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Title:
ANGIOPOIETIN 2-BASED POLYPEPTIDE INHIBITORS AND METHODS OF USE THEREOF
Document Type and Number:
WIPO Patent Application WO/2018/173062
Kind Code:
A1
Abstract:
Described herein is the generation of inhibitory polypeptides based on the Ang2- binding domain (Ang2-BD), but having greater affinity for Tie2 and/or integrin alpha v beta 3 (ανβ3) than the native Ang2-BD. Uses of the generated Ang2-BD variants as cancer and anti- angiogenesis therapeutics are also described.

Inventors:
PAPO NIV (IL)
SHLAMKOVICH TOMER (IL)
Application Number:
IL2018/050331
Publication Date:
September 27, 2018
Filing Date:
March 22, 2018
Export Citation:
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Assignee:
NAT INST BIOTECHNOLOGY NEGEV LTD (IL)
International Classes:
A61K38/18; A61P35/00; C07K14/475; C12N15/11
Foreign References:
Other References:
SHLAMKOVICH, TOMER ET AL.: "Utilizing combinatorial engineering to develop Tie2 targeting antagonistic angiopoetin-2 ligands as candidates for anti-angiogenesis therapy", ONCOTARGET, vol. 8, no. 20, 4 April 2017 (2017-04-04), pages 33571 - 33585, XP055550246, Retrieved from the Internet
BARTON, WILLIAM A. ET AL.: "Structure of the angiopoietin-2 receptor binding domain and identification of surfaces involved in Tie2 recognition", STRUCTURE, vol. 13.5, 10 May 2005 (2005-05-10), pages 825 - 832, XP004910463, Retrieved from the Internet
WITTRUP, K. DANE: "Yeast surface display for protein engineering and characterization", CURRENT OPINION IN STRUCTURAL BIOLOGY, vol. 17, no. 4, 17 September 2007 (2007-09-17), pages 467 - 473, XP022273660, Retrieved from the Internet
Attorney, Agent or Firm:
BEN-DAVID, Yirmiyahu M. et al. (IL)
Download PDF:
Claims:
We claim:

1. An isolated polypeptide comprising an amino acid sequence set forth herein as SEQ ID NO: 23, or a fragment, derivative, or analog thereof.

2. The isolated polypeptide of claim 1, wherein the amino acid sequence is selected from the group consisting of SEQ ID NOs 24-27.

3. An isolated polypeptide comprising an amino acid sequence at least 70% identical to SEQ ID NO: 1, or a fragment, a derivative or analog thereof, wherein the isolated polypeptide differs by at least one amino acid compared to SEQ ID NO: 2.

4. The isolated polypeptide of claim 3, comprising an amino acid sequence is at least 70% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs 3-7.

5. The isolated polypeptide of claim 4, comprising an amino acid sequence at least 70% identical to an amino acid sequence set forth as SEQ ID NO: 8.

6. The isolated polypeptide of claim 5, comprising a polypeptide, or a fragment, a derivative or analog thereof, at least 70% identical to an amino acid sequence set forth as SEQ ID NOs: 9-21 or SEQ ID NOs 31-34.

7. An isolated nucleic acid sequence encoding the isolated polypeptide of any one of claims 1 to 6.

8. An expression vector comprising the isolated nucleic acid of claim 7.

9. A pharmaceutical composition comprising the isolated polypeptide of any one of claims 1-6, or comprising the isolated nucleic acid sequence of claim 7, and at least one of a

pharmaceutically acceptable carrier, salt, or excipient.

10. The pharmaceutical composition of claim 9, for use in the treatment of a Τίε2/ανβ3 integrin-related condition or an Ang2/Tie2 related condition.

11. The pharmaceutical composition of claim 10, wherein the integrin-related

condition or an Ang2/Tie2 related condition is an angiogenesis-related condition or

osteoporosis. 12. The pharmaceutical composition of claim 11, wherein the angiogenesis-related pathology is a cancer or macular degeneration.

13. The pharmaceutical composition of claim 9, for use in inhibition of cancer cell invasion, metastasis, or growth of a vascular tumor.

14. The pharmaceutical composition of any one of claims 9-13, wherein the pharmaceutical composition is formulated for systemic or local administration.

15. A method for treating a integrin-related condition or an Ang2/Tie2 related

condition in a subject, comprising:

administering to a subject in need thereof, a therapeutically effective amount of the polypeptide of any one of claims 1-6 or the nucleic acid of claim 7, thereby treating the

integrin-related condition or Ang2/Tie2 related condition.

16. Use of a polypeptide of any one of claims 1-6 or the nucleic acid of claim 7 in the preparation of a medicament for treatment of a integrin-related condition or an

Ang2/Tie2 related condition.

Description:
ANGIOPOIETIN 2-BASED POLYPEPTIDE INHIBITORS AND METHODS OF USE

THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS Benefit is claimed to US Provisional Patent Application No. 62/475,378, filed March

23, 2017, and to US Provisional Patent Application No. 62/550,709, filed August 28, 2017, the contents of both of which are incorporated by reference herein in their entirety.

FIELD

This disclosure relates to generation of inhibitory polypeptides based on the Ang2- binding domain (Ang2-BD), but having greater affinity for Tie2 and/or integrin alpha v beta 3 (α ν β 3 ) than the native Ang2-BD. Uses of the generated Ang2-BD variants as cancer and anti- angiogenesis therapeutics are also described. BACKGROUND

Angio genesis is associated with structural changes in blood vessels, a process which is mediated via multiple pathways resulting in the formation of new blood vessels, which supports changing tissue requirements (Carmeliet, 2005; Otrock et al., 2007; Pandya et al., 2006; Potente et al., 2011). Both pro- and anti-angiogenic factors effect blood vessels and their constituent endothelial cells, facilitating vascular remodeling during both normal and pathological conditions, namely cancer (Dvorak, 2002; Griffioen and Molema, 2000; Hanahan and Weinberg, 2011).

To obtain blood supply for their growth, tumor cells can tilt the balance toward stimulatory angiogenic factors to drive vascular growth by attracting and activating endothelial cells from within the microenvironment of the tumor (Folkman, 2008; Leenders et al., 2002). The level of the angiogenic response is dictated by the combination of multiple pro-angiogenic signals and their activities on endothelial cells in the tumor microenvironment (Carmeliet and Jain, 2011; Katoh, 2013). The current understanding of how some of these various components are regulated and cross interact has allowed for experimental engineering of potent

antiangiogenic therapies for cancer (Bellou et al., 2013; Oliner et al., 2004; Pandya et al., 2006; Shojaei, 2012).

However, antiangiogenic therapeutic approaches focusing on targeting single components were found to possess limited clinical benefit. The limited clinical benefit of antiangiogenic mono-therapies is largely due to the rapidly acquired resistance mechanisms which enable endothelial cells in the tumor microenvironment to activate compensatory proliferative pathways (Bergers and Hanahan, 2008; Giuliano and Pages, 2013; Kerbel et al., 2001; Shojaei, 2012; Wang et al., 2017). This problem has led to the development of cocktail- therapeutics, many of which have already been introduced in clinical practice (Dorrell et al., 2007; Duffy et al., 2017; Kluza et al., 2010; Rosen et al., 2017; Zirlik and Duyster, 2017).

Given the complexity, redundancy, and cross-interaction of angiogenic signaling pathways, the design of multicomponent therapeutics which are able to interfere with parallel nodes of critical angiogenesis-associated networks has attracted considerable attention as a promising avenue to combat drug resistance in different types of cancers. This is best illustrated, for example, by the highly investigated combined vascular endothelial growth factor receptor-2 (VEGFR2)-a v p3 integrins and VEGFA-angiopoietin-2 (Ang2) inhibitors (Haisma et al., 2010; Kapur et al., 2017; Kienast et al., 2013; Kloepper et al., 2016; Papo et al., 2011; Schmittnaegel et al., 2017; Somanath et al., 2009). Although integrins and growth factor-signaling pathways show crosstalk, this phenomenon has only been recently demonstrated with regard to the Tie2 receptor tyrosine kinase (RTK)-integrin system, which plays a significant role in mediating angiogenesis. Specifically, different studies have identified the Tie2-a 5 Pi integrin axis as a common module in angiogenesis (Cascone et al., 2005; Felcht et al., 2012) but more recently, other studies were able to identify an additional, critical player for this axis, namely the α ν β 3 integrin (Dalton et al., 2016; Thomas et al., 2010). Similar to Tie2, both α 5 βι and α ν β 3 integrins are highly expressed on activated endothelial cells in the tumor neovasculature, but are weakly expressed in resting endothelial cells and in most normal tissues and organs (Avraamides et al., 2008; Desgrosellier and Cheresh, 2010; Martin et al., 2008).

The study of the mechanism of Tie2 involvement in endothelial cell proliferation, invasion and angiogenesis is a new and evolving field. Tie2 triggers tumor- associated endothelial cell progression in the cancer microenvironment and significantly enhances the angiogenic and invasive potential of endothelial cells in vitro (Fukuhara et al., 2008; Venneri et al., 2007; Zhang et al., 2006). Accordingly, Tie2 suppression by RNA interference markedly reduced endothelial cell growth, proliferation, and invasive potential (Santel et al., 2006). The pro-angiogenic and invasive potentials of Tie2 have also been demonstrated in in vivo models of angiogenesis (Hasenstein et al., 2012). For example, ectopic expression of Tie2 correlated well with increased endothelial cell proliferation and migration in vivo (Roviezzo et al., 2005; Sarraf-Yazdi et al., 2008).

While the exact mechanisms downstream of Tie2 remain largely elusive, it was previously shown that integrins (especially α 5 βι integrins) are established mediators of Tie2 functioning. Activation of α 5 βι integrin by its native ligand, fibronectin, results in strong Tie2- α 5 βι integrin interaction and this complex has a slows dissociation rate(Cascone et al., 2005). The formation of the Tie2-a 5 Pi integrin complex results in a high sensitivity of the Tie2 receptor for its native agonistic ligand, angiopoietin-1 (Angl), and enhancement in Angi -dependent Tie2 activation (Cascone et al., 2005; Dalton et al., 2016). Recently, Tie2 was shown to cross- interact with α ν β 3 integrin as well, and putative pathobiological roles of this axis in a diverse array of cancers have been suggested (Dalton et al., 2016).

It was shown that Tie2 readily associates with α 5 βι and α ν β 3 integrins through their respective ectodomains (Dalton et al., 2016). It was further demonstrated that the Tie2 agonistic ligand Angl (Kim et al., 2005; Yuan et al., 2007, 2009), but not Ang2, an

angiopoietin family member with Tie2 antagonistic activity (Seegar et al., 2010), can independently associate with α 5 βι and α ν β 3 (Cascone et al., 2005; Fiedler et al., 2004; Thomas et al., 2010; Yuan et al., 2009). These Ang/integrin and Tie2/integrin interactions were independent of the Arg-Gly-Asp (RGD) tripeptide motif, which facilitates integrin interactions with its natural extracellular matrix (ECM) ligands, including fibronectin, vitronectin, fibrinogen, and osteopontin (Avraamides et al., 2008; Humphries et al., 2006).

Given the network of pathways involved in the Tie2/integrin axis there remains a need to develop inhibitors of Tie 2 and the Tie2/integrin axis to stop development and progression of cancer and which minimize off target inhibition of angiogenesis and invasive cellular activity which can be detrimental and provide unwanted side effects to the patient.

SUMMARY

Provided herein are variants of Ang2, based on the monomeric and Tie2-binding Ang2- binding domain (Ang2-BD), that can bind with increased affinity and inhibit the Tie2 receptor tyrosine kinase. The described polypeptides include an isolated polypeptide having an amino acid sequence at least 70% identical to SEQ ID NOs: 1 or 8, or fragments, a derivatives or analogs thereof, wherein the polypeptide differs by at least one amino acid compared to SEQ ID NO: 2.

Also described herein are isolated polypeptides based on the Ang2-BD and which is adapted to bind to both Tie2 and α ν β 3 integrin. The described polypeptides include an amino acid sequence set forth herein as SEQ ID NO: 23, or a fragment, derivative, or analog thereof, and which in particular embodiments can be any one of SEQ ID NOs 24-27.

Pharmaceutical compositions that include the described isolated polypeptide, for use in the treatment of a Τίε2/α ν β 3 integrin-related condition or an Ang2/Tie2-related condition, and methods of treatment of a Τίε2/α ν β3 integrin-related condition or an Ang2/Tie2-related condition using such compositions are also described.

Additionally described herein are nucleic acid sequences encoding the described isolated polypeptides, which in particular examples are provided in an expression vector that includes the described nucleic acids.

The foregoing and other objects, features, and advantages will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a diagram showing the Ang2-BD construct displayed on yeast. Ang2-BD is presented on the cell surface of yeast as a fusion to agglutinin proteins. Display levels are detected using a primary antibody against the C-terminal cMyc tag (9E10 mouse antibody) and a phycoerythrin (PE)-labeled secondary anti mouse antibody. Binding to Tie2-Fc is measured using a fluorescein isothiocyanate (FITC) conjugated anti human Fc antibody.

Figures 2a-2i: Screening of first- and second-generation Ang2-BD libraries against soluble Tie2. Shown is a FACS analysis of yeast expressing Ang2-BD. Fig. 2a: Negative control. Fig. 2b: Ang2-BDwT expression and binding of Tie2 (10 nM). Fig. 2c: Ang2-BD library expression and binding of Tie2 (10 nM). Fig. 2d: Ang2-BD library expression and binding of Tie2 (10 nM) after five rounds of sorting. Fig. 2e: Ang2-BDci.7o expression and binding of Tie2 (5 nM). Fig. 2f: Ang2-BDci.7o library sort expression and binding of Tie2 (5 nM). Figs. 2g, 2h and 2i: Ang2-BDc 1.70 library expression and binding of Tie2 (5 nM) after sorts 1, 3 and 5, respectively. Sorts 2-5 were conducted using gates similar to the one shown in Fig. 2f.

Figures 3a-3c: Isolated clones with improved binding affinity towards Tie2. Fig.

3a: Seventy individual clones were isolated from sort 5 of the Ang2-BD library, and their affinity (when displayed on yeast) for Tie2 (10 nM) was determined. Clones with improved affinity were sequenced with the aim to identify specific mutations. Fig. 3b: FACS analysis of Ang2-BDwT (red) and isolated clone Ang2-BDci.7o (green) binding to 10 nM Tie2. Fig. 3c: Tie2 binding at different concentrations of Ang2-BDwT (red) and isolated Ang2-BDci.7o (green).

Figure 4 shows clones isolated from the second-generation library, with improved binding affinity towards Tie2. Sixty individual clones were isolated from sort 5 of the Ang2- BDci.7o-based second-generation library, and their affinity for Tie2 at different concentrations was determined

Figure 5: Ang2-BD purification process. Shown is an SDS PAGE gel showing the purified Ang2-BDwT (lane A), Ang2-BDci.7o untreated (lane B) and treated with Endo Hf (lane C), Ang2-BDc2.36 untreated (lane D) and treated with Endo Hf (lane E).

Figure 6: Size exclusion chromatography (SEC) purification of Ang2-BDci.70.

Glycosylated form is shown in blue, and non-glycosylated form in red. Elution volumes for the standards ovalbumin (44 kDa), ribonuclease A (13.7 kDa) and aprotinin (6.5 kDa) are shown. MS analysis of the molecular weights of the purified proteins gave: Ang2-BDci.7o glycosylated 32.7 kDa; Ang2-BDci.7o nonglycosylated 28.1 kDa; Ang2-BD C 2.36 glycosylated 33.2 kDa; Ang2-BD C 2.36 nonglycosylated 28.3 kDa; and Ang2-BD W T 27.7 kDa (Ang2-BD W T wild type was not glycosylated).

Figures 7a-7b: Structural characterization and thermal denaturation of Ang2-BD variants. Fig 7a: CD spectra of Ang2-BDwT and Ang2-BDc 1.70 and Ang2-BDc2.36 in both their glycosylated and non-glycosylated forms. Fig 7b: Thermal denaturation of purified Ang2-BD and the two Ang2-BD variants.

Figures 8a-8c: Binding of Ang2-BD variants to recombinant and cell-expressed human Tie2. Fig. 8a: Representative SPR sensorgrams of binding of Ang2-BD variants to immobilized Tie2. The ranges of protein concentrations analyzed are indicated in parentheses: Ang2-BD W T (62.5 nM - 1 μΜ); Ang2-BD C i.7o (12.5 nM - 200 nM); Ang2-BD C2 .36 (7.5 nM - 120 nM); Ang2-BD C 2.36 glycosylated (7.5 nM - 120 nM). Fig. 8b: Binding of Ang2-BD variants to TIME cell line: lxlO 5 cells were incubated with indicated proteins (Ang2-BDwT, Ang2-BDc 1.70 and Ang2-BDc2.36, red, green and blue, respectively) for 2 hours at 4 °C with a gentle agitation. Mean fluorescence values were determined by flow cytometry using a fluorescently labeled antibody against a FLAG epitope tag. Data shown are the average of triplicate experiments, and error bars represent standard deviations. * indicates P value <0.05 for comparison of results between Ang2-BD variants at the same concentration. Fig. 8c:

Competitive binding assay of Ang2-BDwT, Ang2-BDc 1.70 and Ang2-BDc2.36, without Angl (red, green and blue, respectively) and with 400 ng/ml Angl (pink, brown and yellow, respectively) and 1000 ng/ml Angl for Ang2-BDc2.36 competition (grey). * indicates P value <0.05 for comparison of results between Ang2-BD variants with and without Angl. Data shown is the average of triplicate experiments, and error bars represent standard error of the mean. Figure 9: Expression of Tie2 receptor on TIME cells. Levels of cell surface expression of Tie2 receptor (stained with APC-labeled anti-human Tie2, blue histogram) versus cells only control (unstained, red histogram), on TIME cells. lx10 5 cells were either stained with APC-labeled anti-human Tie2 or unstained and incubated at 4 °C for 30 mintes and analyzed by flow cytometry.

Figures 10a- 10b: Inhibition of Tie2 phosphorylation by Ang2-BD variants.

Fig. 10a: TIME cells were treated with control buffer (basal level, black), 200 ng/ml of Angl (green), 200 ng/ml of Angl and 1 μΜ Ang2-BD W T (brown), 1 μΜ Ang2-BD W T (red), 200 ng/ml of Angl and 1 μΜ Ang2-BD C 2.36 (blue) and 1 μΜ Ang2-BDc2.36 (purple) for 15 min for Tie2 phosphorylation. Fig. 10b: Cell lysates were analyzed by western blot using antibodies against pTie2, Tie2 and β-actin. # indicates P value <0.05 for comparison of results between Ang2-BDwT and Ang2-BDc2.36; ## indicates P value <0.01 for comparison of results between Angl+ Ang2-BDwT and Ang2-BDwT and between Angl+ Ang2-BDc2.36 and Ang2-BDc2.36; * indicates P value <0.05 for comparison of results between Angl and Angl+ Ang2-BDwr; ** indicates P value <0.01 for comparison of results between Angl and Angl+ Ang2-BDc2.36. Data shown is the average of triplicate experiments and error bars represent standard error of the mean.

Figures 11a- lib: Inhibition of tube formation in endothelial cells by Ang2-BD variants. Fig. 11a: TIME cells were treated with the indicated proteins. Fig. lib: Quantitation of tube formation: control buffer (cells only, black), 200 ng/ml of Angl (green), 2 μΜ, 4 μΜ and 8 μΜ Ang2-BDwT (red, pink and brown respectively), 2 μΜ, 4 μΜ and 8 μΜ Ang2- BDc2.36 (blue, yellow and grey respectively). Tube structures were analyzed for the number of generated junctions and the total tube length. * indicates P value <0.05 for comparison of results between cells alone and tested proteins. Data shown is the average of triplicate experiments and error bars represent standard error of the mean. Scale bar, 500 μιη.

Figures 12a- 12b: Inhibition of endothelial cells invasiveness by Ang2-BD variants. Fig. 12a: TIME cells were treated with indicated proteins in Boyden chambers. Fig. 12b: Quantitation of cells/field: control buffer (cells only, black), 2 μΜ and 4 μΜ Ang2-BDwT (red and pink, respectively), 2 μΜ and 4 μΜ Ang2-BDc2.36 (blue and brown, respectively). The invading cells accumulated on the bottom of membrane were counted in 16 frames for each membrane and analyzed for the number of cells. * indicates P value <0.05 for comparison of results between cells only and cells + tested proteins. Data shown is the average of triplicate experiments, and error bars represent standard error of the mean. Scale bar, 200μιη. Figures 13A-13C show binding of bispecific Ang2-BDRGD clones to Tie2 and α ν β 3 integrin. Fig. 13A is a schematic representation of Ang2-BD which is presented on the cell surface of yeast cells as a fusion to agglutinin proteins. Display levels are detected using a primary antibody against the C-terminal cMyc tag (chicken anti-cMyc antibody) and phycoerythrin (PE)-conjugated antichicken antibody. Binding to Tie2-Fc is measured using a fluorescein isothiocyanate (FITC) conjugated anti human Fc antibody. Binding to α ν β 3 integrin is measured by using a (FITC)-labeled mouse anti-a v integrin antibody. Fig. 13B: Binding of isolated yeast displaying bispecific Ang2-BDRGD clones to Tie2 (20 nM). Data was normalized to the yeast surface expression levels of each clone and Tie2 binding of Ang2-BDwT. Fig 13C: Binding of isolated yeast displaying bispecific Ang2-BDRGD clones to α ν β 3 , α 5 βι α ν β 5 , ou$7, ο¾β 3 and α 3 βι integrins (50 nM). Data was normalized to the yeast surface expression levels of each clone. Data shown is the average of triplicate experiments, and error bars represent standard error of the mean.

Figures 14A-14F show the affinity maturation of the bispecific Ang2-BDRGD-based library against α ν β 3 integrin in a FACS analysis of bispecific Ang2-BD based library. Fig. 14A: Negative control. Fig. 14B: Ang2-BDwT expression and α ν β 3 integrin binding (10 nM). Fig. 14C: Bispecific Ang2-BDRGD based library expression and α ν β 3 integrin binding (10 nM) presorting. Figs 14D-14F: Bispecific Ang2-BD based library expression and α ν β 3 integrin binding (10 nM) post sorts 1, 3 and 5 respectively.

Figures 15A-15B show production and purification of soluble Ang2-BD bispecific variants. Fig. 15A: SDS-PAGE analyses of purified Ang2-BDwT, Ang2-BDBC5, Ang2-BDBC6 and Ang2-BDBcio. Fig 15B: Size exclusion chromatography (SEC) was used to purify Ang2- BD bispecific variants. Shown is a representative separation of Ang2-BDBcio with standards elution volume for Ovalbumin (44 KDa), Ribonuclease A (13.7 KDa) and Aprotinin (6.5 KDa).

Figures 16A-16C show surface plasmon resonance (SPR) sensorgram analyses.

Fig. 16A: Representative SPR sensorgrams of Ang2-BDwT, Ang2-BDBC5, Ang2-BDBC6 and Ang2-BDBcio (31.25 nM - 500 nM). Each variant was flowed over a Tie2-immobilized chip and binding was detected as increase in response units Fig. 16B: Ang2-BDBC5, Ang2-BDBC6 and Ang2-BD B cio (12.5 nM - 200 nM) binding to immobilized α ν β 3 integrin Fig. 16C: Ang2- BD B c5 (red, 1 μΜ), Ang2-BD B c6 (green, 1 μΜ) and Ang2-BD B cio (black, 1 μΜ) binding to immobilized α ν β 3 , α ν β 5 and α 5 βι integrins. No binding to θ4β7, ο¾β 3 and α 3 βι integrins was observed. Figures 17A-17C show binding of bispecific Ang2-BD variants to recombinant and cell-expressed human Tie2 and α ν β 3 integrin. Fig. 17A:Representative SPR sensorgram of a dual binding of bispecific Ang2-BD variants (400 nM) to both α ν β 3 integrin (immobilized) and soluble Tie2 (400 nM). Fig. 17B: Binding of bispecific Ang2-BD variants to TIME cell line: l x10 5 cells were incubated with indicated proteins (Ang2-BDwT (blue), Ang2-BDBC5 (green), Ang2-BDBC6 (yellow) and Ang2-BDBcio (red)) for 2 hrs at 4 °C with a gentle agitation. Mean fluorescence values were determined by flow cytometry using a fluorescently labeled antibody against a FLAG epitope tag. Data shown is the average of triplicate experiments, and error bars represent standard error of the mean. * indicates P value <0.05 for comparison of results between Ang2-BD variants at the same concentration. Fig. 17C: Competitive binding assay of Ang2-BDwT (violet), Ang2-BDBC5 (green), Ang2-BDBC6 (yellow) and Ang2-BDBcio (red) at 1 μΜ either alone or with a combination of 1000 ng/ml Angl (squared bars), 10 μΜ cRGD peptide (horizontal lined bars) and both 1000 ng/ml Angl and 10 μΜ cRGD peptide (vertical lined bars). Mean fluorescence values were determined by flow cytometry using a

fluorescently labeled antibody against a FLAG epitope tag. Data shown is the average of triplicate experiments, and error bars represent standard error of the mean. * indicates P value <0.05 for comparison of results between Ang2-BD variants alone and with the competitors Angl and cRGD peptide.

Figures 18A-18B show expression of Tie2 receptor and α ν β 3 integrin on TIME cells. Fig. l8A: Cell surface expression of Tie2. Fig. 18B: Cell surface expression of α ν β 3 : integrin, cells only (red). l x 10 5 TIME cells were stained with APC-labeled anti-human Tie2 (FITC)- labeled or mouse anti-human CD51 (integrin a v ) and incubated at 4 °C for 30 min.

Figure 19 shows inhibition of TIME cells adhesion to vitronectin-coated wells. 5x l0 4 TIME cells were incubated with 1 μΜ of cRGD peptide (purple), Ang2-BDwT (blue), Ang2- BDBCS (green), Ang2-BDBC6 (yellow) and Ang2-BDBcio (red) for 2 hrs on vitronectin coated 96 well plate. * indicates P value <0.05 for comparison of results between cells only control and the tested proteins/peptide. Data shown is the average of triplicate experiments, and error bars represent standard error of the mean.

Figures 20A-20B show endothelial cell tube formation inhibition by bispecific Ang2- BD variants. Fig. 20A: 3.25x l0 4 TIME cells were treated with indicated proteins overnight. Cells were washed pictured using 2x Objective, Scale bar, 500 μιη. Fig. 20B: Images of Tube structures were analyzed for the number of generated junctions and number of meshes.

Control buffer (cells only, black), 500 ng/ml of Angl (grey), combination of 500 ng/ml of Angl with 1 μΜ of cRGD peptide (purple), 1 μΜ of Ang2-BD W T (blue), 1 μΜ of Ang2-BD B c5 (green), 1 μΜ of Ang2-BDBC6 (yellow) and 1 μΜ of Ang2-BDBcio (red). * indicates P value <0.05 for comparison of results between Angl alone and combination of Angl with the tested proteins. Data shown is the average of triplicate experiments, and error bars represent standard error of the mean.

Figures 21A-21B show inhibition of endothelial cells invasiveness by bispecific Ang2-

BD variants. Fig. 21A: TIME cells were treated with indicated proteins in Boyden chambers, Scale bar, 200 μιη. Fig. 21B: The invading cells accumulated on the bottom of membrane were counted in 16 frames for each membrane and analyzed for the number of cells. Control buffer (cells only, black), 500 ng/ml of Angl (grey), combination of 500 ng/ml of Angl with 1 μΜ of cRGD peptide (purple), 1 μΜ of Ang2-BD W T (blue), 1 μΜ of Ang2-BD B c5 (green), 1 μΜ of Ang2-BDBC6 (yellow) and 1 μΜ of Ang2-BDBcio (red). * indicates P value <0.05 for comparison of results between Angl alone and combination of Angl with the tested proteins. Data shown is the average of triplicate experiments, and error bars represent standard error of the mean.

Figures 22A-22B: is a docking solution cluster analysis. The distribution of the abundant clusters is presented as a percentage of all docking solutions. Fig. 22A: BC10 angiopoietin mutant. Fig. 22B: WT angiopoietin.

Figures 23A-23B show the docking model of angiopoietin-avP3 complex.

Angiopoietin is shown in pink, av in yellow and β3 in green. YPGRGD PD (amino acids 22- 30 of SEQ ID NO: 26) mutant residues (Fig. 23 A) or TFPNSTEE (amino acids 22-29 of SEQ

ID NO: 2) WT residues (Fig. 23B) are presented in red. Fig. 23 A: BC10 mutant-avP3 complex.

Fig 23B: WT Ang2- ανβ3 complex. In each panel zoom of the binding interface is shown.

Figures 24A-24C show the interaction residues between ανβ3 integrin and the mutant

Angiopoietin. Fig. 24A: residues 301-303 of BC10. Fig. 24B: residues 304-306. Fig 24C:

residues 307-309. ανβ3 residues are shown in pink and Angiopoietin residues in cyan. Closest interactions (in the range of up to 5 A) are drawn by a dotted line.

Figure 25: shows the superposition of BC10 RGD with crystallized RGD from 1L5G.

The RGD sequence from the mutant angiopoietin 2 was aligned with the PDB 1L5G crystal structure of ανβ3 in complex with RGD. av in yellow, β3 in green, RGD from BC10 mutant in cyan and RGD from the crystal structure is shown in pink.

BRIEF DESCRIPTION OF THE DESCRIBED SEQUENCES

The amino acid sequences provided herewith are shown using standard letter abbreviations for amino acids, as defined in 37 C.F.R. 1.822. As described herein: SEQ ID NO; 1 is the amino acid sequence of the 1 st generation consensus sequence from the Ang2-BD-based library.

SEQ ID NO: 31 is a summary amino acid sequence of an embodiment of the bispecific Τίε2/α ν β3 integrin inhibitor polypeptides, optimized for Tie2 binding, as described herein, SEQ ID NO: 32 is the amino acid sequence of the Ang2-BDBC5_2.36 polypeptide. SEQ ID NO: 33 is the amino acid sequence of the Ang2-BDBC6_2.36 polypeptide. SEQ ID NO: 34 is the amino acid sequence of the Ang2-BDBC10_2.36 polypeptide.

DETAILED DESCRIPTION

I. Abbreviations

Ang angiopoietin

Ang2-BD angiopoietin 2 binding domain

CD circular dichroism

SPR surface plasmon resonance

TIME telomerase-immortalized human microvascular endothelium

RTK receptor tyrosine kinase

RU response unit

YSD yeast surface display

II. Terms

Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The term "comprises" means "includes." The abbreviation, "e.g." is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example."

In case of conflict, the present specification, including explanations of terms, will control. In addition, all the materials, methods, and examples are illustrative and not intended to be limiting. Administration: The introduction of a composition into a subject by a chosen route. Administration of an active compound or composition can be by any route known to one of skill in the art. Administration can be local or systemic.

Examples of local administration include, but are not limited to, topical administration, subcutaneous administration, intramuscular administration, intrathecal administration, intrapericardial administration, intra-ocular administration, topical ophthalmic administration, or administration to the nasal mucosa or lungs by inhalational administration. In addition, local administration includes routes of administration typically used for systemic administration, for example by directing intravascular administration to the arterial supply for a particular organ. Local administration also includes the incorporation of active compounds and agents into implantable devices or constructs, such as vascular stents or other reservoirs, which release the active agents and compounds over extended time intervals for sustained treatment effects.

Systemic administration includes any route of administration designed to distribute an active compound or composition widely throughout the body via the circulatory system. Thus, systemic administration includes, but is not limited to intra- arterial and intravenous

administration. Systemic administration also includes, but is not limited to, oral, topical, subcutaneous, intramuscular routes, or administration by inhalation, when such administration is directed at absorption and distribution throughout the body by the circulatory system.

Analog or Derivative: An analog is a molecule that differs in chemical structure from a parent compound, for example a homolog (differing by an increment in the chemical structure, such as a difference in the length of an alkyl chain), a molecular fragment, a structure that differs by one or more functional groups, a change in ionization. Structural analogs are often found using quantitative structure activity relationships (QSAR), with well-known techniques such as those disclosed inter alia in Remington (The Science and Practice of Pharmacology, 19th Edition (1995), chapter 28). A derivative is a biologically active molecule derived from the base structure.

Aberrant angiogenesis: The growth and development of blood vessels is angiogenesis. While angiogenesis is a necessary biological process, it can be pathological when uncontrolled or when directed by abnormal angiogenic signaling. As used herein, aberrant angiogenesis refers to such excessive angiogenesis. In particular examples, aberrant angiogenesis is an intrinsic factor in the pathology of tumor growth and metastasis, as well as wet macular degeneration.

Ang2/Tie2 related condition: Polypeptides of the present invention are useful for the treatment of a wide range of medical conditions which are characterized by abnormal activation of the Tie2 receptor or abnormal expression of Ang2. Non-limiting examples of Ang2/Tie2 related conditions include: any angiogenesis related pathologies such as found in diabetes (e.g., diabetic retinopathy and more specifically proliferative diabetic retinopathy and diabetic macular edema), wet age-related macular degeneration, and angiogenesis related cancers such as angiosarcoma in the endothelial layer of the inner lining of vasculature, hemoangiosarcoma (blood vessels), and lymphoangionsarcoma (lymph vessels).

Binding affinity: A term that refers to the strength of binding of one molecule to another at a site on the molecule. If a particular molecule will bind to or specifically associate with another particular molecule, these two molecules are said to exhibit binding affinity for each other. Binding affinity is related to the association constant and dissociation constant for a pair of molecules. The concepts of binding affinity, association constant, and dissociation constant are well known, and their determination is standard in the art, such as by surface plasmon resonance, as well as other fluorescence-mediated binding assays (to measure FRET, fluorescence anisotropy, and the like).

Cellular proliferation: Cell division. Abnormal or hyperproliferation is a factor in the development of cancer.

Cellular survival: Cell death is a natural stage in the lifecycle of many cell types. Cell death (e.g. apoptosis) is also a mechanism by which a cell will self-destruct in response to certain real or perceived damage or stress. In particular hyperproliferative disorders, such as cancer, the normal processes of cell death can be inhibited, and cells which otherwise would undergo apoptosis remain with harmful effects to the patient.

Cancer: The product of neoplasia is a neoplasm (a tumor or cancer), which is an abnormal growth of tissue that results from excessive cell division. A tumor that does not metastasize is referred to as "benign." A tumor that invades the surrounding tissue and/or can metastasize is referred to as "malignant." Neoplasia is one example of a proliferative disorder. A "cancer cell" is a cell that is neoplastic, for example a cell or cell line isolated from a tumor.

Examples of hematological tumors include leukemias. Examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma,

chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers (such as small cell lung carcinoma and non-small cell lung carcinoma), ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, melanoma, and CNS tumors (such as a glioma, astrocytoma, medulloblastoma, craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, neuroblastoma and retinoblastoma).

Chemotherapeutic agent: An agent with therapeutic usefulness in the treatment of diseases characterized by abnormal cell growth or hyperplasia. Such diseases include cancer, as well as diseases characterized by hyperplastic growth such as psoriasis. In one embodiment, a chemotherapeutic agent is a radioactive compound. One of skill in the art can readily identify a chemotherapeutic agent using standard and well-known techniques (for instance, see Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison's Principles of Internal Medicine, 14th edition; Perry et al., Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology 2 nd ed., © 2000 Churchill Livingstone, Inc.; Baltzer L, Berkery R (eds): Oncology Pocket Guide to Chemotherapy, 2nd ed. St. Louis, Mosby-Year Book, 1995; Fischer DS, Knobf MF, Durivage HJ (eds): The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 1993). Chemotherapeutic agents include small molecule agents and biologic agents such as the Tie2 inhibitors and the bispecific Τίε2/α ν β3 integrin inhibitors described herein.

Contacting: Placement in direct physical association. Includes both in solid and liquid form. Contacting can occur in vitro with isolated cells or in vivo by administering to a subject.

Diagnosis: The process of identifying a disease, a predisposition to developing a disease, and accordingly a subject in need of a particular treatment, by its signs, symptoms, and results of various tests and methods. The conclusion reached through that process is also called "a diagnosis." The term "predisposition" refers to an effect of a factor or factors that render a subject susceptible to a condition, disease, or disorder, such as cancer. In particular

embodiments of the described methods of treatment, the subject in need of the described treatment has not been diagnoses with a disease, but is known to have a predisposition to the disease.

Effective amount of a compound: A quantity of compound sufficient to achieve a desired effect in a subject being treated; also referred to as a "therapeutically effective amount." An effective amount of a compound can be administered in a single dose, or in several doses, for example daily, during a course of treatment. However, the effective amount of the compound will be dependent on the compound applied, the subject being treated, the severity and type of the affliction, and the manner of administration of the compound. Encode: A polynucleotide is said to "encode" a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed and/or translated to produce the mRNA for and/or the polypeptide or a fragment thereof.

Expression Control Sequences: Nucleic acid sequences that regulate the expression of a heterologous nucleic acid sequence to which it is operatively linked, for example the expression of a nucleic acid encoding an Ang2-BD variant polypeptide described herein, such as the Tie2 or Τίε2/α ν β3 integrin inhibitory polypeptides described herein. Expression control sequences are operatively linked to a nucleic acid sequence when the expression control sequences control and regulate the transcription and, as appropriate, translation of the nucleic acid sequence. Thus expression control sequences can include appropriate promoters, enhancers, transcription terminators, a start codon (ATG) in front of a protein-encoding gene, splicing signal for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons. The term "control sequences" is intended to include, at a minimum, components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences. Also included are those promoter elements which are sufficient to render promoter-dependent gene expression controllable for cell-type specific, tissue- specific, or inducible by external signals or agents; such elements may be located in the 5' or 3' regions of the gene. Both constitutive and inducible promoters are included. In one embodiment, when cloning in mammalian cell systems, promoters derived from the genome of mammalian cells (such as metallothionein promoter) or from mammalian viruses (such as the retrovirus long terminal repeat; the adenovirus late promoter; the vaccinia virus 7.5K promoter) can be used.

Functional fragments and variants of a polypeptide: Included are those fragments and variants that maintain one or more functions of the Ang2-BD variant polypeptides described herein. It is recognized that the gene or cDNA encoding a polypeptide can be considerably mutated without materially altering one or more the polypeptide's functions. First, the genetic code is well-known to be degenerate, and thus different codons encode the same amino acids. Second, even where an amino acid substitution is introduced, the mutation can be conservative and have no material impact on the essential functions of a protein. See Stryer, Biochemistry 3rd Ed., (c) 1988. Third, part of a polypeptide chain can be deleted without impairing or eliminating all of its functions. Fourth, insertions or additions can be made in the polypeptide chain for example, adding epitope tags, without impairing or eliminating its functions (Ausubel et al. Short Protocols in Molecular Biology, 4 th ed., John Wiley & Sons, Inc., 1999). Other modifications that can be made without materially impairing one or more functions of a polypeptide include, for example, in vivo or in vitro chemical and biochemical modifications or the incorporation of unusual amino acids. Such modifications include, for example, acetylation, carboxylation, phosphorylation, glycosylation, ubiquination, labeling, e.g., with radionucleides, and various enzymatic modifications, as will be readily appreciated by those well skilled in the art. A variety of methods for labeling polypeptides and labels useful for such purposes are well known in the art, and include radioactive isotopes such as 32 P, ligands which bind to or are bound by labeled specific binding partners (e.g., antibodies), fluorophores, and chemiluminescent agents.

Immediate release (of a pharmaceutical compound): A compound that is

immediately biologically/chemically available and active upon administration to a subject and contact with a target site.

Inhibiting protein activity: To decrease, limit, or block an action, function or expression of a protein. The phrase inhibit protein activity is not intended to be an absolute term. Instead, the phrase is intended to convey a wide-range of inhibitory effects that various agents may have on the normal (for example, uninhibited or control) protein activity.

Inhibition of protein activity may, but need not, result in an increase in the level or activity of an indicator of the protein's activity. By way of example, this can happen when the protein of interest is acting as an inhibitor or suppressor of a downstream indicator. Thus, protein activity may be inhibited when the level or activity of any direct or indirect indicator of the protein's activity is changed (for example, increased or decreased) by at least 10%, at least 20%, at least 30%, at least 50%, at least 80%, at least 100% or at least 250% or more as compared to control measurements of the same indicator.

Injectable composition: A pharmaceutically acceptable fluid composition comprising at least one active ingredient, for example, a polypeptide. The active ingredient is usually dissolved or suspended in a physiologically acceptable carrier, and the composition can additionally comprise minor amounts of one or more non-toxic auxiliary substances, such as emulsifying agents, preservatives, pH buffering agents and the like. Such injectable compositions that are useful for use with the compositions of this disclosure are conventional; appropriate formulations are well known in the art.

Isolated: A biological component (such as a nucleic acid molecule or polypeptide) that has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs or in which it has been produced, i.e., other chromosomal and extra-chromosomal DNA and RNA, proteins and organelles. Nucleic acids and proteins that have been isolated include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids and polypeptides.

Label: A detectable compound or composition that is conjugated directly or indirectly to another molecule to facilitate detection of that molecule, such as in SPR or similar assays utilized herein. Specific, non-limiting examples of labels include radioactive isotopes, enzyme substrates, co-factors, ligands, chemiluminescent or fluorescent agents, haptens, and enzymes.

Operably linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.

Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.

Parenteral: Administered outside of the intestine, for example, not via the alimentary tract (a route of administration known as enteral). Generally, parenteral formulations are those that will be administered through any possible mode except ingestion. This term especially refers to injections, whether administered intravenously, intrathecally, intramuscularly, intraperitoneally, or subcutaneously, and various surface applications including intranasal, intradermal, and topical application, for instance.

Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers useful in this disclosure are conventional. Remington 's Pharmaceutical Sciences, by Lloyd V. Allen, Jt. (ed.), 22nd Edition (2012), describes compositions and formulations suitable for pharmaceutical delivery of the compounds herein disclosed.

In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (for example, powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate. Polypeptide: A polymer in which the monomers are amino acid residues which are joined together through amide bonds. When the amino acids are alpha- amino acids, either the L-optical isomer or the D-optical isomer can be used, the L-isomers being preferred. The term polypeptide or protein as used herein encompasses any amino acid sequence and includes modified sequences such as glycoproteins. The term polypeptide is specifically intended to cover naturally occurring proteins, as well as those that are recombinantly or synthetically produced.

The term polypeptide fragment refers to a portion of a polypeptide which exhibits at least one useful epitope. The phrase "functional fragments of a polypeptide" refers to all fragments of a polypeptide that retain an activity, or a measurable portion of an activity, of the polypeptide from which the fragment is derived. Fragments, for example, can vary in size from a polypeptide fragment as small as an epitope capable of binding an antibody molecule to a large polypeptide capable of participating in the characteristic induction or programming of phenotypic changes within a cell. An epitope is a region of a polypeptide capable of binding an immunoglobulin generated in response to contact with an antigen. Thus, smaller peptides containing the biological activity of insulin, or conservative variants of the insulin, are thus included as being of use.

The term substantially purified or isolated polypeptide as used herein refers to a polypeptide that is substantially free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated. In one embodiment, the polypeptide is at least 50%, for example at least 80% free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated. In another embodiment, the polypeptide is at least 90% free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated. In yet another embodiment, the polypeptide is at least 95% free of other proteins, lipids,

carbohydrates or other materials with which it is naturally associated.

Conservative amino acid substitution tables providing functionally similar amino acids are well known to one of ordinary skill in the art. The following six groups are examples of amino acids that are considered to be conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). Variations in the cD A sequence that result in amino acid changes, whether conservative or not, are usually minimized in order to preserve the functional and immunologic identity of the encoded protein. The immunologic identity of the protein may be assessed by determining whether it is recognized by an antibody; a variant that is recognized by such an antibody is immunologically conserved. Any cDNA sequence variant will preferably introduce no more than twenty, and preferably fewer than ten amino acid substitutions into the encoded polypeptide. Variant amino acid sequences may, for example, be 80%, 90% or even 95% or 98% identical to the native amino acid sequence. Programs and algorithms for determining percentage identity can be found at the NCBI website.

Preventing or treating a disease: Preventing a disease refers to inhibiting the full development of a disease, for example inhibiting the development of myocardial infarction in a person who has coronary artery disease or inhibiting the progression or metastasis of a tumor in a subject with a neoplasm. Treatment refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop.

Receptor tyrosine kinase (RTK): Large family of cell surface receptors that dimerize and propagate an intracellular signal when bound by functional ligand. Tiel and Tie2 are particular examples of RTKs, and are bound by angiopoietin ligands in the regulation of angiogenesis. The abnormal activation of Tiel and Tie2 has been linked with aberrant angiogenesis and the pathology of multiple cancer types, including glioblastomas, breast, ovarian, and liver cancers.

Sequence identity: The similarity between two nucleic acid sequences, or two amino acid sequences, is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Methods of alignment of sequences for comparison are well known in the art. The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al. J. Mol. Biol. 215:403-10, 1990) is available from several sources, including the National Center for

Biotechnology Information (NCBI, Bethesda, MD) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx.

Nucleic acid sequences that do not show a high degree of identity can nevertheless encode similar amino acid sequences, due to the degeneracy of the genetic code. It is understood that changes in nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid molecules that all encode substantially the same protein. Subject: Living multi-cellular organisms, including vertebrate organisms, a category that includes both human and non-human mammals.

Sustained release (of a pharmaceutical compound): A sustained release compound that can be biologically/chemically available and active upon administration to a subject and contact with a target site, and over an extended period of time. Numerous sustained release additives and formulations are known to the art and can be combined with the Ang2-BD variant polypeptides described herein.

integrin related condition: Polypeptides of the present invention are useful

for the treatment of a wide range of medical conditions which are characterized by abnormal activation of the Tie2 receptor or abnormal expression of ανβ3 integrin. Non-limiting examples of integrin related conditions include: metastatic and non-metastatic cancer, osteoporosis, and any angiogenesis related pathologies such as found in diabetes (e.g., diabetic retinopathy and more specifically proliferative diabetic retinopathy and diabetic macular edema), wet age-related macular degeneration, and angiogenesis related cancers such as angiosarcoma in the endothelial layer of the inner lining of vasculature, hemoangiosarcoma (blood vessels), and lymphoangionsarcoma (lymph vessels).

Vector: A nucleic acid molecule as introduced into a host cell, thereby producing a transfected host cell. Recombinant DNA vectors are vectors having recombinant DNA. A vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector can also include one or more selectable marker genes and other genetic elements known in the art. Viral vectors are recombinant DNA vectors having at least some nucleic acid sequences derived from one or more viruses.

III. Overview of Several Embodiments

Provided herein are Ang2-BD variants that can bind with increased affinity and inhibit the Tie2 receptor tyrosine kinase. The described polypeptides include an isolated polypeptide having an amino acid sequence at least 70% identical to SEQ ID NOs: 1 or 8, or fragments, a derivatives or analogs thereof, wherein the polypeptide differs by at least one amino acid compared to SEQ ID NO: 2.

In particular embodiments, the described isolated polypeptide has an amino acid sequence at least 70% identical to an amino acid sequence selected from SEQ ID NOs 3-7. In other embodiments, the described isolated polypeptide has an amino acid sequence at least 70% identical to an amino acid sequence set forth herein as SEQ ID NO: 8. In yet other embodiments, the described isolated polypeptide has an amino acid sequence at least 70% identical to an amino acid sequence set forth herein as SEQ ID NOs: 9-21 or SEQ ID NOs: 31- 34.

In some embodiments, the described isolated polypeptide is glycosylated, in other embodiments, the described isolated polypeptide is non- glycosylated.

in particular embodiments, the described isolated polypeptide binds to the Tie2 receptor tyrosine kinase (RTK) with a binding affinity at least two-fold greater than the wildtype Ang2-

BD polypeptide (SEQ ID NO: 2).

In particular embodiments, the described isolated polypeptides and/or nucleic acids are formulated as pharmaceutical compositions that include at least one pharmaceutically acceptable carrier, salt or excipient.

Further described herein are methods for treating cancer or an Ang2/Tie2 related condition by administering to a subject in need thereof, an effective amount of a composition that includes one of the described isolated polypeptides thereby treating the cancer or

Ang2/Tie2 related condition. In particular embodiments, the composition is a pharmaceutical composition further including at least one of a pharmaceutically acceptable carrier, salt, or excipient. Compositions for use in such treatments and for use in the preparation of a medicament for such treatments are also described.

In particular embodiments of the described treatments, the cancer or Ang2/Tie2 related condition is associated with abnormal activation of the receptor tyrosine kinase (RTK) Tie2. In other embodiments, the treatments include inhibition of at least one of cancer metastasis, cellular proliferation, and cellular survival. In particular embodiments, the cancer is a glioblastoma or, breast, ovarian, or liver cancer.

Also described herein is an isolated polypeptide based on the Ang2-BD and which is adapted to bind to both Tie2 and α ν β 3 integrin. The described polypeptides include an amino acid sequence set forth herein as SEQ ID NO: 23, or a fragment, derivative, or analog thereof, and which in particular embodiments can be any one of SEQ ID NOs 24-27. Other particular embodiments include polypeptides further optimized for Tie2 and α ν β 3 integrin binding, and which combine the optimized sequences described herein. Particular examples of such polypeptides include those described herein as SEQ ID NOs. 31-34.

Also provided herein are isolated nucleic acids sequences encoding the described polypeptides. In particular embodiments, the isolated nucleic acid is included in an expression vector. In particular embodiments, the described polypeptides and/or nucleic acids are included in a pharmaceutical composition that also includes at least one of a pharmaceutically acceptable carrier, salt, or excipient.

In some embodiments, the described pharmaceutical compositions are used in the treatment of a integrin-related condition, including an angiogenesis-related pathology

or osteoporosis. Particular examples of angiogenesis-related pathologies include a cancer or macular degeneration.

In some embodiments, the described pharmaceutical compositions can be used in inhibition of cancer cell invasion or metastasis. In other embodiments, the described pharmaceutical compositions can be used for inhibiting the growth of a vascular tumor.

Also described herein are methods for treating an Τίε2/α ν β3 integrin-related condition in a subject, by administering to a subject in need thereof, a therapeutically effective amount of the described polypeptides and/or nucleic acids, thereby treating the Τίε2/ανβ3 integrin-related condition.

In a particular embodiment of the described methods, the Τίε2/α ν β3 integrin-related condition is an angiogenesis-related pathology or osteoporosis. In particular examples, the angiogenesis-related pathology is a cancer or macular degeneration.

In further examples of the described methods, the cancer to be treated is selected from the group consisting of breast, lung, hepatocarcinoma, melanoma, lymphoma, myeloma, cervical, pancreas, colon, prostate, ovary, osteoma, and metastatic cancer.

Further described herein are methods for inhibiting growth of vascular tumors in a subject, by administering to a subject in need thereof, a therapeutically effective amount of the described polypeptides or nucleic acids, thereby inhibiting growth of the vascular tumor.

Additionally described are methods for inhibiting metastasis of a cancer in a subject, by administering to a subject in need thereof, a therapeutically effective amount of the described polypeptides or the nucleic acids, thereby inhibiting the metastasis.

IV. Ang2-BD Based Tie2 Inhibitors and bispecific inhibitors of Tie2 and αδβι/ α ν β3 integrins

Described herein is the mutagenic generation, and subsequent identification of inhibitors of the Tie2 RTK, based on the Angiopoietin-2 binding domain (Ang2-BD), which can bind Tie2, but not multimerize. The described Ang2-BD variants have been optimized for enhanced Tie2 binding affinity, and as demonstrated herein, effectively inhibit activation and subsequent function of Tie2. In a particular embodiment, the described Ang2-BD variants include polypeptides, variants, and fragments thereof, of a polypeptide including the following sequence, with variable positions X1-X14, and in which the polypeptide is at least one amino acid different from the wildtype Ang2-BD sequence (set forth herein as SEQ ID NO: 2):

In particular embodiments the polypeptide includes the following sequence described herein as

was used as the basis for a second generation mutagenesis and screening

of Ang2-BD variants. From the variants identified in that second generation screening, several additional Ang2-BD variant polypeptides have been identified which bind and inhibit Tie2. In a particular embodiment, the additionally-described Ang2-BD variants include polypeptides, variants, and fragments thereof, of a polypeptide including the following sequence, with variable positions X1-X9:

In particular embodiments the polypeptide includes the following sequence described herein as

In particular embodiments the polypeptide includes the following sequence described herein as

In particular embodiments the polypeptide includes the following sequence described herein as

In particular embodiments the polypeptide includes the following sequence described herein as g

Also described herein is the mutagenic generation, and subsequent identification of synthetic variants of Ang2-BD which have been optimized for bispecific enhanced binding to integrins. The identified synthetic Ang2-BD variants are generally

described herein as "bispecific integrin inhibitors."

In a particular embodiment, the described bispecific integrin inhibitors include polypeptides, variants, and fragments thereof, of a polypeptide including the following summary sequence, with variable positions

In particular embodiments the bispecific integrin inhibitor polypeptide includes the following sequence described herein as SEQ ID NO: 24, and which is the amino acid sequence of the Ang2-BDBC5 polypeptide:

In further embodiments the bispecific integrin inhibitor polypeptide includes

the following sequence described herein as SEQ ID NO: 25, and which is the amino acid sequence of the Ang2-BDBC6 polypeptide:

In other embodiments the bispecific integrin inhibitor polypeptide includes

the following sequence described herein as SEQ ID NO: 26, and which is the amino acid sequence of the Ang2-BDBC10 polypeptide:

In particular embodiments the bispecific ntegrin inhibitor polypeptide

includes the following sequence described herein as SEQ ID NO: 27, and which is the amino acid sequence of the Ang2-BDBC14 polypeptide:

In still further embodiments the bispecific integrin inhibitor polypeptide

includes the following sequence described herein as SEQ ID NO: 28, and which is the amino acid sequence of the Ang2-BDBC35 polypeptide:

Variants, fragments, and analogs of the described polypeptides are included in the current disclosure. Such polypeptides include polypeptides that share about 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% sequence identity with the described Ang2-BD variants. In particular embodiments, sequence variance from wildtype (WT) is particularly found in those amino acids apart from those described herein as involved in Tie2 binding (e.g. X1-X14 in SEQ ID NO: 1, X1-X9 in SEQ ID NO: 8, and in SEQ ID NO: 23 above).

For example, the Tie2-optimized binding peptides can be modified by a substitution of the eight amino acids from position 22 to 29 of SEQ ID NO: 1 or SEQ ID NO: 8 with the nine amino acid "RGD loop" (including the six variable residues flanking the RGD sequence) of SEQ ID NO: 23. Such polypeptides are accordingly optimized for both Tie 2 and

integrin binding.

A summary sequence of one embodiment of the peptides that are optimized for both Tie 2 and integrin binding is provided herein as SEQ ID NO: 31:

A particular embodiment of the further optimized bispecific integrin inhibitor

polypeptide includes the following sequence described herein as SEQ ID NO: 32, and which is the amino acid sequence of the Ang2-BDBC5_2.36 polypeptide:

In another particular embodiment of the further optimized bispecific integrin

inhibitor polypeptide includes the following sequence described herein as SEQ ID NO: 33, and which is the amino acid sequence of the Ang2-BDBC6_2.36 polypeptide:

In a further particular embodiment of the further optimized bispecific integrin

inhibitor polypeptide includes the following sequence described herein as SEQ ID NO: 34, and which is the amino acid sequence of the Ang2-BDBC10 J2.36 polypeptide:

In other embodiments the variation from the sequences expressly provided herein results from conservative sequence substitutions that one of skill will not expect to

significantly alter the shape or charge of the polypeptide. The described polypeptides also include those polypeptides that share 100% sequence identity to those indicated, but which differ in post-translational modifications from the native or natively-produced sequence, such as glycosylation, or which are artifacts from synthetic procedures such as acetylation.

Accordingly, in particular embodiments, the described isolated polypeptides are glycosylated, while in other embodiments they are non-glycosylated. Likewise, in particular embodiments the described polypeptides include a terminal acetyl group, while in other embodiments, they do not.

As indicated, the described Ang2-BD variants have been selected for improved binding to Tie2 and/or α ν β 3 integrin. Such increased binding affinity can be measured by standard methods (e.g. fluorescence mediated assays such as SPR and the like). In particular embodiments the Ang2-BD variant binds Tie2 and/or α ν β 3 integrin with at least 2-fold greater affinity in comparison to WT, such as 2, 3, 4, 5, 6, 7, 8, 9, 10-fold or greater affinity.

In particular embodiments, the described Ang2-BD variants are provided as a discrete biomolecule. In other embodiments, the described polypeptides are a domain of a larger polypeptide, such as an independently-folded structural domain, or an environment-accessible functional domain.

The Ang2-BD polypeptides described herein inhibit Tie2 and/or α ν β 3 integrin activity (e.g. Tie2-mediated signal transduction), but also bind with increased affinity and specificity to Tie2 and/or α ν β 3 integrin, and which allow for strong and specific targeting of a Tie2- expressing cell and/or a integrin-expressing cell, such as a cancer cell that is

overexpressing Tie2. In some embodiments, the described polypeptides are conjugated at the N- and/or C-terminus to at least one of a variety of small molecules, chemotherapeutic agents, labels, and solid substrates, which are thereby targeted to a Tie2 expressing cell or a

integrin-expressing cell.

In particular embodiments, the described polypeptides are conjugated to any chemotherapeutic agent as described herein and as known to the art. Such agents can include small molecule agents, antibodies, and the like which are designed, for example, to inhibit cell proliferation and/or metastatic cell migration and/or promote cell death (i.e. inhibit cellular survival). Particular non-limiting examples of such agents include cytotoxic radioisotopes, chemotherapeutic agents and toxins.

In other embodiments, a detectable label, such as a detectable chemical, fluorescent, or radioactive label can be conjugated to the described polypeptides. Antibodies that can be conjugated to Ang2-BD variant polypeptides, and which themselves are or can be labeled, are standard in the art and are included in the current disclosure. Such labeled polypeptides can serve as diagnostic reagents for detecting cells that are expressing Tie2 and/or α ν β 3 integrin, and can identify patterns of Tie2 and/or α ν β 3 integrin overexpression, if present.

In still other embodiments, the described Ang2-BD variants can be conjugated to any suitable affinity tag for specific isolation of Tie2 and/or α ν β 3 integrin expressing cells through standard affinity chromatographic methods. Particular non-limiting examples of such tags include myc, glutathione- s-transferase (GST), maltose binding protein (MBP), hemagglutinin (HA), and polyhistadine.

Also provided herein are nucleic acids encoding the described Ang2-BD variant polypeptides. Due to degeneracy of the genetic code, the sequences of the Ang2-BD variant- encoding nucleic acids can vary significantly without any change in the encoded polypeptide. Other and/or additional mutations in the described polypeptides, such as conservative amino acid mutations, and those not expected to significantly alter polypeptide shape or charge can also be included without an appreciable difference.

In particular embodiments, the described nucleic acid sequences are contained within a DNA cloning and/or expression plasmid as are standard in the art. It will be appreciated that any standard expression plasmid can be used to express one or more of the described Ang2-BD variant-encoding nucleic acids. Such plasmids will minimally contain an origin of replication, selection sequence (such as, but not limited to an antibiotic resistance gene), and expression control sequences operably linked to the sequence encoding the particular Ang2-BD variant polypeptide. In particular embodiments, the expression plasmids include post-translational sequences (e.g. signal sequences to direct polypeptide processing and export) that are encoded in-frame with the Ang2-BD variant-encoding nucleic acids.

Particular non-limiting examples of bacterial expression plasmids include IPTG- inducible plasmids, arabinose-inducible plasmids and the like. Other non-limiting examples of expression induction include light induction, temperature induction, nutrient-induction, and autoinduction, and mammalian- specific DNA expression plasmids. Custom-made expression plasmids are commercially available from suppliers such as New England Biolabs (Ipswich, MA) and DNA 2.0 (Menlo Park, CA). In a particular embodiment, a Ang2-BD variant- expressing plasmid can be designed for specific localized induction in response to a local cellular micro environment, such as the micro environment of a specific tissue or tumor type.

In particular embodiments, the peptides and nucleic acids described herein can be supplied in any pharmaceutically acceptable composition. In such embodiments, one or more Ang2-BD variant polypeptide or polypeptide-expressing nucleic are provided in a

pharmaceutical formulation having a therapeutically effective amount of each therapeutic agent, as described herein, and including standard pharmaceutically acceptable salts, excipients, carriers and the like.

Various delivery systems are known and can be used to administer polypeptides and nucleic acids as therapeutic agents. Such systems include, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the therapeutic molecule(s), construction of a therapeutic nucleic acid as part of a retroviral or other vector, and the like. Methods of administration of the therapeutic agents include, but are not limited to, intrathecal, intradermal, intramuscular, intraperitoneal (ip), intravenous (iv), subcutaneous, intranasal, epidural, and oral routes. The therapeutics can be formulated for administration by any convenient route, including, for example, infusion or bolus injection, topical, absorption through epithelial or mucocutaneous linings (e.g. , oral mucosa, rectal and intestinal mucosa, and the like) ophthalmic, nasal, and transdermal, and may be administered together with other biologically active agents. Pulmonary administration can also be employed (e.g., by an inhaler or nebulizer), for instance using a formulation containing an aerosolizing agent.

In a specific embodiment, it may be desirable to administer the described

pharmaceutical compositions by injection, catheter, suppository, or implant (e.g., implants formed from porous, non-porous, or gelatinous materials, including membranes, such as sialastic membranes or fibers), and the like. In another embodiment, therapeutic agents are delivered in a vesicle, in particular liposomes.

In particular embodiments, the described polypeptides and nucleic acids can be formulated for immediate release, whereby they are immediately accessible to the surrounding environment, thereby providing an effective amount of the active agent(s), upon administration to a subject, and until the administered dose is metabolized by the subject.

In yet another embodiment, the described polypeptides and nucleic acids can be formulated in a sustained release formulation or system. In such formulations, the therapeutic agents are provided for an extended duration of time, such as 1, 2, 3, 4 or more days, including 1-72 hours, 24-48 hours,. 16-36 hours, 12-24 hours, and any length of time in between. In particular embodiments, sustained release formulations are immediately available upon administration, and provide an effective dosage of the therapeutic composition, and remain available at an effective dosage over an extended period of time. In other embodiments, the sustained release formulation is not immediately available within the subject and only becomes available, providing a therapeutically effective amount of the active compound(s), after the formulation is metabolized or degraded so as to release the active compound(s) into the surrounding environment.

In one embodiment, a pump may be used. In another embodiment, the sustained released formulations include polymeric materials commonly used in the art, such as in implants, gels, capsules, and the like. By way of example, polymers such as bis(p- carboxyphenoxy)propane-sebacic-acid or lecithin suspensions may be used to provide sustained localized release.

In particular embodiments, the described polypeptides and nucleic acids are formulated in pharmaceutically acceptable compositions of the compounds, using methods well known to those with skill in the art. For instance, in some embodiments, the compounds are formulated with a pharmaceutically acceptable carrier, salt, or excipient. The term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or a state government or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia world-wide for use in animals, and, more particularly, in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such

pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Saline solutions, blood plasma medium, aqueous dextrose, and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. The medium may also contain conventional pharmaceutical adjunct materials such as, for example, pharmaceutically acceptable salts to adjust the osmotic pressure, lipid carriers such as cyclodextrins, proteins such as serum albumin, hydrophilic agents such as methyl cellulose, detergents, buffers, preservatives and the like.

Examples of pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. The described compositions can, if desired, also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The described compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, all in immediate and sustained-release formulations as understood in the art. The therapeutic can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like.

Therapeutic preparations will contain a therapeutically effective amount of at least one active ingredient, preferably in purified form, together with a suitable amount of carrier so as to provide proper administration to the patient. The formulation should suit the mode of administration.

The ingredients of the described formulations can be supplied either separately or mixed together in unit dosage form, for example, in solid, semi-solid and liquid dosage forms such as tablets, pills, powders, liquid solutions, or suspensions, or as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Kits comprising the described Ang2-BD variant polypeptides or encoding nucleic acids are accordingly also contemplated herein.

In particular embodiments, the described Ang2-BD variants are produced by standard chemical synthesis procedures (i.e. ex vivo). In other embodiments, the described polypeptides are produced from the described encoding nucleic acids (i.e. in vivo) and subsequently isolated. Such synthesis procedures are described herein and are well known to the art.

V. Methods of treatment of Tie2-associated diseases and integrin-associated

diseases

As indicated above, aberrant overexpression of Tie2 and/or α ν β 3 integrin has been implicated in a variety of diseases and conditions, in particular cancers, for example with relation to roles in metastasis and aberrant angiogenesis. The Ang2-BD variant polypeptides described herein provide Tie2 and/or α ν β 3 integrin inhibitory polypeptides with increased Tie2 and/or α ν β 3 integrin binding affinity, and resultant Tie2 and/or α ν β 3 integrin inhibitory activity (e.g. phosphorylation, angiogenesis, and cellular invasiveness). Accordingly, the described polypeptides (collectively, also referred to herein as "Ang2-BD" variant polypeptides, and nucleic acids expressing the described polypeptides, can be used in pharmaceutical

compositions in methods for treating diseases and conditions associated with aberrant overexpression and/or activation of Tie2 and/or α ν β 3 integrin. Such diseases include cellular hyperproliferative diseases, including cancer, diseases resultant from or related to aberrant angiogenesis (also described herein as angiogenesis-related pathologies), osteoporosis, and macular degeneration. In particular embodiments, the described methods include administering a therapeutically effective amount of an Ang2-BD variant polypeptide described herein to a subject in need thereof, thereby treating the disease or condition associated with aberrant expression of Tie2 and/or α ν β 3 integrin, such as a cancer, including a glioblastoma, breast, ovarian, or liver cancer and any angiogenesis related pathologies such as found in diabetes (e.g. diabetic retinopathy and more specifically proliferative diabetic retinopathy and diabetic macular edema), wet age-related macular degeneration, and angiogenesis related cancers such as angiosarcoma in the endothelial layer of the inner lining of vasculature, hemoangiosarcoma (blood vessels) and lymphoangionsarcoma (lymph vessels).

Any of the compositions described herein which include the described Ang2-BD variant polypeptides or polypeptide-encoding nucleic acids, can be used in the described methods of treatment, and similarly can be used in the preparation of a medicament for use in any of the described methods of treatment.

In particular embodiments, the disease related to aberrant Tie2 and/or α ν β 3 integrin expression is a cancer, such as a fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers (such as small cell lung carcinoma and non-small cell lung carcinoma), ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, melanoma, and CNS tumors (such as a glioma, astrocytoma, medulloblastoma, craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, neuroblastoma and retinoblastoma).

In some embodiments, the described Ang2-BD variants inhibit cell proliferation. In other embodiments, the described variants inhibit cell migration and or invasion, thereby inhibiting cancer metastasis. In still other embodiments, the described variants promote cell death or conversely limit cellular survival (e,g, by inhibiting cell death blockades), such as by promotion of apoptosis.

In other particular embodiments, the disease is related to or resultant from aberrant angiogenesis. Aberrant angiogenesis has been implicated in multiple facets of cancer pathology, including tumor promotion/survival and metastasis, as well as the transition to malignancy. The anti-angiogenic Ang2-BD variants described herein can therefore be used for treatment of cancers, and in particular embodiments, treatments aimed at slowing or inhibiting tumor metastasis. Aberrant angiogenesis is also a key contributor to progression of wet macular degeneration, and it will be appreciated that the Ang2-BD variant polypeptides described herein can also be used for such treatment.

In particular embodiments, the Ang2-BD variant (i.e. the Tie2 and/or α ν β 3 integrin inhibitor polypeptide) is administered to the subject as a polypeptide. In other embodiments, the Ang2-BD variant is administered to the subject by way of an expression vector containing a Ang2-BD variant-encoding nucleic acid. It will be appreciated that in such embodiments, expression of the polypeptide can be constitutive or induced, as is well-known in the art. In some embodiments, inducible expression systems can allow for specific targeting of an area, which contains the inducing signal.

In some embodiments, the Ang2-BD variant is administered to the subject in combination with other pharmaceutical agents for treatment of the disease or condition under treatment. For example, in methods for treating breast cancer the Ang2-BD variant can be combined with trastuzumab (Herceptin) therapy. In other examples of cancer treatment, administration of the Ang2-BD variant can be combined with surgery and/or radiation therapy. The one or more therapies in combination with the Ang2-BD variant can be administered to the subject in sequence (prior to or following) or concurrent. Where applicable, in particular embodiments, combinations of active ingredients can be administered to a subject in a single or multiple formulations, and by single or multiple routes of administration.

The amount of each therapeutic agent for use in the described methods, and that will be effective, will depend on the nature of the disorder or condition to be treated, as well as the stage of the disorder or condition. Therapeutically effective amounts can be determined by standard clinical techniques. The precise dose to be employed in the formulation will also depend on the route of administration, and should be decided according to the judgment of the health care practitioner and each patient's circumstances. The specific dose level and frequency of dosage for any particular subject may be varied and will depend upon a variety of factors, including the activity of the specific compound, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of

administration, rate of excretion, drug combination, and severity of the condition of the host undergoing therapy. The therapeutic compounds and compositions of the present disclosure can be administered at about the same dose throughout a treatment period, in an escalating dose regimen, or in a loading-dose regime (e.g., in which the loading dose is about two to five times the maintenance dose). In some embodiments, the dose is varied during the course of a treatment based on the condition of the subject being treated, the severity of the disease or condition, the apparent response to the therapy, and/or other factors as judged by one of ordinary skill in the art. In some embodiments long-term treatment with the drug is contemplated. The following examples are provided to illustrate certain particular features and/or embodiments. These examples should not be construed to limit the disclosure to the particular features or embodiments described.

EXAMPLES

Example 1: Methods

Preparation of YSD Ang2-BD constructs and libraries

The construct for Ang2-BDwT (amino acids 281 to 496 of human Angiopoietin-2, Uniprot Ref. No. 015123) was obtained by custom gene synthesis (Integrated DNA

Technologies). Amplification of the gene was performed using primers containing Nhel and BamHI restriction sites and a 12-amino-acid linker (LPDKPLAFQDPS; SEQ ID NO: 22) that connects the C-terminus of the Ang2-BDwT protein with a c-Myc epitope. The amplified gene was then introduced into the pCTCON yeast display vector (a generous gift from the laboratory of Dane Wittrup, MIT). The first generation library was prepared using the Ang2-BDwT construct as the template, and the library was generated by error-prone PCR and homologous recombination into Saccharomyces cerevisiae EBY100 cells, as previously described Chao, 2016). The library size was 6x l0 6 transformants, as estimated by dilution plating on selective SDCAA medium (2% dextrose, 1.47% sodium citrate, 0.429% citric acid monohydrate, 0.67% yeast nitrogen base and 0.5% casamino acids, pH 4.5). The second-generation library was prepared as described above using the Ang2-BDci.7o clone, isolated from first-generation library sort 5, as the template. The library size was about 8x l0 6 transformants, as estimated by dilution plating on selective SDCAA medium. Screening of YSD Ang2-BD libraries

The yeast-displayed Ang2-BD libraries growing in selective SDCAA medium were induced for expression with 2% w/v galactose at 20°C overnight until an OD of 3.0 was reached, according to established protocols (Chao, 2016). The first-generation library underwent five rounds of screening using high-throughput flow cytometric sorting to isolate clones with high affinity for recombinant human Tie2 (Ala23-Lys745). Initial and final sorts were performed using 100 nM and 5 nM Tie2-Fc, respectively. A diagonal sorting gate including 1% of the entire yeast pull was used to select Ang2-BD mutants that bind strongly to Tie2, relative to their expression. For each round of sorting, yeast cells of approximately 10 times the library size were labeled as described below to facilitate fluorescent detection by flow cytometry. Binding of the yeast-displayed Ang-BD library to Tie2 was detected using soluble Tie2-Fc (R&D Systems) in Tie2 binding buffer [20 mM Hepes, pH 7.0, 150 mM NaCl, and 1% bovine serum albumin (BSA)], and Ang-BD expression levels were detected using 1:50 dilution of mouse anti c-Myc 9E10 antibody (Abeam) in a 1 hour reaction at room

temperature. Cells were washed and resuspended in ice-cold PBS A (phosphate buffered saline + 1% BSA) containing a 1:50 dilution of anti-human Fc fluorescein isothiocyanate (FITC) conjugated antibody (Sigma) and a 1:50 dilution of phycoerythrin (PE) conjugated anti-mouse IgG (Sigma). After 25 minutes on ice, yeast cells were washed in PBS A and sorted using iCyt Synergy FACS (fluorescence-activated cell sorting ) [Proteomics Unit, National Institute for Biotechnology in the Negev (NIBN), Ben-Gurion University of the Negev (BGU)]. Plasmid DNA was extracted from the yeast clones using a Zymoprep kit (Zymo Research) and transformed into electrocompetent Escherichia coli cells for plasmid miniprep (RBC

Bioscience Corp, Taiwan) and DNA sequencing (DNA Microarray and Sequencing Unit, NIBN, BGU). The second-generation library was subjected to five rounds of sorting using the method described above, where the initial and final sorts were performed with 20 nM and 500 pM Tie2, respectively. Sixty clones from the two final sorts were sequenced (DNA Microarray and Sequencing Unit, NIBN, BGU) and evaluated for their binding affinity towards Tie2-Fc by dividing the mean fluorescence intensity (MFI) of the Tie2 binding signal by the MFI of expression levels. The values obtained were normalized to Ang2-BDwT.

Purification of Ang2-BD variants

The Multi-Copy Pichia Expression Kit (Invitrogen K1750-01) was used to produce the soluble proteins, as previously described (Moore, 2013). The Ang2-BD variants were cloned into the pPIC9K vector for expression in P. pastoris yeast strain GS 115 using EcoRI and Avrll restriction sites. Plasmid DNA (approximately 20 μg) was linearized by digestion with Sacl (New England Biolabs) and electroporated into P. pastoris. Proteins were prepared with an N- terminal FLAG epitope tag and a C-terminal hexahistidine tag as handles for cell binding studies and protein purification, respectively. Transformed yeast cells were allowed to recover on RDB plates (18.6% sorbitol, 2% agar, 2% dextrose, 1.34% yeast nitrogen base, 0.001% biotin and 0.005% L-glutamic acid/L methionine/L-leucine/L-lysine/L-isoleucine) for two days at 30 °C and were then selected for growth on YPD plates (1% yeast extract, 2% peptone and 2% dextrose) containing 4 mg mL Geneticin (Gibco). Several Geneticin-resistant colonies were grown in BMGY (1% yeast extract, 2% peptone, 0.23% potassium phosphate monobasic, 1.18% potassium phosphate dibasic, 1.34% yeast nitrogen base, 0.00004% biotin and 1% glycerol), followed by induction in BMMY (1% yeast extract, 2% peptone, 0.23% potassium phosphate monobasic, 1.18% potassium phosphate dibasic, 1.34% yeast nitrogen base, 0.00004% biotin and 1% methanol) for four days, with the methanol concentration being maintained at 1% throughout. Protein expression was detected by Western blot analysis of the culture supernatants, using an antibody against the FLAG epitope tag (Sigma). The highest expressing colony for each individual mutant was scaled up for expression by growing the yeast cultures in baffled base shake flasks. Ang2-BD variants were purified from yeast culture supernatants by metal chelating chromatography using a 5-ml HisTrap FF column (GE

Healthcare) with 10 mM imidazole and eluted with 250 mM imidazole. Eluted protein fractions were concentrated and buffer exchanged to 20 mM Hepes, pH 7.0, 150 mM NaCl buffer using a 10-kDa cutoff Vivaspin ® concentrator (GE Healthcare). Approximately 4 mg of purified protein was treated with Endo Hf (3,000 Units, New England Biolabs) overnight at room temperature to remove N-linked glycosylation. Gel filtration chromatography was performed using a Superdex 75 column (GE Healthcare) equilibrated with 20 mM Hepes, pH 7.0, 150 mM NaCl at a flow rate of 0.4 ml/min on AKTA pure instrument (GE Healthcare).

Proteins were analyzed by SDS-PAGE under non-reducing conditions. Protein concentrations were determined by UV-Vis absorbance at 280 nm and an extinction coefficient of 66,500 M " for all Ang2-BD variants. The molecular weights of the purified proteins were

determined using MALDI-TOF REFLEX-IV (Bruker) mass spectrometry instrument (IKI- BGU).

Far-UV circular dichroism spectroscopy

CD spectra were recorded on a Jasco J-715 spectropolarimeter over a range of 185-260 nm in 20 mM Hepes, pH 7.0, 150 mM NaCl buffer using a quartz cuvette with a path length of 1 mm. Protein spectra were collected at a scanning speed of 50 nm/min and a data interval of 1 nm. Four scans of 30 μΜ protein solutions were averaged to obtain smooth data. All spectra were background corrected with respect to 20 mM Hepes, pH 7.0, 150 mM NaCl buffer and converted to units of mean residue ellipticity. For thermal denaturation studies, ellipticity was monitored at 230 nm using a 1 ° /min scan rate.

Surface plasmon resonance experiments

The binding interactions of Tie2 to Ang2-BDwT, Ang2-BDci.7o, Ang2-BDc2.36

(glycosylated and non-glycosylated) were analyzed (Proteomics Unit, NIBN BGU) in real-time by SPR using a ProteOn XPR36 instrument (Bio-Rad). A ProteOn GLC sensor chip (Bio-Rad) was air initialized and PBST (PBS x 1, 0.005% Tween) buffer was flushed through the instrument prior to binding measurements. The rhTie2 extracellular domain (Sino Biological Inc.) was immobilized on the surface of a GLC sensor chip by using the amine coupling reagents N-hydroxysuccinimide (0.1 M; sulfo-NHS) and l-ethyl-3-(3-dimethylaminopropyl)- carbodiimide (0.4 M; EDC; Bio-Rad). rhTie2 (0.88 μg or 1.76 μg) in 10 mM sodium acetate, pH 5.0, was allowed to flow over two activated GLC sensor chip channel surfaces,

respectively, at a flow rate of 30 μL/min until the target immobilization levels for each channel (1200 and 2,400RU, respectively) were reached. BSA (3 μg) in 10 mM sodium acetate, pH 4.5, was then allowed to flow over the activated surfaces of a control GLC sensor chip channel at a flow rate of 30 μΐ/min until the target immobilization level (3000 RU) was reached. After protein immobilization, the chip surface was treated with 1 M ethanolamine HC1 at pH 8.5 to deactivate excess reactive esters. All binding experiments were performed at 25 °C in degassed Tie2 binding buffer (20 mM Hepes pH 7.0, 150 mM NaCl). A range of concentrations (25 nM to 1 μΜ for Ang2-BD W T, 12.5 nM to 200 nM for Ang2-BD C i.7o, 7.5 nM to 500 nM for Ang2- BDc2.36 non-glycosylated and 7.5 nM to 500 nM for Ang2-BDc2.36 glycosylated) of the protein analytes were allowed to flow over the surface-immobilized rhTie2 at a flow rate of 100 μΐ/min for 3 min, and the binding interactions were monitored. Following association, the dissociation of the various ligand-receptor complexes was monitored for 5 min. After the dissociation of each analyte, a regeneration step with 50 mM NaOH at a flow rate of 100 μΐ/min was performed. Each analyte sensorgram run was normalized by subtracting the BSA-immobilized channel and the zero analyte concentration runs. The binding constant (KD) was determined from the sensogram of the equilibrium binding phase. Binding kinetics of glycosylated and non-glycosylated Ang2-BDc2.36 were also analyzed by fitting to a 1 : 1 Langmuir model. Cell binding assays

TIME cells (ATCC) were cultured in growth-factor-depleted Vascular Cell Basal Medium (ATCC) supplemented with 2% FBS and growth factor supplements (ATCC). For binding assays, 10 5 cells were suspended in different concentrations of Ang2-BD variants in a total volume of 200 μΐ PBS A (PBS and 0.1% BSA), followed by incubation at 4 °C for 2 hours with gentle agitation. Cell suspensions were centrifuged at 150g at 4 °C for 5 minutes and washed in 100 μΐ PBSA followed by centrifugation at 150g at 4 °C for 5 minutes for two additional times. Cells were then resuspended in 100 μΐ PBSA containing a 1:200 dilution of allophycocyanin (APC)-conjugated anti-FLAG antibody (Biolegend). After 20 minutes on ice, cells were washed twice in PBSA and analyzed by flow cytometry with a BD Accuri C6 flow cytometer (BD Biosciences). Mean fluorescence values were generated using Flow Jo software (Treestar). For receptor level detection, 10 5 cells were harvested, resuspended in 100 μΐ PBSA with 1: 100 APC-labeled anti-human Tie2 antibody (Biolegend), incubated at 4 °C for 30 min, and then analyzed by flow cytometry. The data for the cellular assays was analyzed for column statistics with GraphPad Prism version 5.00 for Windows (La Jolla, CA, USA). Statistical significance was determined by column statistics and t-test analysis. P value < 0.05 was considered statistically significant.

Tie2 phosphorylation assays

Confluent TIME cells were cultured in growth-factor-depleted Vascular Cell Basal

Medium supplemented with 0.5% fetal bovine serum for 12 hours at 37 °C/5% C0 2 prior to experimentation. Cells were then washed with PBS, and the medium was exchanged to fresh Vascular Cell Basal Medium depleted of growth factors and serum. After pretreatment with 1 mM sodium orthovanadate (Na 3 V0 4 , Sigma) for 15 minutes, cells were co-incubated for 15 minutes at 37 °C with either commercial full-length rhAngl as a positive control (R&D Systems) or a combination of full-length rhAngl and the Ang2-BD variants. Since rhAngl exists in different oligomeric states, the rhAngl concentration was reported in mass concentration units instead of molar concentration units. Unstimulated cells were used as a negative control. Cells were then washed twice with PBS plus 1 mM Na 3 V0 4 and lysed in ice- cold lysis buffer [20 mM HEPES, pH 7.4, 150mM NaCl, 1% TritonX-100, 1 mM Na 3 V0 4 , and 1 x complete protease inhibitor cocktail tablet (Roche)]. Cells were scraped from the culture plate wells, and the lysates were clarified by centrifugation (13,000 rpm for 30 minutes at 4 °C). Protein concentration was measured by BCA assay (ThermoFisher Scientific), and equivalent amounts of each lysate sample were analyzed by a duplicate 10% SDS-PAGE and transferred to duplicate PVDF membranes (Biorad). Blots were blocked (5% BSA, 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20) for 1 hour at room temperature and probed with a phospho-Tie2 specific rabbit polyclonal antibody (1:500 dilution; Y992-Tie2, R&D Systems) and a specific Tie2 rabbit monoclonal antibody (1: 1000 dilution; Tie2 (D9D10) rabbit mAb, Cell Signaling Technology) overnight at 4 °C. Membranes were washed three times with TBST (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20) and probed with anti-rabbit, HRP-linked antibody (1: 1000 dilution, Cell Signaling Technology) for 1 hour at room temperature. Membranes were washed three times with TBST and then visualized and quantified using chemiluminescence (ECL, Biological Industries) and ImageJ software, respectively. The intensities of the phospho-Tie2 bands were adjusted for the expression of total Tie2 for each experiment. Blots were stripped and re-probed with anti-actin antibody for further normalization. Tie2 phosphorylation assay data was analyzed with GraphPad Prism version 5.00 for Windows (La Jolla, CA, USA). Statistical significance was determined by column statistics and t test analysis. P value < 0.05 was considered statistically significant.

Endothelial cell tube formation assay

Serum-reduced Matrigel (10 mg/ml; BD Biosciences) was thawed overnight at 4 °C, and 150 μΐ were added to each well of a 48-well microtiter plate and allowed to solidify for 1 hour at 37 °C. Wells were incubated with 3.25 10 4 TIME cells with 2 μΜ, 4 μΜ and 8 μΜ of Ang2-BDwT and Ang2-BDc2.36; rhAngl, 200 ng/ml, was added as a positive control. Cells were incubated for 16-18 hours at 37 °C/5% C0 2 . Cells were then washed twice in HBSS (Hanks' balanced salt solution, Sigma), and capillary tube formation was observed using EVOS Cell Imaging Systems microscope (ThermoFisher Scientific). Images were taken with EVOS 2x Objective, phase-contrast. Total length and number of junctions of the tubes were quantified by analysis of digitized images using ImageJ software and Angiogenesis Analyzer plugin of the capillary-like structures. Tube formation assay data was analyzed with GraphPad Prism version 5.00 for Windows (La Jolla, CA, USA). Statistical significance was determined by column statistics and t test analysis. P value < 0.05 was considered statistically significant. Invasion assay

An in-vitro Boyden chamber assay was performed using ThinCert™ 24 well inserts (Greiner Bio-One). ThinCert cell culture insert membranes were coated with Matrigel (Corning) diluted in Vascular Cell Basal Medium (ATCC) to 1:30 ratio. The lower compartment was filled with 600 μΐ of Vascular Cell Basal Medium supplemented with 2% FBS. TIME cells, 2x 10 4 , with or without Ang2-BD variants, were incubated in 200 ul supplement-free Vascular Cell Basal Medium, added to the pre-coated ThinCert cell culture inserts, and incubated for 20 h at 37 °C with 5% C0 2 . Invasive cells were stained with DippKwik stain kit (American MasterTech Scientific) and were detected by EVOS FL Cell Imaging System at x20 magnification. Quantification was accomplished by counting 16 fields of each membrane. Analysis of digitized images was performed using ImageJ software. Data was analyzed with GraphPad Prism version 5.00 for Windows (La Jolla, CA, USA). Statistical significance was determined by column statistics and t test analysis. P value < 0.05 was considered statistically significant.

Example 2: Affinity maturation and isolation of Ang2-BD variants Introduction

This and the following example show the isolation and characterization for Ang2-BD variants for targeting and inhibiting the activity of Tie2. As discussed earlier, in view of research to date, it seems likely that molecules targeting Tie2 would show promise as cancer therapeutics, diagnostics, and inhibition of pathological (aberrant) angiogenesis. Further in view of work to date, approaches that hold the most promise for developing therapeutics are likely those aimed at inhibiting ligand-mediated receptor activation by receptor antagonists derived from natural ligands (Jones, 2008; Nanda,2004; Dupont, 2005; Cretney, 2006; Jones, 2011; Cochran, 2006; Sarkar, 2002; Fuh, 1992; Papo, 2011; Kariolis, 2014; Kariolis, 2016; Levin, 2016 ). Natural ligands, in contrast to antibodies or peptibodies, bind functionally important epitopes and therefore provide an excellent starting point for engineering protein- protein interactions. In addition, ligand-based antagonists are likely to be more specific than kinase domain small molecule inhibitors due to their large interaction surface that involves not only the highly conserved active site but also the different surrounding target-interacting residues. Despite the enormous inherent potential of ligand-based antagonists for generating new cancer biologies, all but a few have been prevented from advancing to clinical trials by limitations in ligand binding affinity and specificity, expression yield, and stability ( Deng, 1996; Boesen, 2002).

Previous work has provided an indication as to how the potential of ligand-based antagonists could be realized: A rational engineering approach was applied to transform the natural multimeric Angl agonist into a monomeric high-affinity Tie2 antagonist (Kim, 2005). The ligand multimerization was abolished by mutating two amino acids on residues critical for Angl multimerization in the superclustering domain (SCD) of the molecules (i.e., C41S and C54S). The Anglc4is,c54s variant could not multimerize and consequently, having reduced ability to bind, could not activate Tie2 ( Kim, 2005). Since the diminished affinity of

Anglc4is,c54s (relative to wild-type Angl) for Tie2 presumably resulted in a low inhibitory effect of the mutant in Angl-mediated processes ( Cho, 2004; Davis, 2003), we reasoned that the development of efficacious monomeric Ang-based antagonists would be contingent on improving their affinity for Tie2.

Taking advantage of the high sequence and structural similarities between Angl and the natural Tie2 antagonist, Ang2, and as described below in greater detail, we began by constructing a monomeric version of Ang2, namely, the Ang2-binding domain (Ang2-BD). The next step in our strategy was to engineer the natural monomeric Ang2-BD for higher affinity binding and subsequently improved antagonism to Tie2. To this end, we screened random mutagenesis Ang2-BD yeast surface display (YSD) libraries ( Gai, 2007), using increasingly stringent sorts against Tie2 and selecting the mutants with the highest stabilities and target affinities. The final steps were to demonstrate that these high-affinity Tie2 antagonists are potent inhibitors of Tie2 signaling in vitro, and were able to inhibit

angiogenesis in cell-based models.

Affinity maturation of Ang2-BD YSD libraries

Ang2-BD was created as the starting point for affinity maturation towards recombinant human (rh)Tie2. It was first necessary to test the compatibility of Ang2-BD with the YSD system that was to be used subsequently as a platform for the creation of the Ang2-BD library and affinity maturation towards Tie2. To this end, Ang2-BD was cloned into an YSD plasmid (pCTCON) and presented on the yeast cell surface as a fusion to agglutinin proteins. High yeast display and Tie2 binding levels were detected for Ang2-BD by staining with

fluorescently labeled antibodies as compared to unstained controls. A 12-amino-acid linker (LPDKPLAFQDPS; SEQ ID NO: 22) was added between the cMyc tag and Ang2-BD to prevent steric hindrance between the two antibodies (Fig. 1). A yeast-displayed library in which random mutations were introduced to the Ang2-BD gene was generated using error- prone PCR, with 2-9 mutations per clone and a yield of approximately 6 X 10 6 transformants. This Ang2-BD first-generation library, enriched for expression, was subjected to four additional rounds of sorting using decreasing concentrations of Tie2 (Figs. 2a-2d).The sorting gates are shown in Fig. 2c for the selection of clones with high affinity relative to their expression. The expression and binding of the YSD library at the beginning and the end of the sorting process are shown in Fig. 2c and 2d, respectively. Isolation of clones from the first-generation library with improved binding affinity towards Tie2

To identify specific Ang2-BD variants with improved Tie2 binding affinity, 70 individual clones were isolated from the fifth sort of the affinity maturation. Most of the clones showed a 50% increase in affinity relative to wild-type Ang2 (Ang2-BDwi), with clone CI.70 showing the highest (2.5-fold) increase in affinity (Figs. 3a-3c). Sequencing analysis of individual clones isolated from this first-generation library revealed mutations, such as K432N, I434T, N467K, F469L, N470D and Y475H, that are located specifically at the Ang2-BD/Tie2 binding interface, 5 A apart alongside mutations located on the Ang2-BD backbone. In particular, clone 70 (CI.70), with the highest affinity towards Tie2, had three mutations in its binding interface and one additional mutation in close proximity to the Ang2-BD-Tie2 interface (Table 1). Table 1: Sequencing analysis of the isolated clones with improved binding affinity towards Tie2. Individual clones isolated after sort 5 were sequenced, and mutations at the Ang2-BD/Tie2 interface were identified (italics).

Isolation from the second-generation library of clones showing further improvement in binding affinity towards Tie2

A second-generation library in which the Ang2-BDci.7o variant was used as a starting point for library construction (Fig. 2e) resulted in a diversity of approximately 8 X 10 6 transformants. Five rounds of sorting starting with library expression enrichment followed by target screening at decreasing concentrations of Tie2 were performed (Fig. 2f-2i). After five rounds of sorting, a significant shift of the library towards high affinity binding was clearly evident (Fig. 2i). With the aim to isolate variants with improved binding affinities from the second-generation library, 60 individual clones from sort 5 were isolated, sequenced and tested for their binding affinity towards Tie2 (shown in Table 2).

Table 2: Sequencing analysis of the isolated clones from the second generation library with improved binding affinity towards Tie2. Individual clones from the Ang2-BDci.7o based library were sequenced and their binding affinity towards Tie2 was tested.

Based on the sequencing results and the increase in Tie2 binding affinity, two clones, Ang2- BDci.7o and Ang2-BDc2.36, were chosen for purification as soluble proteins for their increased binding affinity towards Tie2 (Fig. 4). Example 3: Production, biochemical evaluation, and biological activity of soluble Ang2-

BD variants

Production and biochemical evaluation of soluble Ang2-BD variants

Wild-type Ang2-BD and the mutant variants generated and isolated in Example 2 were produced recombinantly in Pichia pastoris strain GS 115 and purified using affinity

chromatography, followed by treatment with endoglycosidase H (Endo Hf) to remove all relinked glycosylations (Fig. 5) and size-exclusion chromatography (SEC) to remove Ang2-BD multimers (Fig. 6). Circular dichroism (CD) spectra revealed no change in structure of Ang2- BDci.7o and Ang2-BDc2.36 variants both in their glycosylated and non-glycosylated forms in comparison to Ang2-BDwT (Fig. 7a). Thermal denaturation of the purified proteins revealed that the melting temperature (Tm) of Ang2-BDwT and Ang2-BDci.7o was 49 °C, and that of Ang2-BDc2.36 was 47 °C (Fig. 7b). The binding kinetics of Ang2-BD variants to Tie2 were determined by surface plasmon resonance (SPR) (Fig. 8a). The K D values were found to be: Ang2-BDwT 1.5 μΜ, Ang2-BD C i.7o 393 nM, and Ang2-BD C 2.36 200 nM and 333 nM for the non-glycosylated and the glycosylated forms, respectively (shown in Table 3, below). The K D values demonstrate an improvement in binding to Tie2 of 3.8-fold for the first-generation variant (Ang2-BDci.7o) and 7.5-fold for the second-generation variant (Ang2-BDc2.36). The kinetic parameters for the second-generation variant, Ang2-BDc2.36, were K and

for the non-glycosylated form and for the

glycosylated form (shown in Table 3, below). No significant difference in binding affinity was observed between the glycosylated and the non-glycosylated forms of Ang2-BDc2.36. The affinity of Ang2-BD variants to Tie2 showed the same trend that was observed with the Ang2- BD variants when displayed on yeast.

Table 3: Equilibrium binding affinities and kinetic rate constants for Ang2-BD variants to immobilized Tie2. SPR sensorgram curves were fitted to a 1 : 1 Langmuir binding model.

to cell-expressed Tie2

Binding of Ang2-BD variants to cell-expressed Tie2 was evaluated using the human telomerase-immortalized microvascular endothelium (TIME) cell line. These cells expressed Tie2 on their surface (Fig. 9), and Ang2-BD variants were found to bind to these cells in a dose-response manner (Fig. 8a). Ang2-BDc2.36 exhibited the highest affinity towards TIME cells relative to the other Ang2-BD variants and to Ang2-BDwT. A competitive binding assay using Angl demonstrated that the Ang2-BD variants bind to Tie2 at the same epitope as does Angl (Fig. 8b).

Ang2-BD variants inhibit Tie2 phosphorylation of TIME cells

To test the ability of the Ang2-BD variants to inhibit Tie2 phosphorylation, we used a system exploiting the endogenous expression of basal levels of Angl, which induce phosphorylation of Tie2, in TIME cells. This phosphorylation is enhanced by the addition of soluble full length Angl to the cell culture (Yuan, 2009). The results obtained demonstrate that the Ang2-BD variants could significantly inhibit Tie2 phosphorylation induced by both endogenous and soluble Angl and thus act as functional antagonists. In particular, the engineered high affinity binder Ang2-BDc2.36 was found to be a more potent antagonist than Ang2-BD WT (Fig. 10).

Ang2-BD variants inhibit tube formation and invasiveness of endothelial cells

Ang2-BD variants were tested for their ability to inhibit capillary tube formation by

TIME cells grown on Matrigel, an extracellular basement membrane matrix. The Ang2-BD variants were able to inhibit tube formation in a dose-dependent manner, and Ang2-BDc2.36 was found to be a potent inhibitor in comparison to Ang2-BDwT (Figs. 11a- lib). Ang2-BD variants were tested for their ability to inhibit endothelial cells invasiveness. Ang2-BDc2.36 was able to inhibit invading cells in comparison to Ang2-BDwT (Figs. 12a- 12b). Discussion

The success story of protein-based therapeutics centers on monoclonal antibodies ( Carter, 2006; Weiner,2015) that exert their activity either through immune-related effector functions or by inhibiting dysregulated ligand-receptor interactions. In contrast, the

development of monoclonal antibodies that target Ang/Tie2 interactions has met with little success, and only a few human monoclonal antibodies, mainly 3.19.3 and MEDI3617, that target the angiopoietin ligand have entered early-stage clinical trials ( Brown, 2010; Leow, 2012). The results disclosed herein open the way to a more promising approach, namely, a combinatorial methodology for engineering natural ligands to function as alternatives to antibodies. We were able to show that this method is indeed an effective strategy for creating ligand-based RTK receptor inhibitors and molecular tools to study the effects of growth factor/RTK recognition on receptor activation and cell function.

To date, most of the successful attempts to engineer growth factor-based RTK antagonists have used rational methods that rely on natural ligand-receptor interactions ( Fuh, 1992; Boesen, 2002). Despite the relative simplicity and the successes of using rational engineering to develop the initial ligand-based antagonists, several difficulties have arisen during the development and use of these first-generation agents as therapeutics. For example, the monomeric form of a dimeric vascular endothelial growth factor-A (VEGFA) ligand showed a severely diminished (three orders of magnitude) binding affinity for its VEGFR2 receptor, as compared to that of natural dimeric ligand, VEGF (Fuh, 1998). In addition, the stability of a single domain ligand was significantly decreased when this domain was removed from the constraints of other stabilizing domains. Moreover, growth factor ligands and RTKs with clinical relevance are generally complex, multidomain mammalian proteins that suffer from low levels of recombinant expression ( Jenkins, 2009; Zhu, 2012).

In a similar vein, a previous approach for transforming a multimeric Angl agonist to an antagonist, having two amino acid mutations corresponding to residues critical for Angl multimerization at the supercluster domain (SCD) of the molecules (i.e., C41S and C54S), produced an Angl variant that could not multimerize and was unable to activate Tie2, although it did bind to Tie2 weakly ( Davis, 2003; Kim, 2005; Cho, 2004). In addition, this recombinant monomeric Angl variant exhibited low expression yields and low stability (Cho, 2004; Davis, 2003). To overcome these limitations, particularly to generate new Ang-based variants with improved expression yields, stabilities and high affinity to Tie2, we utilized combinatorial Ang2-BD libraries for generating protein diversity and YSD as a quantitative selection platform for enhancing expression, affinity and stability.

Sequencing of 70 and 60 clones selected from the final sorting rounds of the first and second generation Ang2-BD libraries yielded 5 and 14 unique sequences for each library, respectively. We found that two mutations, namely N467K and F469L, in the affinity matured clones were common to the clones selected from the two independent libraries. This result supports the idea that these mutations, which are located in the Ang2-BD/Tie2 binding interface, are indeed involved in the improved affinity between Ang2-BD and Tie2, through direct binding interactions. The fact that three mutations were observed for position 467 (N467Y, N467H and N467K) further suggests that this position stabilizes the Ang2-BD/Tie2 complex. Another common mutation (already identified from the first-generation screens), namely, I413T, was located very close to the Ang2-BD/Tie2 binding interface (Barton, 2006) and could therefore also contribute to the conformational stability of the binding site. None of the first generation Ang2-BDci.7o mutations either reverted to wild-type residues or was replaced with other mutations during the second-generation library screens, demonstrating the strength and effectiveness of the YSD system in affinity maturation.

The most commonly observed mutations fall into two classes: those located within the Ang2-BD/Tie2 binding interface (residues 421, 432, 434, 436, 467, 469, 470 and 475), all located within 5A from Tie2 (Barton, 2006), and those located along the Ang2-BD backbone (residues 302, 304, 324, 330, 343, 353, 359, 386, 389, 393, 402, 407, 413 and 415). The most marked changes in amino acid properties are evident in mutations K432N, N467K, N467H, N470D, Y475H and smaller changes in N421Y, I434T and N467Y. The above mutations are manifested as electrostatic changes (K432N, N467K, N467H, N470D, Y475H), changes in the hydrophobicity (N421Y and N467Y) or altered size (I434T).

Mutations K432N and I434T are clustered together in the first-generation clone Ang2- BDci.7o. These mutations might together contribute to binding. In the second-generation sorts, K436R was added to this cluster in clone Ang2-BDc2.i9- Importantly, a comparison of the second-generation sequences allowed us to identify unique mutations that independently enhance the binding of Ang2-BD to Tie2. These include N421Y, N467K, N467Y and F469L; all have an individual effect on binding affinity.

Stability analysis demonstrated an improvement for the first-generation variants that was lost during the second-generation affinity screens, as shown by the similar shapes of the melting curves of Ang2-BDc2.36 and Ang2-BDwT. At this stage it is not known whether further stability maturation is needed, that is, whether the Tm values of the Ang2-BD variants are sufficiently high for them to be suitable for diagnosis and therapy: a higher Tm could result in increased systemic retention time, which should improve the therapeutic effect and optimize dosing regimen.

The binding of the engineered second-generation Ang2-BD mutant Ang2-BDc2.36 to Tie2 was approximately twice as strong as that the first generation mutant Ang2-BDci.7o, and 7.5-fold stronger than that of the parental Ang2-BDwT. Glycosylation of Ang2-BDc2.36 did not have any influence on its affinity for Tie2, suggesting that the interaction between this variant and Tie2 is mediated solely by the amino acid residues of Ang2-BDc2.36.

Ang2-BDc2.36 bound more strongly than Ang2-BDwT to endothelial cells, with the binding being dose-dependent. This variant was able to compete with full-length Angl for binding to endothelial cells, demonstrating direct interaction with cellular Tie2. Not surprisingly, the ability of Ang2-BDwT and its Ang2-BDc2.36 variant to inhibit Tie2

phosphorylation in TIME cells was highly correlated with their affinity for recombinant soluble Tie2 and to cell-surface-expressed Tie2. As expected, both Ang2-BDwT and its Ang2-BDc2.36 variant inhibited Tie2 phosphorylation, with effect of the variant being stronger. A similar inhibitory trend (but a weaker effect) was observed in the presence of exogenous agonistic Angl, which served as a Tie2 inducer.

Having established that the affinity matured Ang2-BD variants do indeed antagonize

Tie2 in endothelial cells, we examined the effect of the Ang2-BD variants on the angiogenesis process in these cells. An in vitro endothelial tube formation assay examined the formation of capillary-like structures by the endothelial cells, when they were incubated with Ang2-BD on an extracellular basement membrane matrix. In a different assay, the Ang2-BD variants were tested for their ability to inhibit endothelial cells invasiveness. Here again, the ability of Ang2- BDWT and the affinity matured variant Ang2-BDc2.36 to inhibit the formation of capillary-like structures by endothelial cells and endothelial cells invasiveness was correlated with the affinity of the variants to Tie2.

The results from the phosphorylation and tube formation assays further define the roles of Angl and Ang2-BD as Tie2 agonists and antagonists, respectively. Accordingly, our approach provides new tools for studying the molecular mechanisms that mediate Ang- and Tie2-dependent angiogenesis. The significance of this study stems from its potential to provide insight into the sequence-structure-function relationships and mechanism of action of the antagonistic Ang mutants and it supports engineering of further improved Ang variants. Moreover, the approach of using a natural protein ligand as a molecular scaffold for engineering high affinity agents can be applied to other ligands and create functional protein antagonists against additional biomedical targets. Example 4: Methods - Isolation and Characterization of Ang2-BD bispecific proteins Preparation of YSD Ang2-BD constructs and RGD loop library

The construct for Ang2-BD W T (amino acids 281 to 496; SEQ ID NO: 2) was obtained by custom gene synthesis (Integrated DNA Technologies). Amplification of the gene was performed using primers containing Nhel and BamHI restriction sites. The amplified gene was then introduced into the pCTCON yeast display vector (a generous gift from the laboratory of Dane Wittrup, MIT). A loop library was constructed from an Ang2-BDwT loop. The loop library included the RGD integrin binding motif and randomized flanking residues on both sides of the motif. The library was prepared using the NNS degenerate codons where N = A, C, T or G and S = C or G. The loop library was constructed with a RGD sequence that is flanked by 3 random residues from each side of the RGD motif (GenScript) and homologous recombination into Saccharomyces cerevisiae EBYIOO cells, as previously described (Chao et al., 2006). The library size was approximately 10x 10 6 transformants, as estimated by dilution plating on selective SDCAA media (2% dextrose, 1.47% sodium citrate, 0.429% citric acid monohydrate, 0.67% yeast nitrogen base and 0.5% casamino acids, pH 4.5).

Screening of YSD Ang2-BDRGD libraries

According to known protocols, yeast-displaying Ang2-BDRGDloop libraries were grown in selective media and induced for expression with 2% w/v galactose at 30 °C overnight until an OD of 10 was achieved. (Chao et al., 2006). The library underwent five rounds of screening using high-throughput flow cytometry sorting which isolated clones with high affinity for recombinant α ν β 3 integrin (R&D Systems). The bispecific Ang2-BDRGD based library was subjected to 5 rounds of sorting using decreasing concentrations of α ν β 3 integrin (sort 1 was for positive expression and Tie2 binding (100 nM); sort 2 - 250nM α ν β 3 integrin; sort 3 - ΙΟΟηΜ α ν β 3 integrin; sort 4 - 30nM α ν β 3 integrin and sort 5 - ΙΟηΜ α ν β 3 integrin). A diagonal sorting gate, which included 1% of the entire yeast pull, was used to select Ang2-BD mutants that strongly bonded to α ν β 3 integrin, relative to their expression. In each round of sorting, approximately 10 times the library size of the yeast cells were labeled with a solubilized α ν β 3 integrin (R&D Systems) and a 1:200 dilution of chicken anti-cMyc antibody (Invitrogen) in an integrin binding buffer (IBB, 20 mM Tris pH 7.5, 100 mM NaCl, 1 mM MnC12, 2 mM CaC12 and 1% BSA) for lh at room temperature in order to facilitate fluorescent detection through flow cytometry. Cells were then washed and resuspended in ice-cold PBSA, which contained a 1:25 dilution of fluorescein isothiocyanate (FITC)-labeled mouse anti-a v integrin (Bio Legend) and a 1: 100 dilution of phycoerythrin (PE)-conjugated antichicken IgY (Santa Cruz

Biotechnology). After 25min on ice, yeast cells were washed in PBSA and sorted using iCyt Synergy FACS (fluorescence-activated cell sorting) [Proteomics Unit, National Institute for Biotechnology in the Negev (NIBN), Ben-Gurion University of the Negev (BGU)]. Sixty isolated clones from the two final sortings were sequenced by extraction of plasmid DNA from the yeast clones using a Zymoprep kit (Zymo Research) and transformed into electrocompetent Escherichia coli cells for plasmid miniprep (RBC Bioscience Corp, Taiwan) and DNA sequencing (DNA Microarray and Sequencing Unit, NIBN, BGU). These clones were evaluated for their binding affinity towards α ν β 3 integrin through dividing the mean fluorescent intensity (MFI) of the α ν β 3 integrin binding signal by the MFI expression level. The isolated clones were evaluated for their binding affinity towards Tie2-Fc (R&D Systems) through dividing the mean fluorescence intensity (MFI) of the Tie2 binding signal by the MFI expression level. The values obtained were normalized to Ang2-BDwT. Data shown is the average of triplicate experiments, and error bars represent standard error of the mean.

Specific integrin binding assay

A flow cytometry analysis of lx 10 6 cells of each of the 5 isolated clones of the RGD loop library was carried out. This was performed by using a 1:200 dilution of chicken anti- cMyc antibody (Invitrogen), solubilized 50 nM of

integrins (R&D Systems) and 20 nM of soluble Tie2-Fc (R&D Systems) simultaneously for lh at room temperature. Cells were washed and re-suspended in ice-cold PBSA containing a 1:25 dilution of fluorescein isothiocyanate (FITC)-labeled mouse integrin and 1:25 dilution of allophycocyanin (APC)-labeled mouse integrin (BioLegend) and a 1: 100 dilution of phycoerythrin (PE)-conjugated antichicken IgY (Santa Cruz Biotechnology). After 25min on ice, yeast cells were washed in PBSA and analyzed using BD Accuri C6 flow cytometer (BD Biosciences). These clones were evaluated for their binding affinity towards integrins integrin by dividing the mean fluorescent

intensity (MFI) of the α ν β 3 integrin binding signal by the MFI expression level. Data shown is the average of triplicate experiments, and error bars represent standard error of the mean. Soluble purification of Ang2-BD proteins

The Multi-Copy Pichia Expression Kit (Invitrogen K1750-01) was used to produce the soluble Ang2-BDRGD variants, as previously described (Shlamkovich et al., 2017). Ang2- BD RGD variants were purified from yeast culture supernatants by metal chelating

chromatography using a 5-ml HisTrap FF column (GE Healthcare) with 10 mM imidazole and then eluted by 500 mM imidazole. Eluted protein fractions were concentrated and buffer exchanged to 20 mM Hepes, 150 mM NaCl, pH 7.2 buffer using a 5-kDa cutoff Vivaspin ® concentrator (GE Healthcare). Gel filtration chromatography was performed using a Superdex 75 column (GE Healthcare) and were equilibrated with 20 mM Hepes, 150 mM NaCl, pH 7.2 buffer at a flow rate of 0.5 ml/min on an AKTA pure instrument (GE Healthcare). Proteins were analyzed by SDS-PAGE under non-reducing conditions. For all Ang2-BDRGD variants, protein concentrations were determined by UV-Vis absorbance at 280 nm and an extinction coefficient of 66,500 The molecular weights of the purified proteins were determined

by using a MALDI-TOF REFLEX-IV (Bruker) mass spectrometer (Use Katz Institute for Nanoscale Science & Technology, BGU).

Surface plasmon resonance experiments

The binding interactions of Tie2 to Ang2-BDwT, Ang2-BDBC5, Ang2-BDBC6 and Ang2- were analyzed as previously described (Shlamkovich et al., 2017) (Proteomics Unit, NIBN, BGU) in real-time by SPR using a ProteOn XPR36 instrument (Bio-Rad). The binding interactions of integrins to Ang2-BDBC5, Ang2-BDBC6 and Ang2-BDBcio were analyzed in a similar way using recombinant human

and α 3 βι integrins extracellular domain (R&D Systems). All integrins were immobilized on the surface of a GLC sensor chips (Bio-Rad) using a amine coupling reagents sulfo-NHS (0.1 M N-hydroxysuccinimide) and EDC (0.4 M l-ethyl-3-(3-dimethylaminopropyl) -carbodiimide, Bio-Rad). The intergrins (5.6 μg), in 10 mM sodium acetate pH

4.0, were flowed over the activated surfaces of the GLC sensor chip channel at a flow rate of 30 uL/min until the target immobilization level (4,300, 7,800, 3,400, 7, 100 and 4,200 RU respectively) was reached. BSA, (3 μg) in 10 mM sodium acetate pH 4.5, was then flowed over the activated surfaces of a control GLC sensor chip channel six at a flow rate of 30 μί/ιηίη until the target immobilization levels (3000 RU) was reached. After protein immobilization, chip surface was treated with 1 M ethanolamine-HCl at pH 8.5 in order to deactivate the excess of reactive esters. All binding experiments were performed at 25 °C in degassed integrin binding buffer (IBB, 20 mM Tris pH 7.5, 100 mM NaCl, 1 mM MnC12, 2 mM CaC12). No suitable regeneration conditions were found for the surface with immobilized α ν β 3 integrin, therefore, a separate channel was used to test the binding of each Ang2-BD protein. The range of concentrations used for α ν β 3 integrin binding interaction, was 12.5 nM to 200 nM of Ang2- BD variants and for was used. The protein analytes were flowed over the surface-immobilized integrins at a flow rate of 30 μΙ/min for 10 min and binding interactions were monitored. Following association, the dissociation of the various ligand-receptor complexes was monitored for 10 min. Each analyte sensogram run was normalized by subtracting the BSA channel (channel six) run and the zero analyte concentration run. Sensorgram binding data for all Ang2-BD variants were analyzed by equilibrium binding and a 1: 1 Langmuir model was used for binding kinetics evaluation.

Dual receptor binding experiments

A ProteOn GLC sensor chip was prepared, as described above, with an immobilized α ν β 3 integrin extracellular domain (R&D Systems). α ν β 3 integrin (5.6 ug) was in 10 mM sodium acetate, pH 4.0, until immobilization levels of 4,400 RU was obtained. The

experiments were performed at a temperature of 25° C in degassed integrin binding buffer (integrin binding buffer, 20 mM Tris pH 7.5, 100 mM NaCl, 1 mM MnC12, 2 mM CaC12). A single concentration of Ang2-BDwT, Ang2-BDBC5, Ang2-BDBC6 and Ang2-BDBcio (400 nM) was flowed over the integrin immobilized surface at a flow rate of 30 μL/min for 7 min.

Recombinant human Tie2 (rhTie2) extracellular domain (400 nM) was then flowed over the surface for nine minutes. The dissociation of the complex was monitored for ten minutes. The negative control used was the injection of running buffer followed by rhTie2 found in the integrin binding buffer. . Cell binding assays

The human telomerase-immortalized microvascular endothelium (TIME) cells (ATCC) were cultured in growth-factor-depleted Vascular Cell Basal Medium (ATCC) and were supplemented with 2% FBS and growth factor supplements (ATCC). For the binding assays, 10 5 cells were suspended in different concentrations of Ang2-BD variants in a total volume of 200 μΐ of PBS A (PBS and 0.1% BSA), followed by incubation at 4 °C for 2 hours with gentle agitation. Cell suspensions were centrifuged at 150 g at 4 °C for 5 minutes and washed in 100 μΐ of PBSA followed by centrifugation at 150 g at 4 °C for 5 minutes for two additional times. Cells were then re-suspended in 100 μΐ of PBSA containing a 1:200 dilution of

allophycocyanin (APC)-conjugated anti-FLAG antibody (Biolegend). After 20 minutes on ice, cells were washed twice in PBSA and analyzed by flow cytometry with a BD Accuri C6 flow cytometer (BD Biosciences). Mean fluorescence values were generated using Flow Jo software (Treestar). For a competitive binding assay, cells were treated as described above with the addition of Angl, cRGD peptide or the combination of both. Mean fluorescent intensity was detected using phycoerythrin (PE) -conjugated anti-FLAG antibody and then analyzed by flow cytometry with a BD Accuri C6 flow cytometer (BD Biosciences). For receptor level detection, lxlO 5 cells were harvested and re-suspended in 100 μΐ of PBSA with 1: 100 APC-labeled anti- human Tie2 antibody (Biolegend). They were then incubated at 4°C for 30 minutes and analyzed by flow cytometry. The data for the cellular assays was analyzed for column statistics with GraphPad Prism version 5.00 for Windows (La Jolla, CA, USA). Data shown is the average of triplicate experiments, and error bars represent standard error of the mean.

Statistical significance was determined by column statistics and t-test analysis. P value < 0.05 was considered statistically significant. Computational model of ανβ3 and Angiopoietin 2 complex

Molecular coordinates for the ανβ3 binding domains were taken from the 1L5G PDB structure (Xiong 2002) (residues 1-438 of the αv subunit and 55-432 of the β3 subunit).

Binding domain of Angiopoietin 2 coordinates were obtained from the 1Z3S PDB structure (Barton 2005) (residues 280-495). BC10 mutant of Angiopoietin 2 was created by replacing residues 301-308 of the native protein to the residues YPGRGDNPD (amino acids 22-30 of SEQ ID NO: 26) using PyMOL Molecular Graphics System, Version 1.8 Schrodinger, LLC. (De Lano). Each structure was minimized in energy using Gromacs 4.6.7 package of programs (Van Der Spoel et al.). Receptor-ligand docking procedure was performed by a PatchDock server (Schneidman-Dubovny et al.). To avoid irrelevant structures, potential binding sites both for the receptor and the ligand were defined, according to PatchDock recommendations. Slight variations in the interaction restraints yielded in five experiments for both of the WT and the mutant angiopoietin. Using Gromacs, the best docking solutions of all structures were clustered with 0.6 nm cutoff, based on the variations of the whole structure and not the protein backbone alone. Most abundant clusters were analyzed using Gromacs tools and the snapshots were prepared by VMD program (Humphrey et al.). The distances were measured by both g-mindist tool of Gromacs and in VMD. Cell adhesion assays

Inhibition of TIME cells adhesion to vitronectin was performed using human vitronectin coated, 96-well Microplates (R&D Systems). The peptides Ang2-BDwT, Ang2- BDBC5, Ang2-BDBC6, Ang2-BDBcio and cRGD (Merck Millipore), at a concentration of ΙμΜ, were mixed with 5x l0 4 TIME cells and subsequently plated on vitronectin-coated wells. They were then incubated at 37 °C/5% C0 2 for 2 hours and washed twice with PBS. A solution of 0.2% crystal violet in 10% ethanol was added to the wells for 10 min, and then washed out three times with PBS. A solubilization buffer (a 1 : 1 mixture of 0.1 M NaH2P04 and ethanol) was added and the plate was gently shaken for 15 minutes. Absorbance was measured at 600nm using a microtiter plate reader (BioTek Instruments), data was background subtracted with a negative control containing no cells. Data shown is the average of triplicate experiments, and error bars represent standard error of the mean. Statistical significance was determined by column statistics and t test analysis. P value < 0.05 was considered statistically significant. Matrigel endothelial cell tube formation assay

Serum-reduced Matrigel (10 mg/ml; BD Biosciences) was thawed overnight at 4 °C, and 150 μΐ was added to each well of a 48-well microtiter plate and left to solidify for 1 hour at 37 °C. To each well was added with 3.25x l0 4 TIME cells, which were incubated with 500 ng/ml rhAngl alone/or with 1 μΜ of Ang2-BDwT, Ang2-BDBC5, Ang2-BDBC6, Ang2-BDBcio, and cRGD peptide (Merck Millipore). Cells were incubated for 16-18 hours at 37 °C/5% C0 2 . Cells were then washed twice in HBSS (Hanks' balanced salt solution, Sigma), and capillary tube formation was observed using EVOS Cell Imaging Systems microscope (ThermoFisher Scientific). Images were taken with EVOS 2x Objective. The total number of tubal meshes and junctions were quantified by analysis of digitized images using ImageJ software and

Angiogenesis Analyzer plugin of the capillary-like structures. Tube formation assay data was analyzed with GraphPad Prism version 5.00 for Windows (La Jolla, CA, USA). Data shown is the average of triplicate experiments, and error bars represent standard error of the mean.

Statistical significance was determined by column statistics and t test analysis. P value < 0.05 was considered statistically significant, and t test analysis. P value < 0.05 was considered statistically significant.

Boyden Chamber-Invasion assay

An in-vitro Boyden chamber assay was performed using ThinCert™ 24 well inserts (Greiner Bio-One). ThinCert cell culture insert membranes were coated with Matrigel (Corning) diluted (1:30) in Vascular Cell Basal Medium (ATCC). The lower compartment was filled with 600 μΐ of Vascular Cell Basal Medium supplemented with 2% FBS, 2 10 4 of TIME cells, , with/without Angl, and Ang2-BD variants were incubated in 200 μΐ of supplement- free Vascular Cell Basal Medium. The treatments were added to the pre-coated ThinCert cell culture inserts and incubated for 20 hours at 37 °C with 5% C0 2 . Invasive cells were stained with DippKwik stain kit (American MasterTech Scientific) and were detected by EVOS FL Cell Imaging System at x20 magnification. Quantification was done by counting 16 fields of each membrane. Analysis of digitized images was performed using ImageJ software and Cell Colony Edge Analyser. Data was analyzed with GraphPad Prism version 5.00 for Windows (La Jolla, CA, USA). Data shown is the average of triplicate experiments, and error bars represent standard error of the mean. Statistical significance was determined by column statistics and t test analysis. P value < 0.05 was considered statistically significant.

Example 5 Development of bispecific integrin inhibitors

Construction and screening of bispecific Ang2-BD library that bind both Tie2 and

integrin

In order to develop a bispecific Ang-BD protein antagonist, we generated a library in which one of the Ang2-BD exposed loops was replaced with a RGD motif which is flanked by three random amino acids from each side. This was done with the purpose of generating a loop that would be able to bind α ν β 3 integrin without disrupting the binding of the resulting Ang2- BDRGD variant to its native receptor, Tie2 (Fig. 13A). The bispecific Ang2-BDRGD based library was subjected to five rounds of sorting using decreasing concentrations of α ν β 3 integrin (Figs. 14D-14F). Sorts 2-5 were performed using the gate shown in Fig. 14C. As expected, Ang2-BDwT did not bind to α ν β 3 integrin (Fig. 14B).

Isolation of bispecific clones that bind both Tie2 and integrin

Sequence analysis of the bispecific Ang2-BDRGD based library clones isolated from the fifth sort showed that the integrin binding loop contains the RGD motif flanked by random amino acids. There is no consensus sequence but proline appeared in all clones and the RGD motif was located in the middle of the loop sequence (Table 4):

The isolated yeast displayed variants that maintained their binding affinity towards Tie2 despite the introduction of the RGD epitope into Ang2-BD (Fig. 13B). The isolated clones were found to bind α ν β 3 integrin in contrast to Ang2-BDwT which did not bind to integrin at all (Fig. 13C). Since the RGD motif can bind different integrins other than integrin, the

yeast displayed bispecific variants were tested against other integrins. Given that some integrins have several biological functions which are not associated with cancer, several integrins were selected for this assay. Integrins α 5 βι and α ν β 5 are similar to the integrin in relation to their involvement in pathological angiogenesis. α 3 βι integrin promotes tumor cell adhesion, migration and invasion. integrin is involved in platelet aggregation and integrins function in leukocyte recruitment. It is important that the bispecific variant will not interfere with non-angiogenesis relevant integrins function. The binding results showed that the isolated clones bound weakly to other angiogenesis relevant integrins (α 5 βι and α ν β 5 ) and not at all to other integrin and all bound strongly to α ν β 3 integrin (Fig. 13C).

Purification and evaluation of soluble Ang2-BD bispecific proteins

Based on the results obtained from the YSD, three variants, namely Ang2-BDBC5, Ang2-BDBC6 and Ang2-BDBcio were chosen as the most suitable variants to be produced as soluble polypeptides (Figs. 15A-15B). Using Surface plasmon resonance (SPR), we were able to show that the bispecific variants retained their ability to bind the original target receptor Tie2 (Fig. 16A), however, their binding affinity towards Tie2 was decreased relative to Ang2- BDWT. These bispecific variants bind α ν β 3 integrin (Fig. 16B) while Ang2-BDwT does not. The K D values for Tie2 were found to be: Ang2-BD W T 0.66±0.04 μΜ, Ang2-BD B cs 1.36±0.68 μΜ, Ang2-BD Bee 1.26±0.18 μΜ and Ang2-BD BCIO 0.95±0.1 μΜ. The K D values for α ν β 3 integrin were found to be: Ang2-BD B cs 14.9±1.14 nM, Ang2-BD B c62.97±0.34 nM and Ang2-BD BCIO 4.17±0.54 nM. Binding of Ang2-BDwT to α ν β 3 integrin was not detected. Binding kinetic rate constants are summarized in Table 5:

Table 5: Kinetic Binding Rates

hese rate constants demonstrate that the engineered RGD loop grafted into Ang2-BD result in strong affinity towards α ν β 3 integrin. The high affinity towards α ν β 3 integrin also resulted in a reduction in Tie2 affinity to varying degrees. Nevertheless, the dual SPR binding experiment showed that all Ang2-BD bispecific variants were able to bind both Tie2 and α ν β 3 integrin simultaneously, demonstrating their great potential as bispecific agents (Fig. 17A) Yeast which displayed Ang2-BD bispecific variants were additionally tested for other integrin binding, and, the binding of the bispecific soluble Ang2-BD variants to other integrins was tested as well. The results demonstrated that the Ang2-BD bispecific variants can bind other integrins that are overexpressed in cancer and tumor vasculature such as α ν β 5 integrin and α 5 βι integrin, however, they do not bind to integrins (such as α 3 βι, ο¾β 3 and θ4β?) that are less highly- expressed in cancer and tumor vasculature (Fig. 16C). These results demonstrate the great potential of the engineered Ang2-BD variants to function as cancer specific agents with potentially reduced undesirable side effects often associated with anti-cancer drugs.

Example 6: In-Vitro characterization of bi-specific Ang2-BD

binding to Tie2 and integrins

Bi-specific Ang2BD variants bind and inhibit Tie! and integrin on TIME cell line

The binding capability of Ang2-BD bispecific variants to cells-expressing Tie2 and integrin was evaluated using the human telomerase-immortalized microvascular endothelium (TIME) cell line. These cells expressed both Tie2 and α ν β 3 integrin on their surface (Figs. 18A- 18B). Strong binding to TIME cells was observed in a dose-response manner (Fig. 17B). All of the Ang2-BD bispecific variants displayed high affinity to TIME cells in comparison to Ang2- BDW T . In order to evaluate if the Ang2-BD bispecific variants indeed bind to both Tie2 and α ν β 3 integrin a competitive binding assay was employed, in which Angl and cRGD peptide competed for the binding of Tie2 and α ν β 3 integrin respectively. When a competitive combination of both Angl and cRGD peptide was tested, significant decrease in binding of Ang2-BD bispecific variants was observed. These results demonstrate that Ang2-BD bispecific variants indeed bind to both targets (Fig. 17C). Ang2-BD bispecific variants inhibit integrin mediated adhesion of TIME cells

Cancer cells rely heavily on their adhesion properties for the continued growth. The effects of Ang2-BD variants on TIME cell adhesion was thus examined. Adhesion assay of TIME cells to vitronectin-coated plates demonstrated that the Ang2-BD bispecific variants can more strongly inhibit integrin-mediated adhesion via their RGD integrin-binding loops in comparison to Ang2-BDwT which was found to mildly inhibit adhesion. (Fig. 19). Ang2-BD bispecific variants inhibit capillary tube structure formation and invasiveness of endothelial cells

Endothelial cell invasion is a crucial step in angiogenesis. Because the invasiveness of cancer cells is influenced by integrins, Ang2-BD bispecific variants were evaluated though the Boyden chamber Matrigel invasion assay for their ability to inhibit capillary tube structure formation by endothelial TIME cells grown on Matrigel, an extracellular basement membrane matrix. Ang2-BD bispecific variants were tested for their ability to inhibit tube formation in the presence of Angl which is the natural agonist ligand of Tie2. The Ang2-BD bispecific variants inhibited tube formation in comparison to Ang2-BDwT and commercial cRGD peptide demonstrating their advantage in binding to both Tie2 and integrin over the mono- specific controls (Figs. 20A-20B). Ang2-BD bispecific variants were also tested for their ability to inhibit the invasiveness of endothelial TIME cells. The Ang2-BD bispecific variants were found to be potent inhibitors in comparison to Ang2-BDwT and commercial cRGD peptide (Figs. 21A-21B).

Example 7: Structural Modeling of Bispecific Peptides Computational model of ανβ3 and Angiopoietin 2 complex

A molecular docking procedure was independently performed for both WT Ang2 and the Ang2BDBcio mutant. Each angiopoietin structure was used to prepare a complex with the binding domains of ανβ3 integrin. Clustering was performed on the docking solutions and the most abundant structure (first cluster) was used for the proceeding analysis (Figs. 22A-22B).

In the produced mutant complex, a dominant structure was observed in 39% of all docking solutions. The second-most abundant cluster was present in 21% of all docking solutions. These percentages were significantly lower in the other modeled structures.

Likewise, modeling of the WT complex resulted in the most prominent structure in 34% of the solutions and a second-most prominent structure 28% of the time. Again other docking solutions were present at lower values.

As observed in the docking solutions, the binding interface for the Ang2BDBcio- vP3 complex is composed mainly Of the RGD motif and its flanking residues from Ang2 with both subunits of the integrin (Fig. 23A). As shown in the figure, the red-colored mutant residues protrude into the cleft between the av and the β3 domains. The 9-residue mutant loop takes on a sickle shape with the ends of both residues 301 and 309 touching β3 and the middle of the loop, and with 303 and 304 residues interacting with av. The WT residues (Fig. 23B), labeled red, take on a slightly different position in the space between the integrin subunits. While the mutant loop has an open sickle form and interacts with both av and β3 subunits, the WT residues have a denser structure and are closely associated with β3.

In view of the observed binding surfaces, it can be concluded that the interaction surface in the most abundant cluster is 1843 A 2 for the mutant, and 1622.8 A 2 for the WT ligand.

Interaction interface of the ανβ3 and Ang2BDBCio complex

To characterize the specific interactions between the receptor and the ligand, the distances between the interaction residues from the both sides was measured. Snapshots of the closest interactions are presented in Figures 24A-24C and the distances are summarized in Table 6: Table 6: Interaction residues between ανβ3 integrin and BC10 mutant of Angiopoietin 2

In the mutant loop of angiopoietin, the RGD sequence is flanked by YPG residues (numbered 301-303) on the one side and by NPD (numbered 307-309) on the other side. On the YPG side, Tyrosine 301 makes hydrogen bonds with β3 D251 and hydrophobic interaction through its aromatic ring with side chain carbon atom of β3 S123. Proline 302 is not close enough to the receptor residues, while glycine, which is located precisely in the cleft between the av and the β3 subunits (Fig. 23A) make interactions with Y178 of av (Fig. 24A). The RGD sequence, which belongs to the native integrin ligands, is also located between the two subunits. R304 contacts both av and β3, whether G and D interact with β3 subunit only (Fig. 24B). RGD-flanking residues 307-309 are located far away from the av subunit, also interact with β3 only. Asparagine 307 is buried inside the cleft comprised of Tyrl22, Arg214, Metl80 and S I 23, making hydrogen and polar bonds with them. Proline 308 interacts with the nearby Serl23, while Aspartic 309 is oriented towards the outer surface of the integrin (Fig. 23A), and makes a close-range salt bridge with K125. The differences in RGD orientation in comparison to the crystal structure are shown in Fig. 25. The D side chain location is almost identical to that of the crystal structure in our model. But, starting from Glycine, the orientation of the RGD fragment undergoes a change and bends towards the surface of the integrin. Thus, the Arginine side chain in the crystal is buried deep in the av structure, whereas in the docking model, R side chain points to the solution.

Discussion

Research into the discovery of Tie2 inhibitors is still an emerging field, in spite of the plethora of work done on related human receptor tyrosine kinases (RTKs). The development of specific Tie2 inhibitors has been challenging due to toxicity or off-target effects (Luke et al., 2009), and until now, the discovery of highly potent and selective Tie2 inhibitors has remained elusive. Our team has recently employed a combinatorial engineering approach which allowed us to transform the Ang2 binding domain (Ang2-BD) into a highly potent Tie2 inhibitor with enhanced anti angiogenic and anti-invasive cellular activity on endothelial cells. A potential risk with the therapeutic targeting of Tie2 (and of all other RTK multi-families) is that the latter do not work in isolation, but as part of complex enzymatic cascades, in which each RTK may cross-activate other molecules, such as integrins, and the latter may compensate for its loss of function. To circumvent this potential limitation, we present herein the development of Ang2- BD-based bispecific inhibitors that simultaneously target both Tie2 and its immediate in vivo targets, α 5 βι and α ν β 3 integrins.

Moreover, enhanced Tie2 expression was shown to be associated with increased proteolytic cleavage of cell-cell adhesion molecules, in particular α ν β 3 integrin (Thomas et al., 2010). Tie2-integrin association was significantly enhanced in the presence of the extracellular matrix component and the α 5 βι/ α ν β 3 integrin ligand fibronectin. It was also demonstrated that cooperative Tie2/integrin interactions selectively stimulate ERK/MAPK signaling on endothelial cells in the presence of both Angl and fibronectin, respectively (Dalton et al., 2016).

The structural modeling described herein indicates that the 301-308 mutant loop of the angiopoietin BC10 mutant obtains an open sickle form, with the Arginine of the RGD sequence located in the middle of this structure. This structure enables both ends of the loop to interact with β3 subunit, while the middle part is available to contact av. The observed mutant structure is in contrast to that of WT angiopoietin, which has an inferior fit to the ανβ3 interface, resulting in a reduced interaction surface and a different conformation of the 301- 308 loop.

As described, superposition with RGD-bound ανβ3 showed different orientation of the ligand in our model in comparison to the crystal structure. We believe that this discrepancy is due to the bulky angiopoietin that contains the RGD sequence in the docking model. There is not enough space on the ανβ3 interface to accommodate more of the ligand, so RGD could not be deeply inserted into the integrin structure. The main difference accounts for the spatial orientation of the R residue, which is pointing towards the integrin surface, rather than being buried inside the αv subunit. Still, a guanidium side chain group can create a salt bridge with the carboxylate of av D150, as in the crystal structure. In addition, R makes several other points of contact with both av and β3 residues. It has been shown that R adopts various conformations during a series of conformational changes that integrin undergoes upon

RGD binding (Zhu et al., 2013). The same work also proposed that D serves as a key factor in RGD binding to integrins, while the orientation of R could be disordered and flexible. Indeed, the interactions of D with β3 in our model are almost identical to the described in different crystal structures (Xiong, 2002; Zhu et al., 2013). In the RGD-bound integrin, D interacts with R214, which thus cannot contact D179, as in the ligand-free form (Xiong, 2002). In our model, the distance between amino side chain of R214 to either carboxylate of D179 or D of the RGD was equal (-3.5 A) and, thus, could serve as a basis for the competition between them. The G residue from the RGD sequence, makes a contact with β3 N215, just several angstroms away from its crystal structure position (Xiong, 2002). The interaction surface between ανβ3 and Ang2BDBcio is 1843 A 2 ; for cyclic RGD a 355 A 2 surface area was reported (Xiong, 2002). Binding of the RGD ligand induces the conformational change in the integrin by the induced-fit mechanism, in which the headpiece is opened and the RGD ligand binds further in and with a higher affinity (Zhu et al., 2013). We could propose that in the native state Angiopoietin's RGD fit to the ανβ3 interface would induce a further change in the receptor structure, resulting in a better fit in the receptor-ligand configuration. This could lead to the headpiece opening and result in integrin activation. Integrin headpiece opening, as a part of its activation mechanism, is due to RGD binding which has been demonstrated for all integrins tested to this point (Shi et al., 2011; Springer and Dustin, 2012; Yu et al., 2012).

The RGD flanking residues of the Ang2BDBcio mutant account for multiple interactions with the integrin. Because of the sickle structure of the mutant loop, only Ang2BDBcio 's middle fragment, e.g. G303 and R304, could contact the av subunit, as they are located in the bend or the knee of the sickle shape. Other flanking residues interact only with β3, contacting some of the MIDAS residues (D251, S 123 of β3) and with M180 from the ligand-specificity region. Several other residues, adjacent to the RGD binding site, are also involved in the interaction, such as Y178 of av, R214, R216, Y122 and K125 of β3. Just one flanking residue is located too far from the integrin subunits and was not found to make a contact, P302. The carboxylate of D309 is open to the bulk and could alternatively make a salt bridge with K125, stabilizing the structure, or in the native state contacting surrounding water or ions in a physiologic solution. The distance between the D309 and K125 charged groups is very close (2.7 A), thus classifying this interaction as a "stabilizing salt bridge", according to (Kumar and Nussinov, 2002).

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In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.