Y / S-WG (SEQ ID NO: 454), with reference to scaffold structure I. In certain embodiments, the non-naturally occurring VEGF-A binding CKP 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 VEGF-A binding CKP further comprises: L2 comprising the amino acid sequence GRDFE (SEQ ID NO: 97). In certain embodiments, the non-naturally occurring VEGF-A binding CKP further comprises: L3 comprising the amino acid sequence L / V / M / F-A / Q / E / S / N / Y / I / T-G / Q / R / D / T / N / E. In certain embodiments, the non-naturally occurring VEGF-A binding CKP further comprises: L4 comprising the 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).

[0250] In certain embodiments, the non-naturally occurring VEGF-A binding CKP 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 VEGF-A binding CKP 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 VEGF-A binding CKP further comprises: an L2 comprising the amino acid sequence GRDFE (SEQ ID NO:97). In certain embodiments, the non-naturally occurring VEGF-A binding CKP further comprises: an 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 VEGF-A binding CKP further comprises: an L4 comprising the amino acid I. In certain embodiments, the non-naturally occurring VEGF-A binding CKP further comprises: an L5 comprising the amino acid sequence QYYQS (SEQ ID NO:45).

[0251] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises an amino acid sequence selected from the group consisting of SEQ ID NO:103 - 114. The amino acid sequences of SEQ ID NO:103 - 114 are provided in Table 12 below.

[0252] Table 12

[0253]

[0254]

[0255] In certain embodiments, the non-naturally occurring VEGF-A binding CKP 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 VEGF-A binding CKP further comprises: L2 comprising the amino acid sequence K / G / D / A / E-Q / E / R / V / P / D / M / G / N / L / A / F-D / N / Y / S-S / F / L / I / M / Y / V / N / E-D / L / Q / S / E / T / L / A / N (SEQ ID NO:121). In certain embodiments, the non-naturally occurring VEGF-A binding CKP further comprises: L3 comprising the amino acid sequence LAG. In certain embodiments, the non-naturally occurring VEGF-A binding CKP further comprises: L4 comprising the amino acid V. In certain embodiments, the non-naturally occurring VEGF-A binding CKP further comprises: L5 comprising the amino acid sequence HWYNS (SEQ ID NO:46).

[0256] In certain embodiments, the non-naturally occurring VEGF-A binding CKP 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 VEGF-A binding CKP 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 VEGF-A binding CKP further comprises: L3 comprising the amino acid sequence LAG. In certain embodiments, the non-naturally occurring VEGF-A binding CKP further comprises: L4 comprising the amino acid V. In certain embodiments, the non-naturally occurring VEGF-A binding CKP 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.

[0257] Table 13

[0258]

[0259] 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.

[0260] Table 14

[0261] 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)

[0262] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises: L1 comprising the amino acid sequence D-V-M / L-Q / K / D-P-Y / M / T / L-W-G (SEQ ID NO: 130), with reference to scaffold structure I. In certain embodiments, the non-naturally occurring VEGF-A binding CKP further comprises:

[0263] L2 comprising the amino acid sequence KQDSD (SEQ ID NO: 93). In certain embodiments, the non-naturally occurring VEGF-A binding CKP further comprises: L3 comprising the amino acid sequence LAG. In certain embodiments, the non-naturally occurring VEGF-A binding CKP further comprises: L4 comprising the amino acid V. In certain embodiments, the non-naturally occurring VEGF-A binding CKP further comprises: L5 comprising the amino acid sequence H / L / Q / R-W-Y-N-S (SEQ ID NO: 134).

[0264] In certain embodiments, the non-naturally occurring VEGF-A binding CKP 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 VEGF-A binding CKP further comprises: L2 comprising the amino acid sequence KQDSD (SEQ ID NO: 93). In certain embodiments, the non-naturally occurring VEGF-A binding CKP further comprises: L3 comprising the amino acid sequence LAG. In certain embodiments, the non-naturally occurring VEGF-A binding CKP further comprises: L4 comprising the amino acid V. In certain embodiments, the non-naturally occurring VEGF-A binding CKP 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:

[0265] Table 15

[0266] 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)

[0267] 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: 135 - 137. The amino acid sequences of SEQ ID NOs: 135 - 127 are provided in Table 16 below:

[0268] Table 16

[0269] GCDVMKPMWGCKQDSDCLAGCVCQWYNSCG (SEQ ID NO:135) GCDVLDPTWGCKQDSDCLAGCVCLWYNSCG (SEQ ID NO:136) GCDVLQPLWGCKQDSDCLAGCVCRWYNSCG (SEQ ID NO:137)

[0270] In certain embodiments, the non-naturally occurring VEGF-A-binding CKP 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 VEGF-A-binding CKP further comprises: L2 comprising the amino acid sequence GPDID (SEQ ID NO:118). In certain embodiments, the non-naturally occurring VEGF-A-binding CKP further comprises: L3 comprising the amino acid sequence L / F-A / V / S-G / R / N. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP further comprises: L4 comprising an amino acid selected from V, I, and L. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP further comprises: L5 comprising the amino acid sequence HWYNS (SEQ ID NO:46).

[0271] In certain embodiments, the non-naturally occurring VEGF-A-binding CKP 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 VEGF-A-binding CKP further comprises: L2 comprising the amino acid sequence GPDID (SEQ ID NO:118). In certain embodiments, the non-naturally occurring VEGF-A-binding CKP further comprises: L3 comprising the amino acid sequences FVR and LSN. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP further comprises: L4 comprising an amino acid selected from V, I, and L. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP further comprises: L5 comprising the amino acid sequence HWYNS (SEQ ID NO:46).

[0272] In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence GCDVMQPYWGCGPDIDCFVRCLCHWYNSCG (SEQ ID NO:139). In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence GCDVMQPYWGCGPDIDCLSNCICHWYNSCG (SEQ ID NO:140).

[0273] In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises L1, L2, L3, L4, and / or L5 of any of the non-naturally occurring VEGF-A-binding CKPs disclosed herein. Thus, in certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises: an 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 VEGF-A-binding CKP further comprises: an 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 VEGF-A-binding CKP further comprises: an 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 VEGF-A-binding CKP further comprises: an L4 comprising the amino acid V, F, I, or L. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP further comprises: an L5 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 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 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 modified (such as capped). In certain embodiments, the C-terminal carboxyl 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 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.

[0274] In certain embodiments, at least one amino acid is deleted from the VEGF-A binding CKP provided herein. 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 both 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 VEGF-A binding CKP comprises 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, both the C- and N-termini of the non-naturally occurring cystine knot peptide (CKP) that binds 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 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 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.

[0275] In certain embodiments, the non-naturally occurring VEGF-A binding CKP 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 both the N-terminus and the C-terminus.

[0276] In certain embodiments, two amino acids are added to the N-terminus of the non-naturally occurring VEGF-A binding CKP provided herein. In certain embodiments, two amino acids are added to the N-terminus of the non-naturally occurring VEGF-A binding CKP, the CKP being

[0277] as 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 / L-H / 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 VEGF-A-binding CKP comprises

[0278] the amino acid sequence shown in GCNIMLPFWGCGRDFECLAGCVCQYYQSCGFH (SEQ ID NO:379). In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence shown in GCNIMLPFWGCGRDFECLAGCVCQYYQSCGIA (SEQ ID NO:380). In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence shown in GCNIMLPFWGCGRDFECLAGCVCQYYQSCGGS (SEQ ID NO:381). In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence shown in GCNIMLPFWGCGRDFECLAGCVCQYYQSCGTR (SEQ ID NO:382). In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence shown in GCNIMLPFWGCGRDFECLAGCVCQYYQSCGVH (SEQ ID NO:383). In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises

[0279] the amino acid sequence shown in GCNIMLPFWGCGRDFECLAGCVCQYYQSCGLS (SEQ ID NO:384).

[0280] In certain embodiments, two amino acids are added to the N-terminus of the non-naturally occurring VEGF-A-binding CKP, which is

[0281] as 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 / S-T / 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 VEGF-A-binding CKP comprises the amino acid sequence shown in GCDVLQPYWGCGPDIDCLSNCICHWYNSCGRT (SEQ ID NO:387). In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence shown in GCDVLQPYWGCGPDIDCLSNCICHWYNSCGWK (SEQ ID NO:388). In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises

[0282] the amino acid sequence shown in GCDVLQPYWGCGPDIDCLSNCICHWYNSCGPL (SEQ ID NO:389). In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence shown in GCDVLQPYWGCGPDIDCLSNCICHWYNSCGDE (SEQ ID NO:390). In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence shown in GCDVLQPYWGCGPDIDCLSNCICHWYNSCGQF (SEQ ID NO:391). In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence shown in GCDVLQPYWGCGPDIDCLSNCICHWYNSCGEQ (SEQ ID NO:392). In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence shown in GCDVLQPYWGCGPDIDCLSNCICHWYNSCGPT (SEQ ID NO:393). In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises

[0283] 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 shown in GCDVLQPYWGCGPDIDCLSNCICHWYNSCGSL (SEQ ID NO:395).

[0284] In certain embodiments, three amino acids are added to the N-terminus of the non-naturally occurring VEGF-A binding CKP provided herein. In certain embodiments, two amino acids are added to the N-terminus of the non-naturally occurring VEGF-A binding CKP, which CKP is

[0285] as 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 / W-L / Y / F / H / D / P-I / 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 VEGF-A binding CKP comprises

[0286] the amino acid sequence shown in GCNIMLPFWGCGRDFECLAGCVCQYYQSCGPLI (SEQ ID NO:369). In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence shown in GCNIMLPFWGCGRDFECLAGCVCQYYQSCGNYQ (SEQ ID NO:370). In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence shown in GCNIMLPFWGCGRDFECLAGCVCQYYQSCGPLQ (SEQ ID NO:371). In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence shown in GCNIMLPFWGCGRDFECLAGCVCQYYQSCGTFQ (SEQ ID NO:372). In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises

[0287] The amino acid sequence shown in GCNIMLPFWGCGRDFECLAGCVCQYYQSCGDLV (SEQ ID NO:373). In certain embodiments, the non-naturally occurring VEGF-A-binding CKP 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 shown in GCNIMLPFWGCGRDFECLAGCVCQYYQSCGYLS (SEQ ID NO:375). In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises

[0288] The amino acid sequence shown in GCNIMLPFWGCGRDFECLAGCVCQYYQSCGWDY (SEQ ID NO:376). In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence shown in GCNIMLPFWGCGRDFECLAGCVCQYYQSCGWPH (SEQ ID NO:377). In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence shown in GCNIMLPFWGCGRDFECLAGCVCQYYQSCGPHQ (SEQ ID NO:378).

[0289] 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, both the C- and N-termini of the non-naturally occurring cystine knot peptide (CKP) that binds 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 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 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.

[0290] In certain embodiments, the non-naturally occurring VEGF-A-binding CKP is a variant of the non-naturally occurring VEGF-A-binding CKPs described herein. In certain embodiments, such variant comprises 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:93-97, 117-120, 211, 298-309, and 322-339; the 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 substitutions are conservative amino acid substitutions. In certain embodiments, the amino acid substitutions do not substantially reduce the ability of the non-naturally occurring VEGF-A-binding CKP to bind human VEGF-A. For example, conservative changes (e.g., conservative substitutions as provided herein) can be made that do not substantially reduce the VEGF-A binding affinity. Using the methods described in the following examples, the binding affinity of variants of the non-naturally occurring VEGF-A-binding CKP can be evaluated.

[0291] Conservative substitutions are shown in Table 17 below 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 the non-naturally occurring VEGF-A-binding CKP and the products screened for the desired activity (e.g., retained / improved VEGF-A binding).

[0292] Table 17: Conservative Substitutions

[0293]

[0294]

[0295] Non-conservative substitutions would require exchanging a member of one of these classes for a member of another class.

[0296] Exemplary substitution variants are affinity matured non-naturally occurring CKP that bind VEGF-A and can be readily generated, for example, using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more residues in L1, L2, L3, L4, and / or L5 are altered (i.e., added, deleted, or substituted), and the VEGF-A-binding CKP of the variant is displayed on a phage and screened for VEGF-A 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, such as error-prone PCR, loop shuffling, or oligonucleotide-directed mutagenesis. A secondary library is then established. The library is then screened to identify any non-naturally occurring CKP variants that have the desired affinity for VEGF-A. In certain embodiments, introducing diversity involves a loop-directed protocol in which 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 the target ligand can be identified, for example, using alanine-scanning mutagenesis or modeling.

[0297] In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIMLPYWGCGQSFECLAGCVCQYYQSCG (SEQ ID NO:215).

[0298] In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIMLPYWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO:216).

[0299] In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIMLPYWGCGRDFECLAKCVCQYYQSCG (SEQ ID NO:542).

[0300] In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIMLPYWGCGRDFECMSDCICQYYQSCG (SEQ ID NO:363).

[0301] In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence GCNIMLPYWGCGRDFECMSDCICQYYQSCG (SEQ ID NO:364).

[0302] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNIMLPYWGCGRDFECMNQCICQYYQSCG (SEQ ID NO:222).

[0303] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNIMLPYWGCGRDFECFYECICQYYQSCG (SEQ ID NO:223).

[0304] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNIMLPYWGCGRDFECMEQCICQYYQSCG (SEQ ID NO:142).

[0305] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNILLPFWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO:405).

[0306] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNILLPYWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO:406).

[0307] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNIMSPFWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO:407).

[0308] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNIMTPFWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO:408).

[0309] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNIMQPFWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO:409).

[0310] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNIMNPFWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO:410).

[0311] In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence GCNIMEPFWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 411).

[0312] In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence GCNIMDPFWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 412).

[0313] In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence GCNIMLPSWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 415).

[0314] In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence GCNIMLPFWGCGRDFECLSGCVCQYYQSCG (SEQ ID NO: 421).

[0315] In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence GCNIMLPFWGCGRDFECLTGCVCQYYQSCG (SEQ ID NO: 422).

[0316] In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence GCNIMLPFWGCGRDFECLEGCVCQYYQSCG (SEQ ID NO: 423).

[0317] In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence GCNIMLPYWGCGRDFECLAGCLCQYYQSCG (SEQ ID NO: 424).

[0318] In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence GCNIMLPYWGCGRDFECLAGCICQYYQSCG (SEQ ID NO: 425).

[0319] In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence GCNIMLPYWGCGRDFECLAGCVCQYYQSCS (SEQ ID NO: 431).

[0320] In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence GCNILLPYWGCGRDFECMEQCICQYYQSCG (SEQ ID NO:435).

[0321] In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence GCDVLQPYWGCGPDIDCLSNCICHWYNSCG (SEQ ID NO:386).

[0322] In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence GCNILLPFWGCGRDFECVSQCICQYYQSCG (SEQ ID NO:547).

[0323] In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence GCNILQPFWGCGRDFECVSQCICQYYQSCG (SEQ ID NO:548).

[0324] In certain embodiments, one or more amino acids in the sequence of the non-naturally occurring VEGF-A-binding CKP provided herein are replaced with non-natural amino acids. In certain embodiments, the one or more amino acids are replaced with the same non-natural amino acid. In certain embodiments, the one or more amino acids are each replaced with a different non-natural amino acid. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises a non-natural amino acid at any amino acid position in L1, L2, L3, L4, and / or L5, with reference to scaffold structure I.

[0325] 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 C(=O)-oxetan-3-yl.

[0326] 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.

[0327] 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.

[0328] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNIMLPXWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO:362), where X is sulfotyrosine. In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNIMLPXWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO:362), where X is 3,4-difluoro-L-phenylalanine. In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNIMLPXWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO:362), where X is 3,4-dichloro-L-phenylalanine. In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNIMLPXWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO:362), where X is 4-chloro-L-phenylalanine. In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNIMLPXWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO:362), where X is 3-F,4-Cl-L-phenylalanine. In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNIMLPXWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO:362), where X is 2-pyridone (NH para)-L-alanine. In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNIMLPXWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO:362), where X is pyridone (NH meta)-L-alanine.

[0329] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNIXLPFWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO:218), where X is norleucine.

[0330] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNIMLPFXGCGRDFECLAGCVCQYYQSCG (SEQ ID NO:219), where X is 1-naphthylalanine.

[0331] 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.

[0332] 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.

[0333] 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.

[0334] 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.

[0335] 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.

[0336] 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 VEGF-A binding CKP comprises the amino acid sequence

[0337] GCNIXLPFWGCGRDFECVSKCICQYYQSCG (SEQ ID NO:545), wherein X is norleucine. In certain embodiments, the non-naturally occurring VEGF-A binding CKP 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 VEGF-A binding CKP comprises the amino acid sequence

[0338] GCNIX1LPFWGCGRDF-N / K / X2-CVS-D / E-CICQYYQSCG (SEQ ID NO:541), where X1 is norleucine and X2 is ornithine.

[0339] In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence GCNIXLPFWGCGRDFKCVS-D / E-CICQYYQSCG (SEQ ID NO:561), where X is norleucine. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence CNIXLPFWGCGRDFKCVSDCICQYYQSCG (SEQ ID NO:562), where X is norleucine. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence

[0340] CNIXLPFWGCGRDFKCVSECICQYYQSCG (SEQ ID NO:563), where X is norleucine. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP 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 VEGF-A-binding CKP comprises the amino acid sequence

[0341] GCNIX1LPFWGCGRDFX2CVSDCICQYYQSCG (SEQ ID NO:565), where X1 is norleucine and X2 is ornithine. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence

[0342] GCNIX1LPFWGCGRDFX2CVSECICQYYQSCG (SEQ ID NO:566), where X1 is norleucine and X2 is ornithine.

[0343] In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence GCNIXLPYWGCGRDFECMEQCICQYYQSCG (SEQ ID NO:146), where X is norleucine.

[0344] In certain embodiments, the non-naturally occurring VEGF-A binding CKP 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 VEGF-A binding CKP comprises the amino acid sequence

[0345] GCNIXLPYWGCGRDFECMEKCICQYYQSCG (SEQ ID NO:543), wherein X is norleucine. In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNIX1LPYWGCGRDFECMEX2CICQYYQSCG (SEQ ID NO:544), wherein X1 is norleucine and X2 is ornithine.

[0346] In certain embodiments, the non-naturally occurring VEGF-A binding CKP 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 VEGF-A binding CKP comprises the amino acid sequence

[0347] GCNIXLPYWGCGRDFKCME-D / E-CICQYYQSCG (SEQ ID NO:555), wherein X is norleucine. In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNIXLPYWGCGRDFKCMEDCICQYYQSCG (SEQ ID NO:556), wherein X is norleucine. In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence

[0348] GCNIXLPYWGCGRDFKCMEECICQYYQSCG (SEQ ID NO:557), wherein X is norleucine. In certain embodiments, the non-naturally occurring VEGF-A binding CKP 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 VEGF-A binding CKP comprises the amino acid sequence

[0349] GCNIX1LPYWGCGRDFX2CMEDCICQYYQSCG (SEQ ID NO:559), 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

[0350] GCNIX1LPYWGCGRDFX2CMEECICQYYQSCG (SEQ ID NO:560), where X1 is norleucine and X2 is ornithine.

[0351] 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), 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

[0352] GCDVXQPYWGCGPDIDCLSKCICHWYNSCG (SEQ ID NO:536), where 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), 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

[0353] GCDVX1QPYWGCGPDI-N / K / X2-CLS-D / E-CICHWYNSCG (SEQ ID NO:535), 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

[0354] GCDVXQPYWGCGPDIDCLSNCICHWYNSCG (SEQ ID NO:224), where 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), where X is norleucine. In certain embodiments, the non-naturally occurring CKP that binds VEGF-A comprises the amino acid sequence

[0355] GCDVXQPYWGCGPDIKCLSDCICHWYNSCG (SEQ ID NO:550), where X is norleucine. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence GCDVXQPYWGCGPDIKCLSECICHWYNSCG (SEQ ID NO:551), where X is norleucine. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence

[0356] GCDVX1QPYWGCGPDIX2CLS-D / E-CICHWYNSCG (SEQ ID NO:552), where X1 is norleucine and X2 is ornithine. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence

[0357] GCDVX1QPYWGCGPDIX2CLSDCICHWYNSCG (SEQ ID NO:553), where X1 is norleucine and X2 is ornithine. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence

[0358] GCDVX1QPYWGCGPDIX2CLSECICHWYNSCG (SEQ ID NO:554), where X1 is norleucine and X2 is ornithine.

[0359] In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence GCNIMLXFWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO:413), where X is γ-benzyl-L-proline. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence GCNIMLXFWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO:413), where X is γ-(4-fluoro-benzyl)-L-proline. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence

[0360] GCNIMLXFWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO:413), where X is 4-OH-L-proline. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence GCNIMLXFWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO:413), where X is 4-fluoro-L-proline. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence

[0361] GCNIMLXFWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO:413), wherein X is 4-[4-(trifluoromethyl)benzyl]-L-proline.

[0362] In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence GCNIMLPYXGCGRDFECLAGCVCQYYQSCG (SEQ ID NO:417), wherein X is N-methylindole. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence GCNIMLPYXGCGRDFECLAGCVCQYYQSCG (SEQ ID NO:417), wherein X is N-ethylindole. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence

[0363] GCNIMLPYXGCGRDFECLAGCVCQYYQSCG (SEQ ID NO:417), wherein X is N-isopropylindole. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence GCNIMLPYXGCGRDFECLAGCVCQYYQSCG (SEQ ID NO:417), wherein X is 5-aza-indole.

[0364] In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence GCNIMLPYWGCGRDXECLAGCVCQYYQSCG (SED ID NO:419), wherein X is 4-methyl-L-phenylalanine. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence

[0365] GCNIMLPYWGCGRDXECLAGCVCQYYQSCG (SED ID NO:419), wherein X is 2-naphthyl-L-alanine. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence GCNIMLPYWGCGRDXECLAGCVCQYYQSCG (SED ID NO:419), wherein X is 2-quinolyl-alanine. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence

[0366] GCNIMLPYWGCGRDXECLAGCVCQYYQSCG (SED ID NO:419), wherein X is 4,4'-biphenyl-L-alanine. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence GCNIMLPYWGCGRDXECLAGCVCQYYQSCG (SED ID NO:419), wherein X is 3-(3-quinolyl)-L-alanine. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence

[0367] GCNIMLPYWGCGRDXECLAGCVCQYYQSCG (SED ID NO:419), wherein X is 3-(2-quinolyl)-L-alanine. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP 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 VEGF-A-binding CKP 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 VEGF-A-binding CKP comprises the amino acid sequence

[0368] GCNIMLPYWGCGRDXECLAGCVCQYYQSCG (SED ID NO:419), wherein X is 4-ethyl-2-pyridyl-L-alanine. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence

[0369] GCNIMLPYWGCGRDXECLAGCVCQYYQSCG (SED ID NO:419), wherein X is benzothiazole-L-alanine. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence GCNIMLPYWGCGRDXECLAGCVCQYYQSCG (SED ID NO:419), wherein X is benzothiophene-L-alanine. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence

[0370] GCNIMLPYWGCGRDXECLAGCVCQYYQSCG (SED ID NO:419), wherein X is 3-(3-isoquinolyl)-L-alanine.

[0371] In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence GCNIMLPYWGCGRDFECXAGCVCQYYQSCG (SEQ ID NO: 420), where X is tert-butyl-L-alanine (also known as L-neopentylglycine). In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence

[0372] GCNIMLPYWGCGRDFECXAGCVCQYYQSCG (SEQ ID NO: 420), where X is 3-cyclobutyl-L-alanine. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence GCNIMLPYWGCGRDFECXAGCVCQYYQSCG (SEQ ID NO: 420), where X is 3-cyclopentyl-L-alanine. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence

[0373] GCNIMLPYWGCGRDFECXAGCVCQYYQSCG (SEQ ID NO: 420), where X is 5,5,5-trifluoro-L-leucine.

[0374] In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence GCNIMLPYWGCGRDFECLAGCXCQYYQSCG (SEQ ID NO: 426), where X is L-tert-leucine. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence GCNIMLPYWGCGRDFECLAGCXCQYYQSCG (SEQ ID NO: 426), where X is tert-butyl-L-alanine (also known as L-neopentylglycine). In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence GCNIMLPYWGCGRDFECLAGCXCQYYQSCG (SEQ ID NO: 426), where X is L-cyclopentylglycine. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence GCNIMLPYWGCGRDFECLAGCXCQYYQSCG (SEQ ID NO: 426), where X is 3-cyclopentyl-L-alanine. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence

[0375] GCNIMLPYWGCGRDFECLAGCXCQYYQSCG (SEQ ID NO:426), where X is L-cyclobutyl-L-glycine. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence GCNIMLPYWGCGRDFECLAGCXCQYYQSCG (SEQ ID NO:426), where X is 3-cyclobutyl-L-alanine. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence

[0376] GCNIMLPYWGCGRDFECLAGCXCQYYQSCG (SEQ ID NO:426), where X is 5,5,5-trifluoro-L-leucine.

[0377] In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence GCNIMLPYWGCGRDFECLAGCVCQXYQSCG (SEQ ID NO:428), where X is 2-pyridone. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence GCNIMLPYWGCGRDFECLAGCVCQXYQSCG (SEQ ID NO:428), where X is 3,4-hydroxy-L-phenylalanine. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence

[0378] GCNIMLPYWGCGRDFECLAGCVCQXYQSCG (SEQ ID NO:428), where X is 3,4-fluorophenylalanine. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence GCNIMLPYWGCGRDFECLAGCVCQXYQSCG (SEQ ID NO:428), where X is 3-fluoro,4-OH-L-phenylalanine.

[0379] In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence GCNIMLPYWGCGRDFECLAGCVCQYXQSCG (SEQ ID NO:430), where X is 2-chloro-L-tyrosine. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence GCNIMLPYWGCGRDFECLAGCVCQYXQSCG (SEQ ID NO:430), where X is 2-methyl-L-tyrosine. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence

[0380] GCNIMLPYWGCGRDFECLAGCVCQYXQSCG (SEQ ID NO:430), wherein X is 2-ethyl-L-tyrosine or 4-(naphthalen-1-yl)-L-alanine.

[0381] 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 with its carboxyl terminus converted to glycerol ester, glycine with its carboxyl terminus converted to ethylene glycol ester, glycine with its carboxyl terminus converted to oxetane ester, or glycine morpholine amide.

[0382] 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.

[0383] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCNIX1LPYWGCGRDFECX2EQCICQYYQSCG (SEQ ID NO:437). In certain embodiments, X1 and X2 are the same non-natural amino acid. 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.

[0384] 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.

[0385] In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence GCNIX1LPYWGCGRDFECX2EQCX3CQYYQSCG (SEQ ID NO: 439). In certain embodiments, X1, X2, and / or X3 are the same non-natural amino acid. In certain embodiments, X1, X2, and / or X3 are not the same non-natural amino acid. 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.

[0386] In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence CNIX1QPYWGCGRDFECX2EQCX3CQYYQSCG (SEQ ID NO: 440). In certain embodiments, X1, X2, and / or X3 are the same non-natural amino acid. In certain embodiments, X1, X2, and / or X3 are not the same non-natural amino acid. 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.

[0387] In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence GCNILLPYWGCGRDFECXEQCICQYYQSCG (SEQ ID NO: 441), where X is 3-cyclobutyl-L-alanine or tert-butyl-L-alanine (also known as L-neopentylglycine).

[0388] In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence GCNILLPYWGCGRDFECMEQCXCQYYQSCG (SEQ ID NO: 442), where X is cyclobutyl-L-glycine or 3-cyclobutyl-L-alanine.

[0389] In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence X1CNIX2LPYWGCGRDFECMEQCICQYYQSCX3 (SEQ ID NO: 443). In certain embodiments, X1, X2, and / or X3 are the same non-natural amino acid. In certain embodiments, X1, X2, and / or X3 are not the same non-natural amino acid. In certain embodiments, X1 is N-acetylglycine, X2 is norleucine, and X3 is glycylamide.

[0390] In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence X1CNILLPYWGCGRDFECMEQCICQYYQSCX2 (SEQ ID NO: 444). In certain embodiments, X1 and X2 are the same non-natural amino acid. In certain embodiments, X1 and X2 are different non-natural amino acid. In certain embodiments, X1 is N-acetylglycine and X2 is glycylamide.

[0391] In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence X1CNILQPYWGCGRDFECMEQCICQYYQSCX2 (SEQ ID NO: 445). In certain embodiments, X1 and X2 are the same non-natural amino acid. In certain embodiments, X1 and X2 are different non-natural amino acid. In certain embodiments, X1 is N-acetylglycine and X2 is glycylamide.

[0392] In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence X1CNILQPYWGCGRDFECLEQCICQYYQSCX2 (SEQ ID NO: 446). In certain embodiments, X1 and X2 are the same non-natural amino acid. In certain embodiments, X1 and X2 are different non-natural amino acid. In certain embodiments, X1 is N-acetylglycine and X2 is glycylamide.

[0393] In certain embodiments, the non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence GCDVLQPYWGCGPDIDCXSNCICHWYNSCG (SEQ ID NO: 447), where X is 3-cyclobutyl-L-alanine or tert-butyl-L-alanine (L-neopentylglycine).

[0394] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCDVLQPYWGCGPDIDCLSNCXCHWYNSCG (SEQ ID NO:448), where X is cyclobutyl-L-glycine.

[0395] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCDVX1QPYWGCGPDIDCX2SNC2X3CHWYNSCG (SEQ ID NO:449). In certain embodiments, X1, X2, and / or X3 are the same non-natural amino acid. In certain embodiments, X1, X2, and / or X3 are not the same non-natural amino acid. 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.

[0396] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence GCDVLQPYWGCGPDIDCX1SNC2X2CHWYNSCG (SEQ ID NO:450). In certain embodiments, X1 and X2 are the same non-natural amino acid. In certain embodiments, X1 and X2 are different non-natural amino acid. 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.

[0397] In certain embodiments, the non-naturally occurring VEGF-A binding CKP 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 acid. In certain embodiments, X1, X2, X3, and / or X4 are not the same non-natural amino acid. In certain embodiments, X1 is N-acetylglycine, X2 is 3-cyclobutyl-L-alanine, X3 is cyclobutyl-L-glycine, and X4 is glycine amide. In certain embodiments, X1 is acetylglycine, X2 is cyclobutyl-L-alanine, X3 is cyclobutyl-L-alanine, and X4 is glycine amide.

[0398] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence X1CDVX2QPYWGCGPDIDCLSNCICHWYNSCX3 (SEQ ID NO: 452). In certain embodiments, X1, X2, and / or X3 are the same non-natural amino acid. In certain embodiments, X1, X2, and / or X3 are not the same non-natural amino acid. In certain embodiments, X1 is N-acetylglycine, X2 is norleucine, and X3 is glycylamide.

[0399] In certain embodiments, the non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence X1CDVLQPYWGCGPDIDCLSNCICHWYNSCX2 (SEQ ID NO: 453). In certain embodiments, X1 and X2 are the same non-natural amino acid. In certain embodiments, X1 and X2 are different non-natural amino acids. In certain embodiments, X1 is N-acetylglycine and X2 is glycylamide.

[0400] 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 modified (such as capped). In certain embodiments, the C-terminal carboxyl 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 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.

[0401] Structural characteristics

[0402] In certain embodiments, the structure of the non-naturally occurring VEGF-A-binding CKP provided herein has disulfide linkages different from those of the WT EETI-II protein, i.e., different from the C1-C4, C2-C5, and C3-C6 disulfide bond patterns characteristic of WT EETI-II. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP provided herein has C1-C4, C2-C3, and C5-C6 disulfide linkages. Methods for determining the disulfide linkages of, for example, non-naturally occurring VEGF-A-binding CKPs include, for example, solving and analyzing the co-crystal structure of the non-naturally occurring VEGF-A-binding CKP complexed with VEGF-A, via mass spectrometry after partial reduction alkylation, or via mass spectrometry after proteolytic digestion, and performing structure calculations as described in Sampoli et al. (2000) Proteins Struct Funct Gen. 40, 168-174, etc.

[0403] In certain embodiments, the non-naturally occurring VEGF-A-binding CKP has an α-helix 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 range 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 VEGF-A-binding CKP form an α-helix. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP has C1-C4, C2-C3, and C5-C6 disulfide linkages and an α-helix 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 range between these values. In certain embodiments, the α-helix content of the non-naturally occurring VEGF-A-binding CKP is determined via mass spectrometry after partial reduction alkylation, or via mass spectrometry after proteolytic digestion, by solving and analyzing the co-crystal structure of the non-naturally occurring VEGF-A-binding CKP complexed with VEGF-A, by, for example, circular dichroism (CD), optical rotatory dispersion (ORD), nuclear magnetic resonance (NMR).

[0404] In certain embodiments, the non-naturally occurring VEGF-A-binding CKP provided herein competes with a second non-naturally occurring VEGF-A-binding CKP for binding to VEGF-A, wherein the second non-naturally occurring VEGF-A-binding CKP comprises: comprising the amino acid sequence

[0405] L1 of 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 the amino acid V, and L5 comprising the amino acid sequence QYYQS (SEQ ID NO:45), with reference to scaffold structure I. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP provided herein competes with a second non-naturally occurring VEGF-A-binding CKP for binding to VEGF-A, wherein the second non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence

[0406] GCNIMLPFWGCKQDSDCLAGCVCQYYQSCG (SEQ ID NO:52).

[0407] In certain embodiments, the non-naturally occurring VEGF-A-binding CKP provided herein binds 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 the amino acid V, and L5 comprising the amino acid sequence QYYQS (SEQ ID NO:45), with reference to scaffold structure I. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP provided herein binds 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).

[0408] In certain embodiments, the non-naturally occurring CKP that binds VEGF-A provided herein competes with a second non-naturally occurring CKP that binds VEGF-A, wherein the second 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 the amino acid V, and L5 comprising the amino acid sequence QYYQS (SEQ ID NO:45), with reference to scaffold structure I. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP provided herein competes with a second non-naturally occurring VEGF-A-binding CKP for binding to VEGF-A, wherein the second non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence GCNIMLPFWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO:102).

[0409] In certain embodiments, the non-naturally occurring VEGF-A-binding CKP provided herein binds 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 the amino acid V, and L5 comprising the amino acid sequence QYYQS (SEQ ID NO:45), with reference to scaffold structure I. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP provided herein binds 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).

[0410] In certain embodiments, the non-naturally occurring VEGF-A-binding CKP provided herein competes with a second non-naturally occurring VEGF-A-binding CKP for binding to VEGF-A, wherein the second non-naturally occurring VEGF-A-binding CKP comprises: comprising the amino acid sequence

[0411] L1 of 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 the amino acid V, and L5 comprising the amino acid sequence HWYNS (SEQ ID NO:46), with reference to scaffold structure I. In certain embodiments, the non-naturally occurring VEGF-A binding CKP provided herein competes with a second non-naturally occurring VEGF-A binding CKP for binding to VEGF-A, wherein the second non-naturally occurring VEGF-A binding CKP comprises the amino acid sequence

[0412] GCDVMQPYWGCKQDSDCLAGCVCHWYNSCG (SEQ ID NO:55).

[0413] In certain embodiments, the non-naturally occurring VEGF-A binding CKP provided herein binds 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 the amino acid V, and L5 comprising the amino acid sequence HWYNS (SEQ ID NO:46), with reference to scaffold structure I. In certain embodiments, the non-naturally occurring VEGF-A binding CKP provided herein binds 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).

[0414] In certain embodiments, the non-naturally occurring VEGF-A binding CKP protein competes with a second non-naturally occurring VEGF-A binding CKP for binding to VEGF-A, wherein the second 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 the amino acid V, and the amino acid sequence

[0415] L5 of HWYNS (SEQ ID NO:46), with reference to scaffold structure I. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP provided herein competes with a second non-naturally occurring VEGF-A-binding CKP for binding to VEGF-A, wherein the second non-naturally occurring VEGF-A-binding CKP comprises the amino acid sequence

[0416] GCDVMQPYWGCGPDIDCLAGCVCHWYNSCG (SEQ ID NO:123).

[0417] In certain embodiments, the non-naturally occurring VEGF-A-binding CKP provided herein binds 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 the amino acid V, and L5 comprising the amino acid sequence HWYNS (SEQ ID NO:46), with reference to scaffold structure I. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP provided herein binds 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).

[0418] In certain embodiments, the non-naturally occurring VEGF-A-binding CKP provided herein binds to an epitope of VEGF-A, the epitope comprising 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 VEGF-A-binding CKP provided herein binds to an epitope of VEGF-A, the epitope comprising K48, N62, and D63. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP provided herein binds to an epitope of VEGF-A, the epitope comprising H86. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP provided herein binds to an epitope of VEGF-A, the epitope comprising Y21, Y25, and P106. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP provided herein binds to an epitope of VEGF-A, the epitope comprising M81, D19, and Q22. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP provided herein binds to an epitope of VEGF-A, the epitope comprising F17, M81, and I91. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP provided herein binds 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, the epitope comprising Q22 and Y25.

[0419] In certain embodiments, the non-naturally occurring VEGF-A-binding CKP provided herein binds to an epitope on VEGF-A that overlaps with the epitope of VEGF-A 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 VEGF-A-binding CKP provided herein binds to an epitope on VEGF-A that overlaps with the epitope of VEGF-A bound by Flt-1. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP provided herein binds to an epitope on VEGF-A that overlaps with the epitope of VEGF-A bound by bevacizumab.

[0420] Functional characteristics

[0421] In certain embodiments, non-naturally occurring CKP that "specifically binds" VEGF-A, such as human VEGF-A, murine VEGF-A, and / or rat VEGF-A, has a binding affinity (Kd) value of no more than about 1×10 -7 M, preferably no more than about 1×10 -8 and most preferably no more than about 1×10 -9 M), but the binding affinity for a VEGF-A homolog or other growth factor is at least about 50-fold weaker, or at least about 500-fold weaker, or at least about 1000-fold weaker than its binding affinity for VEGF-A.

[0422] In certain embodiments, the non-naturally occurring CKP that binds VEGF-A provided herein binds to, for example, non-target proteins (e.g., VEGF-A homologs such as VEGF-B, VEGF-C, and VEGF-D) or other growth factors (such as PlGF, EGF, NGF, IGF, and PDGF) to an extent less than about 10% of the binding of the non-naturally occurring CKP that binds 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 relative 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, e.g., an excess of unlabeled target. In this case, if the binding of the labeled target to the probe is competitively inhibited by the excess unlabeled target, specific binding is indicated. Other methods for assessing the binding of non-naturally occurring CKP that "specifically binds" VEGF-A are described in the Examples.

[0423] As used herein, the term "specifically binds" to a particular polypeptide or an epitope on a particular polypeptide target, or "binds specifically with" or "is specific for" an epitope on a particular polypeptide or a particular polypeptide target can be demonstrated, 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 -12A Kd of M or greater. In one embodiment, the term "specifically binds" refers to a molecule where the molecule binds to a specific polypeptide or an epitope on a specific polypeptide and does not substantially bind to any other polypeptide or polypeptide epitope.

[0424] In certain embodiments, the non-naturally occurring CKP that binds VEGF-A binds VEGF-A with a Kd of from 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 from about 1 pM to about 50 pM, from about 50 pM to about 250 pM, from about 250 pM to about 500 pM, from about 500 pM to 750 pM, from about 750 pM to about 1 nM, from about 1 nM to about 25 nM, from about 25 nM to about 50 nM, 50 nM to about 100 nM, from about 100 nM to about 250 nM, or from about 250 nM to about 500 nM.

[0425] In certain embodiments, the non-naturally occurring CKP that binds VEGF-A binds human VEGF-A, murine VEGF-A, and / or rat VEGF-A. In certain embodiments, the non-naturally occurring CKP that binds human VEGF-A, murine 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 the amino acid V, and L5 comprising the amino acid sequence QYYQS (SEQ ID NO:45), with reference to scaffold structure I. In certain embodiments, the non-naturally occurring CKP that binds human VEGF-A, murine VEGF-A, and rat VEGF-A comprises the amino acid sequence GCNIMLPFWGCKQDSDCLAGCVCQYYQSCG (SEQ ID NO:52).

[0426] In certain embodiments, the non-naturally occurring CKP that binds VEGF-A binds human VEGF-A, murine VEGF-A, and rat VEGF-A, and comprises: comprising the amino acid sequence

[0427] NIMLPFWG (SEQ ID NO:33) of L1; L2 comprising the amino acid sequence GRDFE (SEQ ID NO:97); L3 comprising the amino acid sequence LAG; L4 comprising the amino acid V, and L5 comprising the amino acid sequence QYYQS (SEQ ID NO:45), with reference to scaffold structure I. In certain embodiments, the non-naturally occurring CKP that binds human VEGF-A, murine VEGF-A, and rat VEGF-A comprises the amino acid sequence

[0428] GCNIMLPFWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO:102).

[0429] In certain embodiments, a non-naturally occurring VEGF-A-binding CKP that binds human VEGF-A, murine 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 the amino acid V, and L5 comprising the amino acid sequence HWYNS (SEQ ID NO:46), with reference to scaffold structure I. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP that binds human VEGF-A, murine VEGF-A, and rat VEGF-A comprises the amino acid sequence

[0430] GCDVMQPYWGCKQDSDCLAGCVCHWYNSCG (SEQ ID NO:55).

[0431] In certain embodiments, a non-naturally occurring VEGF-A-binding CKP that binds human VEGF-A, murine 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 the amino acid V, and L5 comprising the amino acid sequence HWYNS (SEQ ID NO:46), with reference to scaffold structure I. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP that binds human VEGF-A, murine VEGF-A, and rat VEGF-A comprises the amino acid sequence

[0432] GCDVMQPYWGCGPDIDCLAGCVCHWYNSCG (SEQ ID NO:123).

[0433] In certain embodiments, the non-naturally occurring VEGF-A-binding CKP described herein has an IC 50 value 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, including any range between these values.

[0434] In certain embodiments, the non-naturally occurring VEGF-A-binding CKP does not inhibit trypsin protease activity, as in a peptide substrate cleavage assay (such as the peptide substrate cleavage assay described in the Examples).

[0435] In certain embodiments, the non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A is resistant to trypsin digestion. In certain embodiments, after incubation with trypsin at 37 °C for 24 h, 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.

[0436] Also contemplated are nucleic acid molecules encoding the non-naturally occurring CKP that binds VEGF-A described herein, expression vectors comprising the nucleic acid molecules encoding the non-naturally occurring CKP that binds VEGF-A, and cells comprising the nucleic acid molecules. Also provided herein are methods for producing the non-naturally occurring CKP that binds VEGF-A described herein by culturing the cells, expressing the non-naturally occurring CKP that binds VEGF-A, and recovering the non-naturally occurring CKP that binds VEGF-A from the cell culture.

[0437] In certain embodiments, the non-naturally occurring CKP that binds VEGF-A is produced via in vitro translation, as described elsewhere herein.

[0438] In certain embodiments, the non-naturally occurring CKP that binds VEGF-A is produced via chemical peptide synthesis, e.g., 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.

[0439] A non-naturally occurring cystine knot peptide (CKP) that binds to human low density lipoprotein receptor (LDL)-related protein 6 (LRP6)

[0440] 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 numbers: NM_002336 (mRNA) and NP_002327 (protein); UniProtKB: O75581) functions as a receptor, or functions together with Frizzled (a co-receptor for Wnt), and thereby transduces 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.

[0441] doi:10.1371 / journal.pone.0036565; Liu et al. (2010) Proc Natl Acad Sci USA 107:5136-5141).

[0442] Wnt signaling is involved in many biological pathways. In terms of diseases, it is associated with cancer and metastatic diseases, 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 utilities. Bone loss is a serious medical problem, not only in the course of postmenopausal osteoporosis, but also in rheumatoid arthritis. Bone degradation occurs in multiple myeloma and in bone metastases. Therefore, therapeutic strategies aimed at strengthening bone, preventing fractures or restoring damaged bone are of very high 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). Because of their role in inhibiting new bone formation, the Wnt pathway inhibitors DKK1 and SOST are regarded as 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).

[0443] Aberrant regulation of Wnt signaling is involved 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 extended 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). There is a particularly clear link between mutations in the protein adenomatous polyposis coli (APC), which prevent 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 result in the autosomal recessive disorder osteoporosis-pseudoglioma syndrome (OPPG), which is characterized by low bone mass, ocular defects, and a propensity to fracture (Gong et al. (2001) Cell 107:513-23).

[0444] Provided herein are non-naturally occurring CKP that bind to human low density lipoprotein receptor-related protein 6 (LRP6), wherein the non-naturally occurring CKP comprises the following cystine scaffold structure (i.e., scaffold structure I):

[0445] Z1C1L1C2L2C3L3C4L4C5L5C6Z2(I)

[0446] wherein:

[0447] Z1 and Z2 are any amino acids;

[0448] L1 is loop 1 and has a structure selected from the group consisting of: X1X2X3X4X5X6, X1X2X3X4X5X6X7, X1X2X3X4X5X6X7X8, X1X2X3X4X5X6X7X8X9, and X1X2X3X4X5X6X7X8X9X 10 , wherein each of X1-X 10 is any amino acid;

[0449] L2 is loop 2 and has the structure: X1X2X3X4X5, wherein each of X1–X5 is any amino acid;

[0450] L3 is Loop 3 and has the structure: X1X2X3, where each of X1–X3 is any amino acid;

[0451] L4 is Loop 4 and has the structure: X1, where X1 is any amino acid; and

[0452] L5 is Loop 5 and has the structure: X1X2X3X4X5, where each of X1–X5 is any amino acid.

[0453] In certain embodiments, Z1 and / or Z2 of a 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.

[0454] In certain embodiments, a non-naturally occurring CKP that binds LRP6 comprises L1, where X1 is an amino acid selected from R, V, M, A, G, N, S, and E; where X2 is an amino acid selected from T, N, S, G, R, and A; where X3 is an amino acid selected from N, R, H, V, K, S, G, I, and Y; where X4 is an amino acid selected from R, V, N, I, K, S, and T; where X5 is an amino acid selected from V, R, K, I, T, S, L, and N; and where X6 is an amino acid selected from K, G, A, I, R, N, S, and V. In certain embodiments, a non-naturally occurring CKP that binds LRP6 comprises L1, where X7 is an amino acid selected from G, R, K, E, P, and T. In certain embodiments, a non-naturally occurring CKP that binds LRP6 comprises L1, where X8 is an amino acid selected from G, R, K, Q, A, and S. In certain embodiments, a non-naturally occurring CKP that binds LRP6 comprises L1, where X9 is an amino acid selected from R or G. In certain embodiments, a non-naturally occurring CKP that binds LRP6 comprises L1, where X 10is an amino acid selected from E, W, and G. In certain embodiments, the non-naturally occurring CKP that binds 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 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 LRP6 comprises L3, wherein X1 is L, X2 is A, and X3 is G. In certain embodiments, the non-naturally occurring CKP that binds LRP6 comprises L4, wherein X1 is V.

[0455] 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.

[0456] Non-naturally occurring CKPs that bind to overlapping or similar regions on a target can be identified by competitive inhibition / binding assays. Such assays are well known in the art and are described, for example, in S.J. 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.

[0457] In certain embodiments, the non-naturally occurring CKP that binds LRP6 binds the same human LRP6 epitope bound by a second non-naturally occurring CKP that binds LRP6, the second non-naturally occurring CKP that binds LRP6 comprising: comprising the amino acid sequence

[0458] V / R / N / S / E / G - N / S / G / R - R / V / K / S / N / I / Y - V / N / I / R / S / T - R / K / I / N - G / I / R / K / S / A (SEQ ID NO: 185) or

[0459] A / R / M / V / G / S-N / T / S / A-R / N / H-V / R / K-K / V / I-R / K / A / N / S / V-T / G / R / K / P-S / G / R / A (SEQ ID NO:186) or

[0460] L1 of R / A / Q-S / A-G / S / N / I-N / K-T / S / L / R-I / R / V-R / E / K-K / Q / A / R-R / G / Q-E / W / G / R (SEQ ID NO:187); L2 containing the amino acid sequence KQDSD (SEQ ID NO:93); L3 containing the amino acid sequence LAG; L4 containing the amino acid V; and L5 containing the amino acid sequence G / S / N / Y / A / R-P / G / S / V / E / R / F / D-N / G / S / E / P / K / H / R-G / R / H / S / Q / V / D-F / D / N / R / G / Y / S / T (SEQ ID NO:188), with reference to scaffold structure I.

[0461] In certain embodiments, the non-naturally occurring CKP that binds to LRP6 comprises: containing the amino acid sequence

[0462] V / R / N / S / E / G-N / S / G / R-R / V / K / S / N / I / Y-V / N / I / R / S / T-R / K / I / N-G / I / R / K / S / A (SEQ ID NO:185) or

[0463] L1 of A / R / M / V / G / S-N / T / S / A-R / N / H-V / R / K-K / V / I-R / K / A / N / S / V-T / G / R / K / P-S / G / R / A (SEQ ID NO:186) or R / A-S-G / S / N-N / K-T / S / L-I / R-R / E-K / Q / A-R / G-E / W / G (SEQ ID NO:187). In certain embodiments, the non-naturally occurring CKP that binds to LRP6 further comprises: L2 containing the amino acid sequence KQDSD (SEQ ID NO:93). In certain embodiments, the non-naturally occurring CKP that binds to LRP6 further comprises: L3 containing the amino acid sequence LAG. In certain embodiments, the non-naturally occurring CKP that binds to LRP6 further comprises: L4 containing the amino acid V. In certain embodiments, the non-naturally occurring CKP that binds to LRP6 further comprises: containing the amino acid sequence

[0464] G / S / N / Y / A / R-P / G / S / V / E / R / F / D-N / G / S / E / P / K / H / R-G / R / H / S / Q / V / D-F / D / N / R / G / Y / S / T (SEQ ID NO:188) of L5.

[0465] In certain embodiments, the non-naturally occurring LRP6-binding CKP comprises L1 and / or L5 of any of the non-naturally occurring LRP6-binding CKPs disclosed herein. In certain embodiments, the non-naturally occurring LRP6-binding CKP comprises: L1 comprising the amino acid sequence shown in any of SEQ ID NOs: 147-168 and 367, with reference to scaffold structure I. In certain embodiments, the non-naturally occurring LRP6-binding CKP further comprises: L2 comprising the amino acid sequence shown in SEQ ID NO: 93. In certain embodiments, the non-naturally occurring LRP6-binding CKP further comprises: L3 comprising the amino acid sequence LAG. In certain embodiments, the non-naturally occurring LRP6-binding CKP further comprises: L4 comprising the amino acid V. In certain embodiments, the non-naturally occurring LRP6-binding CKP further comprises: L5 comprising the amino acid sequence shown in any of SEQ ID NOs: 19 and 169-184.

[0466] The L1 and L5 amino acid sequences described above are provided in Table 18 below:

[0467] Table 18

[0468]

[0469]

[0470] 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 the amino acid V; and L5 comprising the amino acid sequence GPNGF (SEQ ID NO: 19), according to reference scaffold structure I.

[0471] 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 the amino acid V; and L5 comprising the amino acid sequence SGGRD (SEQ ID NO: 169), according to reference scaffold structure I.

[0472] In certain embodiments, a non-naturally occurring CKP that binds LRP6 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 the amino acid V; and L5 comprising the amino acid sequence GPNGF (SEQ ID NO:19), with reference to scaffold structure I.

[0473] In certain embodiments, a non-naturally occurring CKP that binds LRP6 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 the amino acid V; and L5 comprising the amino acid sequence GSSRN (SEQ ID NO:170), with reference to scaffold structure I.

[0474] In certain embodiments, a non-naturally occurring CKP that binds LRP6 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 the amino acid V; and L5 comprising the amino acid sequence GVEGR (SEQ ID NO:29), with reference to scaffold structure I.

[0475] In certain embodiments, a non-naturally occurring CKP that binds LRP6 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 the amino acid V; and L5 comprising the amino acid sequence SVGHG (SEQ ID NO:172), with reference to scaffold structure I.

[0476] In certain embodiments, a non-naturally occurring CKP that binds LRP6 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 the amino acid V; and L5 comprising the amino acid sequence GPNGF (SEQ ID NO:19), with reference to scaffold structure I.

[0477] In certain embodiments, the non-naturally occurring CKP that binds to LRP6 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 the amino acid V; and L5 comprising the amino acid sequence NESRG (SEQ ID NO:173), with reference to scaffold structure I.

[0478] In certain embodiments, the non-naturally occurring CKP that binds to LRP6 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 the amino acid V; and L5 comprising the amino acid sequence GGPGG (SEQ ID NO:174), with reference to scaffold structure I.

[0479] In certain embodiments, the non-naturally occurring CKP that binds to LRP6 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 the amino acid V; and L5 comprising the amino acid sequence GPKSN (SEQ ID NO:175), with reference to scaffold structure I.

[0480] In certain embodiments, the non-naturally occurring CKP that binds to LRP6 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 the amino acid V; and L5 comprising the amino acid sequence YGHGD (SEQ ID NO:176), with reference to scaffold structure I.

[0481] In certain embodiments, the non-naturally occurring CKP that binds to LRP6 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 the amino acid V; and L5 comprising the amino acid sequence GSRQD (SEQ ID NO:177), with reference to scaffold structure I.

[0482] 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 the amino acid V; and L5 comprising the amino acid sequence SRGVN (SEQ ID NO:178), with reference to scaffold structure I.

[0483] 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 the amino acid V; and L5 comprising the amino acid sequence GPNDF (SEQ ID NO:179), with reference to scaffold structure I.

[0484] 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 the amino acid V; and L5 comprising the amino acid sequence GRGDY (SEQ ID NO:180), with reference to scaffold structure I.

[0485] 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 the amino acid V; and L5 comprising the amino acid sequence ASGSS (SEQ ID NO:181), with reference to scaffold structure I.

[0486] 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 the amino acid V; and L5 comprising the amino acid sequence SPGGR (SEQ ID NO:182), with reference to scaffold structure I.

[0487] 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 the amino acid V; and L5 comprising the amino acid sequence GPNGF (SEQ ID NO:19), with reference to scaffold structure I.

[0488] 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 the amino acid V; and L5 comprising the amino acid sequence GPNGF (SEQ ID NO:19), with reference to scaffold structure I.

[0489] 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 the amino acid V; and L5 comprising the amino acid sequence GFRGT (SEQ ID NO:183), according to reference scaffold structure I.

[0490] 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 the amino acid V; and L5 comprising the amino acid sequence RDRVG (SEQ ID NO:184), with reference to scaffold structure I.

[0491] 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 the amino acid V; and L5 comprising the amino acid sequence GPNGF (SEQ ID NO:19), with reference to scaffold structure I.

[0492] In certain embodiments, a non-naturally occurring CKP that binds LRP6 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 the amino acid V; and L5 comprising the amino acid sequence GPNGF (SEQ ID NO:19), with reference to scaffold structure I.

[0493] In certain embodiments, a non-naturally occurring CKP that binds LRP6 comprises the amino acid sequence shown in any of SEQ ID NOs: 189-210 and 366. SEQ ID NOs: 189-210 and 366 are provided below.

[0494]

[0495]

[0496] In certain embodiments, a non-naturally occurring CKP that binds LRP6 is a variant of the non-naturally occurring CKP that binds LRP6 described herein. In certain embodiments, such a variant comprises one or more of the sequences shown in SEQ ID NOs: 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 substitutions in the amino acid sequence LAG. In certain embodiments, the amino acid substitutions are conservative amino acid substitutions. In certain embodiments, the amino acid substitutions do not substantially reduce the ability of the non-naturally occurring CKP that binds LRP6 to bind human LRP6. For example, conservative changes (e.g., conservative substitutions as provided herein) can be made that do not substantially reduce the LRP6 binding affinity. Using the methods described in the following examples, the binding affinity of variants of the non-naturally occurring CKP that binds LRP6 can be evaluated.

[0497] Conservative substitutions are shown in Table 17 above 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 the non-naturally occurring CKP that binds LRP6 and the products screened for the desired activity (e.g., retained / improved LRP6 binding).

[0498] Non-conservative substitutions would require the exchange of a member of one of these classes for a member of another class. Exemplary substitution variants are non-naturally occurring CKP that bind LRP6 and are affinity matured, which can be conveniently generated, for example, using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more residues in L1, L2, L3, L4, and / or L5 are altered (i.e., added, deleted, or substituted), and the variant CKP that binds LRP6 is displayed on a 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, loop shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then established. The library is then screened to identify any non-naturally occurring CKP variants that have the desired affinity for LRP6. In certain embodiments, introducing diversity involves a loop-directed protocol, where 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 that participate in binding the target ligand can be identified, for example, using alanine-scanning mutagenesis or modeling.

[0499] In certain embodiments, the non-naturally occurring CKP "specifically binds" human LRP6 (i.e., has a binding affinity (Kd) value of no more than about 1×10 -7 M, preferably no more than about 1×10 -8 and most preferably no more than about 1×10 -9 M), but the binding affinity for another LRP protein is at least about 50-fold weaker, or at least about 500-fold weaker, or at least about 1000-fold weaker than its binding affinity for LRP6.

[0500] In certain embodiments, the non-naturally occurring CKP that binds LRP6 binds to non-target proteins (e.g., LRP6 homologs such as LRP1, LRP1B, LRP2, LRP3, LRP4, LRP5, LRP8, LRP10, LRP11, and LRP12) to an extent less than about 10% of the binding of the non-naturally occurring CKP that binds LRP6 to human LRP6, as determined by methods known in the art such as ELISA, fluorescence-activated cell sorting (FACS) analysis, or radioimmuno-precipitation (RIA). Specific binding can be measured, for example, by determining the binding of a molecule relative 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, e.g., an excess of unlabeled target. In such cases, if the binding of the labeled target to the probe is competitively inhibited by the excess unlabeled target, specific binding is indicated. As used herein, the term "specifically binds" to an epitope on a particular polypeptide or a particular polypeptide target, or "specifically binds with" an epitope on a particular polypeptide or a particular polypeptide target, or "is specific for" an epitope on a particular polypeptide or a particular polypeptide target can be demonstrated by, for example, a molecule that has a Kd of 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 M or greater. In one embodiment, the term "specifically binds" refers to a molecule that binds to an epitope on a particular polypeptide or a particular polypeptide and does not substantially bind to any other polypeptide or polypeptide epitope.

[0501] In certain embodiments, the non-naturally occurring CKP that binds LRP6 binds to human LRP6 with a Kd of from about 1 pM to about 500 nM. In certain embodiments, the non-naturally occurring CKP protein that specifically binds LRP6 binds to human LRP6 with a Kd of from about 1 pM to about 50 pM, from about 50 pM to about 250 pM, from about 250 pM to about 500 pM, from about 500 pM to 750 pM, from about 750 pM to about 1 nM, from about 1 nM to about 25 nM, from about 25 nM to about 50 nM, 50 nM to about 100 nM, from about 100 nM to about 250 nM, or from about 250 nM to about 500 nM, including any range therebetween.

[0502] In certain embodiments, a non-naturally occurring CKP that binds LRP6 inhibits Wnt1 signaling, such as as determined using the methods described in the following examples.

[0503] Also contemplated are nucleic acid molecules encoding the non-naturally occurring CKP that binds LRP6, expression vectors comprising the nucleic acid molecules encoding the non-naturally occurring CKP that binds LRP6, and cells comprising the nucleic acid molecules. Also provided herein are methods for producing a non-naturally occurring CKP that binds LRP6 by culturing the cells, expressing the non-naturally occurring CKP that binds LRP6, and recovering the non-naturally occurring CKP that binds LRP6 from the cell culture.

[0504] In certain embodiments, the non-naturally occurring CKP that binds LRP6 is produced via in vitro translation, as described elsewhere herein.

[0505] As described elsewhere herein, the non-naturally occurring CKP that binds LRP6 is produced via chemical peptide synthesis, for example by grafting chemically synthesized L1, L2, L3, L4, and / or L5 peptides onto an EETI-II framework, or by chemically synthesizing the entire non-naturally occurring CKP that binds LRP6.

[0506] In certain embodiments, the non-naturally occurring CKP that binds LRP6 is used as a therapeutic agent in the treatment of diseases or conditions in which excessive LRP6 activity is involved.

[0507] Production methods

[0508] In certain embodiments, non-naturally occurring CKP that binds VEGF-A or non-naturally occurring CKP that binds LRP6 are produced by genetic engineering. A variety of methods for mutagenesis (along with appropriate screening or selection methods) have been previously described. Such mutagenesis methods include, but are not limited to, for example, error-prone PCR, DNA shuffling or oligonucleotide-directed mutagenesis, random nucleotide insertion, or other methods prior to recombination. Additional details regarding 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. Accordingly, in certain embodiments, non-naturally occurring CKP that binds VEGF-A or non-naturally occurring CKP that binds LRP6 produced by genetic engineering techniques are provided.

[0509] In certain embodiments, non-naturally occurring CKP that binds VEGF-A or non-naturally occurring CKP that binds LRP6 are produced by in vitro translation. Briefly, 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. Desired variant proteins can be simply produced by altering the cloned protein-coding sequences. Many mRNAs can be efficiently translated in wheat germ extract or in rabbit reticulocyte lysate. Additional 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.

[0510] Accordingly, nucleic acid molecules encoding non-naturally occurring CKP that binds VEGF-A or non-naturally occurring CKP that binds LRP6 as described herein are provided. Expression vectors operably linked to the nucleic acid molecules encoding non-naturally occurring CKP that binds VEGF-A or non-naturally occurring CKP that binds LRP6 are also provided. Host cells (including, for example, prokaryotic host cells such as Escherichia coli (E. coli), eukaryotic host cells such as yeast cells, mammalian cells, CHO cells, etc.) containing nucleic acids encoding non-naturally occurring CKP that binds VEGF-A or non-naturally occurring CKP that binds LRP6 are also provided.

[0511] In certain embodiments, non-naturally occurring CKP that binds VEGF-A or non-naturally occurring CKP that binds LRP6 is produced by in vitro translation. Briefly, 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 altering the cloned protein coding sequence. Many mRNAs can be efficiently translated in wheat germ extract or in rabbit reticulocyte lysate. Additional details regarding in vitro translation are described, for example, in 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.

[0512] In certain embodiments, non-naturally occurring CKP that binds VEGF-A or non-naturally occurring CKP that binds LRP6 is generated chemically. 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 generate non-naturally occurring CKP that binds VEGF-A or 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 of solid-phase and liquid-phase peptide synthesis are well known in the art and are described in detail, for example, in Methods of Molecular Biology, 35, Peptide Synthesis Protocols, (M.W. Pennington and B.M. Dunn eds.), Springer, 1994; Welsch et al. (2010) Curr Opin Chem Biol 14, 1-15; Methods of Enzymology, 289, Solid Phase Peptide Synthesis, (G.B. 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, (W.C. Chan, P.D. White eds.), Oxford University Press, 2000; Solid Phase Synthesis, A Practical Guide, (S.F. 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, general editor, A. Felix, L. Moroder, C. Tmiolo eds.), Thieme, 2002; N.L.In Benoiton, 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, (edited by A. B. Hughs), Wiley-VCH, 2011.

[0513] Chimeric molecules comprising non-naturally occurring EETI-II proteins

[0514] If desired, the non-naturally occurring CKP described herein (such as non-naturally occurring CKP that binds VEGF-A or non-naturally occurring CKP that binds LRP6) can also be modified in a manner that forms a chimeric molecule, the 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 CKP that binds VEGF-A or non-naturally occurring CKP that binds LRP6) with an antibody to form, for example, a bivalent or bispecific molecule.

[0515] In certain embodiments, the chimeric molecule comprises a fusion of a non-naturally occurring CKP described herein (such as non-naturally occurring CKP that binds VEGF-A or non-naturally occurring CKP that binds LRP6) with a second moiety (such as a protein transduction domain), the second moiety targeting the chimeric molecule for delivery to a different tissue or, for example, across the blood-brain barrier, where a protein transduction domain such as the human immunodeficiency virus TAT protein is used (Schwarze et al., 1999, Science 285:1569-72).

[0516] In certain embodiments, the non-naturally occurring CKP provided herein can be used as a bispecific or multispecific (against different target ligands or different epitopes on the same target ligand) in multimeric form. For example, a dimeric bispecific non-naturally occurring CKP has one subunit specific for a first target protein or epitope and a second subunit specific for a second target protein or epitope. The non-naturally occurring CKP protein subunits can be linked in a variety of conformations that can increase avidity and thus increase the affinity of binding to the target ligand.

[0517] In certain embodiments, the chimeric molecules provided herein comprise two or more (such as three, four, five, six, seven, eight, nine, ten or more than ten) non-naturally occurring CKP proteins. In certain embodiments, nucleic acids can be engineered to encode two or more copies of a single non-naturally occurring CKP, which are transcribed and translated in tandem to produce covalently linked multimers of the same subunit. In certain embodiments, nucleic acids can be engineered to encode two or more different non-naturally occurring CKPs, which are transcribed and translated in tandem to produce covalently linked multimers of different subunits, which bind to different epitopes of, for example, a single target ligand or, for example, different target ligands.

[0518] 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) with a tag polypeptide that provides 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 an antibody against the tag polypeptide, the presence of this epitope-tagged form of the non-naturally occurring CKP protein can be detected. Also, the provision of the epitope tag enables the non-naturally occurring CKP to be readily purified by affinity purification using an anti-tag antibody or another class of affinity matrix that binds 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; the influenza HA tag polypeptide and its antibody 12CA5 (Field et al. (1988) Mol. Cell. Biol. 8, 2159-2165); c-myc tags and their antibodies 8F9, 3C7, 6E10, G4, B7, and 9E10 (Evan et al. (1985) Mol. Cell. Biol. 5, 3610-3616]; and herpes simplex virus glycoprotein D (gD) tags and its antibody (Paborsky et al. (1990) Protein Eng., 3, 547-553). Other tag polypeptides include the Flag-peptide (Hopp et al. (1988) BioTechnology, 6, 1204-1210); the KT3 epitope peptide (Martin et al. (1992) Science, 255, 192-194]; the α-tubulin epitope peptide (Skinner et al. (1991) J. Biol. Chem. 266, 15163-15166); and the T7 gene 10 protein peptide tag (Lutz-Freyermuth et al. (1990) Proc. Natl. Acad. Sci. USA 87, 6393-6397).

[0519] In certain embodiments, the chimeric molecule can 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) with an immunoglobulin or a specific region of an immunoglobulin. For a bivalent form of the chimeric molecule (e.g., an "immunoadhesin"), such a fusion can be to the Fc region of an IgG molecule. The Ig fusions provided herein include polypeptides that comprise human residues approximately or only 94-243, residues 33-53, or residues 33-52 at the location of at least one variable region within the Ig molecule. In particularly preferred embodiments, the immunoglobulin fusion comprises the hinge, CH2, and CH3, or the hinge, CH1, CH2, and CH3 regions of an IgG1 molecule. Also see, U.S. Patent No. 5,428,130, issued June 27, 1995, regarding the production of immunoglobulin fusions. 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 fused, for example, at the N- or C-terminus to the constant region (Fc) of IgG. In certain embodiments, the non-naturally occurring CKP / Fc fusion molecule activates complement components 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, 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) is fused (such as recombinantly) at the N- or C-terminus, for example, to a complement protein such as Clq. A variety of publications describe methods for obtaining non-naturally occurring proteins with altered half-lives: 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 in which the FcRn-binding affinity is retained but the affinity for other Fc receptors has been greatly reduced (WO 1999 / 43713), or to the FcRn-binding domain of an antibody (WO 2000 / 09560, US 4703039). Specific techniques and methods for increasing the half-life of a physiologically active molecule (e.g., a non-naturally occurring CKP) can also be found in US7083784. In certain embodiments, a non-naturally occurring CKP protein provided herein (such as a non-naturally occurring CKP that binds VEGF-A or a non-naturally occurring CKP that binds LRP6) is fused to an Fc region from IgG that comprises the following amino acid residue mutations (numbered according to the EU index in Kabat): M252Y / S254T / T256E or H433K / N434F / Y436H.

[0520] In certain embodiments, the non-naturally occurring CKP proteins described herein (such as non-naturally occurring CKP that binds VEGF-A or non-naturally occurring CKP that binds LRP6) are fused to a molecule that increases or extends the in vivo or serum half-life. In certain embodiments, the non-naturally occurring CKP described herein (such as non-naturally occurring CKP that binds VEGF-A or non-naturally occurring CKP that binds LRP6) is fused to albumin (such as human serum albumin (HSA)), polyethylene glycol (PEG), polysaccharide, immunoglobulin molecule (IgG), complement, hemoglobin, binding peptide, lipoprotein, or other factors to increase its half-life in the bloodstream and / or its tissue penetration.

[0521] Techniques such as gene shuffling, motif shuffling, exon shuffling, and / or codon shuffling (collectively referred to as "DNA shuffling") can be used to generate additional chimeric molecules that contain non-naturally occurring CKP that binds VEGF-A or non-naturally occurring CKP that binds LRP6. DNA shuffling can be employed to alter the activity of the non-naturally occurring CKP (e.g., non-naturally occurring CKP with higher affinity and lower dissociation rate). Generally, see, US 5605793, US5811238, US 5830721, US5834252, 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.

[0522] 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 CKP that binds VEGF-A or non-naturally occurring CKP that binds LRP6 provided herein. One or more portions of the polynucleotide encoding the scaffold that binds to a specific target can be recombined with one or more components, motifs, segments, portions, domains, fragments, etc. of one or more heterologous molecules.

[0523] Any of these fusions can be produced by standard techniques, e.g., by expressing a fusion protein from a recombinant fusion gene constructed using publicly available gene sequences or by chemical peptide synthesis.

[0524] Conjugates comprising non-naturally occurring CKP that binds VEGF-A or non-naturally occurring CKP that binds LRP6.

[0525] The present disclosure provides immunoconjugates comprising an unnatural CKP described herein (such as an unnatural CKP that binds VEGF-A or an unnatural CKP that binds LRP6) conjugated to a cytotoxic agent such as a chemotherapeutic agent, a toxin (e.g., an enzymatically active toxin from bacterial, fungal, plant, or animal sources, or a fragment thereof), or a radioisotope (i.e., a radio conjugate).

[0526] Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Saponaria officinalis inhibitor, 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 radio-conjugated unnatural CKPs. Examples include 212 Bi, 131 I, 131 In, 90 Y, and 186 Re.

[0527] Conjugates of the non-naturally occurring CKP described herein (such as non-naturally occurring CKP that binds VEGF-A or non-naturally occurring CKP that binds LRP6) with, for example, cytotoxic agents are prepared using a variety of bifunctional protein conjugating agents such as N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidates (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), di-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), diazo derivatives (such as bis-(p-diazo benzoyl)-ethylenediamine), diisocyanates (such as 2,6-tolylene diisocyanate) and bifunctional fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins are 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 the non-naturally occurring CKP provided herein. See WO94 / 11026.

[0528] In another embodiment, the non-naturally occurring CKP described herein (such as non-naturally occurring CKP that binds VEGF-A or non-naturally occurring CKP that binds LRP6) can be conjugated to a "receptor" (such as streptavidin) for use in "pre-targeting" in the eye, wherein the non-naturally occurring EETI-II scaffold protein-receptor conjugate is administered to an eye patient, followed by removal of unbound conjugate from the circulation using a scavenger, and then a "ligand" (such as avidin) conjugated to a cytotoxic agent (e.g., a radionuclide) or therapeutic agent is administered.

[0529] In certain embodiments, the non-naturally occurring CKP provided herein (such as non-naturally occurring CKP that binds VEGF-A or non-naturally occurring CKP that binds LRP6) can be used in multimeric form as bis- or multi-specific (for different target ligands or different epitopes on the same target ligand). The linkage can be covalent or non-covalent. For example, a dimeric bis-specific non-naturally occurring CKP has one subunit that is specific for a first target protein or epitope and a second subunit that is specific for a second target protein or epitope. The non-naturally occurring CKP subunits can be linked in a variety of conformations that can increase potency and thus increase the affinity of binding to target ligands or bind multiple target ligands.

[0530] In certain embodiments, the non-naturally occurring CKP provided herein is engineered to provide reactive groups for conjugation. In certain embodiments, the N-terminus and / or C-terminus can also be used to provide reactive groups for conjugation. In certain embodiments, the N-terminus is conjugated to one moiety (e.g., but not limited to PEG), while the C-terminus is conjugated to another moiety (e.g., but not limited to biotin), or vice versa.

[0531] Provided are the non-naturally occurring CKPs described herein (such as non-naturally occurring CKP that binds VEGF-A or non-naturally occurring CKP that binds LRP6) conjugated to one or more moieties, said moieties including but not limited to, peptides, polypeptides, proteins, fusion proteins, nucleic acid molecules, small molecules, mimetics, synthetic drugs, inorganic molecules, and organic molecules. Also provided is the use of the non-naturally occurring CKPs described herein (such as non-naturally occurring CKP that binds VEGF-A or non-naturally occurring CKP that binds LRP6) chemically conjugated (including both covalent and non-covalent conjugation) to a heterologous protein or polypeptide (or a fragment thereof, a polypeptide 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 sequences described herein.

[0532] In certain embodiments, the non-naturally occurring CKPs described herein (such as non-naturally occurring CKP that binds VEGF-A or non-naturally occurring CKP that binds LRP6) or analogs or derivatives thereof can be conjugated to diagnostic or detectable reagents. 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, said detectable substances including but not limited to: various enzymes, such as but not limited to horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; cofactors, 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), gallium( 68 Ga, 67 Ga), palladium( 103 Pd), molybdenum( 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 Ru, 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; metals that emit positrons using various positron emission tomography, non-radioactive paramagnetic metal ions, and molecules radiolabeled or conjugated to specific radioisotopes.

[0533] Also provided are non-naturally occurring CKP conjugated to a therapeutic moiety (such as non-naturally occurring CKP that binds VEGF-A or non-naturally occurring CKP that binds LRP6). In certain embodiments, the non-naturally occurring CKP can be conjugated to a therapeutic moiety such as a cytotoxin, e.g., a cell growth inhibitory or cytocidal agent, a therapeutic agent, or a radioactive metal ion, e.g., an α-emitter. Cytotoxins or cytotoxic agents include any agent that is harmful to cells.

[0534] In certain embodiments, the non-naturally occurring CKP described herein (such as non-naturally occurring CKP that binds VEGF-A or 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 (b) or macrocyclic chelating agents that can be used to conjugate radio-metallic ions (including but not limited to, 131 In, 131 Lu, 131 Y, 131 Ho, 131 Sm) to a polypeptide. In certain embodiments, the macrocyclic chelating agent is 1,4,7,10-tetraazacyclododecane-N,N’,N”,N”’-tetra-acetic acid (DOTA), which can be linked to the non-naturally occurring CKP via a linker molecule. Such linker molecules are generally known in the art and are described, for example, in 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.

[0535] Techniques for conjugating therapeutic moieties to antibodies are well known and can be applied to the non-naturally occurring CKP 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 Radio labeled Antibody 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 CKP provided herein.

[0536] 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 prophylactic or therapeutic effect against a specific disorder in a subject. When deciding which therapeutic moiety or drug to conjugate to the scaffold, a clinician or other healthcare provider should consider the following: the nature of the disease, the severity of the disease, and the condition of the subject.

[0537] 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) can also be linked to a solid support that is particularly useful for immunoassays or purification of a target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

[0538] Covalent modification

[0539] Covalent modification of 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 also contemplated. One class of covalent modifications involves reacting a targeting amino acid residue of the non-naturally occurring CKP with an organic derivatizing reagent that is capable of reacting with a selected side chain or N- or C-terminal residue of the non-naturally occurring CKP. Derivatization using bifunctional reagents can be used, for example, to crosslink the non-naturally occurring CKP to a water-insoluble support matrix or surface used in methods for purifying a target ligand, and vice versa. Commonly used crosslinking agents include, for example, 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters (such as the ester formed with 4-azidosalicylic acid), homobifunctional imidoesters (including disuccinimidyl esters such as 3,3'-dithiobis(succinimidyl-propionate)), bifunctional maleimides such as di-N-maleimidyl-1,8-octane, and reagents such as methyl-3-[(p-azidophenyl)-dithio]propioimidate.

[0540] Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, hydroxylation of proline and lysine, phosphorylation of the hydroxyl groups of serine or threonine residues, methylation of the α-amino groups of lysine, arginine, and histidine side chains (T.E. Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)), acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

[0541] Another class of covalent modifications of non-naturally occurring CKP includes attaching the non-naturally occurring CKP to one of a variety of non-proteinaceous polymers (e.g., polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylene) in the manner set forth in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192; or 4,179,337.

[0542] The term "polyethylene glycol" or "PEG" means a polyethylene glycol compound or derivative thereof, with or without a coupling agent, coupling or activation moiety (e.g., having a thiol, triflate, tresylate, aziridine, oxirane, N-hydroxysuccinimide, or maleimide moiety). The term "PEG" is intended to denote polyethylene glycols having a molecular weight in the range of 500 to 150,000 Da, including analogs thereof in which, for example, the terminal -OR group has been replaced by a methoxy group (referred to as mPEG).

[0543] 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 having two terminal hydroxyl groups. PEGs are classified according to their molecular weight, which generally ranges from about 500 daltons to about 40,000 daltons. In presently preferred embodiments, the PEG employed 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). PEGs are 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-tresylate (MePEG-TRES), and monomethoxypolyethylene glycol-imidazolyl-carbonyl (MePEG-IM).

[0544] In certain embodiments, the hydrophilic polymer employed (e.g., PEG) is capped at one end with a non-reactive group such as a methoxy or ethoxy group. Thereafter, the polymer is activated at the other end by reaction with a suitable activator such as cyanuric halides (e.g., cyanuric chloride, cyanuric bromide, or cyanuric fluoride), diimidazole, anhydride reagents (e.g., dihalosuccinic anhydrides such as dibromosuccinic anhydride), acyl azides, p-diazo benzyl ether, 3-(p-diazo phenoxy)-2-hydroxypropyl ether), etc. The activated polymer is then reacted with a non-naturally occurring CKP herein (such as a non-naturally occurring CKP that binds VEGF-A or a non-naturally occurring CKP that binds LRP6) to produce a non-naturally occurring CKP derivatized with the polymer. Alternatively, a functional group in the non-naturally occurring CKP provided herein can be activated for reaction with the polymer, or known coupling methods can be used to link the two groups in a concerted coupling reaction. It will be readily appreciated that many other reaction schemes known and used by those skilled in the art can be used to derivatize the non-naturally occurring CKP provided herein with PEG.

[0545] Liposomes

[0546] The non-naturally occurring CKP disclosed herein (such as a non-naturally occurring CKP that binds VEGF-A or a non-naturally occurring CKP that binds LRP6) can also be formulated as liposomes. Liposomes containing the non-naturally occurring EETI-II scaffold protein described herein are prepared by methods known in the art, such as those described in Epstein et al., Proc Natl Acad Sci USA, 82:3688 (1985); Hwang et al., Proc Natl Acad Sci USA, 77:4030 (1980); and U.S. Patent Nos. 4,485,045 and 4,544,545. Liposomes with increased circulation times are disclosed in U.S. Patent No. 5,013,556.

[0547] Particularly useful liposomes can be produced by the reverse evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). The liposomes are extruded through a filter of a defined pore size to produce liposomes of the desired diameter. A second therapeutic agent is also optionally included in the liposomes. See Gabizon et al., J. National Cancer Inst., 81(19):1484 (1989). Pharmaceutical compositions and formulations comprising a non-naturally occurring cystine knot peptide (CKP) that binds human vascular endothelial growth factor A (VEGF-A).

[0548] In certain embodiments, provided herein are pharmaceutical compositions comprising a non-naturally occurring VEGF-A-binding CKP and a pharmaceutically acceptable excipient. In certain embodiments, the composition may further contain a buffer, a carrier, a stabilizer, a preservative, and / or a bulking agent to render the composition suitable for ocular administration to a patient to achieve a desired effect or outcome. In certain embodiments, the pharmaceutical composition comprises one or more permeation enhancers that permit the non-naturally occurring VEGF-A-binding CKP to penetrate the cornea. Examples of such permeation enhancers include, for example, surfactants, bile acids, chelating agents, preservatives, cyclodextrins (i.e., cylindrical oligonucleotides having a hydrophilic outer surface and a lipophilic inner surface that form complexes with lipophilic drugs), and the like. Such permeation enhancers increase chemical stability and bioavailability and reduce local irritation. In certain embodiments, the pharmaceutical compositions provided herein further comprise agents that increase the absorption and distribution of the non-naturally occurring VEGF-A-binding CKP in various ocular compartments. In certain embodiments, the pharmaceutical compositions provided herein comprise cross-linked polyacrylic acid, by means of whose mucoadhesive properties ocular bioavailability can be enhanced. In certain embodiments, the pharmaceutical compositions provided herein comprise a bioadhesive polymer.

[0549] In certain embodiments, the pharmaceutical compositions provided herein are formulated as in situ gelling systems, such as viscous polymer-based liquids that exhibit a sol-to-gel phase transition on the ocular surface due to changes in specific physicochemical parameters (particle strength, temperature, pH, or solvent exchange) when the composition contacts the tear fluid. In certain embodiments, the pharmaceutical compositions provided herein are formulated as ocular sprays. In certain embodiments, the provided pharmaceutical compositions are formulated as liposomes. In certain embodiments, the pharmaceutical compositions provided herein are formulated as niosomes (i.e., nonionic surfactant-based vesicles containing, for example, cholesterol as an excipient). In certain embodiments, the pharmaceutical compositions provided herein are formulated as pharmacosomes (i.e., vesicles formed from amphiphilic drugs). In certain embodiments, the pharmaceutical compositions provided herein are formulated as microemulsions. Additional details regarding 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 other literature.

[0550] In certain embodiments, a pharmaceutical composition comprising a non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A described herein can be stable at room temperature (such as at about 20-25 °C) for about 0.5 weeks, about 1.0 week, 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 range between these values. In certain embodiments, a pharmaceutical composition comprising a non-naturally occurring cystine knot peptide (CKP) that binds VEGF-A described herein can be stable under accelerated conditions (such as stored at about 37 °C) for about 0.5 weeks, about 1.0 week, 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 range between these values.

[0551] Methods of treatment using a non-naturally occurring cystine knot peptide (CKP) that binds vascular endothelial growth factor A (VEGF-A)

[0552] Vascular endothelial growth factor (VEGF-A), a dimeric glycoprotein of approximately 40 kDa, is a potent endothelial cell mitogen that stimulates proliferation, migration, and tube formation, giving rise to 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 the abnormal (such as excessive or otherwise inappropriate) growth of leaky blood vessels contributes to the pathology of several debilitating ocular 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, pathologic myopia, von Hippel-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 redness. VEGF-A-induced angiogenesis is detrimental, and retinal, trabecular, or corneal neovascularization can ultimately lead to vision loss.

[0553] In certain embodiments, provided herein are methods of treating an ocular disease or disorder in a subject, the methods comprising administering to the subject an effective amount of a non-naturally occurring VEGF-A-binding CKP described herein or a composition (such as a pharmaceutical composition) comprising a non-naturally occurring VEGF-A-binding CKP described herein. In certain embodiments, provided is the use of a composition (such as a pharmaceutical composition) comprising a non-naturally occurring VEGF-A-binding CKP described herein for the treatment of an ocular disease or disorder in a subject. In certain embodiments, provided is the use of a non-naturally occurring VEGF-A-binding CKP (or a composition comprising such a non-naturally occurring CKP) described herein in the preparation of a medicament for treating an ocular disease and disorder in a subject.

[0554] In certain embodiments, the subject to be treated is a mammal (e.g., a human, non-human primate, rat, mouse, cow, 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 ocular disease or disorder characterized by abnormal angiogenesis and / or abnormal vascular permeability (such as those described herein) or is at risk of developing such an ocular disease or disorder. In certain embodiments, the subject has been diagnosed with an ocular disease or disorder characterized by abnormal angiogenesis and / or abnormal vascular permeability (such as those described herein).

[0555] 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, pathologic myopia, von Hippel-Lindau disease, ocular histoplasmosis, retinal vein occlusion (RVO) (both retinal branch vein occlusion (BRVO) and central retinal vein occlusion (CRVO)), corneal neovascularization, retinal neovascularization, and redness. In certain embodiments, corneal neovascularization results in 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 herpes simplex keratitis, trachoma, or onchocerciasis.

[0556] In certain embodiments, an effective amount of the non-naturally occurring VEGF-A-binding CKP described herein (or a composition comprising such non-naturally occurring VEGF-A-binding CKP described herein) is administered directly to the eye of the subject (such as intravitreally or topically), as further described in detail elsewhere herein.

[0557] In certain embodiments, the non-naturally occurring VEGF-A-binding CKP described herein (or a composition comprising such non-naturally occurring CKP) is administered in combination with a second agent. For patients whose ocular disease or disorder is triggered by an inflammatory response, combination therapy using an anti-inflammatory agent may be considered. For example, the combined use of a steroid and the non-naturally occurring VEGF-A-binding CKP described herein (or a composition comprising such non-natural CKP) to respectively reduce inflammation and prevent neovascularization may be particularly advantageous in patients presenting, for example, with corneal neovascularization. Patients suffering from an ocular disease or disorder secondary to a bacterial, viral, fungal or acanthamoebal infection may benefit from the administration of a combination of the non-naturally occurring VEGF-A-binding CKP described herein (or a composition comprising such non-naturally occurring CKP) with an antimicrobial agent and optionally an anti-inflammatory agent. Patients with corneal stromal blood vessels due to an ocular disease or disorder are at an increased risk of immune rejection following corneal transplantation. Thus, administration of the non-naturally occurring VEGF-A-binding CKP described herein (or a composition comprising such non-naturally occurring CKP) before (and optionally also after) corneal transplantation may be particularly beneficial for patients with corneal stromal blood vessels, as successful reduction of corneal vascularization will reduce the risk of transplant rejection. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP described herein (or a composition comprising such non-naturally occurring CKP) is administered in combination with a second anti-angiogenic agent. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP described herein (or a composition comprising such non-naturally occurring CKP) is administered in combination with a matrix metalloproteinase (MMP) inhibitor.

[0558] In certain embodiments, the non-naturally occurring VEGF-A-binding CKP described herein (or a composition comprising such 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 create a controlled lesion in the retina, thereby producing a beneficial therapeutic effect. Short pulses of laser can seal leaking blood vessels, destroy abnormal blood vessels, seal retinal tears, or destroy abnormal tissue in the posterior part of the eye. It is rapid, non-invasive, and generally does not require anesthesia other than anesthetic eye drops. LPT techniques and devices are readily available to ophthalmologists (see Lock et al. (2010) Med J Malaysia 65:88-94). Further details regarding LPT can be found, for example, in WO 2014 / 033184.

[0559] In certain embodiments, the second therapy is photodynamic therapy (PDT). PDT uses photoactivatable molecules to cause local damage to the neovascular endothelium, resulting in vascular occlusion. The light is delivered to the retina as a single circular spot (laser treatment) via a fiber optic cable and a slit lamp using a suitable ophthalmic magnifier. The photoactivatable compound is injected into the circulation prior to laser treatment, and damage is inflicted by photoactivation of the compound in the area troubled by neovascularization. A commonly used photoactivatable compound is verteporfin. Verteporfin is mainly transported in plasma by lipoproteins. Once verteporfin is photoactivated in the presence of oxygen, highly reactive short-lived singlet oxygen and reactive oxygen free radicals are generated, thereby damaging the endothelium surrounding the blood vessels. It is known that damaged endothelium releases procoagulant factors and vasoactive factors via the lipoxygenase (leukotrienes) and cyclooxygenase (eicosanoids, such as thromboxane) pathways, leading to platelet aggregation, fibrin clot formation, and vasoconstriction. Verteporfin seemingly preferentially accumulates in neovascular tissue to some extent. The laser wavelength for photoactivation of the photoactivatable compound can vary depending on the particular photoactivatable compound used. Other details regarding PDT can be found, for example, in WO 2014 / 033184.

[0560] In certain embodiments, the second therapy is diathermy and cauterization, where the blood vessels are occluded by applying a coagulation current via a monopolar diathermy unit or by performing thermal cauterization using an electrolytic needle inserted into the feeder vessel at the limbus.

[0561] Administration

[0562] In certain embodiments, a non-naturally occurring CKP that binds VEGF-A (or a composition comprising such non-naturally occurring CKP) is administered, for example, by injection (such as subconjunctival injection, intracorneal injection, or intravitreal injection). Administration in 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, the non-naturally occurring CKP that binds VEGF-A (or a composition comprising such 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 prior to administration can be avoided. The pre-filled syringe also provides easier handling for the ophthalmologist. For additional details regarding 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.

[0563] In certain embodiments, a non-naturally occurring CKP that binds VEGF-A (or a composition comprising such non-naturally occurring CKP) is administered topically (such as in the form of eye drops). Additional details regarding topical drug delivery to the eye can be found in, for example, 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.

[0564] In certain embodiments, a non-naturally occurring VEGF-A-binding CKP (or a composition comprising such non-naturally occurring CKP) is continuously delivered to the eye using an intravitreal device. In certain embodiments, the non-naturally occurring VEGF-A-binding CKP (or a composition comprising such non-naturally occurring CKP) is administered via an ocular 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 shield). See, for example, Gaikwad et al. (2013) Indo Amer J Pharm Res. 3, 3216-3232). In certain embodiments, the non-naturally occurring VEGF-A-binding CKP (or a composition comprising such non-naturally occurring CKP) is administered as a sustained release depot, ocular plug / reservoir, ocular implant (such as a scleral or vitreous implant). Various scleral 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 for active administration via a pressure differential. Alternatively, active delivery can be achieved by an implantable micropump. An example of an intravitreal delivery system using a micropump to deliver a therapeutic agent is the Ophthalmic MicroPumpSystem sold by Replenish, Inc. TM, which can be programmed to deliver a set amount of therapeutic agent a predetermined number of times. In certain embodiments, a non-naturally occurring VEGF-A-binding CKP (or a composition comprising such non-naturally occurring CKP) is loaded into a capsule-like container (e.g., a silicone elastomer cup). The container is typically implanted above the iris in the eye. The container includes a release opening. Release of the non-naturally occurring VEGF-A-binding CKP (or a composition comprising such non-naturally occurring CKP) can be controlled by a membrane located between the non-naturally occurring VEGF-A-binding CKP (or a composition comprising such non-naturally occurring CKP) and the opening or by means of a micropump attached to the container. Alternatively, the non-naturally occurring VEGF-A-binding CKP (or a composition comprising such non-naturally occurring CKP) can be deposited in a slow-release matrix, which prevents rapid diffusion of the antagonist out of the container. Preferably, the intravitreal device is designed to release the non-naturally occurring VEGF-A-binding CKP (or a composition comprising such non-naturally occurring CKP) at a higher initial rate in the first month. The release rate, for example, slowly decreases to a rate that is about 50% lower than the initial rate over the course of the first month after implantation. The container can be sized to hold a supply of the non-naturally occurring VEGF-A-binding CKP (or a composition comprising such non-naturally occurring CKP) that lasts for about four to six months. Since a reduced dose of the non-naturally occurring VEGF-A-binding CKP (or a composition comprising such non-naturally occurring CKP) may be sufficient to achieve effective treatment when administered continuously, the supply in the container can last for one year or longer, preferably about two years, more preferably about three years. Since only a minor surgery is required to implant the delivery system and intravitreal injections are avoided, patient compliance issues associated with repeated intravitreal injections can be avoided. The intravitreal concentration of the non-naturally occurring VEGF-A-binding CKP (or a composition comprising such non-naturally occurring CKP) is reduced, and thus the risk of potential side effects due to entry of the non-naturally occurring VEGF-A-binding CKP (or a composition comprising such non-naturally occurring CKP) into the circulation is reduced.

[0565] In certain embodiments, a non-naturally occurring VEGF-A-binding CKP (or a composition comprising such non-naturally occurring CKP) is administered by iontophoresis. Iontophoresis is a non-invasive technique in which a small electric current is applied to enhance the penetration of an ionized drug into tissue (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). The drug is administered using an electrode carrying the same charge as the drug, and a ground electrode having the opposite charge is placed at another location on the body to complete the circuit. The drug acts as a conductor of the electric current through the tissue.

[0566] Additional details regarding drug administration to the eye are provided, for example, in Kuno et al. (2011) Polymers 3, 193-221; Short (2008) Toxicologic Path. 36, 49-62; Ghate et al. (2006) Expert Opin DrugDeliv 3, 275-87; Davis et al. (2004) Curr Opin Mol Therap 6, 195-205; Gaudana et al. (2010) AAPS J. 12, 348-360; and other literature.

[0567] Sustained release / long-acting delivery formulations

[0568] In certain embodiments, a non-naturally occurring VEGF-A-binding CKP (or a composition comprising such non-naturally occurring CKP) is provided as a sustained release formulation. Sustained release formulations are generally obtained by mixing a therapeutic agent with a biodegradable polymer or encapsulating it into microparticles.

[0569] The sustained release formulations according to the present invention generally comprise a non-naturally occurring VEGF-A-binding CKP (or a composition comprising such non-naturally occurring CKP), a polymeric carrier, and a release regulator for regulating the release rate of the non-naturally occurring VEGF-A-binding CKP (or a composition comprising such non-naturally occurring CKP) from the polymeric carrier. By varying the preparation conditions of the polymer-based delivery composition, the release kinetic properties of the resulting composition can be adjusted. The polymeric carrier generally comprises one or more biodegradable polymers or copolymers or combinations thereof. For example, the polymeric carrier can be selected from polylactic acid (PLA), polyglycolic acid (PGA), poly(lactic-co-glycolic acid) (PLGA), polyesters, poly(orthoesters), poly(phosphazines), poly(phosphates), polycaprolactone, or combinations thereof.

[0570] In certain embodiments, the polymeric carrier is PLGA. The release modifier is generally a long-chain fatty alcohol, preferably comprising from 10 to 40 carbon atoms. Commonly used release modifiers include octanol, nonanol, decanol, lauryl alcohol, myristyl alcohol, cetyl alcohol, palmityl alcohol, stearyl alcohol, isostearyl alcohol, elaidyl alcohol, oleyl alcohol, linoleyl alcohol, polyunsaturated elaidyl alcohol, polyunsaturated linoleyl alcohol, elaidyl alcohol, polyunsaturated ricinoleyl alcohol, arachidyl alcohol, behenyl alcohol, brassidyl alcohol, lignoceryl alcohol, montanyl alcohol, octacosanyl alcohol, melissyl alcohol, ceryl alcohol, and dotriacontanyl alcohol.

[0571] In certain embodiments, a non-naturally occurring VEGF-A-binding CKP (or a composition comprising such non-naturally occurring CKP) is incorporated into a microsphere-based sustained release composition. In certain embodiments, the microspheres are prepared from PLGA. The amount and the release rate of the non-naturally occurring VEGF-A-binding CKP (or a composition comprising such non-naturally occurring CKP) incorporated into the microspheres can be controlled by varying the conditions used to prepare the microspheres. Methods for producing such slow release formulations are described in US2005 / 0281861 and US2008 / 0107694.

[0572] In certain embodiments, a non-naturally occurring VEGF-A binding CKP (or a composition comprising such non-naturally occurring CKP) is incorporated into a biodegradable implant, such as a microneedle. Matrix implants, such as microneedles, are commonly used to treat ocular diseases that require a loading dose, followed by a decreasing drug dose over a period of 1 day to 6 months (Davis et al. (2004) Curr Opin Mol Therap 6, 195–205). They are most commonly made of the copolymers polylactic acid (PLA) and / or poly-lactic-co-glycolic acid (PLGA) that degrade into water and carbon dioxide. The rate and e...

Claims

1. A non-naturally occurring peptide that binds to vascular endothelial growth factor A (VEGF-A), wherein said peptide comprises a cystine scaffold structure: Z1C1L1C2L2C3L3C4L4C5L5C6Z2 Wherein: Z1 and Z2 are any amino acids; L1 is Ring 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 X1X2X3X4X5X6X7X8X9X 10 (SEQ ID NO:6), where each of X1-X 10 is any amino acid; L2 is loop 2 and has the structure: X1X2X3X4X5 (SEQ ID NO:7), wherein each of X1–X5 is any amino acid or non-natural amino acid; L3 is loop 3 and has the structure: X1X2X3, wherein each of X1–X3 is any amino acid or non-natural amino acid; L4 is loop 4 and has the structure: X1, wherein X1 is any amino acid or non-natural amino acid; L5 is loop 5 and has the structure: X1X2X3X4X5 (SEQ ID NO:7), wherein each of X1–X5 is any amino acid or non-natural amino acid; The unnatural amino acids are 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, glycylamide, glycerol glycinate, ethylene glycol glycinate, oxetan-3-yl glycinate, and glycine morpholine amide; wherein the peptide binds VEGF-A with an affinity of 500 pM or better.

2. The peptide according to claim 1, wherein, 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 peptide has an altered disulfide bond connectivity; wherein the altered disulfide bond connectivity is C1-C4, C2-C3, and C5-C6.

3. The peptide according to claim 1 or 2, wherein Z1 and / or Z2 is G.

4. The peptide according to any one of claims 1-3, wherein in L1: X3 is not I; X5 is not M; and / or X6 is not R.

5. The peptide according to any one of claims 1-4, wherein 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 K, N, F, P, L, Y, T, D, M, and X; X7 is an amino acid selected from Q, W, H, and / X; and / or X8 is an amino acid selected from Y, A, G, D, E, W, S, and X, wherein X is a non-natural amino acid and is selected from the group consisting of: L-propargylglycine-PEG6-, L-sulfo 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 (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, glycylamide, glycerol glycine ester, ethylene glycol glycine ester, oxetan-3-yl glycine ester, and glycine morpholine amide.

6. The peptide according to claim 5, wherein in L1: wherein X9 is an amino acid selected from L, I, V, D, E, and X, where 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, 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, glycylamide, glycerol glycinate, ethylene glycol glycinate, oxetan-3-yl glycinate, and glycine morpholine amide.

7. The peptide according to claim 5 or 6, wherein in L1: wherein X 10 is an amino acid selected from Y, T, M, N, F, 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, 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-L-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, glycylamide, glycerol glycine ester, ethylene glycol glycine ester, oxetan-3-yl glycine ester, and glycine morpholine amide.

8. The peptide according to any one of claims 1-7, wherein in L5, each of X1–X5 is any amino acid, except that X2 is not proline (P).

9. The peptide according to any one of claims 1-7, wherein in L5, each of X1–X5 is any amino acid, except that X4 is not glycine (G).

10. The peptide according to any one of claims 1-7, wherein 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.

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