De novo designed tie2 mini binder acting as an agonist or antagonist for tie2 receptor

Abstract

Polypeptides are provided comprising an ammo acid sequence at least 50% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 1-17, not including any amino acid insertions at identified insertion sites, and not including residues noted as dispensable in Tables 2-7, wherein the polypeptide binds to Tie2, fusion proteins and scaffold including such polypeptides, and methods for their use.

Description

[0001]

[0002] UW 50163.02W02

[0003] De novo designed Tie2 mini binder acting as an agonist or antagonist for Tie2 receptor

[0004] Federal Funding Statement

[0005] This invention was made with government support under Grant No. T90DE021984, awarded by the National Institute of Dental and Craniofacial Research. The government has certain rights in the invention.

[0006] Sequence Listing Statement

[0007] A computer readable form of the Sequence Listing is filed with this application by electronic submission and is incorporated into this application by reference in its entirety. The Sequence Listing is contained in the file created on November 20, 2025, having the file name “24-1583-WO” and is 43,151 bytes in size.

[0008] Background

[0009] The angiopoietin-Tie2 pathway regulates blood vessel stability, remodeling, and permeability. Angiopoietins (Angl and Ang2) are the natural ligands for Tie2. Angl promotes pAKT activation and blood vessel stabilization, while Ang2 inhibits pAKT and induces leaky vasculatures despite sharing nearly identical protein structures. Previously, we demonstrated that angiopoietin-1 and -2 activities fall into two broad phenotypic classes distinguished by the number of presented Angl F-domains that describe the mechanism of Tie2 signaling output9. We found that ligands presenting six or more copies of F-domains behaved like Angl and activated pAKT while trimer ligands behaved like Ang2 and inhibited pAKT9. Interestingly, we also found high F-domain valency ligands, like Angl, accelerated cell migration and promoted vascular stability9. It is unclear how Tie2 performs these opposing cellular functions. In the present study, we investigated the formation and function of these Tie2 complexes. We utilized de novo designed, self-assembling, two-dimensional protein sheets conjugated with F-domains (Fd-sheet) to produce large Tie2 clusters for analyzing Tie2 complexes that drive cell migration or vascular stability.

[0010] Both Tie2 and a5pi have been implicated as critical vascular stability modulators. Angl, a5, and tight junction molecule (claudin-5 and ZO1) protein expressions were upregulated in a timely manner at the same rupture sites in mice that received an induced-ischemic brain injury, suggesting that Tie2 and a pi may be essential for maintaining the BBB integrity' post-injury. However, it is unclear how these proteins interact at the molecular level. Contrary, α5β1 was also argued to inhibit vascular stability such that the inhibition of a5 promoted a tighter blood-brain barrier via the upregulation of CLDN5 expression.

[0011] Disruption of the a5 and p i heterodimers has been shown to enrich E-Cadherin (CD144) and ZO1 at cell-cell j unctions. These contradicting results illustrated a big knowledge gap regarding the role α5β1 in Tie2 signaling for vascular integrity.

[0012] Summary

[0013] In a first aspect, the disclosure provides polypeptides comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 1-17, not including any amino acid insertions at identified insertion sites (i.e., any insertions are not considered when determining percent identity to the reference polypeptide), and not including residues noted as dispensable in Tables 2-7, wherein the polypeptide binds to Tie2.

[0014] In some embodiments the polypeptides comprise an amino acid sequence at least 75% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 1-17, not including any amino acid insertions at identified insertion sites, and not including residues noted as dispensable in Tables 2-7. In other embodiments, the polypeptides comprise an amino acid sequence at least 90% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 1-17, not including any amino acid insertions at identified insertion sites, and not including residues noted as dispensable in Tables 2-7.

[0015] In other embodiments, the polypeptides comprise an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 1-17, not including residues noted as dispensable in Tables 2-7.

[0016] In further embodiments, the polypeptides comprise an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 1-17.

[0017] In another embodiment, the polypeptides comprise an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 1-4, residues 10-88. In another embodiment, the polypeptides comprise an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 1-4. In one embodiment, substitutions relative to SEQ ID NO: 1-4 are selected from substitutions listed in Options 1 or 2 of Table 2; in a further embodiment, substitutions relative to SEQ ID NO: 1-4 are selected from substitutions listed in Option 1 of Table 2.

[0018] In another embodiment, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or all 22 identified interface residues are identical (not substituted), or conservatively substituted, relative to SEQ ID NO: 1-4. In another embodiment, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or all 30 core residues are identical (not substituted), or conservatively substituted, relative to SEQ ID NO: 1-4. In a further embodiment, all identified interface residues and core residues are identical (not substituted), or conservatively substituted, relative to SEQ ID NO: 1-4. In one embodiment, the polypeptide comprises an insertion in one or more insertion sites relative to SEQ ID NO: 1-4. In other embodiments, the polypeptides do not comprise an insertion in one or more insertion sites relative to SEQ ID NO: 1-4.

[0019] In another embodiment, the polypeptides comprise an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO:5-8, residues 34-88. In one embodiment, the polypeptide comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 5-8. In another embodiment, substitutions relative to SEQ ID NO:5-8 are selected from substitutions listed in Options 1 or 2 of Table 3. In a further embodiment, substitutions relative to SEQ ID NO:5-8 are selected from substitutions listed in Option 1 of Table 3.

[0020] In one embodiment, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or all 22 identified interface residues are identical (not substituted), or conservatively substituted, relative to SEQ ID NO:5-8 In a further embodiment, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or all 29 core residues are identical (not substituted), or conservatively substituted, relative to SEQ ID NO:5-8. In one embodiment, all identified interface residues and core residues are identical (not substituted), or conservatively substituted, relative to SEQ ID NO:5-8. In a further embodiment, the polypeptide comprises an insertion in one or more insertion site relative to SEQ ID NO:5-8. In another embodiment, the polypeptides do not comprise an insertion in one or more insertion site relative to SEQ ID NO:5-8.

[0021] In one embodiment, the polypeptide comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:9, residues 38-76. In another embodiment, the polypeptide comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:9. In a further embodiment, substitutions relative to SEQ ID NO: 9 are selected from substitutions listed in Options 1 or 2 of Table 4. In one embodiment, substitutions relative to SEQ ID NO:9 are selected from substitutions listed in Option 1 of Table 4.

[0022] In one embodiment, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or all 22 identified interface residues are identical (not substituted), or conservatively substituted, relative to SEQ ID NO:9. In another embodiment, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or all 26 core residues are identical (not substituted), or conservatively substituted, relative to SEQ ID NO:9. In a further embodiment, all identified interface residues and core residues are identical (not substituted), or conservatively substituted, relative to SEQ ID NO:9. In one embodiment, the polypeptides comprise an insertion in one or more insertion site relative to SEQ ID NO:9. In another embodiment, the polypeptides do not comprise an insertion in one or more insertion site relative to SEQ ID NO:9.

[0023] In one embodiment, the polypeptides comprise an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 10-11, residues 11-74. In another embodiment, the polypeptides comprise an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 10-11. In a further embodiment, substitutions relative to SEQ ID NO: 10-11 are selected from substitutions listed in Options 1 or 2 of Table 5.

[0024] In one embodiment, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or all 17 identified interface residues are identical (not substituted), or conservatively substituted, relative to SEQ ID NO:10-11. In another embodiment, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or all 13 core residues are identical (not substituted), or conservatively substituted. relative to SEQ ID NO:10-11. In a further embodiment, all identified interface residues and core residues are identical (not substituted), or conservatively substituted, relative to SEQ ID NO: 10-11. In one embodiment, the polypeptides comprise an insertion in one or more insertion site relative to SEQ ID NO: 10-11. In another embodiment, the polypeptides do not comprise an insertion in one or more insertion site relative to SEQ ID NO: 10-11

[0025] In one embodiment, the polypeptide comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 12-13, residues 28-95. In another embodiment, the polypeptide comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 12-13. In a further embodiment, substitutions relative to SEQ ID NO: 12-13 are selected from substitutions listed in Options 1 or 2 of Table 6. In one embodiment, substitutions relative to SEQ ID NO: 12-13 are selected from substitutions listed in Option 1 of Table 6.

[0026] In one embodiment, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or all 19 identified interface residues are identical (not substituted), or conservatively substituted, relative to SEQ ID NO: 12-13 In another embodiment, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or all 29 core residues are identical (not substituted), or conservatively substituted, relative to SEQ ID NO: 12-13. In a further embodiment, all identified interface residues and core residues are identical (not substituted), or conservatively substituted, relative to SEQ ID NO: 12-13. In one embodiment, the polypeptides comprise an insertion in one or more insertion site relative to SEQ ID NO: 12-13. In another embodiment, the polypeptides do not comprise an insertion in one or more insertion site relative to SEQ ID NO: 12-13,

[0027] In one embodiment, the polypeptide comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 14-17, residues 29-91. In another embodiment, the polypeptide comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 14-17. In a further embodiment, substitutions relative to SEQ ID NO: 14-17 are selected from substitutions listed in Options 1 or 2 of Table 7. In one embodiment, wherein substitutions relative to SEQ ID NO:14-17 are selected from substitutions listed in Option 1 of Table 7.

[0028] In one embodiment, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or all 21 identified interface residues are identical (not substituted), or conservatively substituted, relative to SEQ ID NO: 14-17. In another embodiment, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or all 28 core residues are identical (not substituted), or conservatively substituted, relative to SEQ ID NO: 14-17. In a further embodiment, all identified interface residues and core residues are identical (not substituted), or conservatively substituted, relative to SEQ ID NO: 14-17. In one embodiment, the polypeptides comprise an insertion in one or more insertion site relative to SEQ ID NO: 12-13. In another embodiment, the polypeptides do not comprise an insertion in one or more insertion site relative to SEQ ID NO: 12-13.

[0029] In another embodiment, the disclosure provides fusion proteins, comprising a polypeptide of any embodiment or combination of embodiments herein, fused to one or more functional domains, optionally wherein the polypeptide and the one or more functional domains are linked by an amino acid linker.

[0030] In a further embodiment, the disclosure provides conjugates, comprising:

[0031] (a) the polypeptide of any embodiment or combination of embodiments herein; and

[0032] (b) a scaffolding moiety.

[0033] In one embodiment, the disclosure provides fusion proteins, comprising;

[0034] (a) the polypeptide of embodiment or combination of embodiments herein; and (b) a protein scaffolding moiety;

[0035] optionally wherein the polypeptide and the protein scaffolding moiety

[0036] In another embodiment, the disclosure provides scaffolds, comprising the conjugate or fusion protein of any embodiment herein, wherein the scaffold comprises 6 7, 8, 9, 10, or more copies of the polypeptide of any embodiment or combination of embodiments herein.

[0037] In another aspect the disclosure provides nucleic acids encoding the polypeptide or fusion protein of any embodiment or combination of embodiments of the disclosure. In a further aspect, the disclosure provides expression vectors comprising the nucleic acid of any aspect of the disclosure operatively linked to a suitable control sequence, such as a promoter. In another aspect, the disclosure provides host cells that comprise the polypeptide, fusion protein, scaffold, nucleic acid or expression vector (i.e.: episomal or chromosomally integrated) disclosed herein, wherein the host cells can be either prokaryotic or eukaryotic. In another aspect, the disclosure provides pharmaceutical compositions, comprising the polypeptide, fusion protein conjugate, scaffold, the recombinant nucleic acid, the expression vector, or the recombinant host cell of any of any embodiment or combination of embodiments, and a pharmaceutically acceptable carrier. In a further aspect, the disclosure provides methods for using, or a use of the polypeptide, conjugate, fusion protein, nucleic acid, expression vector, host cell, and / or the pharmaceutical composition of any of the preceding claims, for any suitable purpose including but not limited to those disclosed herein. In various embodiments, the purpose includes, but is not limited to, treating or limiting development of limit development of diabetic vasculopathy, vascular dysfunctions, cancer angiogenesis, and wound healing in a subject in need thereof.

[0038] Description of the Figures

[0039] Figure 1. Two classes of Tie2 complexes. A) F-domain fused with SpyTag™ (abbreviated Fd-st) and GFP-Spytag™ (GFP-st) were conjugated to two-dimensional protein sheets to make Fd-sheets for clustering Tie2 receptors on serum starved HUVECs for 30 minutes before fixation for immunofluorescence staining. B) Super-resolution OMX images of immunofluorescence staining (left 3 panels) and Imaris 3D reconstruction (right panels) of Tie2 colocalizing with a5, pCas, ZO1, or claudin-5 (CLDN5) forming two clusters of Tie2 complexes: Tie2-a5 / pCAS and Tie2-Z01 / CLDN5. C-F) Quantifications of the Tie2 complexes under the Fd-domain sheets. G) Super-resolution OMX images of immunofluorescence staining images of HUVECs pre-treated with siRNA against ITGA5 before starvation and Fd-sheet administration. H-J) Quantifications showing the reduction of Tie2-a5 complex upon knockdown of a5, but the formation of Tie2-Z01 / CLDN5 complexes were not inhibited, two-tailed t-test was performed to evaluate the level of statistical significance, p-value < 0.05 is annotated with *, p-value < 0.0001 is annotated with ****, and ns means not significant. At least 30 sheets were counted in each biological replicate, each data point is a biological repeat for all experiments.

[0040] Figure 2. Tie2 minibinder tw1102. A) The design model of tw1102 (Tmb) in complex with the entire Tie2 molecule (PDB 2GY5) showing the binding mode at the same location as Angiopoietin. B) Close up of the tw1000 (tw1102's parent design) designed complex showing the per-position sequence entropy from the SSM experiment. A scale shows the variability of each position within the SSM data (from the 500nM Sort). C). The SSM heatmap for all buried interface residues of tw1000. This maps shows the results of the SSM YSD experiment as quantified by SC50 (the concentration where 50% of the expressing cells are collected). The majority of the core positions have strong preferences that "make sense" when viewed by hand and the positions that showed better affinity were selected for the combinatorial optimization process. D) Size exclusion chromatography of the tw1102 minibinder shows a mono-dispersed peak. E) Surface plasmon resonance analysis was performed to estimate the affinity of tw1102 for Tie2 receptors. KD= 0.65 nM. F) Flow cytometry was performed on the wildtype or Tie2 knockout EA.hy926 cell lines to asses tw1102 binding capacity.

[0041] Figure 3. De novo designed Tie2 mini binder at different valency exhibit super agonistic and antagonistic properties. A) Structural prediction using RosettaFold™ of Tie2 mini binder, Tmb overlayed with Ang1 F-domain binding to the Ig2 domain on a Tie2 receptor. B) Quantification showing competition experiment of H8F vs Tmb in which pAKT activity was attenuated in the presence of Tmb. Serum starved HUVECs treated with 10 nM of H8-Fd with or without Tmb at 0.1-1000 nM before protein lysate collection for pAKT and S6 analysis using western blot. pAKT signals were quantified and normalized to S6, then all samples were normalized to H8F samples) (C) H8-SpyCatcher™ conjugated with Tmb-SpyTag™ (H8T) activated pAKT in serum starved HUVECs. pAKT signals were quantified and normalized to S6, then all samples were normalized to H8F samples. Quantifications in Fig 8E were re-plotted and fitted using FindEC anything in GraphPad Prism to estimate the EC50. D) Confocal images of immunofluorescence staining of FOXO1 in serum starved HUVECs stimulated with H8F + / - Tmb, H8T, or PBS. E) Quantification of FOXO1 images, at least 100 cells were counted in each biological replicate. One-way anova with bonferroni post hoc test were used to analyze level of significance, / ?-value < 0.001 is annotated *** and p-value < 0.0001 is annotated ****. Tmb-st were conjugated to GFP tagged sheets for figures F-G. Quantifications of Tie2 colocalizing with F) a5 or G) Claudin-5 under the Tmb sheets in serum starved HUVECs. H) FACS binding analysis of GFP sheets, Fd-sheets, and Tmb-sheets on MCF10A cells, which do not express Tie2, demonstrating the promiscuity of F-domain binding.

[0042] Figure 4. Tie2 forms different complexes to regulate cell migration and tight junctional assembly. A) Competition experiments of serum starved HUVECs were treated with 10 nM of H8F with or without 1μM of α5β1mb and protein lysates were collected for western blot staining to analyze pAKT activity. B) Quantification of western gels with all samples’ pAKT signal normalized to actin and then normalized to H8F samples. C) Confocal images of FOXO1 immunofluorescence staining of serum starved HUVECs treated with 50 nM of H8F + / - 1μM of α5β1mb or PBS or HUVECs pre-treated with siRNA before starvation and H8F administration. D) Quantification of FOXO1 images, at least 100 cells were counted in each biological replicate. E) Schematic of scratch assay in which a wound was made in the middle of a monolayer of HUVECs before adding H8F + / - a5plmb for 12 hours. F) Representative brightfield images of scratched area at 0 and 12-hour time point for cells treated with lOnM of H8F + / - 500 nM of a5plmb. G) Images of cells at 0 and-12 hour timepoints were quantified to evaluate the level of cell migration. Each data point is a biological replicate. H) Schematic of tight junction disruption using latrunculin-A (latA). I) Confocal images of claudin-5 (CLDN5) immunofluorescence staining of HUVECs after latA washout and recovery with or without H8F addition. J-K) Quantification showing the percentage of cells stained with a junctional ZO1 or claudin-5 lining, at least 100 cells were counted in each biological replicate. One-way anova with bonferroni post hoc test were used to analyze level of significance, p-value < 0.05, 0.01, 0.0001 are annotated *, **, ****, respectively.

[0043] Figure 5. Activating Tie2 rescues diabetic vasculopathy. A) Schematic of differentiating iPSCs into 3D blood vessels organoids (BVOs) and treating day 23 mature BVOs with high glucose and cytokines to induce diabetic condition. Day 37 BVOs were then fixed and stained with antibodies for confocal imaging. B) BVOs were stained with PDGFRb, (VE-Cadherin) CD 144, and Dapi, C) day 37 BVOs’ confocal images were processed using Imaris 3D reconstruction. D) Representative images showing level of Col-IV expression under different conditions. E) Representative images of BVOs stained with ZO1 and FOXO1 antibody under different conditions, F) Quantifications of percentage of cells in BVOs that expressed nuclear FOXO localization, 200-300 cells were counted in each BVO from multiple Z-stack, each data point is one BVO. G) Representative images of day 37 BVOs were stained with ZO1, Claudin-5, and Dapi for tight junction organization assessment, H) Quantification of junctional ZO1 integrity using TiJOR56method. One-way anova with bonferroni post hoc test were used to analyze level of significance, -value < 0.05, 0.01, 0.001, 0.0001 is annotated *, **, *** and ****, respectively.

[0044] Figure 6. A) Serum starved HUVECs were treated with 20 nM of Fdsheet-GFP or sheet-GFP for 30 minutes before one PBS wash and then fixed with PFA for FACS analysis. FITC were used to detect GFP signal in both samples. B) Quantification summarizing the percentage of HUVECs that are bound with sheet-GFP + - F-domains in FACS experiments. C) Summary of Tie2 clusters found under Fdsheet over many experiments, including experiments shown in figure 1. Each count represents one Fdsheet. D-E) Representative confocal images of Fdsheet cluster Tie2 receptors that recruit and activate integrin shown by the presences of active pi subunit not inactive pi staining. F-G) Quantification of Tie2-activepl or Tie2 -inactivepl colocalization under the Fdsheet. H-I) Representative OMX images and quantification of Tie2-occludin localization under the Fdsheet. J) Representative confocal images of Tie2 colocalizing with either activepi or claudin-5 under the Fdsheet using 5 color immunofluorescence imaging. K) Quantification of panel J summarizing the different Tie2 complexes found under the Fdsheet. L-M) Western blot staining of HUVECs pre-treated with 40nM of siRNA against ITGA5 or TEK to knockdown (KD) α5 integrin or Tie2 expression, respectively before starvation for Fdsheet experiments. N) Quantification showing the reduction of Fdsheet binding to Tie2 KD compared to wildtype, a5 KD did not affect Fdsheet binding to HUVECs. O) Quantifications showing the reduction of Tie2-a5 cluster and the significant increase in a5-only cluster under the Fdsheet in Tie2 KD compared to wildtype. P) OMX images of Tie2-CD144 colocalization in wildtype (top) vs a5 KD (bottom) cells. Q) Quantifications showing the percentage of Tie2-CD144 colocalization under the Fdsheet in wildty pe and R) the significant reduction of Tie2-CD144 colocalization under the Fdsheet in o5 KD cells. S) FACS analysis showing Fd-sheet binding to MCF10A cells is inhibited when increasing concentrations of α5β1 minibinder are added.

[0045] Figure 7. A) Representations of the parent design for each family of computationally designed Tie2 minibinders. B) Table with the measured KD values (nM) obtained from Surface Plasmon Resonance experiments conducted with each of the top candidates for Tie2 binders. C) Site Saturation Mutagenesis (SSM) data from the parent binder (tw1000) of the final binder candidate tw1102, The letter in each column show's the original sequence of the parent binder and the scaled boxes shown which residues were more favored from the SSM sort. D-E) SSM charts of the parent twTOOO binder with the core and surface residues selected out.

[0046] Figure 8. A) Representative western blot staining of pAKT and pERK signaling in competition experiment between of H8F at 10 nM + / - Tmb at 1-1000 nM in serum starved HUVECs. B) Quantification of pAKT / S6 level in competition experiments normalized to10 nM of H8F samples as positive control. C-D) Representative images of pAKT activation upon 10 nM of H8F, H8T or H3T administration in serum starved HUVECs. E) Quantification of pAKT / S6 level upon H8T treatment normalized to H8F samples. F) Immunofluorescence images of FOXO1 staining in HUVECs treated with H8T. G-H) Quantifications of percentage of Tie2-ZO1 or Tie2-pCAS colocalization under the Tmb-sheets in HUVECs. I-J) Western blot staining showing Tmb does not interact with other RTKs, thus Tmb was not able to inhibit FGF or NGF ligand activity in their respective receptor overexpression CHO lines.

[0047] Figure 9. A) Western blot images of pAKT inhibition upon treating with H8F and α5β1mb in HUVECs. B) Western blot images of pAKT activation of HUVECs treated with 10 nM of H8F + / - αvβ6mb, αvβ8mb. or fibronectin (FN) at 100-1000 nM. C) Quantifications of pAKT / S6 level normalized to 10 nM of H8F in competition experiments from B. D-E) Immunofluorescence images of FOXO1 staining upon α5β1mb administration, F) Quantification of nuclear FOXO1 in HUVECs upon the administration with Icos1 + / - avp6mb or αvβ8mb. G) Brightfield images of HUVECs at 0 and 12 timepoints in scratch assay with 20 nM of H8F or Icos1 + / - FN, α5β1mb, αvβ6mb, or avp8mb at 20 nM. H) Quantifications of cell migration assay after 12 hours.

[0048] Figure10. A) Confocal images of day 32 BVOs at different conditions stained with PDGFRP, Col-IV, CD144, and Dapi. B) Confocal images of day 32 healthy BVOs treated with C6 or bFGF and stained for CD144 (VE-Cad) and EPHRIN2B to mark arterial blood vessels. C) Confocal images of FOXO1 stains in day 32 arterial BVOs under different conditions. D) Quantifications of nuclear FOXO per cells in each condition.

[0049] Detailed Description

[0050] All references cited are herein incorporated by reference in their entirety. Within this application, unless otherwise stated, the techniques utilized may be found in any of several well-known references such as: Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press), Gene Expression Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, San Diego, CA), “Guide to Protein Purification” in Methods in Enzymology: (M. P. Deutshcer, ed., (1990) Academic Press, Inc.); PCR Protocols: A Guide to Methods and Applications (Innis, et al. 1990. Academic Press, San Diego, CA), Culture of Animal Cells: A Manual of Basic Technique, 2ndEd. (R. I. Freshney. 1987. Liss, Inc. New York, NY), Gene Transfer and Expression Protocols, pp, 109-128, ed, E. J. Murray, The Humana Press Inc,, Clifton, N. J,), Dang, B. et al. SNAC-tag for sequence-specific chemical protein cleavage. Nat. Methods 16, 319- 322 (2019), and the Ambion 1998 Catalog (Ambion, Austin, TX).

[0051] As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

[0052] As used herein, the amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gin; Q), glycine (Gly; G), histidine (His: H), isoleucine (He; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Vai; V).

[0053] Any N-terminal methionine residue in any polypeptide of the disclosure may be present or may be deleted. In all embodiments of the polypeptides disclosed herein, 1, 2, 3, 4, or 5 residues may be deleted from the N-terminus and / or the C-terminus of the polypeptide while retaining activity.

[0054] All embodiments of any aspect of the disclosure can be used in combination, unless the context clearly dictates otherwise.

[0055] Unless the context clearly requires otherwise, throughout the description and the claims, the words ‘comprise’, ‘comprising’, and the like are to be construed m an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”. Words using the singular or plural number also include the plural and singular number, respectively. Additionally, the words “herein,” “above,” and “below” and words of similar import, when used in this application, shall refer to this application as a whole and not to any particular portions of the application.

[0056] In a first aspect, the disclosure provides polypeptides comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 1-17, not including any amino acid insertions at identified insertion sites (i.e., any insertions are not considered when determining percent identity to the reference polypeptide), and not including residues noted as dispensable in Tables 2-7, wherein the polypeptide binds to Tie2.

[0057] Angiopoietin-1 receptor also known as CD202B is a protein that in humans is encoded by the TEK gene. Also known as TIE2, it is an angiopoietin receptor. The full name for Tie2 protein is Tyrosine Kinase with Immunoglobulin-Like and Epidermal Growth Factor-Like Domains 2 (TIE2), The amino acid sequences for SEQ ID NO: 1-17 are provided in Table 1.

[0058] Table 1

[0059] > ie2__twl 666: ’ SBEEQIILVEEAIEKE TKILEELAKKYGEKMKE

[0060]

[0061] LKLTGDEEAALELAKLSAKLFEYAGDADGAVKALKQSGIGEEAEKIAEEIRKKAA SEQ ID NO:1 > Tie2_twllO2 SEEEQKLVEEAIEKLTKILEELAKKYGEKMKEPKEYYLRMSEKIKKNEQPEQDMAILIHNAGKEV LKLTGDEEAALELAKLSAKLFQYAGDTDGAVRALKQSGIGEEAEKIAEEIRKKAA SEQ ID NO: 2 >Tie2_tw1202 SEEEQKLVEEAIEKL LKVTGDEEAALELAKLSAKLFQYAGDTAGAVRALKQSGIGEEAEKIAEEIRKKAA SEQ ID NO: 3 >Tie2_tw1301 SEEEQKLVEEAIEKLTKILEELAKKYGEKMKEPKEYYHRMSEKIKKNEQAEQDMAILLHNACKEV MKLTGDEEAALELAKLSAKLFEYAGDTDGAVRALKQSGIGEEAEKIAEEIRKKAA SEQ ID NO:4 >Tie2_tw2000 DRLREIIELAREAAEEGLSPAVAALAARRATGD GGSSVRHQDIALAVGRAVLYRLRGDEEEAEFYEKLALKIAKREEDRKLVKEILEE SEQ ID NO: 5 >Tie2_tw2101 DRLREIIELAREAAEEGQSPEEASRRARRATGNGVAAIIVHLMAHAGGENAERVARVVWETSERL GGSSEKHQDIALTVGRAVLYRLRGDEEEAEYYEKLALKIAKREEDRKLVKEILEE SEQ ID NO: 6 > Tie2_tw2106 DRLREI lEIAREAAEEGLSPELASRRARRATGESGVAAl’lVHLMAHAGGENAERVARW GGSSERHQDIALTVGRAVLYRLRGDEEEAEYYEKLALKIAKREEDRKLVKEILEE SEQ ID NO: 7 >Tie2_tw2202 DRLREIIELAREAAEEGLSPAEAALRARRATGNGVAAIIAHLMAHAGGENAERVARVVWETSERL GGSSERHQDIALTVGRAVLYRLRGDEEEAEYYEKLALKIAKREEDRKLVKEILEE SEQ ID NO: 8 > Tie2__tw3000 NSEKVLKFIAEEVAKKTGSETAKKVLENIKEDIKNG LDRDADVLE VYVYGRKLYEETGS EE I RKAAEAETLAVALGR I TAEEALKRLEELV SEQ ID NO: 9 >Tie2_tw4000 SEEEKERGEKMVEQYAENLRKLAEEYIERGEPPEEILRRIEKEAEAYLEDLETIFEGSELKEEIL ELAEKKFEEVKEEVEERL SEQ ID NO: 10 >Tie2_tw4104 SEEEKERGEKMVEQYSENLRKTAAENIDRGEPPEEIWRRRIEKEAEAFLEDLETFFEGRELKEEIL ELAEKKFEEVKEEVEERL SEQ ID NO:11 >Tie2_tw5000 GVEEPIRELEEYGRRRIERVEKALPGNRLAVLAMRSLAEHLIARVRYAAERGKDVEGLVEAAKLA LDVAEAAILKNGLVPGDVADKVLLAFREAEEDPKHAKERLEELKEEV SEQ ID NO: 12

[0062]

[0063] > Tie2__tw5104 GVEEPIRELEEYGRRRIERVEKALPGNRLAVESTRSLAKHFISRVRYDAERGKDVEGEVEAAKLA LDVADAAIHKNGLVPGDVADKVLLAEREAEEDPKHAKERLEELKEEV SEQ ID NO: 1.3 >Tie2_tw6000 GSEELIEELERYGKEVIERIKKALPGNRLAVLAMESFVEHLLKLLEYAAEKGLDVEGITKVAKLG LEVFEKAILKNGSVPGDIADKVLLAVREAEKDPEEALKRLEEILKEV SEQ ID NO:14 > Tie2_tw6103 GSEELIEELERYGKEVIERIKKALPGNRMGVLAMESFVKHLLKLLEYAAEKGLDVEGITKVDKLG LEVFEKAILKNGSMPGDIADKVLLAMREAEKDPEEALKRLEEILKEV SEQ ID NO: 15 > Tie2__tw610b GSEELIEEEERYGKEVIERIKKALPGNRLGVLAMQSFVKHLRKSLKYAAEKGLDVEGITKVAKLG LEVFEKAIHKNGSVPGDIADKVLLAVREAEKDPEEALKRLEEILKEV SEQ ID NO: 16 >Tie2_tw6107 GSEELIEELERYGKEVIERIKKALPGNRMAVLSMQSFVEHLRKSLEYAAEKGLDVEGITKVDKLG

[0064]

[0065] LEVFEKANHKHGSVPGDIADKVLLAVREAEKDPEEALKRLEEILKEV SEQ ID NO:17

[0066] As a monomer, or when conjugated to a scaffold with low valency at 5 or less, the polypeptides act as antagonists of Tie2 signaling and downstream pAKT / FOXOl at high efficiency, and can be used, for example to treat vascular leakiness such as diabetic vasculopathy described in the examples that follow and figure 5. When the polypeptides are conjugated onto a scaffold, they act as agonists (when present at a valency of 6 or more) to activate the pAKT / FOXOl pathway and promote tight junction re-assembly and can be used, for example, to treat or limit development of vascular dysfunctions, cancer angiogenesis, and wound healing,

[0067] Tables 2-7 annotate residues that are dispensable for activity in the polypeptides of SEQ ID NO: 1 -17. These residues were predicted to be dispensable because they do not contribute significantly to the protein interface or fold. The protein that remains once these residues are removed still appears to be a strong Tie2-binding protein. Thus, in some embodiments, all of the dispensable residues are deleted. For example, Table 2 shows that, relative to SEQ ID NO: 1-4, residues 1-9 and 89-120 are dispensable for activity in binding Tie2. Thus, in some embodiments, the polypeptide comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 1-4, residues 10-88. It will be well with the level of skill in the art to understand other such embodiments in view of the Tables provided herein.

[0068] Similarly, the polypeptides may comprise an insertion in one or more insertion sites of the polypeptide. The insertion may be any one or more amino acid, and may comprise a functional domain as described herein, or one or more amino acids for additional spacing or for any other purpose. Tables 2-7 identify the position of possible insertion sites relative to SEQ ID NO: 1-17.

[0069] In some embodiments the polypeptides comprise an amino acid sequence at least 75% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 1-17, not including any amino acid insertions at identified insertion sites, and not including residues noted as dispensable in Tables 2-7, In other embodiments, the polypeptides comprise an amino acid sequence at least 90% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 1-17, not including any amino acid insertions at identified insertion sites, and not including residues noted as dispensable in Tables 2-7.

[0070] In other embodiments, the polypeptides comprise an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 1-17, not including residues noted as dispensable in Tables 2-7. In this embodiment, no insertions are present within the polypeptides at insertion site noted in Tables 2-7.

[0071] In further embodiments, the polypeptides comprise an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 1-17. In these embodiments, dispensable residues identified in Tables 2-7 are present in the polypeptides.

[0072] In another embodiment, the polypeptides comprise an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 1-4, residues 10-88. In another embodiment, the polypeptides comprise an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 1-4. In one embodiment, substitutions relative to SEQ ID NO: 1-4 are selected from substitutions listed in Options 1 or 2 of Table 2; in a further embodiment, substitutions relative to SEQ ID NO: 1-4 are selected from substitutions listed in Option 1 of Table 2. Site saturation mutagenesis studies were conducted on Tie2_twl 000 (SEQ ID NO: 1), and Table 2 provides a list of exemplary substitutions (Option 1; increased or maintained activity), and tolerable substitutions (Option 2: retained activity, though at a lower level than SEQ ID NO:1), that in this embodiment can be incorporated into any of SEQ ID NO: 1-4 while retaining activity. Tables 3-7 provide similar information relative to other reference sequences.

[0073] In another embodiment, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or all 22 identified interface residues are identical (not substituted), or conservatively substituted, relative to SEQ ID NO: 1-4. Interface residues (i.e., at the binding interface between the polypeptide and the Tie2) relative to SEQ ID NO: 1-4 are provided in column 4 of Table 2.

[0074] As used throughout the application, conservative amino acid substitutions involve replacing a residue by a residue having similar physiochemical characteristics, e.g., substituting one aliphatic residue for another (such as Ile, Val, Leu, or Ala for one another), or substitution of one polar residue for another (such as between Lys and Arg; Glu and Asp; or Gin and Asn). Other such conservative substitutions, e.g., substitutions of entire regions having similar hydrophobicity characteristics, are known. Amino acids can be grouped according to similarities in the properties of their side chains (in A, L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)): (1) non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gin (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (H). Alternatively, naturally occurring residues can be divided into groups based on common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation:

[0075] In another embodiment, at least I, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or all 30 core residues are identical (not substituted), or conservatively substituted, relative to SEQ ID NO: 1 -4. Residues that are present at the polypeptide core are shown in column 5 of Table 2, These residues help maintain structure of the polypeptide.

[0076] In a further embodiment, all identified interface residues and core residues are identical (not substituted), or conservatively substituted, relative to SEQ ID NO: 1-4.

[0077] In one embodiment, the polypeptide comprises an insertion in one or more insertion sites relative to SEQ ID NO: 1-4. Table 2, column 6 shows the position of potential insertion sites relative to SEQ ID NO: 1-4. The insertion may be any one or more amino acids. In other embodiments, the polypeptides do not comprise an insertion in one or more insertion sites relative to SEQ ID NO: 1-4. Table 2: Relative to SEQ ID NO: 1-4

[0078] pos Opt ion 1 Opt ion 2 at__inte prote loop / i di sp it i rface in__co ins er | ensa on re t ion i bl e N, T, S P, G, Q, I i X P, A, K, I-I, S, G, V, T, L, W, 0 1 X N, I, M, E

[0079] 3 W, N, E H, P, S, I, Q, Y, R, G i X 4 D E P, W, Q 1 X 5 G, W, A, L, S, R, T, D, 1 X F, K, E, M, Y, Q, I-I, I,

[0080] N, V, P Ft- 6 Y, L, M, W, R, Q, D, V, ii X K, T, E, A, H

[0081] 7 V, T, W, R, I, P, L Y, E, K, H, M, Q, N i x 8 Y, F, A, W, V 1, L, P, S X 1 x 9 F, N, H, E P, R, V, T, A, O L ) ni ii X 10 K, R, T, S, N, E Q c Q

[0082] 11 S, H, A o £* X

[0083] 12 T, I £

[0084] 13 F, L, Q, E Y, N, I, D, > E iQ i iS CiI--, I-I, G

[0085] 14 I, F, H, L, Y, Q, N, S, Q

[0086] Pi - K, M, V, A, E o []X:: '

[0087] - 15 P, V, T, G, S, A, L D, N J X

[0088] K - 16 L, M, V, I, N, K, A, T > ~

[0089] 17 H, L, N, K M, T, E, W, R

[0090] 18 W, A, I F, V, D, G, Q, K, Y, S X

[0091] 19 I, V, L P, H, Q, M, W, F X

[0092] 20 0, L, N, W, R, A W, E K, H, M, F, Y, V

[0093] 21 W, I, I-I, R, oE ' D, S, N, F, A, Q, K

[0094] 22 V, I, L XI C K, G, F, R, E, Q X

[0095] 23 I, A £ Q, P, M, L, V, R, K, Y, F

[0096] , W, H, N, E, G, T, D, S

[0097] 24 £ P, R, K I, W, F, Y, M, H, G, Q, T

[0098] 25 P, W, K R, I, D, V, L, G, T, A, N

[0099] , Q, F, E, S

[0100] 26 Q, I, V, Y

[0101] 27 x

[0102] G

[0103] 28 D, P, E F

[0104] 2 9 L, I, F, S, V, Y, K

[0105] 3 0 Y, H, I, V, T, P, F, L, N, S, A, G, D, Q X

[0106] M

[0107] 3 1 L, D, K Y, P, N, I, F, H, T, V, M

[0108] , G, W, A

[0109] 32 G, P, E D

[0110] 3 3 P, G, A, S, T V, E, D, X

[0111] 34 I, K L, E), P, H, Q, V, N, E, W

[0112] , R, T, Y, S, M

[0113] 3 5 P, E I-I, R, Y, S, G, T, 0, V, K

[0114] , W, A, F, L, N, I

[0115] 3 6 Y X

[0116] 3 7 R, Q, L, N X

[0117] 3 8 W, P, H, G, S, Y, R, T, F, N, K, Q, I, M, L

[0118] A

[0119]

[0120] 3 9 R P, T, G, H, S, K

[0121]

[0122] 93 1 A, M Q, E, N, S, W, H, P, L, D 1 X 1 X 94 1 R, W, A, G, S V 1 X 1 X 95 I A 1 X 96: I, V 1 X 9'7: R Q, A, N, K, H 1 X X 98 1 A 1 X X 99 1 L, W 24 X 100 I Y, K R, H 1 X 101 i A, S, Q r x i X i X 102: L, T, S V X X X 103 1 G 1 X. 24 104 1 L, I I H 24 24 X 105: G: X 106 i A, K, M, 1, L, Q, E: P, G, R, Y, V X 107 i K, A, R, E: Q, I, L, H, P X 108 1 A 1 24 1 x 109 1 L, F, M, E i 1 x 110 | K I 1 X 111 1 L, I X 1 X 112 U, A:X 113 1 R, E W, Q, K 1 x 114 1 N, A, M, G, E, K, T, S, X

[0123] 1 R

[0124] 115 | I 24 X 116 1 K, R, I, L, M X 117 1 Y, Q, M, L, A, I, N, K, X

[0125] 1 R, S, H, V, T

[0126] 118 | Y, K, H, R X 119 1 W, 0, Y, I-I, F, N, S, A K, G, L, V, M, E, I, T 24 X

[0127]

[0128] 120 I R, G, F, W, T, Y, H, A S, K X

[0129] In another embodiment, the polypeptides comprise an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the ammo acid sequence selected from the group consisting of SEQ ID NO:5-8, residues 34-88. In one embodiment, the polypeptide comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO:5-8. In another embodiment, substitutions relative to SEQ ID NO: 5-8 are selected from substitutions listed in Options 1 or 2 of Table 3. In a further embodiment, substitutions relative to SEQ ID NO:5-8 are selected from substitutions listed in Option 1 of Table 3. Site saturation mutagenesis studies were conducted on Tie2__tw2000 (SEQ ID NO:5), and Table 3 provides a list of exemplary substitutions.

[0130] (Option 1; increased or maintained activity), and tolerable substitutions (Option 2: retained activity, though at a lower level).

[0131] In one embodiment, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or all 22 identified interface residues are identical (not substituted), or conservatively substituted, relative to SEQ ID NO:5-8. Interface residues (i.e., at the binding interface between the polypeptide and the Tie2) relative to SEQ ID NO:5-8 are provided in column 4 of Table 3. In a further embodiment, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or all 29 core residues are identical (not substituted), or conservatively substituted, relative to SEQ ID NO:5-8. Residues that are present at the polypeptide core are shown in column 5 of Table 3. These residues help maintain structure of the polypeptide. In one embodiment, all identified interface residues and core residues are identical (not substituted), or conservatively substituted, relative to SEQ ID NO: 5-8. In a further embodiment, the polypeptide comprises an insertion in one or more insertion site relative to SEQ ID NO:5-8. Table 3, column 6 shows the position of potential insertion sites relative to SEQ ID NO:5-8. In another embodiment, the polypeptides do not comprise an insertion in one or more insertion site relative to SEQ ID NO:5-8.

[0132] Table 3: Relative to SEQ ID NO:5-8

[0133] pos Opt: ion 1 Option 2 at_inter protei n | loop / | di sp it i fa ce _core I ins er I ens a on I t ion | ble M,F,G,S,I,D P,T,A,E,N,K,Q,Y H,K,R M,G,E 3 I, L Y, F i x 4 H, S, K, T, R, F Y, 0, G, N, V, A, L, E il x 5 Y,E A,F,M,H,R,L,I,S,G,W,K,Q,V,T,D G, W, K, Q, V, T, D

[0134] 6 V, L, I, M i x ‘7 M, V, I, T, W il x 8 R, I, K, E Q, D, S, W, T, N, G l x 9 V,F,M,H,I,L,R Q 10 G, T, A, V, I X | x 11 K, H, R Q 1 X 12 W, S, V, E Y, N, H, L, Q, F, M, I, 1 X R, A, T

[0135] 13 Y, F, A Q, W, T, H X I X 14 1, Y, L, A T, R, H, S, F, M, N, V, x |i x Q, W

[0136] 15 D, H, G, Q, R, N, K ii X, E, Y, A, L, S, I

[0137] 16 A, S, R, L, E D, G, F, Q, M, K, H, W |l X! X 17 R, G, K Q l x l x 18 V, Q, M, S K, Y, Li X 1 X 1 X 19 P,S,D,A,Q,H,T,N,E,W 20 p 1 X 21 V,G,E,L S,F,M,A,Q,D 22 Q, E, A, M, G, L, D R, Y, V, S, H X Il X 23 A X II X 24 S G, A X 1 x 25 R, A Y, H, G, V, W, M, Q, N, X 1 X

[0138]

[0139] T,K,L,S,F,I

[0140]

[0141] 73 Q X

[0142] 74 D X

[0143] 7 F i u T-, I, V X

[0144] 76 L Q o ', A - M, F, Y, V, I, S, T X

[0145] 77 Y, L X

[0146] 78 T L, V, A, S, Q X

[0147] 73 M, V PI X

[0148] 80 X

[0149] 81 X

[0150] 82, A Y, H

[0151] 83 T, V K, Q, I, S, E, A, G, R, X

[0152] M

[0153] 84 S, D

[0154] 85 Y Q

[0155] 86 H, R, K

[0156] 87 E, D, V, S, L G, M

[0157] 88 <2 X 8 9 K, R, G, A, E, ID, F X X, S, Q, L, H, M, Y

[0158] Q E1"

[0159] 90 N, D I, V X X ] <:

[0160] 91 V, K, L, Q, F, H, S X, E

[0161] 92 R, T o i' X Q hH- 93 E i PFP 01 I X X 94 R, A, F, T Q, > 0 iK? X 95 K o? PF I, H, S X 96 W, Y, F X X 97 Y D X X 98 W, T, Y, N, R, S, V L X, Q, E, F £, K, G

[0162] 99 T, A, N p? F, H, K, G, E X, D i PI, I, Q, R, V, L

[0163]

[0164] 10 0 l 0e! 0 Lt: X X ioi. A. 1. i. i ”x. ii. ii x 102 M, A, D, Y, L, Q, H Ii W, S, T i x 103 K, R, D, G Ii X 104 I, L' 1 L I X i x 10 5 T, A, V x I X I X 10 6 E, A, K |l Q, H, S, G, T, F, D, W, l x II X li I, Y, V, N, L, M, R, P

[0165] 10 7 E, Y, F, R l| K, V, H, A, N, D, T, G, Ii X |l X Il I, P

[0166] 10 8 K, G, I, Q, E |l V, F, L, H, T, R, S, M, 1 X ii A, P, N

[0167] 10 9 Ii X 11 0 H, K, T, D, E l| N, R i x 111 N, I, R, Y ii H, K, A, E, S, 0, F, T |l X 112 W, Q, M, L, K, G, R |l F, S ii X, E, N, Y, I, D, V

[0168] 11 3 T, V, A, L 1 R, Q, I, Y, H, K 1 x 114 V, I x l x 115 F, N, K 1 G, Q, H, T, W, E, R, M 1 X 116 Q, E |l S, T, D, F, G, 1 X i V, R, A

[0169]

[0170] 117 x l x 118 F, I, L M, V X 113 I-I, E F, K, Y, I, X T, Q, S, G, R

[0171] 120 S, D, W, K, H, N, E Q, L, R, M X

[0172]

[0173] , T

[0174] In one embodiment, the polypeptide comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:9, residues 38-76. In anotehr embodiment, the polypeptide comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:9. In a further embodiment, substitutions relative to SEQ ID NO:9 are selected from substitutions listed in Options 1 or 2 of Table 4. In one embodiment, substitutions relative to SEQ ID NO:9 are selected from substitutions listed in Option 1 of Table 4. Site saturation mutagenesis studies were conducted on Tie2__tw3000 (SEQ ID NO:9), and Table 4 provides a list of exemplary substitutions (Option 1; increased or maintained activity), and tolerable substitutions (Option 2: retained activity, though at a lower level).

[0175] In one embodiment, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or all 22 identified interface residues are identical (not substituted), or conservatively substituted, relative to SEQ ID NO:9. Interface residues (i.e., at the binding interface between the polypeptide and the Tie2) relative to SEQ ID NO:9 are provided in column 4 of Table 4. In another embodiment, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or all 26 core residues are identical (not substituted), or conservatively substituted, relative to SEQ ID NO: 9. Residues that are present at the polypeptide core are shown in column 5 of Table 4. These residues help maintain structure of the polypeptide. In a further embodiment, all identified interface residues and core residues are identical (not substituted), or conservatively substituted, relative to SEQ ID NO:9. In one embodiment, the polypeptides comprise an insertion in one or more insertion site relative to SEQ ID NO:9. Table 4, column 6 show the position of potential insertion sites relative to SEQ ID NO:9. In another embodiment, the polypeptides do not comprise an insertion in one or more insertion site relative to SEQ ID NO:9.

[0176] Table 4. Relative to SEQ ID NO:9 pos Opt ion 1 Opt i on 2 at_in te protein l oop di s;pensabl it i rface _core / ins

[0177] on erti

[0178] on

[0179] 1 P, K, W, Q, R, G, A, T 24

[0180] , 1.,, I, E, V, S, N, Y,

[0181] D, F, ’4, 24

[0182] 2 H, I, E, N, Q, M, A, R X, V, G, K, W, L, S, T,

[0183] Y, D, P, F

[0184] 3 X

[0185] L, T, S, H, A, R, W, K Y X, E, M, V, I, Q, F, G

[0186] 5 A, V, I 24 24 o K, L, Q, R M X

[0187] 7 D, E, M, Q, S, T, G, Y N X

[0188] , R, K, W, F, H, L, V

[0189] 3 T, I, F, Q, M, Y, W, L 24 X

[0190] 9 A X X

[0191] 10 M, F, L, W, T, I, G, S Q X

[0192] , E, R, V, D, K, A, Y,

[0193] N

[0194] 11 M, A, T, H, V, D, F, Q X, R, G, L, E, I, K, S,

[0195] W, N

[0196] 12 T, V X X

[0197] 13 K, A N, D, F, H, W, S, X

[0198] s i R, T, E, M, Y, Q,

[0199] V

[0200] 14 H, Q, V, T, M, o F ', K D, Y, E 24

[0201] 15 S, H, V, N, G, A, L, M X

[0202] , K, R, WQ C, D, E, Y, Q,

[0203] F, T, 1

[0204] 16 Y, E, A, M, I, S, D, T X

[0205] , P i- V, F, Q, L, H, G, N,

[0206] R, W

[0207] 17 R, S, E, K, L, T, A, P o X X

[0208] , D, G, Y, H, N, M, V

[0209] 18 T, K, S, Q N, I, V / D, F X 24

[0210] 19 W, S, L, H, Q, P, V, E I, R, Y, D, G X

[0211] 20 X X

[0212] 21 S, A 24 X z 2 14, F, H, L, R, I, E, S <2 X

[0213] , K, G, V, Y

[0214] 23 M, Q, T, V, A, H, L, N X 24

[0215] , K, S, I, R, D, E, Y,

[0216] G, F

[0217] 24 V, I X X

[0218] 25 I, Y, L w X

[0219] 26 Y, Q, A, R, M, N, T, I X

[0220] , V, G, K, E, S, F, FJ,

[0221] z 7 24 X

[0222] 28 T, I A X X

[0223] 23 I, A, S, K, T, E, M, H X, R, L, Y, D, W

[0224] 30 W, K, Y, G, V, E, N, F D X X

[0225]

[0226] , T, Q, A, S, H, L, R 39

[0227] > s ' M ' a ' X X ii N ' H ' 3 'V 'W ' O 'X ' A 99

[0228] IQ X A 'N ' l ' O 'O ' H ' S 39 X X 3 ' W ' T ' I 'N 'A f / 9

[0229] I 'O 'X i

[0230] i ' A ' B V S TY i 3 ' X £9

[0231] X ‘ A ‘ I ' 3 ' ii W ' A ' H ' 3 ' 0 ' H ' A ' N £9 X A ' 3 T 9

[0232] O ' W ' X H ' 3 i 3 ' M ' Q 'X ' 1 ' 3 'V ' S 09

[0233] 63 H 'M ' H ' X ' N ' 3 ' Q ' S ' W ' A ' 3 '! T 'l 'V ' A 'Q 'O 'd ' l 83 X A ' O i V ' S 33

[0234] A ' N ' H ' X i S 'X 'O 'V 93

[0235] V 'M 'N ' A ' 3 ' 3 ' A ' 0 ' X ' d ' N ' CQ 1 H 'O ', L ' S ' 3 ' T 'x ' a S3

[0236] 3 ' 3 'V ' A ' S ' A ' d ' N ' 3 ' 0 ' 1 M ' a 'x ' i 'w 'x 'o ' i f / S 3 to' 3 ' 1 'X ' H ' A ' M ' X ii 3 ' a ' 0 ' S 'W 'V ' N ' X

[0237] to - X 3 i T ' S ' 3 'X to “ 'N 'A ' H ' M S3

[0238] 0 Ei -- X i J O ■ -« H 'X TS - OS X I 'A 'N 'D ' 3 ' A ~ ~ 0 to -' A s to to - ■*h' M 6 v X i A ' 3 Q '' < QI 87 X i 3 ' 1 3 v X W ' l 'V ' S 'D 9 ^ X i 3 'W ' 1 'V 'A ' 3 ' S ' I S t7

[0239] X i a TT X i O 'W ' 3 ' I ' A £7

[0240] 3 'W 'V ' S ' A ' I ' A Sv 0 ' 3 ' A ' N ' 3 ' A ' S ' X ' X ' X 1 A ' O ' H ' Q 'V ' 3 ' d ' H T T V ' 0 ' d ' X i S i 3 'M 'W 'A 'A ' A ' I ' 3 O r 3 ' A ' X ii ii N ' O ' H ' I ' I 'A 'X ' S 6 E V ' S ' 0 ' X X ii A ' l, ' I ' 3 ' d 'A 'W ' 3 8 £ X X X ii D ii A ' Q ' S ' d ' 0 'N 3 £

[0241] V ' A ii

[0242] X X ii ' 3 ' 3 ' N ' O ' H ' 0 ii 3 'A 'M 9 £ X X H ii O ' Q ' H 'N ' 3 ' 0 S £ X X H ' A ' X ' M ' A 'V ii N ' 3 1>£ d ' A ' M ' X ' A 'X ', L ' X N ' G ii O ' S ' I ' 3 ' 3 ' 0 ' 3 'W £ £ X V ' 3 ' H ii I 'A 'W 3 £

[0243] 3 ' 1 ' N ' W ' CI ' A '

[0244]

[0245] X ii H ' 0 ' 3 ' O 'A ' l ' S ' 3 T £ S, G, H, A, R, M Li X

[0246] N, D 24

[0247] i AdO G P ', G

[0248] M, Y| c Lr-- 'j, W, L, F, A, E, T

[0249] , D,; H< i *«, I, N, S, G, V,

[0250] T, V C X

[0251] V, I, L u X

[0252] O G '

[0253] N, R, Q, E: s •* A, T, K, L, M, H

[0254] A, I, L, T, V, M - X

[0255] > O - ':

[0256] E, F, W, Q, H, M, Y, L i i X

[0257] I, L, V, Q, R, A 24 X A, F, Q, V, E, T, I, G j M X, R, Y, L', L, H, N, S,

[0258] W, K

[0259] S, E, G, Q, H 1 A X i X R X

[0260] L, W, R, G, I, H, Q, A I E X, K, Y, V, T, F

[0261] G, A, F, R, E, Q, N, L | X I X, Y, W, V, I, S, M, K,

[0262] T

[0263] N, V, K, F, Y, R, M, L | I X, Q, S, W, I, T, H, A,

[0264] G, D

[0265] Y, T, K, F, l, A, i4, S 1 W i X, Q, G, H, R, D, E, V, I

[0266] R, Y, K, H, P, Q, L, E | T, F, I, G, V i x L, N, Q, Y, H, W, T i K, I, G, F i x N, A, E, L, P, H, K, R! T, D, S, w X 1 X R, A, T, M, N, Q, Y, V | X i X, F, D, I, H, W, G, S,

[0267] K, E, L

[0268] R, K, V, T, L, I, F, M | i x, Y, N, H, S, P, Q, A,

[0269] G, E, W, D

[0270] I, K, A, Yvi x, F, L, P, R, Q, G, E,

[0271] W

[0272] F 24 1 x

[0273] K, H, R i N ii X I X

[0274] S, I, Y, E, N, K, A F, H, R, T, Q, W, | I X D, M

[0275] M, T, N, S, L, A, F, E ii X, W, I, H, V, K

[0276] S, T, V, N, Y, G, H, I K, A X, P, R, Q, E

[0277] K, M, G, E, R, Y, N, A X, V, S, Q, H, F, I

[0278] G, D, A, K, W, E, M, Q T, N, S, Y, H X X, V, E, R

[0279] Q, T i G, A, S, H X

[0280]

[0281] I, M, L | V | X X 101 R, G, S, T, L, F, A, N X

[0282] , Q, I, Y, M

[0283] 102 T, A, V 2X X

[0284] 103 T 2 L1", G, A, V, S

[0285] Q - X X

[0286] 104 S, R* A «, L, W, F, V, M

[0287] pd ** X X X 105 F, M, E - -, T, R, S, A, E)

[0288] Hl X X, Q, N, I, G, K, W, H,

[0289] Q

[0290] Y, L, V HJ - Q1 pd 5 '

[0291] 106 K H - X X X

[0292] O3 i-,

[0293] 107 E, L, N, S, K, Q, R, M X X

[0294] , H, I, G, T, W

[0295] 108 H, Q, N, I, D, R, G, Y K X X

[0296] , P, M, T, V, A, E, L,

[0297] 109 P, F, V, K, R., A W, N, Q, G, S, M, X

[0298] 110 D, W, P, K, L, M, T, R Y, Q, F X

[0299] , A, E, S

[0300] 111 S, N X

[0301] 112 G, A, S, V X X

[0302] 113 N, Y, S, M, I, F, T, G R X. L. V X Q X A. E,

[0303] K

[0304] 114 W, I X

[0305] 115 A X

[0306] H- 116 1, V, L, M F X X

[0307] 117 I X

[0308] Rd

[0309] - O

[0310] 118 A, Y, K jjO, V, R, I, Q, T X

[0311] , F w, M *, N, G, S, E, W,

[0312] D, L, P, H

[0313] 119 T, Y, D, W, N, V, S, E G, F X X

[0314] , K, A, Q, L, I-I, P

[0315] 120 T, M, Q, A, K, S, D, P X

[0316]

[0317] , R, I, L, V, H, F, E

[0318] In one embodiment, the polypeptides comprise an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the ammo acid sequence selected from the group consisting of SEQ ID NO: 10-11, residues 11-74. In another embodiment, the polypeptides comprise an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO:10-11. In a further embodiment, substitutions relative to SEQ ID NO: 10-11 are selected from substitutions listed in Options 1 or 2 of Table 5. Site saturation mutagenesis studies were conducted on Tie2_tw4000 (SEQ ID NO: 10), and Table 5 provides a list of exemplary substitutions. (Option 1; increased or maintained activity), and tolerable substitutions (Option 2: retained activity, though at a lower level). In another embodiment, substitutions relative to SEQ ID NO: 10-11 are selected from substitutions listed in Option 1 of Table 5.

[0319] In one embodiment, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or all 17 identified interface residues are identical (not substituted), or conservatively substituted, relative to SEQ ID NO: 10-11. Interface residues (i.e., at the binding interface between the polypeptide and the Tie2) relative to SEQ ID NO: 10-11 are provided in column 4 of Table 5. In another embodiment, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or all 13 core residues are identical (not substituted), or conservatively substituted, relative to SEQ ID NO:10-11.

[0320] Residues that are present at the polypeptide core are shown in column 5 of Table 5. These residues help maintain structure of the polypeptide. In a further embodiment, all identified interface residues and core residues are identical (not substituted), or conservatively substituted, relative to SEQ ID NO: 10-11. In one embodiment, the polypeptides comprise an insertion in one or more insertion site relative to SEQ ID NO: 10-11. Table 5, column 6 show the position of potential insertion sites relative to SEQ ID NO: 10-11. In another embodiment, the polypeptides do not comprise an insertion in one or more insertion site relative to SEQ ID NO: 10-11

[0321] Table 5. Relative to SEQ ID NO:10-II

[0322] pos iOption 1: Opt ion 2 at _inte protei loop / ins di spen tion rf ace n core ertion sable 1 L, S, D, K W, F, N, E X 2 G, T, L, Q, F, A, E, R | x

[0323] ,v

[0324] 3 G, V, E, T, K, L, Q, F H, D X 4 S, G, R, F, N, V, H, E i M, L, P X X 5 L, A, S, E, R, K, I, Y V x, N, G, D, W, T, Q, M

[0325] 6 F, V, Q, E 1 L, D, G, S, H, T X

[0326] ■"7 M, R X 8 I, V, T, Q, R, G, E i Y X X 9 W, N, E S, I, T, G, A, R, K X

[0327] 1, Y, P

[0328] 10 M, W, L, K, Y, R i F X 11 L, M, T X

[0329] 12 L, R, V 1 K, I X

[0330] 13 Y, 1, S, G, R, E, Q, V 1

[0331] 14 H, Q, M, L, V, R, S X

[0332] 15 P, I-I, E, Y, G, D, S, F | X

[0333] , T, N, A, W, Q, I, K

[0334] 16 F, G, N, S 1 I, R, K, A

[0335] 17 R, G, T, E i M, F

[0336] 18 N, R X

[0337] 19 A, Q, L: N, S, K, G, M, F, T X

[0338] 1, I

[0339] 20 V, Y, I, G, E, F, D, H L, P

[0340]

[0341] , N, S, A, T, R l)x

[0342] 21 H, M, K, W X

[0343] x

[0344] 23 F, T, A V, G, Q, M, P, L X

[0345] Q L

[0346] 241"

[0347] M, N, K, V, I

[0348] 25 N, W, F, V, Y, E H, P, S, M, T, G

[0349] 26 Q, G, N F, A, Y X

[0350] D, A, K, r1I, G, W N, H, L

[0351] 28 M, D

[0352] 29 W, R X X 30 N, S, T, I, F, V, Y, A K, P, H

[0353] , G

[0354] 31 N, P, E, T R X 32 D, P, Q L, I, N, F X 33 H, A, S

[0355] 34 M, V, D, G, H, P, S, E F, I

[0356] 35 S, P, E, I-i, Y

[0357] 36 N, 1 W, G, S, Q, R, F

[0358] 37 W N, L

[0359] 38 A, V, N, P, R G

[0360] 39 Q, N, V, P, S, R H, K, M, G, T, D X

[0361] 40 D, P, N, A, I-I, I T, E, G, F, V, K, W X

[0362] , R, S} O,' Y

[0363] 41 F, K, S, H, R, Q, E, L A, N

[0364] , D, V iQ

[0365] 42 M, K o i I X

[0366] 43 E is: X

[0367] 44 M, E, W, Q, R, K, H, L V

[0368] , Y, N, G, A1 0-, D

[0369] 45 I, A, Y, T ti-, E, G D, V, M

[0370] 46 A s - X

[0371] Q

[0372] 47 F, A - W, S, T, N, G, Y X

[0373] < o - '

[0374] 48 T\ K - G, HfVfSfD, L K, W, I, R, E

[0375] TvT

[0376] 49 G, E Q, M X

[0377] 50 Y, D X

[0378] 51 A, Q, L, Y, I-I X

[0379] 52 F, Q, E G, N, IJ

[0380] 53 F, E, S, I, T, L, Q, Y g X

[0381] , A

[0382] 54 W, T, I X

[0383] 55 X X 56 D, E X 57 D, N, I, G, E P X 58 H, K, R Y, L, I, G, Q, T, E X, P, S

[0384] 59 S, T, E R, F, W, A, Y X 60 W, L S, A, V X 61 Q, Y, L

[0385] 62 F, N, W, H, K, 1, A, E g

[0386] 63 W, L, Q, S, G, K, P, E V, R

[0387] 64 V, R, Q, W, I, T, Y, A X

[0388] , S, P, N

[0389] 65 T, N, E, W, F, S, A, Y V, D, H

[0390] , L

[0391]

[0392] 66 Y, S, P, E N, F, I.,, W, Q, G 67 A, W, D, P, N, L, M: K

[0393] 68 E, E, A: W, R, Q, K, S X

[0394] 69 D, A, E, R, L, 1, G, M 1 T

[0395] , W, Y, Q, F

[0396] 70

[0397] 71

[0398] 72 X

[0399] 73

[0400] 74 W, Q, P, E, I: N, G

[0401] 75 N, K, S, A, V G, R, Q, T, W, P, F X A, M, H, E, L

[0402] i i QO 71 1

[0403] 76 Y, N, K E, T

[0404] > IlOX, Q, D, F, V, S X I

[0405] " L1CO"

[0406] 77 W, H, V, E, N, P, K, G I X

[0407] £

[0408] , D, L, Y

[0409] 78 F, V, S, E, D, W, L G, Y, Ml~ i (J --: X 79 E, R, M, L, T, V, N Q, G x: X 80 F, G, V, E, T A, K, Nx81 P, E X 82 G, H, D, W, Y, R, P, N S X 83 F, V, M, W?S ' A' Y. L E 0 X

[0410]

[0411] fT7G Q *

[0412] O'

[0413] In one embodiment, the polypeptide comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,

[0414] Q

[0415] 99%, or 100% iden i Rfttical to the ammo acid sequence selected from the group consisting of SEQ ID NO: 12-13, residues 28-95. In another embodiment, the polypeptide comprises an i oo"

[0416] ammo acid < l:u- sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, Q E

[0417] 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 12-13. In a further embodiment, substitutions relative to SEQ ID NO: 12-13 are selected from substitutions listed in Options 1 or 2 of Table 6. In one embodiment, substitutions relative to SEQ ID NO: 12-13 are selected from substitutions listed in Option 1 of Table 6. Site saturation mutagenesis studies were conducted on Tie2__tw5000 (SEQ ID NO: 12), and Table 6 provides a list of exemplary substitutions (Option 1; increased or maintained activity), and tolerable substitutions (Option 2: retained activity, though at a lower level).

[0418] In one embodiment, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or all 19 identified interface residues are identical (not substituted), or conservatively substituted, relative to SEQ ID NO: 12-13. Interface residues (i.e., at the binding interface between the polypeptide and the Tie2) relative to SEQ ID NO: 12-13 are provided in column 4 of Table 6. In another embodiment, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or all 29 core residues are identical (not substituted), or conservatively substituted, relative to SEQ ID NO: 12-13. Residues that are present at the polypeptide core are shown in column 5 of Table 6. These residues help maintain structure of the polypeptide. In a further embodiment, all identified interface residues and core residues are identical (not substituted), or conservatively substituted, relative to SEQ ID NO: 12-13.

[0419] In one embodiment, the polypeptides comprise an insertion in one or more insertion site relative to SEQ ID NO: 12-13. Table 6, column 6 show the position of potential insertion sites relative to SEQ ID NO: 12-13. In another embodiment, the polypeptides do not comprise an insertion in one or more insertion site relative to SEQ ID NO: 12-13.

[0420] Table 6. Relative to SEQ ID NO: 12-13

[0421] pos Opt ion 1 Opti on 2 a t_inter protein loop / in di s;pen it i face __core i sert ion sable on

[0422] 1 F, Q, G A, N, R, P, H, V, D, W: X

[0423] , I

[0424] K, H, R, Q, M, N, P D, F X:: X, Li, G, T, V

[0425] 3 F, Y, K, L, S, D, H T, W: X, Q, R, E

[0426] 4 V, N, G, P, R, E: S, M, H, T, Q: X D, R, M, K, P: W, I, A, Y, H, N: X K, I: F, D, P, G, A, H: X 7 3, Y, K, I, N, P, D | W 1 X

[0427] , V, T, R, L, F, S

[0428] 3 M, S, P, A, Y, Q, R T, G, V, F, L, K, W: X

[0429] , I, N, E

[0430] 9 A, G, V, L F, N, E X i i X 10 H, R, K, Q, L, I, Y P: X

[0431] , S, A, T, N, G, V,

[0432] W, E

[0433] 11 0, W, N, 3, A, V, R M, S, L, Y: X, K, 1, E

[0434] 12 N, G, Q, S, Y: 0, I, T, V, K, E i X 13 R, K, G: P, A: X 14 G, S, R L, A, I: X 15 F, R N, V, E, A, D, K, I.i, G: X 16 L, D, V, I, Y, I-I, K P X:: X

[0435] , M, T, R

[0436] 17 Q, K, R, L, W, T, P: X

[0437] , Y, H, 1, F, N

[0438] 18 Q, R, G, Y, F, E W, H, S, L, A, K, V, N: X

[0439] , P, T, I

[0440] 19 W, 3, K, I, R Y, P: f x 20 T, N, R, Q, E, H, A I, Y, F X i i X, S, W, V

[0441] 21 R, P, ™, V, Q, I, T: X

[0442] , N, K, Y, F, A, H,

[0443] L, E, G

[0444] 22 P, R, K i H, Y, T, Q, F, A, N: X

[0445]

[0446] 22 M, I, F, N, E, T, A R, V, L, H X X s V ' O ' A S 9

[0447] A ' 3 ' N ' B ' X ' O ' O ' W ' T ' S ' X X ' J / 3 ' V ' d ' A ' M O ' 3 ' W ' H ' 0 ' a ' 3 ' A X ' N ' M S 9 X w Z 9 i x V ' z\ T 9 v ' 3 ' a '

[0448] O ' H ' O ' X ' S ' X ' I ' 1 3 ' A ' W 09

[0449] 3 ' N 3 ' 0 ' A ' l 'V ' D 6 S X 3 ' 3 ' 0 8 S a O ' N ' S A S a ' A ' a ' i ' N 3 ' 3 ' X ' O ' T 9 S N ' l ' H A ' 3 ' I S S W ‘ A ‘ cl '

[0450] X X ' O ' O ' S ' A ' H ' X ' I a ' v t-S X X ' O ' A ' M ' H 'c S J O ' X L <j S 'X ' H 'W ' A X ' I ' N ' M ' d ' A ' A ss W ' K S ' O ' M ' X ' X ' 3 ' d T 9 3~ > G- A ' A '

[0451] Q '. L 'V ' O ' O ' X 0 ' N ' S 3 'X ' H O S A ' I O3- ' k 11 — ' d V ' X ' A ' D X V7M1S1N ‘ J ‘ X7a —7- ° £ 0 A ' A ' W ' 3 ' 3 ' I ' Q X A ' M T f / s X ' M 9 ^ i x S ' T 9 ^

[0452] X 3 ' A iv y V ' X ' N ' H 'X s c 17

[0453] A ' O ' S '

[0454] X ' N ' H ' A ' 3 ' M ' D ' X I 'X >1 '

[0455] X 3 ' V ' S ' A ' H ' N ' l ' W 3 T T7

[0456] X H 0? cn co 3 ' 1 ' a ' a ' M '

[0457] 3 ' A ' D ' A ' 3 ' H ' O 'V S ' X ' H ' N ' A ' X 6 € X d ' 0 ' D ' S V ' A ' H ' X 8 £

[0458] X 3 L ‘c X s 9 £

[0459] 1 X ' M ' d Si E H ' 0 '

[0460] X A ' 3 ' 3 ' l 'V ' O ' X ' d S ' A ' N

[0461] X V ' D ' d s r r X X ' H ' A ' A ' X 3 ' W ' I Z. Z 3 ' M ' I ' A '

[0462] d ' 3 ' 3 'V ' O ' l 'X ' W A ' H ' G ' S ' X ' N T £

[0463] N 'LL ' S 'O V ' B O S X 3 ' 3 6 S X X 82

[0464] N ' H ' A ‘ 3 ' X X d ' 0 ' M ' 0 ' 3 ' W V ' S ' X ' A ' A ' I ' a L Z X X S ' M ' 3 ' 1 ' A ' 3 ' 1 ' A O 'V ' W 92 X X d ' I 92

[0465] I ' 3 ' d ' 1 '

[0466]

[0467] X X: X a ' o ' w 'v ' x ' A ' x ' s S ' X ' A ' M ' O ' H ' N t-2 GxJLJ X

[0468] N, Q, D I, W, T, G, H

[0469] W, V I

[0470] HI ' X

[0471] F ra -, A I, W

[0472] Id: X

[0473] G, W u, •* Y, D, N, F, H T, L, E

[0474] , R, 1 7,. K

[0475] T, A M, I

[0476] H, V, A S, Y, N, W, Q, T X

[0477] ^7

[0478] T, S, I E, Q, A, V, G, H, D, N

[0479] - | X

[0480] ! f?; L

[0481] E, D, N, A, W, F, H S, L

[0482] , G, Q, K, R, Y

[0483] K

[0484] W, Y, N L, F, I, H, M, E

[0485] G X

[0486] H, S, L N, R, M, V X i X L, M, F, V I X X

[0487] x X

[0488] G X

[0489] K, T, S, P, Y, W, D

[0490] , A, E

[0491] T, V I X

[0492] G, S, A X

[0493] D X

[0494] N, R, S, E, K G, Q

[0495] I, V A X

[0496] L X

[0497] 1, 1, X

[0498] £4 i1

[0499] K, M, V, S, A, T, G X

[0500] , I, R, L

[0501] V, K, M, F & ' X i

[0502] H - F, H, R

[0503] L, W,-iF", H, E

[0504] A X

[0505] T7 x

[0506] T, S, V, D, H, E, E X X, N, I, R, F

[0507] R, L, F, S, H, D Q, W, Y X X Y, D, Q, L, P, A, F X, v

[0508] V, L, R, G, N, K, H D X S, W, N, H D, F X G, A X i X L, F, I, K S X X

[0509] Y, W X X X

[0510] Q, K, N, I, H, E A, V, G, T, M, F, W, R X M, E H, K, Q, S, W, Y, L, R X, D

[0511] 1, 1, X X L, Y, K F X A, E Y, L, X, S; K

[0512] N, M, K, R, A, I, F X

[0513]

[0514] , Q, L, E

[0515]

[0516] A, V X X

[0517] In one embodiment, the polypeptide comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 14-17, residues 29-91. In another embodiment, the polypeptide comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 14-17. In a further embodiment, substitutions relative to SEQ ID NO: 14-17 are selected from substitutions listed in Options 1 or 2 of Table 7. In one embodiment, wherein substitutions relative to SEQ ID NO: 14- 17 are selected from substitutions listed in Option 1 of Table 7. Site saturation mutagenesis studies were conducted on Tie2__tw6000 (SEQ ID NO: 14), and Table 7 provides a list of exemplary- substitutions (Option 1; increased or maintained activity-), and tolerable substitutions (Option 2: retained activity-, though at a lower level).

[0518] cJ In one embodiment, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or all 21 identified interface residues are identical (not substituted), or conservatively-substituted, relative to SEQ ID NO: 14-17. Interface residues (i.e., at the binding interface between the polypeptide and the Tie2) relative to SEQ ID NO: 14-17 are provided in column 4 of Table 7. In another embodiment, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or all 28 core residues are identical (not substituted), or conservatively substituted, relative to SEQ ID NO: 14-17. Residues that are present at the polypeptide core are shown in column 5 of Table 7. These residues help maintain structure of the polypeptide. In a further embodiment, all identified interface residues and core residues are identical (not substituted), or conservatively substituted, relative to SEQ ID NO: 14-17.

[0519] In one embodiment, the polypeptides comprise an insertion in one or more insertion site relative to SEQ ID NO:12-13. Table 7, column 6 show the position of potential insertion sites relative to SEQ ID NO: 14-17. In another embodiment, the polypeptides do not comprise an insertion in one or more insertion site relative to SEQ ID NO: 12-13.

[0520] Table 7. Relative to SEQ ID NO: 14-17

[0521] pos be s t: tolerable at_inter protein loop / I n dispen it i f <3 C O _core sert ion sable on

[0522] 1 L, I, Y, Q, F, A, N X

[0523]

[0524] , P, R, G, S, E, D W S

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[0526] ' V ' a ' W ' Cl ' S A ' N T £ X V ' 0 ' 3 ' 1 O S X M ' 3 ' W ' 3 ' a 63 V X a ' 3 8 Z W

[0527] ' O ' N ' d ' H ' T ' 3 ' V ' y x ' 3 ' M ' i ' s ' A ' a ' a £□ C v ' s ' a

[0528] 'X ' H ' A ' S ' N

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[0530] >3 ' 1 ' S ' W ' N ' H ' O ' X A ' V ' l 'X ' a ' 3 ' A ' d I E X ' 3 ' 3 ' W ' V ' S ' a ' I ' S ' X X H ' l ' N ' O ' A ' A ' M ' 3 03

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[0532] X X ' s ' i ' a ' w ' a A ' N ' O 'X ' I ' 3 9T ' H ' d ' V ' cr M ' A ' d7X a H ' W7X1JI1N1E)7S7A ST X w ' s ' 3 'V X ' H ' 3 ' a TT X X W ' 3 3 ' 3 ST s 'x ' a

[0533] V ' N ' 3 ' l ' 1 ' 0 A ' M 3T x 'v ' a ' 3 ' A ' Q ' N ' 3 ' A ' d ' y I ' s ' w ' O ' a ' M ' H ' l TT 3 ' 3 ' H ' 3 ' X 3 ' 1 A ' a ' a ' A ' O 'x ' i ' M OT X X W ' S 3 ' H ' 3 ' M 6 X d ' O ' A 3 'V ' 3 ' a ' l 'X ' l g M ' A ' X ' 3 ' a ' N ' 3 ' 3 ' s ' W ' X V ' A ' 3 ' 0 ' 3 ' d ' H ' l L i ' a ' I ' X M ' S W ' V ' O ' d ' A ' S ' 3 ' 3 9 s ' A ' a ' 3 ' V ' W ' H ' 0 ' a ' 3 ' y 1 ' d 'X ' N ' l ' 3 ' A ' M X N ' S A 'X ' S ' A ' S ' d ' M V a ' N ' B ' a ' O ' s ' X V ' A I 'X ' S ' W ' l ' M ' 3 ' H s A ' S ' 3 ' H '

[0534]

[0535] X D v ' 3 ' a ' O ' N 'x ' a ' w 7 Cri

[0536] 33 P, S A X

[0537] 34 3, P, L, Q, V, M, F, N 1 X

[0538] 3 5 G, K, F, R, N, Q, A, W

[0539] , LI, H, S, T, D, M, Y,

[0540] V, P, E, I

[0541] 3 6 s X

[0542] 3 7 v, I, F x

[0543] 3 8 H, R, A, L, F, S, V G, M, Y, T, K,

[0544] D, P

[0545] 3 9 L, K

[0546] u

[0547] 40 W, Y, H H L, F x

[0548] S m QD X

[0549] 41 Y, A,, V, I, Li, G, N X

[0550] S M

[0551] , H, M

[0552] 42 H, R, K, S Q, Y, T, V, L

[0553] in w

[0554] 43 V, H, R, K, S, I, Y x

[0555] 44 T, S, V, A, N, H E, M, L X

[0556] S, E, L T, N, D X

[0557] 46 K, P, Y, G, R, M, 1, F H, Q, N, S, T,

[0558] E

[0559] 47 F, W, Y, R X

[0560] 48 G, R, E, Q, N, A, D, H X

[0561] , I, Y, S, V, M, T, L,

[0562] K, P

[0563] 4 9 H, M, V, A, S, F, N, E L

[0564] , W

[0565] 50 H, I, G, R, F, T, E M, K, L

[0566] 51 E, M, N, T, L, R, H, A

[0567] , D, Q, W, S, G, V, I,

[0568] K, Y

[0569] H, N, G, Q 1, L, F x 53 S, Y, K, F, L, M, E, W X, Q, R

[0570] 54 G, N, P, D R, K, H X 55 D, G, V

[0571] 56O L i: Q, F, H, V, G, D, K, E

[0572] , W, T, A, S, P, Y, N,

[0573] R

[0574] 57

[0575] 58 F, I L, N, D, R, W, X

[0576] 59 F, T I, W,

[0577] A

[0578] 60 L, N, R, K, Q, E, S, T

[0579] , D

[0580] 61 A, S, V, N, G X

[0581] 62 E, D, M, H, G, N, Y, S m X

[0582] , F, A

[0583] 63 F, Y, H, L, R, K, G, M

[0584] , A, E, P

[0585] 64 G, Li X

[0586] 65 A, G X

[0587] 66 F, V, L I, Y, W, E X

[0588] 67 W, R, K, M, V, L, N, Q

[0589] , H, A, I, T, S, E, D,

[0590] F, G

[0591] 68 I, V X

[0592]

[0593] 6 9 F X Z£

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[0595] X X ' S ' N ' A ' O ' I 3 ' W ' V ' S S OT X ' H ' 3 ' X ' M ' X I ' 0 ' 3 N ' S ' A 'V 'X 'A ' d ' A frOT Q ' N ' S ' 3 ' M 'X ' X ' 3 ' 0 ' A 'V ' H 'W 'O ' A ' 3 ' I £ 0T X 3o u, 3 'V ' 3 30T X X td V ' N ' d T OT 3 co X 'X

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[0598] Y ' 0 'O ' S ' 3 'A 3 'V 'X 'W 66

[0599] D ' a ' 3 ' X A ' A ' S ' d 'V 'M ' H 'A 86

[0600] A ' S ' 3 ' 1 ' 0 ' 3 'N ' 3 ' N ' X X A ' A ' M ' X 'W ' H ' X 'V LG W 'A ' I 'O 'V

[0601] X X ' 3 ' CI ' O ' S ' N X ' A 96 X X TT 96 Y X V ' D 1>6 X ' 0 ' 3 ' H ' G 'V 3 ' 3 £6 X X O 'X Z6

[0602] A 'V ' I

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[0604] X 3 ' 3 'W 'X 'M 'A 68 X 3 88 X A ' A L 8

[0605] 0 'N

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[0607] V 'W ' I-I ' 3 ' M TA A ' 3 ' I ' 3 ' X A 'W ' S ' a 'N 'O ' V ' X ££.

[0608] 3

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[0610]

[0611] A 'M ' O 'N ' H 'X 'X ' Q OL 108 1 X X 109 F, M, Y, L, I, V X

[0612] >

[0613] 110 X

[0614] 111 T, N, F, M, K, E, Y P, L, H, V, A, X

[0615] u

[0616] p 'j Q, G, I, R

[0617]

[0618] 112 M, L, V, F A: X X

[0619] In another embodiment, the disclosure provides fusion proteins, comprising a polypeptide of any embodiment or combination of embodiments herein, fused to one or more functional domains, optionally wherein the polypeptide and the one or more functional domains are linked by an amino acid linker. In these embodiments, any functional domain may be inserted, as an insertion at an insertion site, and / or at the N-terminus and / or at the C- terminus of the polypeptide. In various non-limiting embodiments, the one or more functional domains may comprise, for example, a targeting domain, a detectable domain, a scaffolding domain, an Fc domain, a domain to increase half-life of the polypeptide in the blood stream (including but not limited to an albumin-binding protein), or a further therapeutic peptide domain. In all embodiments, the fusion protein may optionally comprise an amino acid linker separating the polypeptide and the functional domain. Any amino acid s'

[0620] linker may be used as suitable for an intended purpose, including but not limited to a GS-rich linker.

[0621] -l1~

[0622] In a further embodiment, the disclosure provides conjugates, comprising:

[0623] (a) the polypeptide of any embodiment or combination of embodiments herein; and

[0624] (b) a scaffolding moiety.

[0625] As disclosed above, when the polypeptides are conjugated onto a scaffold, they can act as agonists to activate the pAKT / FOXOl pathway and promote tight junction re-assembly (when present at a valency of 6 or more) and can be used, for example, to treat or limit development of vascular dysfunctions, cancer angiogenesis, and wound healing.

[0626] Any scaffolding moiety may be used that can act to present multiple copies (2, 3, 4 5, 6, 7, 8, or more) of the polypeptide on a scaffold formed from the conjugate via SpyCatcherfM-SpyTagfMreaction. In exemplary embodiments, the scaffold moiety comprises H8T, H3T, or H8F proteins, tw1102 is annotated as T. The amino acid sequence of these exemplary embodiments are shown in Table 8 as SEQ ID NO: 18-23. Table 8. Exemplary fusion proteins

[0627] H8T:. i tw1102- SpyTag™ unit (SpyTag™’ underlined): MSGSEEEQKLVEEAIEKLTKILEELAKKYGEKMKEPKEYYLRMSEKIKKNEQPEQDM AILIHNAGKEVLKLTGDEEAALELAKLSAKLFQYAGDTDGAVRALKQSGIGEEAEKI AEEIRKKAAGSRGVPHIVMVDAYKRYK (SEQ ID NO: 18 )

[0628] H8 - SpyCatcher™ unit (SpyCatcher™ underlined): MGDSATHIKFSKRDIDGKELAGATMELRDSSGKTISTWISDGQVKDFYLMPGKYTFV ETAAPDGYEIATAITFTVNEQGQVTVNGKATKGGGSGGSSAEELLRRSREYLKKVKE EQERKAKEFQELLKELSERSEELIRELEEKGAASEAELARMKQQHMTAYLEAQLTAW EIESKSKIALLELQQNQLNLELRHILE (SEQ ID NO: 19)

[0629] H3T: tw1102- SpyTag™ unit (SpyTag™ underlined): MSGSEEEQKLVEEAIEKLTKILEELAKKYGEKMKEPKEYYLRMSEKIKKNEQPEQDM AILIHNAGKEVLKLTGDEEAALELAKLSAKLFQYAGDTDGAVRALKQSGIGEEAEKI AEE I RKKAAGSRGVPHIVMVDAYKRYK (SEQ ID NO: 20) H3 - SpyCatcher™ unit (SpyCatcher™ underlined): MGLVPRGSHMSGAMVDTLSGLSSEQGQSGDMTIEEDSATHlkFSkRDEDGKELAGAT MELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQV TVNGKATKGDAHILEGLNDIFEAQKIEWHEGSGSGTKYELRRALEELEKALRELKKS LDELERSLEELEKNPSEDALVENNRLNVENNKI I VEVLRI I AEVLKI IAKSD (SEQ ID NO: 21)

[0630] H8F: H8 - SpyCatcher™’ unit (SpyCatcher™ sequence underlined): MGDSATHIKFSKRDIDGKELAGATMELRDSSGKTISTWISDGQVKDFYLMPGKYTFV ETAAPDGYEIATAITFTVNEQGQVTVNGKATKGGGSGGSSAEELLRRSREYLKKVKE EQERKAKEFQELLKELSERSEELIRELEEKGAASEAELARMKQQHMTAYLEAQLTAW EIESKSKIALLELQQNQLNLELRHILE (SEQ ID NO: 22 ) F-domain- SpyTag™ unit (SpyTag™’ sequence underlined): GSMAHIVMVDAYKPTKAELASEKPFRDCADVYQAGFNKSGIYTIYINNMPEPKKVFC NMD VNGGGWTVI QHREDGSLD FQRGWKE YKMG FGNPSGE YWLGNE FIFA I TSQRQ YM LRIELMDWEGNRAYSQYDRFHIGNEKQNYRLYLKGHTGTAGKQSSLILHGADFSTKD ADNDNCMCKCALMLTGGWWFDACGPSNLNGMFYTAGQNHGKLNGIKWHYFKGPSYSL

[0631]

[0632] RSTTMMIRPLDF (SEQ ID NO: 23 )

[0633] The polypeptide and the scaffolding moiety may be conjugated via any suitable technique, including but not limited to chemical conjugation of any type, and expression as a fusion protein. For protein conjugation, the polypeptides of the disclosure may, for example be fused with SpyTag™, and scaffold proteins may, for example, be fused with SpyCatcher™, when the two components are combined, they will conjugate via the SpyCatcher™-SpyTag™ reaction as is known to those of skill in the art. Exemplary' such fusion proteins are provided in Table 9 as SEQ ID NO:24-27.

[0634] Table 9. Exemplary fusion proteins

[0635] ; A-Tmb or A-GFP:

[0636]

[0637] A- SpyCatcher™ unit (SpyCatcher™ underlined):

[0638] AM DTLSGL S SEQGQSGDMT I EEDSATHIKFSKRDEDGKELAGATMELRDSSGKTIS TWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAH IGGSGGSGGNDTTTKPDLYYLKNSEAINSLALLPPPPAVGSIAFLNDQAMYEQGRLL RNTERGKLAAEDANLSSGGVANAFSGAFGSPITEKDAPALHKLLTNMIEDAGDLATR SAKDHYMRIRPFAFYGVSTCNTTEQDKLSKNGSYPSGHTSIGWATALVLAEINPQRQ NEILKRGYELGQSRVICGYHWQSDVDAARVVGSAVVATLHTNPEFQAQLIKAKIEFK QHQK (SEQ ID NO: 24) > Tmb- SpyTag™ unit (SpyTag™ underlined): MSGSEEEQKLVEEAIEKLTKILEELAKKYGEKMKEPKEYYLRMSEKIKKNEQPEQDM AILIHNAGKEVLKLTGDEEAALELAKLSAKLFQYAGDTDGAVRALKQSGIGEEAEKI AEE I RKKAAGSRGVPHIVMVDAYKRYK (SEQ ID NO: 25) GFP- SpyTag™ unit (SpyTag™ underlined): MAHIVMVDAYKPTKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKF ICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDD GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNG IKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLYKDPNEKRDH MVLLE F VTAAG I THGMDEL Y (SEQ ID NO: 26)

[0639]

[0640] B domain:

[0641] MGSLITLVELEWLEHQLIVQLSERLKGQIAKVGELLCECLKNGGKILICGNGGSAAD AQHFAAELSGRYKKERKALAGIALTTDTSALSAIGNDYGFEFVFSRQVEALGNEGDV LIGISTSGKSPNVLEALKKARELNMLCIGLSGKGGGKMNDLCDHNLVVPSDDTARIQ EMHILIIHTLCQIIDEAFLE (SEQ ID NO: 27) By way of non-limiting example:

[0642] (a) A- SpyCatcher™ (SEQ ID NO:24) is conjugated to tw1102- SpyTag™ (Tmb- SpyTag™); SEQ ID NO: 25 to make A-Tmb;

[0643] (b) A- SpyCatcher™ (SEQ ID NO:24) is conjugated to GFP- SpyTag™ (SEQ ID NO:26) to make A-GFP

[0644] (c) Combining A-Tmb + A-gfp + B domain at 4: 1:5 molar ratio will allow self-assembly into Tmb-sheets, which can be used, for example, to look at tie2 colocalization with alphas integrin or claudin-5. (see Fig. 3F-G) (d)

[0645] In other embodiments, the scaffolding moiety may comprise a scaffolding protein listed in Table 10 as SEQ ID NO:28-31.

[0646] Table 10. Exemplary scaffolding proteins

[0647] >

[0648] SLLIIAISLLLSSRN (SEQ ID NO: 28) > ( MSAEELLRRSREYLKKV^. ARMKQQHMTAYLEAQLTAWEIESKSKIALLELQQNQLNLELRHI (SEQ ID MSGMIKRHFTLKLTTGASDDMKEVVPEFLAMIKEAAELFDEVTLKLTTENPEMARAL ) MEGVGILIKEGVDVHLEVELGSNVKARVEVLKTLAEELKKIKEEIE (SEQ ID NO: 30) > MSGMMKLIVEITEELKVEDAKklMEEIIELSKY;VDVEVRIETKNVEVALETLKKLAE 1

[0649]

[0650] LLKKEA (SEQ ID NO: 31) >

[0651] Exemplary fusion proteins are shown in Table 11 as SEQ ID NO:32-39, with 1102 (SEQ ID NO:2) as the Tie2 binder. Any of the other binders listed in Table 1 can be substituted for SEQ ID NO: 2. In this table, the oligomer subunit domains are in bold font and linker sequences are underlined. When linked to a scaffolding moiety of 6 or more, 1102 (SEQ ID NO:2) can activate the pAKT pathway at the same concentrations as H8F (see above for H8F). The linker sequences are optional and may be present or absent. When present, they may be any suitable linker. In one embodiment, the underlined linker can be replaced with any (GGS)x flexible linker, or with a rigid linker, including but not limited to one or more EAAK (SEQ ID NO: 42) repeats.

[0652] Table 11

[0653] >

[0654] Name Amino acid sequence H6 C 1102 MTEDEIRKLRKLLEEAEKKLKKLEDKTRRSEEISKTDDDPKAQSLQLIAES LMLIAESLLIIAISLLLSSRNSGSEEEQKLVEEAIEKLTKILEELAKKYGE KMKEPKEYYLRMSEKIKKNEQPEQDMAILIHNAGKEVLKLTGDEEAALELA KE SAKE EQ YAGDTDGAVRADKQS G I GE EAE K I AE E I RKKAA (SEQ ID NO: 32 ) H8 C 1102 MSAEELLRRSREYLKKVKEEQERKAKEFQELLKELSERSEELIRELEEKGA ASEAELARMKQQHMTAYLEAQLTAWEIESKSKIALLELQQNQLNLELRHIG SSGSEEEQKLVEEAIEKLTKILEELAKKYGEKMKEPKEYYLRMSEKIKKNE QPEQDMAILIHNAGKEVLKETGDEEAAEELAKLSAKEFQYAGDTDGAVRAL KQSGIGEEAEKIAEEIRKKAA SEQ ID NO: 33 ) MSGSEEiEiQkLVEEAIEkLTklLEiEiLAkkYGEKMkEpkEYYLRMSEklkKNE QPEQDMAILIHNAGKEVLKETGDEEAAEELAKLSAKEFQYAGDTDGAVRAL KQSGIGEEAEKIAEEIRKKAATEDEIRKLRKLLEEAEKKLKKLEDKTRRSE EISKTDDDPKAQSLQLIAESLMLIAESLLIIAISLLLSSRN SEQ ID NO: 34) MSGSEEiEiQkLVEEAIEkLTklLEiEiLAkkYGEKMkEpkEYYLRMSEklkKNE QPEQDMAILIHNAGKEVLKETGDEEAAEELAKLSAKEFQYAGDTDGAVRAL

[0655]

[0656] KQSGIGEEAEKIAEEIRKKAASAEELLRRSREYLKKVKEEQERKAKEFQEL LKELSERSEELIRELEEKGAASEAELARMKQQHMTAYLEAQLTAWEIESKS KIALLELQQNQLNLELRHI SEQ ID NO: 35) HE0626 rigid 110 MSGMIKRHFTLKLTTGASDDMKEVVPEFLAMIKEAAELFDEVTLKLTTENP EMARALMEGVGILIKEGVDVHLEVELGSNVKARVEVLKTLAEELKKIKEEI EGGS EAAAKEAAAKEAAAKGGS S E E E Q KL VE E A I E KET K I LE E E AKKYGE K MKEPKEYYLRMSEKIKKNEQPEQDMAILIHNAGKEVLKLTGDEEAALELAK LSAKLFQYAGDTDGAVRAEKQSGIGEEAEKIAEE IRKKAA SEQ ID NO: 36 )

[0657]

[0658] MSGMIKRHFTLKLTTGASDDMKEVVPEFLAMIKEAAELFDEVTLKLTTENP EMARALMEGVGILIKEGVDVHLEVELGSNVKARVEVLKTLAEELKKIKEEI EGGSGGSGGSGGSGGSSEEEQKLVEEAIEKLTKILEELAKKYGEKMKEPKE YYERMSEKIKKNEQPEQDMAILIHNAGKEVLKLTGDEEAALELAKLSAKLF QYAGDTDGAVRALKQSGIGEEAEKIAEEIRKKAA SEQ ID NO: 37)

[0659] HE 0675__r ig id__l 10 MSGMMKLIVEITEELKVEDAKKIMEEIIELSKYVDVEVRIETKNVEVALET 2 LKKLAELLKKEAGGSEAAAKEAAAKEAAAKGGSSEEEQKLVEEAIEKLTKI LEELAKKYGEKMKEPKEYYLRMSEKIKKNEQPEQDMAILIHNAGKEVLKLT GDEEAALELAKLSAKLFQYAGDTDGAVRALKQSGIGEEAEKIAEEIRKKAA SEQ ID NO: 38)

[0660]

[0661] HE 0675__f 1 ex_l 102 MSGMMKLIVEITEELKVEDAKKIMEEIIELSKYVDVEVRIETKNVEVALET LKKLAELLKKEAGGSGGSGGSGGSGGSSEEEQKLVEEAIEKLTKILEELAK KYGEKMKEPKEYYLRMSEKIKKNEQPEQDMAILIHNAGKEVLKETGDEEAA LELAKLSAKLFQYAGDTDGAVRALKQSGIGEEAEKIAEEIRKKAA SEQ

[0662]

[0663] ID NO: 39)

[0664]

[0665] In one embodiment, the disclosure provides fusion proteins, comprising;

[0666] (a) the polypeptide of embodiment or combination of embodiments herein; and (b) a protein scaffolding moiety;

[0667] optionally wherein the polypeptide and the protein scaffolding moiety Exemplary scaffolding moieties are discussed above. Any amino acid linker may be used as suitable for an intended purpose, including but not limited to a GS-rich linker.

[0668] In another embodiment, the disclosure provides scaffolds, comprising the conjugate or fusion protein of any embodiment herein, wherein the scaffold comprises 6 7, 8, 9, 10, or more copies of the polypeptide of any embodiment or combination of embodiments herein. These scaffolds are designed to self-associate via hydrophobic interactions to form higher order oligomers. Thus, when conjugated with binders (for example, F-domain or Tmb), the scaffolds present multiple binders on these multivalent scaffolds. For protein conjugation, the design binders are fused with Spy Tag™ and scaffold proteins are fused with SpyCatcher™, when the two components are combined, they will conjugate via the SpyCatcher™-SpyTag™ reaction as described previously (Proc Natl Acad Sci USA 109: E690-E697, 2012). Scaffold proteins are designed to self-associate via hydrophobic interactions that spontaneously form higher-order oligomers. For example, one H8 subunit is one helical protein, but in solution, it will self-interact to form an 8-helical bundle-like scaffold; when linked to a polypeptide binder of the disclosure, one binder to one helix as one subunit, the whole " H8 scaffold" will have 8 polypeptide binders.

[0669] In another aspect the disclosure provides nucleic acids encoding the polypeptide or fusion protein of any embodiment or combination of embodiments of the disclosure. The nucleic acid sequence may comprise single stranded or double stranded RNA or DNA in genomic or cDNA form, or DNA-RNA hybrids, each of which may include chemically or biochemically modified, non-natural, or derivatized nucleotide bases. Such nucleic acid sequences may comprise additional sequences useful for promoting expression and / or purification of the encoded peptide or chimeric molecular construct, including but not limited to poly A sequences, modified Kozak sequences, and sequences encoding epitope tags, export signals, and secretory signals, nuclear localization signals, and plasma membrane localization signals. It will be apparent to those of skill in the art, based on the teachings herein, what nucleic acid sequences will encode the polypeptide or fusion protein of the disclosure.

[0670] In a further aspect, the disclosure provides expression vectors comprising the nucleic acid of any aspect of the disclosure operatively linked to a suitable control sequence, such as a promoter. “Expression vector” includes vectors that operatively link a nucleic acid coding region or gene to any control sequences capable of effecting expression of the gene product. “Control sequences” operably linked to the nucleic acid sequences of the disclosure are nucleic acid sequences such as a promoter) capable of effecting the expression of the nucleic acid molecules. The control sequences need not be contiguous with the nucleic acid sequences, so long as they function to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the nucleic acid sequence and the promoter sequence can still be considered “operably linked” to the coding sequence. Other such control sequences include, but are not limited to, polyadenylation signals, termination signals, and ribosome binding sites. Such expression vectors can be of any type, including but not limited plasmid and viral- based expression vectors. The control sequence used to drive expression of the disclosed nucleic acid sequences in a mammalian system may be constitutive (driven by any of a variety of promoters, including but not limited to, CM V, SV40, RSV, actin, EF) or inducible (driven by any of a number of inducible promoters including, but not limited to, tetracycline, ecdysone, steroid-responsive). The expression vector must be replicable in the host organisms either as an episome or by integration into host chromosomal DNA, In various embodiments, the expression vector may comprise a plasmid, viral-based vector, or any other suitable expression vector. In another aspect, the disclosure provides host cells that comprise the polypeptide, fusion protein, scaffold, nucleic acid or expression vector (i.e.: episomal or chromosomally integrated) disclosed herein, wherein the host cells can be either prokaryotic or eukaryotic. Ihe cells can be transiently or stably engineered to incorporate the expression vector of the disclosure, using techniques including but not limited to bacterial transformations, calcium phosphate co-preci pitati on, electroporation, or liposome mediated-, DEAE dextran mediated-, polycationic mediated-, or viral mediated transfection.

[0671] In another aspect, the disclosure provides pharmaceutical compositions, comprising the polypeptide, fusion protein conjugate, scaffold, the recombinant nucleic acid, the expression vector, or the recombinant host cell of any of any embodiment or combination of embodiments, and a pharmaceutically acceptable carrier. The pharmaceutical compositions of the disclosure can be used, for example, in the methods of the disclosure described herein. The pharmaceutical composition may further comprise (a) a lyoprotectant; (b) a surfactant; (c) a bulking agent; (d) a tonicity adjusting agent; (e) a stabilizer; (f) a preservative and / or (g) a buffer.

[0672] In some embodiments, the buffer in the pharmaceutical composition is a Tris buffer, a histidine buffer, a phosphate buffer, a citrate buffer or an acetate buffer. The pharmaceutical composition may also include a lyoprotectant, e.g. sucrose, sorbitol or trehalose. In certain embodiments, the pharmaceutical composition includes a preservative e.g. benzalkonium chloride, benzethonium, chlorohexidine, phenol, m-cresol, benzyl alcohol, methylparaben, propylparaben, chlorobutanol, o-cresol, p-cresol, chlorocresol, phenylmercuric nitrate, thimerosal, benzoic acid, and various mixtures thereof. In other embodiments, the pharmaceutical composition includes a bulking agent, like glycine. In yet other embodiments, the pharmaceutical composition includes a surfactant e.g., polysorbate-20, polysorbate-40, polysorbate- 60, polysorbate-65, polysorbate-80 polysorbate-85, poloxamer-188, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, sorbitan trilaurate, sorbitan tristearate, sorbitan trioleaste, or a combination thereof. The pharmaceutical composition may also include a tonicity adjusting agent, e.g,, a compound that renders the formulation substantially isotonic or isoosmotic with human blood.

[0673] Exemplary tonicity adjusting agents include sucrose, sorbitol, glycine, methionine, mannitol, dextrose, inositol, sodium chloride, arginine and arginine hydrochloride. In other embodiments, the pharmaceutical composition additionally includes a stabilizer, e.g., a molecule which, when combined with a protein of interest substantially prevents or reduces chemical and / or physical instability of the protein of interest in lyophilized or liquid form. Exemplary stabilizers include sucrose, sorbitol, glycine, inositol, sodium chloride, methionine, arginine, and arginine hydrochloride.

[0674] The polypeptide, conjugate, fusion protein, scaffold, nucleic acid, expression vector, or cell of any embodiment or combination of embodiments herein may be the sole active agent in the pharmaceutical composition, or the composition may further comprise one or more other active agents suitable for an intended use.

[0675] In a further aspect, the disclosure provides methods for using, or a use of the polypeptide, conjugate, fusion protein, nucleic acid, expression vector, host cell, and / or the pharmaceutical composition of any of the preceding claims, for any suitable purpose including but not limited to those disclosed herein. In various embodiments, the purpose includes, but is not limited to, treating or limiting development of limit development of diabetic vasculopathy, vascular dysfunctions, cancer angiogenesis, and wound healing in a subject in need thereof The subject may be any suitable subject, including but not limited to mammalian subjects such as human subjects.

[0676] As used herein, "treat" or "treating" means accomplishing one or more of the following: (a) reducing the severity of the disorder; (b) limiting or preventing development of symptoms characteristic of the disorder(s) being treated; (c) inhibiting worsening of symptoms characteristic of the disorder(s) being treated; (d) limiting or preventing recurrence of the disorder(s) in patients that have previously had the disorder(s); and (e) limiting or preventing recurrence of symptoms in patients that were previously symptomatic for the disorder(s).

[0677] Examples

[0678] Abstract

[0679] The Angiopoietin-Tie2 pathway regulates blood vessel stability, remodeling, and wound healing. Previously, we demonstrated that Angl-like synthetic ligands at high oligomeric states accelerate cell migration and promote vascular stability, but it is unclear how the Angl-Tie2 complex performs these opposing cellular functions. In this study, we found that clustering Tie2 receptors form two classes of complexes: Tie2-a5pi and Tie2-tight junction complexes that regulate endothelial migration and vascular stability, respectively. The Tie2-a5pi complex regulates the focal adhesion complex for cell migration and pAKT / FOXOl signaling axis that promotes cell survival. Conversely, the Tie2-tight junction complex, consisting of ZO1, claudin-5, and occludin, facilitates vascular stability. Furthermore, upon Tie2 activation, there's an acceleration in junctional ZO1 and claudin-5 re-assembly after chemical disruptions in endothelial cells, indicating the recovery of vascular stability through the Tie2 pathway. Since the Angl F-domain binds both Tie2 and ct5p i, to dissect the mechanism of the two Tie2 complexes’ formation and function, a synthetic protein mini binder (Tie2mb) was designed using an artificial intelligence algorithm, RFDiffusion™ to bind Tie2 at high affinity and specificity. Scaffolding Tie2mb to cluster Tie2 receptors is enough to recruit and form the two Tie2 complexes: Tie2-a5pi and Tie2 -tight junction. We have illustrated that modulating Tie2mb at different valencies exhibits super agonist and antagonist activity. As a monomer, Tie2mb can inhibit Tie2 signaling and downstream pAKT / FOXOl at high efficiency. When Tie2mb is conjugated onto an octameric scaffold, H8T, it activates the pAKT / FOXOl pathway and promotes tight junction re-assembly. Utilizing blood vessel organoids (BVOs), we evaluated the role of Tie2 in diabetic vasculopathy. Activating Tie2 using synthetic Tie2 binders in diabetic BVOs shows a significant reduction of pathogenic Col-IV secretion, reduction of nuclear F0X01 localization, and upregulation of tight junctions to a level comparable to healthy BVOs. In conclusion, we have dissected the functions of the two Tie2 complexes in angiogenesis and demonstrated that targeting the Tie2 pathway using the synthetic Tie2 superagonists are a promising therapy for diabetic vasculopathy.

[0680] Introduction

[0681] The angiopoietin-Tie2 pathway regulates blood vessel stability, remodeling, and permeability1–4Angiopoietins (Angl and Ang2) are the natural ligands for Tie2. Angl promotes pAKT activation and blood vessel stabilization, while Ang2 inhibits pAKT and induces leaky vasculatures despite sharing nearly identical protein structures5–8. Previously, we demonstrated that angiopoietin-1 and -2 activities fall into two broad phenotypic classes distinguished by the number of presented Angl F-domains that describe the mechanism of Tie2 signaling output9. We found that ligands presenting six or more copies of F-domains behaved like Angl, activated pAKT, while Ang2 forms trimers inhibited pAKT9.

[0682] Interestingly, we also found high F-domain valency ligands, like Angl, accelerated cell migration and promoted vascular stability9. It is unclear how Tie2 performs these opposing cellular functions. How do migrating cells produce stable endothelial cell-cell interaction? How does Tie2 orchestrate these opposing cellular functions? We hypothesized that upon Angl binding, Tie2 may have formed different complexes to produce these contrasting functional outcomes depending on cellular context. In the present study, we investigated the formation and function of these Tie2 complexes. We utilized de novo designed, selfassembling, two-dimensional protein sheets conjugated with F-domains (Fd-sheet)10to produce large Tie2 clusters for analyzing Tie2 complexes that drive cell migration or vascular stability.

[0683] We have previously shown that Fd-sheet can promote Tie2 clusters to co-localize with a5pl, and this association is critical to pAKT activation9. a.501 is a heterodimer composed of a and P subunits, a5pi is part of the integrin family that regulates cell adhesion and migration via focal adhesion complexes11–13. a5 i is upregulated in newly formed blood vessels13and similar to Tie2 / Angl, genetic ablation of a5 integrin in mice is embryonic lethal to the embryo due to vascular defects and leakage14,15. Activation of α5β1 using fibronectin promotes a5pi-Tie2 interaction and enhances Aug 1 -dependent pERKl / 2 activation16,17, but others also show the inhibition of the a5pl dimers augmented Angl activity in Tie2 phosphorylation18. These conflicting studies show that the role of a5pl in the Tie2 pathway is still unclear.

[0684] Both Tie2 and a5 i have been implicated as critical vascular stability modulators. Angl, a5, and tight junction molecule (claudin-5 and ZO1) protein expressions were upregulated in a timely manner at the same rupture sites in mice that received an induced- ischemic brain injury, suggesting that Tie2 and ct5 i may be essential for maintaining the BBB integrity post-injury19–21. However, it is unclear how these proteins interact at the molecular level. Contrary, a5pl was also argued to inhibit vascular stability22,23such that the inhibition of a.5 promoted a tighter blood-brain barrier via the upregulation of CLDN5 expression23. Disruption of the a5 and pi heterodimers has been shown to enrich E-Cadherin (CD144) and ZO1 at cell-cell junctions18,24. These contradicting results illustrated a big knowledge gap regarding the role a5pl in Tie2 signaling for vascular integrity.

[0685] This study investigated the mechanistic interplay between Tie2, a5pi, and tight junction proteins that drive cell migration and vascular stability. We utilized Al-based RFDiffusion protein design pipeline to produce de novo Tie2 or a5pl mini binder proteins with high affinity and specificity at well-defined oligomeric states or conjugated to 2-dimensional protein sheets to dissect the interactions between Tie2 with a5p i and tight junction proteins (zol and claudin-5) at the molecular level.

[0686] Results

[0687] Two classes of Tie2 complexes: Tie2-«5pi vs. Tie2-tight junction

[0688] We previously demonstrated that Angl F-domain at high valency accelerates cell migration and promote vascular stability9. How does Tie2 orchestrate these opposing cellular functions? How do migrating cells produce stable endothelial cell-cell interaction? In order to answer these questions, we sought to develop scaffolds and design de novo proteins that allowed us to visualize and precisely investigate the activation of Tie2.

[0689] Integrins are known to regulate cell migration and we have shown that clustering Tie2 receptors colocalizes with aSpi9Meanwhile, the tight junction proteins ZO1, claudin-5, and the occludins regulate vascular stability. We analyzed the interactions between Tie2, a5pi integrin, and junctional molecules using micron-scale protein sheets tagged with GFP and conjugated with Angl F-domain (Fd-sheet) to cluster Tie2 receptors (Fig. 1A)10. The efficacy of the F-domain sheets was analyzed using flow' cytometry. Cells treated with GFP-Fd-sheet show a significant higher level of FITC+ signal for GFP compared to GFP-sheet, showing that these GFP-sheets will bind cells only when F-domain is also conjugated (Fig.

[0690] 6A-B).

[0691] To examine the interaction between Tie2 and a5pi integrin serum-starved HUVECs were treated with the Fd-sheet for 30 minutes before fixation for immunofluorescence staining for microscopy analysis. Upon clustering Tie2, we found that a5, active pi, and downstream target pCAS colocalized with Tie2 under the Fdsheet (Fig. 1B-D & Fig. 6 D-F). Integrin activation initiates the assembly of focal adhesion (FA) complexes and recruits pCAS to regulate cell-to-extracellular matrix adhesion and migration25’26. Our data show Tie2 clusters recruited and induced a5pi activation, as illustrated by the presence of a.5, pCAS, and active pi colocalizing with Tie2 under the Fd-sheet while inactive pi is not observed (Fig. 1B-D & 6D-G).

[0692] To analyze the role of Tie2 m vascular stability, we stained HUVECs for tight junction markers, ZO1, claudin-5, and oocludin after Fdsheet treatment. Our data show that Tie2 co-localized with ZO1, claudin-5 (CLDN5), and occludins (OCLN) (Fig. 1E-F and 6 H-I). a5pi and Tie2 were previously suggested to play a role in tight junction formation for vascular repair21; thus, we investigated whether the Tie2-a5pl clusters are involved in the recruitment of these tight junction molecules. Using 5-color confocal microscopy we stained cells for CLDN5, Tie2, and active Bl, our quantifications revealed two classes of Tie2 complexes: Tie2-active Bl or Tie2-CLDN5 with minimal overlaps of the two complexes (Fig. 6 J-K). To investigate whether a5pi affect the formation of the Tie2 -tight junction complex, we knocked down ITGA5 using siRNA before treating the cells with Fd-sheets (Fig.

[0693] 1G and 6 L& N). Our results show that inhibiting the Tie2-a5pl complex, by knocking down the a5 integrin subunit, did not affect the recruitment of ZO1 and claudin-5 into the Tie2 clusters. This result further validated that there are two independent Tie2 complexes: Tie2-a5pl and Tie2 -tight junction components (Fig. 1H-J). Our result suggests that the Tie2-a5pi complex may not be responsible for the direct recruitment of tight junctions, which is consistent with studies that show a5 i inhibition did not affect endothelial tightjunction proteins14’18’23’24, interestingly, our data shows that inhibiting a5pl did significantly reduce the level of Tie2-VE-Cadherin colocalization compared to wild type control suggesting that Tie2-a5pi complex can regulate cell-cell adhesion but not tightjunction protein recruitment (Fig. 6 P-R). In summary, we observe Tie2 forming two classes of Tie2 complexes: Tie2-a5pl and Tie2 -tight junction. We will dissect the functions of the two complexes in more detail below.

[0694] F-domain binds «5pi in the absence of Tie2 receptors

[0695] Previous studies suggested that the Angl F-domain could activate integrin in cell lines that do not express Tie227’28, while others showed that fibronectin, not F-domain, is needed for Tie2-a5pi association. It is unclear whether Tie2-a5pi complexes are formed via a receptor-receptor interaction or shared ligands. To investigate the mode of interaction between Tie2 and a5pi, cells were treated with siRNA against TEK or ITGA5 before Fd-sheets administration (Fig. 6 L-N). Upon Tie2 knockdown using siRNA, we observed a significant reduction of Fdsheets binding to HUVECs, but interestingly the proportion of Fdsheets binding to cells via a5 only increased significantly compared to wildtype (Fig. 6 O).

[0696] F-domain binds Integrin via the RGD binding site

[0697] Consistent with previous studies, we observed F-domain binding and activating a5 i integrin without Tie2, but the F-domain binding site on integrin is unknown. We will now further study the function of the Tie2-a5pl complex using de novo-designed a5pi mini binder (a5plmb) with high specificity and affinity31. We first conjugated the oopimb to sheets (o pi nib-sheet) to see if clustering integrin would recruit Tie2 or not. Our data show's that a5pimb-sheets can recruit a5, active pl, and pCAS, but not Tie2 (data not shown) suggesting Tie2 is the main driver of the Tie2-a5picomplex. To identify the F-domain binding site on integrin, o pimb and Fd-sheet were used in binding competition experiments. MCF10A cells were treated with 20 nM of Fd-sheet and ct5 imb at 20, 200, or 500 nM for 30 minutes before fixation for FACS analysis. Using FACS, we measured the percent of cells bound w ith Fd-sheet using FITC detector. Our data show the percent of cells bound to Fd- sheet reduced as the concentration of a5pimb increased ((Fig. 6S),), suggesting that the a5plmb is competing w ith Fd-sheet and that the F-domain is likely binding to the similar RGD-bmding site where a5pimb is designed to bind. The native ligand, fibronectin binds integrin via the RGD sequence2, but the Angl F-domam does not contain RGD. It was speculated that the QHREDGS (SEQ ID NO: 40) sequence on Angl F-domam may have facilitated the interaction with integrin because QHREDGS (SEQ ID NO: 40) resembles the REDV (SEQ ID NO: 41) motif in fibronectin that bind a4fll28’^, but it is unknown whether this motif participates in a5pi binding. Our result suggests that F-domam was likely binding integrin via the RGD-binding sites in both MCF10A and HUVEC cells. Collectively, we propose that Angl binds and active integrin via F-domain and the Tie2-a5pi complex is likely initiated via F-domain binding to both receptors.

[0698] Modulating the valency of de novo designed Tie2 mini binder exhibits super agonistic and antagonistic properties

[0699] To test this hypothesis, we sought to design a minimum Tie2 binding domain using RFDiffusion™34in order to evaluate whether a5pi could be recruited in the absence of F-domain. The Tie2 mini binder (Tmb) was designed on the Tie2-Angl F-domam binding interface. We created six parent designs of Tie2 binders (PDB 2GY5) (Fig. 7A). Through a senes of mutations on each parent design, we generated subsequent families of binders (Tables 2-7). Among all the mini binders, tw1102 has one of the highest affinities for Tie2 (Fig. 2E and 7B) and expressed robustly and with a single monodisperse peak on SEC (Fig.

[0700] 2D). The full SSM graph of tw1102 with the positions showing better affinity' than the parent tw1000 design is included in Figure 7C. The subset of positions at the interface are shown m Figure 2C. As expected, the vast majority of surface positions for tw1000 do not show' a strong preference in amino acid (Fig. 7E). A close up of the tw1000 (tw1102's parent design) design complex showing the variability of each position from the SSM data (from the 500nM Sort) is shown in Figure 2B. To validate the specificity of tw1102, we knocked (KO) out Tie2 in EA.hy926 cells and demonstrated that tw1102 failed to bind the KO cells compared to the WT cells (Fig. 2F). tw 1102 ( also abbreviated as Tmb) was used in all following cell experiments.

[0701] The average affinity of Tmb (tw1102, 0.65 nM, Fig. 2E) for Tie2 binding is 5-fold higher than the native Angl F-domain (3nM8), even though the binding mode of Tmb is very similar to Angl F-domam (Fig 3 A). To test the activity of Tmb, we performed competition experiments using serum starved HUVECs treated with a Tie2 super agonist, a chimeric designed protein presenting eight copies of F-domain called H8F9. 10 nM H8F activates Tie2 / pAKT9, but starting at 1 nM Tmb significantly inhibited pAKT and pERK activation, indicating that Tmb can compete with the native Angl F-domam (Fig. 3B & 8A-B).

[0702] We have previously demonstrated that F-domain scaffolded at low valency behaved like Ang2, while high valency behaved like Angl9. Thus, we conjugated Tmb to trimeric and octameric scaffolds to make H3T and H8T. Serum starved HUVECs were treated with H3T or H8T at 0.01 to 1000 nM and H8F at 10 nM as positive control. Our result shows that H8T activated pAKT, behaved like Angl and H8F, with an estimated EC50at 0.67 nM (Fig. 3C and 8C-E) while H3T did not activate pAKT and behaved like Ang2. pAKT activation induces FOXO1 translocation out of the nucleus to promote cell survival35,36, thus we tested the capacity of Tmb and H8T to induce nuclear FOXO1 exclusion in HUVECs. Our data shows that H8T and H8F induced nuclear FOXO1 exclusion while Tmb alone does not (Fig.

[0703] 3D-E). Moreover, Tmb inhibits H8F and upregulated FOXO1 in the nucleus.

[0704] To see whether a minimal Tie2 protein binder is able to form the Tie2 complexes described in Figure 1, we conjugated Tmb to sheets (Tmb-sheet). Our data shows that both a.5 integrin and tight junction claudm-5 molecules can still be recruited to Tie2 clusters (Fig. 3F-G). To rule out the possibility of Tmb-sheet binding to integrin non-specifically, we tested the Tmb-sheets in MCF10A cells, which do not express Tie2, using FACS, and our result confirms that Tmb-sheets do not bind MCF10A cells (Figure 3H). Based on our data, we propose that F-domain is not essential to the formation of the Tie2-a5pi complex; a5pl can be recruited by Tie2 receptor via other mechanism and F-domains may have participated in stabilizing the interaction. We will now investigate the function of the Tie2-a5p i complex. Tie2-a5pi complex is critical to pAKT / FOXOl signaling axis.

[0705] We previously demonstrated that a5pl is important for Tie2 / pAKT activation using siRNA to knockdown a5pi expression transiently leading to the inhibition of Tie2 / pAKT9. In the present study, we tested the role of the Tie2-o5pi complex in pAKT / FOXOl signaling using a5pinib to inhibit a5pl. Serum starved HUVECs were treated with 10 nM of H8F + / -a5plmb at 0.1-1000 nM to evaluate pAKT inhibition. Our data shows that a5plmb were able to inhibit H8F and attenuate pAKT activity (Fig. 4A-B & Fig. 9A). a5pimb also inhibited the capacity of H8F to induce FOXO translocation out of the nucleus (Fig. 4C-D & Fig. 9D). To illustrate that a5pi integrin is critical to Tie2 signaling,ie examined the effect of inhibiting other integrins using mini binders against avP6 and avP8j7in competition experiments. avP6 and avP8 are known to play a role in angiogenesis13, hence w?e investigated their role in the Tie2 pathway. Cells were treated with another Tie2 super agonist, Icos19+ avP6mb or avP8mb, and our results show that neither minibinder w ere able to inhibit pAKT activity while the native integrin ligand, fibronectin, mildly downregulated pAKT (Fig. 9C-D). Similarly, inhibiting avP6mb or avpSmb did not affect Tie2-dependent FOXO1 translocation out of the nucleus (Fig. 9E-F). Collectively, we have illustrated that the Tie2-a5pi complex is critical to the activation of the pAKT / FOXOl signaling axis.

[0706] Tie2-a5pi complex regulates endothelial cell migration.

[0707] We have demonstrated that the Tie2-o5pl recruits pCAS (Fig. 1B-D & Fig. 6 D& F), a key component in the focal adhesion complex that regulate cell migration25,26. To validate the role of the Tie2-a5pl complex in endothelial cell migration, we tested whether a5plmb could down regulate Tie2-dependent endothelial cell migration using scratch assay9, j8.

[0708] Briefly, a scratch were made in the monolayer of HUVECs, then treated with Tie2 agonists (H8F or Icos1) with or without integnn mini binders for 12 hours to evaluate the change in wound area (Fig. 4E). Previously we have shown that H8F and Icos1 upregulated cell migration9, here we show that when cells were treated with a5plmb and H8F or Icos1, cell migration is significantly reduced to a level similar to PBS control (Fig. 4F-G). On the other hand, avP6mb and avP8mb were not able to inhibit Tie2-dependent cell migration (Fig. 9G-H). In conclusion, our data strongly suggests that a5pl is critical to Tie2 mediated endothelial cell migration.

[0709] Tie2 activation accelerates tight junction assembly

[0710] We previously demonstrated in vivo Tie2 activation using superagonist, Icos1 accelerated the repair of hemorrhagic vessels after injury in mouse brains9, but how Tie2 regulates this process at the molecular level is unclear. We found that clustering Tie2 forms two classes of complexes: Tie2-α5β1 and Tie2-tight junction (ZO1 / CLDN5 / OCLN) (Fig. 1B-H). To evaluate whether Tie2 modulates tight junction assembly, we destroyed endothelial tight junctions using Latrunculin-A (LatA)-’9,40in HUVECs orHBMECs. Briefly, cells were treated with 0.25 uM of LatA for 30 minutes then LatA was washed off before adding agonistic or antagonistic F-domain scaffolds9for another 30 minutes to allow tight junction recovery (Fig. 4H). Impressively, our data show both H8F and H8T significantly accelerated the recovery of junctional claudin-5 and ZO1 compared to PBS in just 30 minutes of recovery, while Ang2-like ligand, H3F behaved similar to PBS (Fig 4I-K).

[0711] Two functionally distinct classes of Tie2 complexes

[0712] To summarize our findings thus far, we found that clustering Tie2 receptor using high valency ligands form two functionally distinct classes of Tie2 complexes that regulate cell migration and vascular stability; Tie2-a5pl and Tie2 -tight junction. The Tie2-a5pl complex regulates cell migration by recruiting VE-Cadherin out of cell junction to promote cell mobility' and inducing the formation of focal adhesions to promote cell migration. We also illustrated that the Tie2-a5pl complex is essential to the pAKT activation and nuclear exclusion of FOXO1, which promotes cell survival9. In contrast, we demonstrated that the other Tie2 clusters without a5pi recruit ZO1, claudin-5, and occludin to assemble tight junction complex in the endothelial paracellular space to regulate vascular stability and permeability.

[0713] Tie2 activations repair diabetic vascular damages

[0714] Tirus far, we have found that agonizing Tie2 promotes positive vascular outcomes and exhibits reparative capacity. We next evaluated the functional role of the Tie2-a5pi and Tie2 -tight junction complexes in a disease context. We established a diabetic vasculopathy (DV) organoid model that features DV phenotypes, namely, upregulated Col-IV expression, loss of cell-cell junctions, and nuclear FOXO1 enrichment. We adapted a previously developed iPSC-denved blood vessel organoid (BVO) protocol43. At day 23, when organoids are mature, we created three different conditions: a control non-diabetic condition; a diabetic condition that contained 4.5 fold more glucose than controls and TNFa and IL-6, cytokines which are commonly upregulated in diabetic individuals; and a rescue condition in which BVOs were cultured in the diabetic media for one week, and then were supplemented with 20nM H8F or H8T for an additional 7 days (Fig. 5A). Intricate vascular networks were fomied in day 23 mature BVOs with high level of pericyte association (Fig. 5B). After 2 weeks of diabetic induction, the vascular network degraded in diabetic BVOs whereas diabetic BVOs supplemented H8F on day 30 shows re-establishment of vasculature (Fig. 5C).

[0715] 3D renderings of the vascular organoids demonstrated large differences in the vascular networks under normal and diabetic conditions; a reduction in CD144 (VE-cadherin) and PDGFRp colocalization m the diabetic condition indicates the dissociation of endothelial ceils and pericytes which then results in vascular instability (Fig. 10A), We observed that only 7 days of H8F supplementation resulted in upregulation of CD31 and PDGFRP expression and co-localization, suggesting the reestablishment of vascular stability (Fig. 10A). Upregulated collagen IV secretion is commonly seen in diabetic vasculopathy and we also observed an increase in ColIV expression, secreted around diabetic vessels (Fig. 5D & 0A). Treatment with H8F for 7 days reversed this phenotype, restoring localization of CD31 and PDGFR, and reducing ColIV levels (Fig. 5D& Fig. 10A).

[0716] FOXO1 transcription factor enrichment in the nucleus is also a marker for diabetic vasculopathy. Nuclear FOXO1 upregulates gene expression that promotes inflammation and destabilization of blood vessels. When we examined the expression of nuclear FOXO1 in these blood vessel organoids, we observed a significantly high level of nuclear FOXO1 in the diabetic organoids compared to the normal control (Fig. 5E-F). The addition of H8F and H8T to diabetic organoids reduced nuclear F0X01 significantly compared to the untreated diabetic organoids (Fig. 5E-F). Diabetic vasculopathy oftentimes first manifests in arterial blood vessels. We generated arterial vascular organoids44to test the efficacy of H8F in rescuing diabetic arterial blood vessels. Treating BVOs with FGFR super agonist C6 instead of bFGF on day 5-13 of the protocol promoted more arterial endothelial cell marker Epherin2B expression (Fig. 10B). H8F treated arterial BVOs significantly down regulated nuclear FOXOl in diabetic arterial vascular organoids (Fig. 10C-D), suggesting that H8F have the potential to prevent the early onset of diabetic vasculopathy.

[0717] We have demonstrated that Tie2 super agonist H8F and H8T could accelerate tight junction formation (Fig. 4H-K), and junctional instability is a major contributing factor to diabetic vasculopathy4'47Leaky vessels are hallmark of diabetic vasculopathy; thus, we evaluated the organization of tight junctions that regulate vascular stability and permeability in our diabetic organoid model with or without H8F or H8T. While non-diabetic samples exhibited crisp ZO1 and CLDN5 membrane organization, culturing organoids in diabetic media resulted m their complete disruption (Fig 5G-H). However, following 7 days of treatment with H8F and H8T, organoids showed evidence of rebuilding tight junctions, with clearly recovered organization of ZO1 and ongoing reorganization of CLDN5 with H8F being more powerful than H8T in this case. Quantification of junctional ZO1 shows significantly higher levels of junctional ZO1 in H8F-treated diabetic organoids compared to untreated diabetic organoids. In summary, activating Tie2 using H8T and H8F holds promising therapeutic benefits for rescuing diabetic vasculopathy.

[0718] DISCUSSION

[0719] Activating the Angl-Tie2 signaling axis promotes cell migration and vascular stability (citation), but it is unclear how Tie2 elicits these opposing cellular functions. Our present study dissected the underlying mechanism that governed Tie2-mediated cell migration and endothelial junctional stability. We utilized micron-size 2D protein sheets conjugated with F-domain or synthetic Tie2 mini binder (Tmb) to cluster Tie2 to probe the potential associating molecular partners. Upon clustering Tie2 receptors, we found that Tie2 forms two classes of complexes: Tie2-a5pi and Tie2 -tight junction regulates cell migration and vascular stability, respectively. Surprisingly, while many suggest a5pl important for vascular stability and permeability (citations), we found a5 i is not needed for the formation of the Tie2-tight junction complex, but a5pi is needed for adhesion molecule CD144 recruitment, which is consistent with previous studies showing that a5pi is essential to CD144 proper localization in endothelial cells48,49. Adherens junctions (i.e. CD144) formation is a known prerequisite for tight junction formation; however, we saw that downregulated Tie2-CD144 complexes did not affect TJ recruitment to Tie2 in a5pi knockdown. One possible explanation for this is that the Tie2-a5pl complex recruits CD144 out of the cell-cell junction to increase cell mobility for migration, thus when the Tie2-a5pi complex is inhibited, CD144 is re-localized to promote cell-cell attachment allowing Tie2 to assemble tight junction.

[0720] Upon further characterization of the Tie2-a5pi complex, we observed that a5 i is in its activated form. We detected active pi accumulation with the Tie2 clusters and integnn target, pCAS is also recruited to this complex, Integrin signaling regulates cell attachment and migration by assembling focal adhesion complexes that consist of paxillin, talin, vinculin, FAK, and pCAS25,26’29’30”52Thus, we tested whether a5pi is needed for Tie2 to upregulate cell migration. Our data show that a5pl inhibition using a5pl mini binders halted endothelial cell migration, reinforcing that the Tie2-a5pi complex promotes cell migration. Angl was previously shown to promote Tie2 and a5pi association16,17’20, but structural analysis of this interaction has not yet been thoroughly investigated. Our data demonstrated the Angl F-domain is the driver bringing Tie2 and a5pl into a complex. We found endothelial cells treated with siRNA to knockdown Tie2 enable Fd-sheets binding to endothelial cells via a5 only. We validated this observation in experiments using MCF10A cells that lack Tie2 expression. We found Fd-sheets w’ere able to bind to these cells via a5pl. We also demonstrated aSpimb - designed to bind the RGD-bmding site on a5pi- competed with Fd-sheets in MCF10A cells suggesting that F-domain is likely bind to a5pi near the RGD binding site. Fibronectin binds integrin via the RGD sequence on the FN-III10domain, and the synergy site on FNIII9stabilizes this interaction32. Angl F-domam had been suggested to bind and activate the integrin pathway, but F-domam does not contain RGD; thus, it was speculated that the QHREDGS (SEQ ID NO: 40) sequence on Angl F-domain may have facilitated the interaction with integrin because QHREDGS (SEQ ID NO: 40) resembles the REDV motif in fibronectin that bind adpi28’3-’, but it is unknown whether this motif participates in a5pi binding. The structural basis of Angl-Tie2-integrin interaction still requires more investigation.

[0721] In the effort to dissect the role of Angl F-domain in Tie2 signaling, we produced a synthetic Tie2 mini binder (Tmb) which was made possible with the recent technological advancement in artificial intelligence-guided protein design, RFDiffusionTMWe illustrated that Tmb as a monomer is an antagonist and competed w ith F-domain which abolished H8F-induced pAKT activation. Replacing F-domam with Tmb in H8 and sheet scaffolds, we are able to replicate all signaling outputs of Tie2. Collectively, we have generated a fully synthetic Tie2 super agonist and antagonist that holds tremendous therapeutic value for targeting Tie2 signaling in diseases that features vascular dysfunctions, cancer angiogenesis, and wound healing.

[0722] In addition, we demonstrated the Tie2-a5pl complex is critical for pAKT / FOXOl signaling activation. The pAKT / FOXOl signaling axis promotes cell survival9,53–55and junctional protein gene expression36. Our data show α5β1 inhibition using siRNA against ITGA5 or a5pimb attenuated AKT phosphorylation9and F0X01 nuclear exclusion, interestingly, Angl have been shown to promote the gene expression of tight junctions, Z01, Z02, and CLDN5 via pAKT / FOXOl signaling36.

[0723] Our current study demonstrated that clustering Tie2 receptors recruited tight junction molecules: Z01, CLDN5, and OCLN independent of a5pl in our Fd-sheet experiments. Thus, we investigated the capacity of Tie2 activation to re-assemble tight junctions in cell cultures (HUVECs andHBMECs) after LatA-induced junctional disruption. Notably, Tie2 activation using the Tie2 agonist, H8F and H8T, accelerated TJ (Z01 and CLDN5) re¬ assembly.

[0724] Since H8F is a powerful Tie2 agonist that can accelerate tight junction formation, we examined the capacity of H8F vs H8T to reverse diabetes-induced vasculopathy using vascular organoids. Vascular organoids under diabetic conditions for two weeks displayed junctional Z01 and CLDN5 disruption, but impressively one week of H8F and H8T treatment after diabetic conditions reversed these junctional defects back to a normal level.

[0725] Collectively, we have illustrated that multivalent ligands like Angl cluster Tie2 receptors to form different classes of Tie2 complexes. In this study, we studied the functions of the Tie2-a5pi and Tie2-tight junction complexes. Our results illustrated the Tie2-a501 complex regulates cell migration while the Tie2-tight junction complex promotes vascular stability. The Tie2-a5pi complex may have upregulated endothelial cell migration in the early stage of angiogenesis for the branching and growing of new blood vessels. In contrast, the Tie2-tight junction complex stabilizes the nascent vessels in the later stage. These opposing Tie2 cellular outcomes play an important role in different stages of angiogenesis. Activating Tie2 in diabetic BVOs shows a significant reduction of pathogenic Col-IV secretion, reduction of nuclear F0X01 localization, and upregulation of tight junctions to a level comparable to healthy BVOs. In conclusi on, we have dissected the functions of the two Tie2 complexes in angiogenesis and demonstrated that targeting the Tie2 pathway using the synthetic Tie2 super- agonists are a promising therapy to treat diabetic vasculopathy. REFERENCES

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[0773] METHODS

[0774] Cell culture

[0775] Human umbilical vein endothelial cells (HUVECs) were purchased from Lonza (Catalog #: C2519A) and cultured in EGM2 media described previously9. Briefly EGM2 consist of 20% fetal bovine serum (BioWest, SI 620), 1% penicillin–streptomycin (Gibco, 15140122), 1% GlutaMAX™ (Gibco, 35050061), 1% ECGS (endothelial cell growth factor), 1 mM sodium pyruvate (Gibco, 11360070), 7.5 mMHEPES (Gibco, 15630130), 0.08 mg / ml heparin (Fisher BioReagents, 904108-1), 0.01% amphotericin B (Gibco, 15290018), a mixture of lx RPMI 1640 (Gibco, 1875093) with and without glucose (Gibco, 11879020) to reach 5.6 mM glucose concentration in the final volume. Media was filtered through a 0.2-lm filter. HUVECs were expanded and serially passaged to reach passage 4 before

[0776] cry opreservation. Human brain microvessel endothelial cells (HBMECs) were purchased from Cell Systems (catalog # ACBRI 376) and cultured in EGM™-2 MV Microvascular Endothelial Cell Growth Medium-2 BulletK.it™ (catalog # CC-3202, Lonza). Both cell lines were cultured until passage 7 or 8 before 16 hours of serum starvation in low glucose DMEM (catalog # 11885084, Gibco) prior to experiments. MCF10A cells were cultured in media described previously50'52; briefly, the media consisted of DMEM / F12 (Gibco, 11330032), 5% horse serum (Gibco, 16050130), 20ng / ml EGF (Sigma- Aldrich, SRP3027), 0.5mg / ml hydrocortisone (Sigma-Aldrich, H4001), lOOng / ml cholera toxin (Millipore, C8052), lOug / ml insulin (Sigma- Aldrich, 11070-73-8) and 1% penicillin -streptomycin (Gibco, 15140122). MCF10A cells were starved in the same media without EGF and contain 2% horse serum (assay media) for 16 hours before experiments.

[0777] TJP1-GFP iPSCs line were purchased from the Allen Institute Cell Catalog (AICS-0023 cl.20) and cultured with mTeSRl™ media (STEMCELL, #85850) on plates coated in 2.5% Matrigel™ (Corning, #356231) until differentiation experiment.

[0778] Diabetic blood vessel organoids

[0779] This protocol was adapted from a previous version43. To create iPSC-derived vascular organoids, WTC-11 hiPSCs were passaged, counted, and resuspended in differentiation media (DMEM / F12, 20% KOSR (Gibco, #10828028), IX Glutamax™, IX NEAA (Gibco, #11140050) supplemented with 50pM Y -27632 [Tocris Bioscience, #1254] and distributed into Ultra Low Attachment 6-well culture plates (Corning, #07-200-601 ) at a density of 3 x 105cells / well to allow spheroid formation. At day 3, cells were fed with fresh differentiation media supplemented with 12pM CHIR99021. At days 5, 7, and 9, cells were given with fresh differentiation media supplemented with 30ng / ml recombinant BMP-4, 30ng / ml recombinant VEGFA165 (R& D Systems, #293-VE), and 30ng / ml recombinant FGF2. For Days 11-15, the glucose in the differentiation media was reduced to 2 g / L glucose from 3.15 g / L by using SILAC™ Advanced DMEM / F12 (Gibco, #A2494301) supplemented with 147.5 mg / mL L-Arginine, 91.25 mg / mL L -Lysine, 2 g / L D-Glucose, 20% KOSR, IX Glutamax™ and IX NEAA. On Day 11, cells were given fresh differentiation media supplemented with 30ng / ml VEGFA165, 30ng / mL FGF2, and 10 pM SB431542 (Miltenyi, #130-106-275). On Day 13, cells were transferred to Ultra Low Attachment™ U-Bottom 96 well plates (Coming #CLS3474-24EA) at 1 organoid per well, embedded in 1: 1 Matrigel™: Collagen I matrix, and overlaid with differentiation media supplemented with 100 ng / mL VEGFA165 and 100 ng / mL FGF2. From Days 15 to 23, the glucose in the differentiation media was reduced to 1g / L glucose (physiological glucose) by making it as described for Days 11-15, but supplemented with 1 g / L glucose. Cells were fed on Day 15 and every 3 days thereafter until Day 37. To create diabetic vascular organoids, at d23 (when organoids are mature) organoids were fed with media described above, but containing 4.5g / L D-glucose and InM each of recombinant TNFa and IL-6 inflammatory cytokines. Media was changed every' 3 days until harvest at d37. For organoids treated with H8F, organoids were first cultured in diabetic media for a period of 7 days (through d30) and then treated with 20nM H8F through d37.

[0780] Western blot for signaling analysis

[0781] Serum starved HUVECs were treated with synthetic ligands for 15 minutes at 37 degrees Celsius. Treatment was then removed, and cells were washed once with lx PBS before adding lysis buffer described previously9. Lysates were then collected in tubes and 4X Laemmli sample buffer (BioRad, #161-0747) were added before boiling at 95 degrees Celsius heat block for 10 minutes. 30 pL of protein sample per well was loaded and separated on a 4-10% SDS-PAGE gel for 30 min at 250 Volt. The proteins were then transferred on a nitrocellulose membrane for 12 min using the semi-dry turbo transfer Western blot apparatus (Bio-Rad, USA). Post-transfer, the membrane was blocked in 5% bovine serum albumin for 1 hour. After blocking, the membrane was probed with the respective primary antibodies and incubated at 4°C, overnight on a rocker: pAkt-S473 (Cell Signaling, 9271 S) at 1:2,000 dilution;pERKl / 2 p44 / 42 (Cell signaling, 4370S) at 1:1,000 dilution; Tie2 (Cell Signaling, 4224S) at 1:1,000 dilution; a5 integrin (Millipore, AB1928) at 1:1,000; and p-Actin (Cell Signaling, 3700S), S6 (Cell Signaling, 2217S). Next day, the membranes were washed with 1X TBST (3 times, 10 min interval) before adding respective HRP-conj ugated secondary antibody (Bio-Rad, USA) at 1:10,000 dilution and incubated at room temperature for 1 hour. For pAKT(S473), following washes, the membrane was blocked in 5% milk at room temperature for 1 how and then incubated in its respective HRP-conjugated secondary antibody (1:2,000) prepared in 5% milk for 1 hour. After secondary', the membranes were washed with 1× TBST (3 times, 10 min of interval) and developed using Chemiluminescence developer and imaged using Bio-Rad ChemiDoc™ Imager. Quantifications were done by¬ calculating the peak area for each band on western blot images using ImageJ. The peak area of the target proteins was divided by the peak area of housekeeping proteins. All signaling levels are normalized to H8F levels as an internal positive control.

[0782] F-domain conjugation to design protein scaffolds

[0783] Design protein component genetically fused with SpyCatcher™ was combined with binder genetically fused with SpyTag™ (F-domain-st, Tmb-st, a5plmb, or GFP-st) at 1:1 — 1.5 molar ratio for 4 hours at room temperature or overnight in 4 degrees C on nutation. SDS-electrophoresis gels were run to evaluate the conjugation efficiency. All experiments were done using constructs that have reached at least 90% conjugation efficiency. Two-dimensional sheets assembly were done according to previous protocol10. Briefly, A-GFP, A-(Fd or mb), and B domains were combined at 1:4:5 molar ratio, respectively, and incubated at room temperature for 2 hours or overnight in 4 degrees C on nutation before experiments.

[0784] Immunofluorescence analysis of Tie2 clusters

[0785] Serum starved HUVECs or HBMECs were treated with Fd-sheets or α5β1mb-sheets at 50nM of binding domain for 10-30 minutes in low glucose DMEM before fixation with 4% PF A. The fixed cells were washed three times at 5 minutes each before blocking for 1 hour with 3% BSA (VWR, 0332-500G) and 0.1% Triton X-100 (Sigma, T9284-500ML) in PBS while on nutation. The cells were then incubated with primary antibody diluted at 1: 100 in blocking agent overnight: Tie2 (Cell Signaling, catalog #4224S), FOXO1 (Cell Signaling, 2880), and α5 integrin (Millipore, 1928), active pi integrin 9EG7 (BD Biosciences, 550531), inactive pl integrin mAbl3 (BD Biosciences, 552828), phospho-CAS, CAS (Cell Signaling, 4011S), CD144 (Cell Signaling, 2500S), ZO1 (Invitrogen, 33-9100 or 61-7300), claudin-5 (abeam, abl5106), and occludin (Invitrogen, 710192). After the primary antibody, the cells were washed three times at 5 minutes each with IX PBS while on nutation. The cells were then incubated with secondary antibodies at 1:200 (1: 100 for Tie2) and phalloidin at 1: 100 (Invitrogen, A12380) diluted in blocking agent for 1 hour and 20 minutes at 37° C, Secondary antibodies were then removed, and cells were washed for three times at 10 minutes each with PBS on nutation. Coverslips were sealed using VECTASHIELD™ plus DAPI (Vector laboratories, H-2000-2) upside-down on glass slides for analysis in confocal (Leica SP6 or SP8) or super-resolution (Delta vision OMX SR) microscopy,

[0786] SiRNA knockdown of TEK or ITGA5

[0787] Passage 7 HUVECs (100,000 cells / 1 mL) were first transfected with 40 nM siRNA using LipofectamineTMTransfection Kit (Invitrogen, 13778-075) in 1 mL EGM2 medium suspension without antibiotics. Cell suspensions were then seeded on 0.1% gelatin coated 35-mm plate (for sheet experiments, cells were seeded onto coverslips coated with gelatin). After 18 hours of siRNA incubation, media were changed with fresh EGM2 to allow cell recovery for 6 hours before 16-hour serum starvation with low glucose DMEM. Starved HUVECs w'ere treated with scaffolds at 20 nM of F-domain concentrations for 15 minutes before protein collection for western blot analysis or 30 minutes for sheet experiments before PF A fixation. siRNAs targeting TEK (siRNA: M-003178-03-0005) or ITGA5 (siRNA: L-00800300-0005) mRNA were purchased from Dharmacon.

[0788] F-domain and α5β1 mini binder competition experiments

[0789] Starved MCF10A were incubated with 20 nM Fd-sheetGFPand α5β1mb at 20, 200, or 500 nM for 30 minutes before fixation with 4% PFA for 15 minutes on ice. Fixed cells were spun down at 6000 rpm for 1 minute to remove PFA and washed once with PBS then resuspended in 200 uL of PBS for FACS analysis using FITC detector to measure % GFP+ cells that would indicate % of cells bound with Fd-sheetGFP. 10,000 events were recorded per tube.

[0790] Surface plasmon resonance binding assay for Tie2 mini binder

[0791] SPR experiments were performed using a Biacore™ 8K Cytiva instrument. Binding was measured by capturing approximately 250 response units of recombinant human biotinylated Tie2 protein (Sino Biological #10700-H08H-B) using a Biotin CAPture™ Kit, Series S (Cytiva #28920234). A 6-step five-fold single cycle kinetic dilution series from 150 nM was used to assess affinity with 120 sec association time and 300 sec dissociation time at a flow rate of 30 ul / min at 25°C in IX HBS-EP+buffer (Cytiva #BR100669). The chip was regenerated between runs with 0.25M NaOII and 6M guanidine hydrochloride. KDs were determined by fitting curves assuming a langmuir 1:1 model using the Biacore™ Evaluation software.

[0792] Cell adhesion assay

[0793] MCF10A CASmScarlet™ cells57were starved in assay media for 12 hours before the experiment and detached by Accutase™ treatment. These cells were resuspended in assay media, incubated for 60 min at 37°C in 5% CO2 in suspension and plated on to glass-bottom dish (FluoroDish™, FD35-100, World Precision Instruments) precoated with 50 ug / ml collagen-I (Advance Biomatrix, #5056). For spreading experiment, Fd-sheets were added on to glass-bottom dish before plating cells for live cell imaging. While for the binding assay, 12 hours attached cells were treated with Fd-sheets for 30 min in absence or presence of Mn 2+ and harvested for the immunostaining.

[0794] TIRF live-cell and immunofluorescence microscopy Dual-color live cell imaging was performed immediately after plating cells till 30 min of time period. The images were recorded using TIRF microscope (Nikon Ti, 100x / 1.49 CFI Apo TIRF oil immersion objective) equipped with Perfect Focus™, motorized x-y stage, fast piezo z stage, stage-top incubator with temperature set to 37 C and 5% CO2 control, and Andor™ iXon X3 EMCCD camera with 512 x 512-pixel chip (16-micron pixels). The images were processed and analyzed using imageJ™ For immunofluorescence microscopy, cells were fixed using 4% paraformaldehyde for 15 min followed by permeabilization with 0.1% Triton X-100 for 5 min and blocked for an hour in 5% normal goat serum+2% BSA, all in 1XPBS. Primary antibodies were added in 1:200 dilution for 2-3 hours at room temperature or overnight at 4°C which is then incubated with Alexa™ Fluor conjugated secondary antibodies for 1 hour in dark. These coverslips were mounted in ProLong Gold™ (Invitrogen) for confocal microscopy. These mounted coverslips were left at room temperature for solidification of ProLong Gold™, as suggested by manufacturer and imaged on the Dragonfly™ 200 High-speed Spinning disk confocal imaging platform (Andor Technology Ltd.) on a Leica DMi8 microscope stand equipped with iXon EMCCD and sCMOS Zyla™ camera. Images were taken under 100x / 1.4 oil immersion objective by using Fusion Version 2.3.0.36 (Oxford Instruments) software and deconvolved on associated Imaris software simultaneously. Flow cytometry and competition assay: Cells were detached, harvested in assay media and kept in suspension for 60 min followed by treatment with Fd-sheet in absence or presence of Mn 2+ for 30min. In competition assay with the integrin minibinder (mb), these cells were first incubated with increasing concentration of mb for 15 min followed by Fd-sheet treatment for additional 15 min and fixed with 4% paraformaldehyde in PBS for 15 min. These cells were washed with 1XPBS twice before flow cytometric analysis on BD FACSymphony™ 3. FCS files were analyzed using FlowJo™ software.

[0795] Cell migration assay

[0796] Scratch assay was performed according to previous protocols with modifications9,38. Before seeding cells, one line is drawn on bottom of the plate perpendicular to the scratch to track imaging spots. HUVECs were seeded on 0.1% gelatin coated surface and cultured in homemade EGM2 media (HUVECs) until confluent. Once cells reach 80-90% confluency, a scratch is made on the center of the plate and then washed once with PBS then incubated DMEM low glucose supplemented with 2% FBS + / - treatment for 12 hours. HUVECs were imaged at 0 and 12-hour timepoints. Images were taken above and below the drawn line to get two images per well. ImageJ was used to quantify the images at all timepoints in pixel scale. Percent change of wound area were calculated as below:

[0797] * 100% = % Wound Closure

[0798]

[0799] ^(to)

[0800] A(tO) - wound area at time 0

[0801] A(tx) - wound area at 3, 6, 9, or 12

[0802] Tight junction disruption using Latrunculin-A

[0803] To evaluate the effect of Tie2 activation on tight junction re-assembly, endothelial cells were treated with an action inhibitor Latrunculin-A (Cayman, 10010630) to disturb tight junction proteins40before F -domain scaffold administration. Briefly, confluent HUVECs or HBMECs seeded on coverslips were treated with 0.25 uM of LatA diluted in growth media for 30 minutes. After 30 minutes, LatA were washed out with PBS before adding F-domain scaffolds at 100 or 200 nM diluted in LG DMEM + 10% FBS for 30 minutes or 2 hours. Post treatment, cells were fixed with 4% PF A for immunofluorescence staining with antibodies (detail protocol in immunofluorescence section) to visualize ZO1 (Invitrogen, 1:100) or Claudin-5 (abeam, 1:100). Phalloidin (1:100, Invitrogen, A12380) were added during secondary stain. Stained cells were mounted on slides using VECTASHIELD™ plus DAPI. Phalloidin stains were used to find cell colonies with good contact before switching to ZO1 / CLDN5 stain to count junctional tight junction assembly. At least 100 cells were counted per coverslip.

[0804] Computational design of Tie2 binders

[0805] The Tie2 target structure was obtained from PDB 2GY5 and cropped to a smaller size only containing the Ang binding site. The hydrophobic residues of the interface were selected and given to RFDiffusion™ as hotspots. RFDiffusionIMproduced several thousand outputs which were run through ProteinMPNN to generate sequences and subsequently AIphaFold2™ to evaluate their quality. This did not result in satisfactory results so 300 of the best Tie2 designs were selected and their interface helices extracted. RFDiffusion™ was then used to build new scaffolds around these helices and then those new scaffolds were subjected to Partial Diffusion with RFDiffusion™ in the hope that the new connectivity would allow for better shape complementarity after some wiggling. MPNN and AF2 were used again to design and evaluate and the outputs looked a little better. This grafting process was repeated 2 more times until a set of 7,500 very high quality designs had been generated and were ready for testing. REFERENCES

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Claims

We claim1. A polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 1-17, not including any amino acid insertions at identified insertion sites (i.e., any insertions are not considered when determining percent identity to the reference polypeptide), and not including residues noted as dispensable in Tables 2-7, wherein the polypeptide binds to Tie2.

2. The poly peptide of claim 1, comprising an amino acid sequence at least 75% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 1-17, not including any amino acid insertions at identified insertion sites, and not including residues noted as dispensable in Tables 2-7.

3. The polypeptide of claim 1, comprising an amino acid sequence at least 90% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 1-17, not including any amino acid insertions at identified insertion sites, and not including residues noted as dispensable in Tables 2-7.

4. The polypeptide of claim 1, comprising an amino acid sequence at least 50% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 1-17, not including residues noted as dispensable in Tables 2-7.

5. The poly peptide of claim 1, comprising an amino acid sequence at least 75% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 1-17, not including residues noted as dispensable in Tables 2-7.

6. The polypeptide of claim 1, comprising an amino acid sequence at least 90% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 1-17, not including residues noted as dispensable in Tables 2-7.

7. The polypeptide of claim 1, comprising an amino acid sequence at least 50% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 1-17,8. The polypeptide of claim 1, comprising an amino acid sequence at least 75% identical to the ammo acid sequence selected from the group consisting of SEQ ID NO: 1-17.

9. The polypeptide of claim 1, comprising an amino acid sequence at least 90% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 1-17.

10. The polypeptide of any one of claims 1 -6, wherein the polypeptide comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 1-4, residues 10-88.

11. The polypeptide of any one of claims 1-9, wherein the polypeptide comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 1-4.

12. The polypeptide of any one of claims 10-11, wherein substitutions relative to SEQ ID NO: 1-4 are selected from substitutions listed in Options 1 or 2 of Table 2.

13. The polypeptide of any one of claims 10-11, wherein substitutions relative to SEQ ID NO: 1-4 are selected from substitutions listed in Option I of Table 2.

14. The poly peptide of any one of claims 10-13, wherein at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or all 22 identified interface residues, as shown in column 4 of Table 2, are identical (not substituted), or conservatively substituted, relative to SEQ ID NO: 1-4.

15. The polypeptide of any one of claims 10-14, wherein at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or all 30 core residues, as shown in column 5 of Table 2, are identical (not substituted), or conservatively substituted, relative to SEQ ID NO: 1-4.

16. The polypeptide of any one of claims 10-15, wherein all identified interface residues and core residues are identical (not substituted), or conservatively substituted, relative to SEQ ID NO: 1-4.

17. The polypeptide of any one of claims 1-9, comprising an insertion in one or more insertion site, as shown in Table 2, column 6, relative to SEQ ID NO: 1 -4.

18. The polypeptide of any one of claims 1-6, wherein the polypeptide comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO:5-8, residues 34-88.

19. The polypeptide of any one of claims 1 -9, wherein the polypeptide comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO:5-8.

20. The polypeptide of any one of claims 18-19, wherein substitutions relative to SEQ ID NO: 5-8 are selected from substitutions listed in Options 1 or 2 of Table 3.

21. The polypeptide of any one of claims 18-19, wherein substitutions relative to SEQ ID NO: 5-8 are selected from substitutions listed in Option 1 of Table 3.

22. The polypeptide of any one of claims 18-21, wherein at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or all 22 identified interface residues, as shown in column 4 of Table 3, are identical (not substituted), or conservatively substituted, relative to SEQ ID NO: 5-8.

23. The polypeptide of any one of claims 18-22, wherein at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or all 29 core residues, as shown in column 5 of Table 3, are identical (not substituted), or conservatively substituted, relative to SEQ ID NO:5-8.

24. The polypeptide of any one of claims 18-23, wherein all identified interface residues and core residues are identical (not substituted), or conservatively substituted, relative to SEQ ID NO:5-8.

25. The polypeptide of any one of claims 18-24, comprising an insertion in one or more insertion site, as shown in Table 3, column 6, relative to SEQ ID NO:5-8.

26. The polypeptide of any one of claims 1-6, wherein the polypeptide comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NON, residues 38-76.

27. The polypeptide of any one of claims 1 -9, wherein the polypeptide comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 9.

28. The polypeptide of any one of claims 26-27, wherein substitutions relative to SEQ ID NO:9 are selected from substitutions listed in Options 1 or 2 of Table 4.

29. The polypeptide of any one of claims 26-27, wherein substitutions relative to SEQ ID NO:9 are selected from substitutions listed in Option 1 of Table 4.

30. The polypeptide of any one of claims 26-29, wherein at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or all 22 identified interface residues, as shown in column 4 of Table 4, are identical (not substituted), or conservatively substituted, relative to SEQ ID NO:9.

31. The polypeptide of any one of claims 26-30, wherein at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or all 26 core residues, as shown in column 5 of Table 4, are identical (not substituted), or conservatively substituted, relative to SEQ ID NO:

9.

32. The polypeptide of any one of claims 26-31, wherein all identified interface residues and core residues are identical (not substituted), or conservatively substituted, relative to SEQ ID NO: 9.

33. The polypeptide of any one of claims 26-32, comprising an insertion in one or more insertion site, as shown in Table 4, column 6, relative to SEQ ID NO: 9.

34. The polypeptide of any one of claims 1-6, wherein the polypeptide comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 10-11, residues 11-74.

35. The polypeptide of any one of claims 1 -9, wherein the polypeptide comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 10-11.

36. The polypeptide of any one of claims 34-35, wherein substitutions relative to SEQ ID NO: 10-11 are selected from substitutions listed in Options 1 or 2 of Table 5.

37. The polypeptide of any one of claims 34-35, wherein substitutions relative to SEQ ID NO: 10-11 are selected from substitutions listed in Option 1 of Table 5.

38. The polypeptide of any one of claims 34-37, wherein at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or all 17 identified interface residues, as shown in column 4 of Table 5, are identical (not substituted), or conservatively substituted, relative to SEQ ID NO: 10-11.

39. The polypeptide of any one of claims 34-38, wherein at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or all 13 core residues, as shown in column 5 of Table 5, are identical (not substituted), or conservatively substituted, relative to SEQ ID NO: 10-11.

40. The polypeptide of any one of claims 34-39, wherein all identified interface residues and core residues are identical (not substituted), or conservatively substituted, relative to SEQ ID NO:10-11.

41. The polypeptide of any one of claims 34-40, comprising an insertion in one or more insertion site, as shown in Table 5, column 6, relative to SEQ ID NO: 10-11.

42. The polypeptide of any one of claims 1-6, wherein the polypeptide comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 12-13, residues 28-95.

43. The polypeptide of any one of claims 1 -9, wherein the polypeptide comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 12-13.

44. The polypeptide of any one of claims 42-43, wherein substitutions relative to SEQ ID NO: 12-13 are selected from substitutions listed in Options 1 or 2 of Table 6.

45. The polypeptide of any one of claims 42-43, wherein substitutions relative to SEQ ID NO: 12-13 are selected from substitutions listed in Option 1 of Table 6.

46. The polypeptide of any one of claims 42-45, wherein at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or all 19 identified interface residues, as shown in column 4 of Table 6, are identical (not substituted), or conservatively substituted, relative to SEQ ID NO: 12-13.

47. The polypeptide of any one of claims 42-46, wherein at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or all 29 core residues, as shown in column 5 of Table 6, are identical (not substituted), or conservatively substituted, relative to SEQ ID NO:12-13.

48. The polypeptide of any one of claims 42-47, wherein all identified interface residues and core residues are identical (not substituted), or conservatively substituted, relative to SEQ ID NO: 12-13.

49. The polypeptide of any one of claims 42-48, comprising an insertion in one or more insertion site, as shown in Table 6, column 6, relative to SEQ ID NO: 12-13.

50. The poly peptide of any one of claims 1-6, wherein the polypeptide comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 14-17, residues 29-91.

51. The polypeptide of any one of claims 1 -9, wherein the polypeptide comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 14-17.

52. The polypeptide of any one of claims 50-51, wherein substitutions relative to SEQ ID NO: 14-17 are selected from substitutions listed in Options 1 or 2 of Table 7.

53. The polypeptide of any one of claims 50-51, wherein substitutions relative to SEQ ID NO: 14-17 are selected from substitutions listed in Option 1 of Table 7.

54. The polypeptide of any one of claims 50-53, wherein at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or all 21 identified interface residues, as shown in column 4 of Table 7, are identical (not substituted), or conservatively substituted, relative to SEQ ID NO: 14-17.

55. The poly peptide of any one of claims 50-54, wherein at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or all 28 core residues, as shown in column 5 of Table 7, are identical (not substituted), or conservatively substituted, relative to SEQ ID NO: 14-17.

56. The polypeptide of any one of claims 50-55, wherein all identified interface residues and core residues are identical (not substituted), or conservatively substituted, relative to SEQ ID NO: 14-17.

57. The polypeptide of any one of claims 50-56, comprising an insertion in one or more insertion site, as shown in Table 7, column 6, relative to SEQ ID NO: 12-13.

58. A fusion protein, comprising:(a) the polypeptide of any one of claims 1-57; and(b) one or more functional domains, including but not limited to a targeting domain, a detectable domain, a scaffolding domain, an Fc domain, a domain to increase half-life of the polypeptide in the blood stream (including but not limited to an al bumin- binding protein), or a further therapeutic peptide domain;optionally wherein the polypeptide and the one or more functional domains are linked by an amino acid linker.

59. A conjugate, comprising:(a) the polypeptide of any one of claims 1-57; and(b) a scaffolding moiety.

60. A fusion protein, comprising;(a) the polypeptide of any one of claims 1-57; and(b) a protein scaffolding moiety;optionally wherein the polypeptide and the protein scaffolding moiety are linked by an amino acid linker.

61. A scaffold, comprising the conjugate or fusion protein of claim 59 or 60, wherein the scaffold comprises 6 7, 8, 9, 10, or more copies of the polypeptide of any one of claims 1-57.

62. A nucleic acid encoding the polypeptide or fusion protein of any one of claims 1-58 and 60.

63. Ail expression vector comprising the nucleic acid of claim 62 operatively linked to a promoter.

64. A host cell comprising the polypeptide, fusion protein, scaffold, nucleic acid, or expression vector of any one of claims 1-63.

65. A pharmaceutical composition, comprising the polypeptide, fusion protein, scaffold, nucleic acid, expression vector, or host cell of any of the preceding claims, and a pharmaceutically acceptable carrier.

66. A method for using, or a use of the polypeptide, conjugate, fusion protein, nucleic acid, expression vector, host cell, and / or the pharmaceutical composition of any of the preceding claims, for any suitable purpose including but not limited to those disclosed herein.

67. The method or use of claim 66, wherein the purpose includes, but is not limited to, treating or limiting development of limit development of diabetic vasculopathy, vascular dysfunctions, cancer angiogenesis, and wound healing.

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