WO2019195313A1

WO2019195313A1 - Anti-vegf antagonist and pedf agonist constructs and uses thereof

Info

Publication number: WO2019195313A1
Application number: PCT/US2019/025418
Authority: WO
Inventors: Aryeh L. POLLACK
Original assignee: Pollack Aryeh L
Priority date: 2018-04-04
Filing date: 2019-04-02
Publication date: 2019-10-10

Abstract

Some embodiments comprise a bivalent and/or bispecific construct comprising a human IgG1 or human IgG4 subclass specific for human vascular endothelial growth factor (VEGF-A) and further comprising with a PEDF agonist. In some embodiments the bivalent and/or bispecific antibody comprises, consists essentially of, or consists of bevacizumab (AVASTIN product) or a binding fragment thereof.

Description

ANTI-VEGF ANTAGONIST AND PEDF AGONIST CONSTRUCTS AND USES

THEREOF

INCORPORATION BY REFERENCE TO ANY PRIORITY APPUICATIONS

[0001] Any and all applications for which a foreign or domestic priority claim is identified in the Application Data Sheet as filed with the present application are hereby incorporated by reference under 37 CFR 1.57. The present application claims the benefit of U.S. Provisional Application No. 62/652,472, filed April 4, 2018, which is hereby incorporated by reference in its entirety.

REFERENCE TO SEQUENCE FISTING

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled ARPOOOlWO.txt, created and last modified on April 1, 2019, which is 40,672 bytes in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.

Field

[0003] Some embodiments comprise a protein construct comprising a bivalent and/or bispecific construct comprising a human IgGl or human IgG4 subclass specific for human vascular endothelial growth factor (VEGF-A) and a pigment epithelium-derived factor (PEDF) polypeptide or antibody that binds specifically to PEDF receptor (PEDF R), such as a PEDF agonist. In some embodiments the bivalent and/or bispecific construct comprises, consists essentially of, or consists of bevacizumab (AVASTIN product), ranibizumab (LUCENTIS product), or aflibercept (EYLEA product), or a VEGF binding fragment of any of these. BACKGROUND

[0004] Angiogenesis, described by Folkman et al. (J. Biol. Chem. 267 (1992) 10931-10934), has been implicated in the pathogenesis of a variety of disorders which include solid tumors, retinal vascular diseases such as proliferative diabetic retinopathy, diabetic macular edema, ischemic retinopathy secondary to CRVO - central retinal vein occlusion, BRVO - branch retinal vein occlusion, macular edema secondary to CRVO and BRVO, and choroidal neovascular diseases such as wet age related macular degeneration - ARMD.

[0005] Vascular endothelial derived growth factor (VEGF) has been described as the major protein involved in the induction of angiogenesis, the response to ischemia or hypoxia common to all ischemic retinopathies. Without being limited by theory, the retina responds to hypoxia by generating signals to grow new pathologic blood vessels that leak, hemorrhage, and undergo a fibrovascular response resulting traction retinal detachment and macular edema in the ischemic retinopathies and submacular fibrosis in the ARMD.

SUMMARY

[0006] Some embodiments include protein constructs comprising, consisting essentially of, or consisting of a first domain such as an antibody or binding protein that binds specifically to VEGF, and a second domain that comprises a PEDF agonist fragment or an antibody that binds specifically to PEDF receptor (PEDFR) that is a PEDF agonist. Such a protein construct may be referred to herein as a“VEGF-PEDF construct.” The protein construct can be bispecific and/or bivalent. The antibody or binding protein that binds specifically to VEGF (for example, as part of a first domain as described herein) may also be referred to herein as“anti- VEGF.” The PEDF agonist fragment of the second domain can comprise, consist essentially of, or consist of a PEDF polypeptide, such as a 34 amino-acid fragment of PEDF as described herein (e.g., SEQ ID NO: 2 or SEQ ID NO: 3). The protein construct can comprise one or more PEDF polypeptides (such as PEDF agonist fragments), for example, 1, 2, 3, 4, 5, 6, or more PEDF polypeptides, including ranges between any two of the listed values, for example 1-6, 1-4, 2-4, or 2-6 PEDF polypeptides. The anti- VEGF antibody or binding protein can comprise, consist essentially of, or consist of a human IgGl or human IgG4 subclass specific for human VEGF-A. In some embodiments, the protein construct is a PEDF agonist and VEGF antagonist. In some embodiments, the protein construct or VEGF-PEDF construct comprises, consists essentially of, or consists of a bivalent construct (for example with a first domain that binds specifically to VEGF and a second domain that binds specifically to PEDF receptor). In some embodiments, the protein construct or VEGF-PEDF construct comprises, consists essentially of, or consists of a bivalent construct (for example comprising a first domain that comprises a first binding moiety that binds specifically to VEGF, and further comprising a second binding moiety that also binds specifically to VEGF), and further comprises a second domain comprising one or more PEDF polypeptides such as PEDF agonist fragments (for example, at least 1, 2, 3, 4, 5, 6, or more PEDF polypeptides), so that the protein construct thus has a third binding moiety that binds specifically to PEDF receptor. In some embodiments, the second domain comprises an antibody that binds specifically to PEDFR. The antibody can be a PEDF agonist. In some embodiments, the protein construct comprises an additional PEDF agonist at the Fc terminus of the anti- VEGF antibody, which represents a fourth binding moiety. In some embodiments, the protein construct or VEGF-PEDF construct comprises, consists essentially of, or consists of a bivalent construct (for example comprising a first domain comprising a first binding moiety that binds specifically to VEGF, and further comprising a second binding moiety that binds specifically to another target, such as a angiopoietin II or a T cell surface protein), and further comprises a second domain comprising one or more PEDF agonist fragments (for example, 1, 2, 3, 4, 5, 6, or more), so that the construct thus has a third binding moiety that binds specifically to PEDF. In some embodiments, the protein construct or VEGF-PEDF construct has a first binding specificity to VEGF, and a second binding specificity to PEDF receptor. In some embodiments, the protein construct or VEGF-PEDF construct has anti- angiogenic activity. In methods and uses of some embodiments, the protein construct or VEGF-PEDF construct as described herein can be useful for treating, ameliorating, reducing the risk of, delaying the onset of, or preventing solid cancers or tumors, and/or diseases of the eye associated with angiogenesis such as proliferative diabetic retinopathy, diabetic macular edema, ischemic retinopathy secondary to CRVO - central retinal vein occlusion, BRVO - branch retinal vein occlusion, macular edema secondary to CRVO and BRVO, and choroidal neovascular diseases such as wet age related macular degeneration - ARMD.

[0007] In some embodiments, the protein construct or VEGF-PEDF construct comprises, consists essentially of, or consists of first domain comprising a full-length anti- VEGF antibody and a second domain comprising a dimer of PEDF polypeptides (for example, PEDF agonist fragments such as 34 amino acid agonist fragments such as SEQ ID NOs: 2 or 3) as described herein. The second domain can be bound (e.g., fused or linked, for example via a peptide bond) to the Fc of the full length anti-VEGF antibody. The anti-VEGF antibody can bind specifically to VEGF-A. Accordingly, the first domain can bind specifically to VEGF-A. In some embodiments, the anti-VEGF antibody is monoclonal. In some embodiments, the anti-VEGF antibody is humanized or human. In some embodiments, the protein construct or VEGF-PEDF construct comprises, consists essentially of, or consists of two 34 amino acid PEDF agonist fragments as described herein (comprised by a second domain) bound to a Fab of bevacizumab (comprised by a first domain). In some embodiments, the protein construct or VEGF-PEDF construct comprises, consists essentially of, or consists of a bispecific antibody comprising at least one anti-VEGF binding moiety and that uses knob-hole formats (the bispecific antibody can be comprised by a first domain), and the protein construct further comprises one or more PEDF polypeptides (for example, 1, 2, 3, 4, 5, 6, or more), such as PEDF agonist fragments as described herein (the one or more PEDF polypeptides can be comprised by a second domain). In some embodiments, the protein construct or VEGF-PEDF construct comprises, consists essentially of, or consists of a bispecific construct (for example, comprising a first domain comprising one or more binding portions that bind specifically to VEGF and a second domain comprising one or more binding portions that bind specifically to PEDF, for example, 1, 2, 3,4, 5, 6, or more PEDF agonist fragments, or an antibody that binds specifically to PEDFR).

[0008] In protein constructs, compositions, methods, and uses of some embodiments, the PEDF agonist fragment of the second domain of the protein construct or VEGF-PEDF construct comprises, consists essentially of, or consists of SEQ ID NO: 1 (See FIG. 2A), or a PEDF agonist fragment thereof. In protein constructs, compositions, methods, and uses of some embodiments, the second domain of the PEDF agonist fragment of the VEGF-PEDF construct comprises, consists essentially of, or consists of the amino acids of Ser24-Ala57 of PEDF ( e.g ., SEQ ID NO: 2; See, e.g., FIG. 2B). In proteins, constructs, compositions, methods, and uses of some embodiments, the second domain of the PEDF agonist fragment of the VEGF-PEDF construct comprises, consists essentially of, or consists of the amino acids of Asp44-Asn77 of PEDF (e.g., SEQ ID NO: 3; See, e.g., FIG. 2C). In some embodiments, the first domain of the protein construct or VEGF-PEDF construct comprises the antibody, for example bevacizumab, ranibizumab, or a fragment thereof. For example, the first domain can comprise, consist essentially of, or consist of the variable regions of the antibody, such as the heavy chain variable region and light chain variable region of the antibody, or HCDRs 1-3 and LCDRs 1-3 of the antibody. In some embodiments, the first domain of the protein construct or VEGF-PEDF construct comprises the binding protein, for example aflibercerpt (e.g., SEQ ID NO: 14) or a VEGF-binding fragment thereof. For example, the first domain of the protein construct or VEGF-PEDF construct can comprise a binding protein comprising, consisting essentially of, or consisting of SEQ ID NO: 15. In protein constructs, compositions, methods, and uses of some embodiments, the PEDF polypeptide (e.g., PEDF agonist fragment) of the second domain is fused to the antibody or fragment of the first domain, for example as part of the same polypeptide as an antibody chain, and thus connected by a peptide bond. For example, the PEDF agonist fragment can be fused to a C-terminal portion of the heavy chain of the antibody, optionally with an intervening sequence such as a peptide linker. Optionally, the same nucleic acid can encode the first and second domains of a protein construct as described herein (e.g., an antibody polypeptide and one or more PEDF polypeptide agonist fragments). In protein constructs, compositions, methods, and uses of some embodiments, the PEDF agonist fragment is bound to the antibody via a covalent bond, for example via a disulfide bond to a cysteine of the antibody (such as a cysteine of the constant region), or via a linker such as a maleimide linkage.

[0009] In protein constructs, compositions, methods, and uses of some embodiments, the antibody of the first domain of the protein construct or VEGF-PEDF construct comprises, consists essentially of, or consists of full-length bevacizumab. In protein constructs, compositions, methods, and/or uses of some embodiments, the antibody comprises, consists essentially of, or consists of a binding fragment of bevacizumab, for example a Fab of bevacizumab or an ScFv of bevacizumab. In protein constructs, compositions, methods, and/or uses of some embodiments, the antibody of the protein construct or VEGF-PEDF construct comprises, consists essentially of, or consists of a heavy chain variable region of bevacizumab and a light chain variable region of bevacizumab. Example bevacizumab heavy chain variable regions and light chain variable regions and CDRs suitable for protein constructs, compositions, methods, and uses of some embodiments herein are shown in FIGs. 3A-3F. For example, a heavy chain variable region of bevacizumab can be a heavy chain variable region of SEQ ID NO: 6, and a light chain variable region of bevacizumab can be a light chain variable region of SEQ ID NO: 8. In proteins constructs, compositions, methods, and/or uses of some embodiments, the antibody of the VEGF-PEDF construct comprises heavy chain complementarity determining regions (HCDRs) and light chain complementarity determining regions (LCDRs) of bevacizumab. In protein constructs, compositions, methods, and/or uses of some embodiments, the antibody of the VEGF-PEDF construct comprises a HCDR1 that is an HCDR1 in a heavy chain variable region of bevacizumab, a HCDR2 that is an HCDR2 in a heavy chain variable region of bevacizumab, a HCDR3 that is an HCDR3 in a heavy chain variable region of bevacizumab, a LCDR1 that is a LCDR1 in a light chain variable region of bevacizumab, a LCDR2 that is a LCDR2 in a light chain variable region of bevacizumab, and a LCDR3 that is a LCDR3 in a light chain variable region of bevacizumab (by way of example, the heavy chain variable region can be a heavy chain variable region of SEQ ID NO: 6, and the light chain variable region can be a light chain variable region of SEQ ID NO: 8) (See, e.g., FIGs. 3A and 3C). The CDR’ s as described herein can be defined according to definitions known to one skilled in the art, for example the Rabat, Chothia, AbM, IMGT, and/or contact definitions.

[0010] In protein constructs, compositions, methods, and/or uses of some embodiments, the antibody of the first domain of the VEGF-PEDF construct comprises, consists essentially of, or consists of full-length ranibizumab (LUCENTIS product). In protein constructs, compositions, methods, and/or uses of some embodiments, the antibody comprises, consists essentially of, or consists of a binding fragment of ranibizumab, for example a Fab of ranibizumab or an ScFv of ranibizumab. In protein constructs, compositions, methods, and/or uses of some embodiments, the antibody comprises, consists essentially of, or consists of a heavy chain variable region of ranibizumab and a light chain variable region of ranibizumab. Example ranibizumab heavy chain variable regions and light chain variable regions and CDRs suitable for protein constructs, compositions, methods, and/or uses of some embodiments herein are shown in FIGs. 3G-3J. For example, a heavy chain variable region of ranibizumab can be a heavy chain variable region in SEQ ID NO: 11, and a light chain variable region of ranibizumab can be a light chain variable region in SEQ ID NO: 13. In some embodiments, the antibody comprises HCDRs and LCDRs of ranibizumab. In protein constructs, compositions, methods, and/or uses of some embodiments, the antibody comprises a HCDR1 that is an HCDR1 in a heavy chain variable region of ranibizumab, a HCDR2 that is an HCDR2 in a heavy chain variable region of ranibizumab, a HCDR3 that is an HCDR3 in a heavy chain variable region of ranibizumab, a LCDR1 that is a LCDR1 in a light chain variable region of ranibizumab, a LCDR2 that is a LCDR2 in a light chain variable region of ranibizumab, a LCDR3 that is a LCDR3 in a light chain variable region of ranibizumab (by way of example, the heavy chain variable region can be a heavy chain variable region of SEQ ID NO: 11, and the light chain variable region can be a light chain variable region of SEQ ID NO: 13) (See, e.g., FIGs. 3F and 3H). The CDR’s can be defined according to definitions known to one skilled in the art, for example the Rabat, Chothia, AbM, IMGT, and/or contact definitions.

[0011] Some embodiments include a protein construct or VEGF-PEDF construct comprising, consisting essentially of, or consisting of a bivalent and/or bispecific fusion IgG ScFV antibody comprising bevacizumab. The protein construct can include a first domain comprising the ScFv, which comprises a binding site that specifically binds human VEGF-A and a second domain comprising two additional sites, which are linked to the proximal end of the scFv, each site comprising a PEDF agonist fragment, such as a 34mer sequence of a PEDF agonist or an antibody that binds specifically to PEDFR as described herein.

[0012] Some embodiments include a protein construct comprising a first domain comprising an antibody or binding domain that binds specifically to human vascular endothelial growth factor (VEGF), and a second domain comprising a PEDF polypeptide that is a PEDF agonist fragment or an antibody that binds specifically to PEDF receptor (PEDFR) and is a PEDF agonist. In some embodiments, the first domain comprises (a) an antibody or fragment thereof comprising: a heavy chain variable region comprising a HCDR1 that is a HCDR1 in SEQ ID NO: 6, a HCDR2 that is a HCDR2 in SEQ ID NO: 6, and a HCDR3 that is a HCDR3 in SEQ ID NO: 6; and a light chain variable region comprising a LCDR1 that is a LCDR1 in SEQ ID NO: 8, a LCDR2 that is a LCDR2 in SEQ ID NO: 8, and a LCDR3 that is a LCDR3 in SEQ ID NO: 8; or (b) an antibody or fragment thereof comprising: a heavy chain variable region comprising a HCDR1 that is a HCDR1 in SEQ ID NO: 11, a HCDR2 that is a HCDR2 in SEQ ID NO: 11, and a HCDR3 that is a HCDR3 in SEQ ID NO: 11; and a light chain variable region comprising a LCDR1 that is a LCDR1 in SEQ ID NO: 13, a LCDR2 that is a LCDR2 in SEQ ID NO: 13, and a LCDR3 that is a LCDR3 in SEQ ID NO: 13; or (c) a binding domain comprising a polypeptide of SEQ ID NO: 15. In some embodiments, (a) the heavy chain variable region is a heavy chain variable region of SEQ ID NO: 6 and the light chain variable region is a light chain variable region of SEQ ID NO: 8; or (b) the heavy chain variable region is a heavy chain variable region of SEQ ID NO: 11 and the light chain variable region is a light chain variable region of SEQ ID NO: 13. In some embodiments, the first domain comprises a VEGFR fragment of SEQ ID NO: 14 that binds specifically to human VEGF. In some embodiments, the second domain comprises the PEDF polypeptide. In some embodiments, the second domain comprises the PEDF polypeptide, which comprises at least 20, 25, 30, 31, 32, 33, 34, or, 35 consecutive amino acids of SEQ ID NO: 1. In some embodiments, the second domain comprises the PEDF polypeptide, which comprises SEQ ID NO: 2 or SEQ ID NO: 3. In some embodiments, the second domain comprises the antibody that binds specifically to PEDFR. In some embodiments, the protein construct comprises (i) SEQ ID NO: 16 or 18; and (ii) SEQ ID NO: 20. In some embodiments, the first domain comprises a heavy chain variable region of SEQ ID NO: 16 or 18; and a light chain variable region of SEQ ID NO: 2. The second domain can comprise the PEDF polypeptide that is a PEDF polypeptide in SEQ ID NO: 16 or 18.

[0013] In some embodiments, a protein construct is described. The protein construct can comprise a first domain comprising: (a) a heavy chain variable region comprising a HCDR1 that is a HCDR1 in SEQ ID NO: 6, a HCDR2 that is a HCDR2 in SEQ ID NO: 6, and a HCDR3 that is a HCDR3 in SEQ ID NO: 6 and a light chain variable region comprising a LCDR1 that is a LCDR1 in SEQ ID NO: 8, a LCDR2 that is a LCDR2 in SEQ ID NO: 8, and a LCDR3 that is a LCDR3 in SEQ ID NO: 8; or (b) a heavy chain variable region comprising a HCDR1 that is a HCDR1 in SEQ ID NO: 11, a HCDR2 that is a HCDR2 in SEQ ID NO: 11, and a HCDR3 that is a HCDR3 in SEQ ID NO: 11; and a light chain variable region comprising a LCDR1 that is a LCDR1 in SEQ ID NO: 13, a LCDR2 that is a LCDR2 in SEQ ID NO: 13, and a LCDR3 that is a LCDR3 in SEQ ID NO: 13; or (c) a polypeptide of SEQ ID NO: 15; and a second domain comprising a PEDF polypeptide or an antibody that binds specifically to PEDFR. In some embodiments, (a) the heavy chain variable region is a heavy chain variable region of SEQ ID NO: 6 and the light chain variable region is a light chain variable region of SEQ ID NO:8; or (b) the heavy chain variable region is a heavy chain variable region of SEQ ID NO: 11 and the light chain variable region is a light chain variable region of SEQ ID NO: 13. In some embodiments, the first domain comprises a VEGF binding domain that is a VEGF binding domain of SEQ ID NO: 14. In some embodiments, the second domain comprises the PEDF polypeptide. In some embodiments, the second domain comprises the PEDF polypeptide, which comprises at least 20, 25, 30, 31, 32, 33, 34, or 35 consecutive amino acids of SEQ ID NO: 1. In some embodiments, the second domain comprises the PEDF polypeptide, which comprises SEQ ID NO: 2 or SEQ ID NO: 3. In some embodiments, the second domain comprises the antibody that binds specifically to PEDFR. In some embodiments, the protein construct comprises: (i) SEQ ID NO: 16 or 18; and (ii) SEQ ID NO: 20. In some embodiments, the first domain comprises a heavy chain variable region of SEQ ID NO: 16 or 18; and a light chain variable region of SEQ ID NO: 20. In some embodiments, the second domain comprises a PEDF polypeptide that that is a PEDF polypeptide of SEQ ID NO: 16 or 18. In some embodiment, the first domain binds specifically to human VEGF. In some embodiments, the second domain comprises the PEDF polypeptide, which is a PEDF agonist. In some embodiments, the second domain comprises the antibody that binds specifically to PEDFR, and the antibody that binds specifically to PEDFR is a PEDF agonist. In some embodiments, the first domain comprises an immunoglobulin. In some embodiments, the first domain comprises a construct selected from the group consisting of: a bispecific antibody, a Fab, a bispecific construct comprising a Knob in Hole, a bispecific construct comprising a duobody, a tetravalent construct, a tetravalent dual variable domain (DVD) construct, a tetravalent IgGScv construct, a tetravalent Mbatryn construct, and a composite antibody (e.g., comprising MAT as described herein). In some embodiments, the protein construct comprises, consists essentially of, or consists of an antibody, bispecific T cell engager (BiTE), nanobody, dual affinity retargeting antibody (DART), tandem antibody, triabody, and/or tetrabody. In some embodiments, the protein construct comprises, consists essentially of, or consists of an antibody, bispecific antibody, nanobody, tandem antibody, triomab, kih IgG (common light chain), crossmab, ortho-Fab IgG, DVD-Ig, 2 in l-Ig, IgG-scFv, scFv₂-Fc, bi-Nanobody, BiTE, tandAbs, DART, DART-Fc, scFv-HSA-scFv, DNF-Fab3, minibody, scFv-Fc, scFv- zipper, scFv, Fab, Fab₂ (bispecific), Fab₃ (trispecific), scFab, Bis-scFv (bispecific), sdAb (VH/VHH), tetrabody, triabody, diabody, diabody, camel Ig, IgNAR, IgG, bispecific construct comprising a Knob in Hole, bispecific construct comprising a duobody, a tetravalent multispecific antibody, tetravalent construct, tetravalent dual variable domain (DVD) construct, tetravalent IgGScv construct, tetravalent Mbatryn construct, and/or a composite antibody (e.g., comprising MAT as described herein), or a combination of two or more of any of the listed items. In some embodiments, a pharmaceutical composition comprises any of the protein constructs as described herein. In some embodiments, the pharmaceutical composition comprises the protein construct in a unit dose effective to inhibit angiogenesis. In some embodiments, the pharmaceutical composition comprises the protein construct in a unit dose effective to inhibit cancer cell growth or proliferation. In some embodiments, the pharmaceutical composition comprises the protein construct in a unit dose effective to induce proliferation of retinal pigment epithelial cells.

[0014] In some embodiments, a method of treating a patient suffering from ocular vascular disease is described. The method can comprise administering a protein construct as described herein to the patient. For example, the protein construct can comprise a first domain and a second domain as described herein. For example, the protein construct can be a protein construct of any of the preceding paragraphs. In some embodiments, the protein construct is administered in an amount effective to inhibit angiogenesis. In some embodiments, the protein construct is administered in an amount effective to inhibit cancer cell growth or proliferation. In some embodiments, the protein construct is administered to the patient in an amount effective to induce proliferation of retinal pigment epithelial cells.

[0015] In some embodiment a method of treating, ameliorating, reducing the risk of, delaying the onset of, or preventing a disease of the eye in a patient is described. The method can comprise administering a protein construct as described herein to the patient. For example, the protein construct can comprise a first domain and a second domain as described herein. For example, the protein construct can be a protein construct of any of the preceding paragraphs. In some embodiments, the protein construct is administered in an amount effective to inhibit angiogenesis. In some embodiments, the protein construct is administered in an amount effective to inhibit cancer cell growth or proliferation. In some embodiments, the protein construct is administered to the patient in an amount effective to induce proliferation of retinal pigment epithelial cells. In some embodiments, the disease of the eye is selected from the group consisting of: macular degeneration, diabetes, retinal vascular occlusion, proliferative diabetic retinopathy, diabetic macular edema, ischemic retinopathy secondary to CRVO - central retinal vein occlusion, BRVO - branch retinal vein occlusion, macular edema secondary to CRVO and BRVO, and/or choroidal neovascular diseases such as wet age related macular degeneration - ARMD, or a combination of two or more of any of the listed items.

[0016] In some embodiments, a method of treating, ameliorating, reducing the risk of, delaying the onset of, or preventing a solid cancer or solid tumor is described. The method can comprise administering a protein construct as described herein to the patient. For example, the protein construct can comprise a first domain and a second domain as described herein. For example, the protein construct can be a protein construct of any of the preceding paragraphs. In some embodiments, the protein construct is administered in an amount effective to inhibit angiogenesis. In some embodiments, the protein construct is administered in an amount effective to inhibit cancer cell growth or proliferation.

[0017] In some embodiments, a method of treating, ameliorating, reducing the risk of, delaying the onset of, or preventing a disease of the eye in a patient is described. The method can comprise administering to the patient a protein construct comprising (a) SEQ ID NO: 16 or 18; and (b) SEQ ID NO: 20. In some embodiments, the first domain comprises a heavy chain variable region of SEQ ID NO: 16 or 18; and a light chain variable region of SEQ ID NO: 20. The second domain can comprise the PEDF polypeptide of SEQ ID NO: 16 or 18. In some embodiments, the disease of the eye is selected from the group consisting of: macular degeneration, diabetes, retinal vascular occlusion, proliferative diabetic retinopathy, diabetic macular edema, ischemic retinopathy secondary to CRVO - central retinal vein occlusion, BRVO - branch retinal vein occlusion, macular edema secondary to CRVO and BRVO, and/or choroidal neovascular diseases such as wet age related macular degeneration - ARMD, or a combination of two or more of any of the listed items. In some embodiments, the protein construct is administered to the patient in an amount effective to induce proliferation of retinal pigment epithelial cells. In some embodiments, the protein construct is administered in an amount effective to inhibit angiogenesis. In some embodiments, the protein construct is administered in an amount effective to inhibit cancer cell growth or proliferation.

[0018] In some embodiments, for any of the methods described herein, the protein construct is administered intravitreally, subretinally, subconjuntivally, sub-Tenon’s, or topically to an eye of the patient. In some embodiments, for any of the methods described herein, the protein construct is administered intravitreally to an eye of the patient. In some embodiments, for any of the methods describe herein, the protein construct is administered subretinally to an eye of the patient. In some embodiments, for any of the methods described herein, the protein construct is administered subconjuntivally to an eye of the patient. In some embodiments, for any of the methods described herein, the protein construct is administered sub-Tenon’s or topically to an eye of the patient. In some embodiments, for any of the methods described herein, the protein construct is administered topically to an eye of the patient. In some embodiments, for any of the methods described herein, the protein construct is administered to the patient in no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 administrations.

[0019] Some embodiments include a nucleic acid encoding any of the protein constructs described herein. In some embodiments, the nucleic acid comprises two or more polynucleotide molecules in trans, that together encode the protein construct. In some embodiments, the nucleic acid that encodes the protein construct is a single polynucleotide molecule. In some embodiments, a host cell comprises the nucleic acid.

[0020] Some embodiments include a composition comprising a first nucleic acid comprising SEQ ID NO: 17 or SEQ ID NO: 19; and a second nucleic acid comprising SEQ ID NO: 21. In some embodiments, the first nucleic acid and the second nucleic acid are part of the same polynucleotide molecule. In some embodiments, the first nucleic acid and the second nucleic acid are separate polynucleotide molecules. In some embodiments, the first and second nucleic acid are comprised by a single vector. In some embodiments, the first and second nucleic acid are comprised by separate vectors.

[0021] In some embodiments, a method is described. The method can comprise administering a protein comprising a VEGF binding domain to a patient who has, or is at risk of having, a solid cancer or solid tumor, or a disease of the eye. The method can comprise administering a administering a PEDF polypeptide or an antibody that binds specifically to PEDFR to the patient. By way of example, the method can be for treating, ameliorating, reducing the risk of, delaying the onset of, or preventing a solid cancer or a tumor, or a disease of the eye in a patient having or at risk of the cancer or tumor, or the disease of the eye. In some embodiments, the patient has or is at risk of having the solid cancer or solid tumor, or wherein the patient has or is at risk of having the disease of the eye. In some embodiments, the cancer or solid tumor is treated, ameliorated, reduced in risk, delayed in onset, or prevented, or the disease of the eye is treated, ameliorated, reduced in risk, delayed in onset, or prevented. In some embodiments, the patient has or is at risk of having the solid cancer or solid tumor. In some embodiments, the cancer or solid tumor is treated, ameliorated, reduced in risk, delayed in onset, or prevented. In some embodiments, the patient has or is at risk of having the disease of the eye. In some embodiments, the disease of the eye is selected from the group consisting of proliferative diabetic retinopathy, diabetic macular edema, ischemic retinopathy secondary to CRVO - central retinal vein occlusion, BRVO - branch retinal vein occlusion, macular edema secondary to CRVO and BRVO, and choroidal neovascular diseases such as wet age related macular degeneration - ARMD. In some embodiments, the disease of the eye is treated, ameliorated, reduced in risk, delayed in onset, or prevented. In some embodiments, the protein comprising the VEGF binding domain comprises an antibody against VEGF or a VEGF binding fragment thereof. In some embodiments, the protein comprising the VEGF binding domain comprises bevacizumab, ranibizumab, or aflibercept, or a VEGF binding fragment thereof. In some embodiments, the protein comprising the VEGF binding domain comprises bevacizumab, ranibizumab, or aflibercept. In some embodiments, the PEDF polypeptide is administered. In some embodiments, the PEDF polypeptide is administered, and comprises, consists essentially of, or consists of at least 20, 25, 30, 31, 32, 33, 34, or, 35 consecutive amino acids of SEQ ID NO: 1. In some embodiments, the PEDF polypeptide is administered, and comprises, consists essentially of, or consists of SEQ ID NO: 1. In some embodiments, the PEDF polypeptide is administered, and comprises, consists essentially of, or consists of SEQ ID NO: 2 or SEQ ID NO: 3. In some embodiments, the PEDF polypeptide is administered, and is a PEDF agonist. In some embodiments, the protein comprising the VEGF binding domain and the PEDF polypeptide are part of a single molecule, for example a PEDF- VEGF construct as described herein. In some embodiments, the antibody that binds specifically to PEDFR is administered. In some embodiments, the antibody that binds specifically to PEDFR is administered, and is a PEDF agonist. In some embodiments, the protein comprising the VEGF binding domain and the PEDF polypeptide are administered, and are in a single molecule. In some embodiments, the protein comprising the VEGF binding domain and the PEDF polypeptide are administered, and are in separate molecules. In some embodiments, the protein comprising the VEGF binding domain and the antibody that binds specifically to PEDFR are administered, and are in a single molecule. In some embodiments, the protein comprising the VEGF binding domain and the antibody that binds specifically to PEDFR are administered, and are in separate molecules. In some embodiments, the protein comprising the VEGF binding domain and the PEDF polypeptide or antibody that binds specifically to PEDFR are administered at the same time. In some embodiments the protein comprising the VEGF binding domain and the PEDF polypeptide are administered at different times, for example separate and/or sequentially. In some embodiments, the PEDF polypeptide or antibody that binds specifically to PEDFR is administered to the patient in an amount effective to induce proliferation of retinal pigment epithelial cells. In some embodiments, the protein comprising the VEGF binding domain is administered to the patient in an amount effective to inhibit growth or proliferation of cancer cells. In some embodiments, the VEGF binding domain is administered intravitreally, subretinally, subconjuntivally, sub-Tenon’s, or topically to an eye of the patient. In some embodiments, the PEDF polypeptide or antibody that binds specifically to PEDFR is administered intravitreally, subretinally, subconjuntivally, sub-Tenon’s, or topically to an eye of the patient. In some embodiments, the protein comprising the VEGF binding domain and the PEDF polypeptide or antibody that binds specifically to PEDFR are administered via the same route of administration. In some embodiments, the protein comprising the VEGF binding domain and the PEDF polypeptide or antibody that binds specifically to PEDFR are administered via different routes of administration.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIGs. 1A-1BBB are a set of schematic diagrams illustrating some embodiments of protein constructs. FIG. 1A illustrates antibodies (e.g., bevacizumab) and PEDF polypeptide fragments such as a PEDF agonist fragment. The antibody can comprise a light chain (2) and a heavy chain (4). FIG. IB illustrates an example protein construct comprising a first domain comprising an antibody (e.g., bevacizumab) and a second domain comprising a PEDF polypeptide (6) comprising a PEDF agonist fragment (FIG. IB). FIGs. 1C-1BBB are schematic diagrams of formats of antibodies and fragments thereof that protein constructs of some embodiments can comprise, consist essentially of, or consist of.

[0023] FIGs. 2A-2F are diagrams of PEDF and PEDFR sequences according to some embodiments. For example, residues 24-57 of PEDF (SEQ ID NO: 2) are shown in FIG. 2B and residues 44-77 of PEDF (SEQ ID NO: 3) are shown in FIG. 2C. A sequence of PEDFR is shown in FIG. 2F.

[0024] FIGs. 3A-3J are diagrams of antibody and/or binding protein sequences of some embodiments. FIGs. 3A-3D are diagrams of bevacizumab sequences of some embodiments (SEQ ID NOs: 6-9). In FIGs. 3A and 3C the IMGT CDRs are underlined. FIGs. 3E-3H are diagrams of ranibizumab sequences of some embodiments (SEQ ID NOs: 10-13). In FIGs. 3F and 3H the IMGT CDRs are underlined FIGs. 31-3 J are diagrams of aflibercept sequences of some embodiments (SEQ ID NOs: 14-15). [0025] FIGs. 4A-4B are diagrams of sequences of bevacizumab heavy chain IgG- PEDF (24-57) constructs according to some embodiments.

[0026] FIGs. 5A-5B are diagrams of sequences of bevacizumab heavy chain IgG- PEDF (44-77) constructs according to some embodiments.

[0027] FIGs. 6A-6B - are diagrams of sequences of bevacizumab kappa light chain constructs according to some embodiments.

DETAILED DESCRIPTION

[0028] Described in some embodiments are protein constructs comprising a first domain that binds specifically to vascular endothelial growth factor A (VEGFA, which, for conciseness, may also be referred to herein as simply“VEGF” or“vascular endothelial growth factor”), and a second domain comprising a PEDF polypeptide or antibody that binds specifically to PEDFR that is a PEDF agonist. By way of the example, the first domain can comprise, consist essentially of, or consist of an antibody or fragment thereof, or a binding protein such as a vascular endothelial growth factor receptor (VEGFR) or fragment thereof. Without being limited by theory, the first domain that binds specifically to VEGF is contemplated to reduce, inhibit, or prevent angiogenesis, and the PEDF agonist is contemplated to reduce, inhibit, or prevent vascularization. Accordingly, the protein constructs of some embodiments are potent inhibitors of vascularization, for example in diseases of the eye associated with angiogenesis and/or in cancers. In some embodiments, the protein construct is a PEDF agonist and VEGF antagonist. In some embodiments, methods of treating, ameliorating, reducing the risk of, delaying the onset of, or preventing a disease of the eye or a cancer are contemplated. The methods can comprise administering an effective amount of the protein construct as described herein to reduce, inhibit, or prevent angiogenesis and/or vascularization. The methods can comprise administering an effective amount of the protein construct as described herein to reduce, inhibit, or prevent cancer cell growth or proliferation. The methods can comprise administering an effective amount of the protein construct as described herein to induce retinal cell proliferation.

[0029] Conventional pharmacotherapy can be targeted against vascular endothelial growth factor A known as VEGF - A (which, for conciseness, may also be referred to as herein as “VEGF”). Bevacizumab (AVASTIN product) is a humanized monoclonal antibody (IgGl) against human VEGF - A that selectively inhibits all isoforms. Bevacizumab has been utilized successfully in all forms of retina vascular and choroidal neovascular diseases (Folkman, J., et ah, J. Biol. Chem. 267 (1992) 10931-10934; Klagsbrun, M., et al., Annu Rev. Physiol. 53 (1991) 217-239; Demetrios G et al , Basic Mechanisms of Pathologic Retinal and Choroidal Angiogenesis, in Ryan’s Retina 6^th edition, Joussein, A et al. Mechanisms of Macular Edema and Therapeutic approaches in Ryan’s Retina 6^th ed).

[0030] PEDF - pigment epithelium derived growth factor also known as SERPIN-l as it is encoded by the SERPIN - 1 gene has been described as a natural antagonist to VEGF in that PEDF regulates the and inhibits VEGF signaling. A 34mer sequence of PEDF has been demonstrated to have a potent antiangiogenic effect (Amaral et al IOVS 51:3; 1318 - 26). PEDF sequences are available as UniProt accession P36955. Example PEDF sequences are shown in FIGs. 2A-E (SEQ ID NOs: 1-5). Conventionally, administration of full-length PEDF in rodent models has been observed to have tumor- causing effects, and has induced retinoblastoma tumors in rats. Moreover, conventional PEDF is a large molecule, and is contemplated to exhibit limited internalization into cells. It is contemplated herein that incorporating full-length PEDF into a larger fusion protein or construct is expected to yield a large fusion protein or construct with limited internalization. Accordingly, in some embodiments, a protein construct comprising a second domain comprising a PEDF polypeptide (that is less than full length) is internalized by cells, for example cells of a patient to whom to whom the protein construct is administered. Unexpectedly, it is contemplated herein that functional fragments of PEDF can retain anti vascularization functionality, and, especially in combination with anti- VEGF moieties as described herein, exhibit anti-angiogenic and/or anti-tumor properties rather than pro-tumor properties. Accordingly, it is contemplated herein that protein constructs or VEGF-PEDF constructs as described herein can be used to inhibit angiogenesis, and thus treat, ameliorate, delay the onset of, prevent, or reduce the risk of solid tumors or cancers, as well as diseases of the eye associated with angiogenesis such as proliferative diabetic retinopathy, diabetic macular edema, ischemic retinopathy secondary to CRVO - central retinal vein occlusion, BRVO - branch retinal vein occlusion, macular edema secondary to CRVO and BRVO, and choroidal neovascular diseases such as wet age related macular degeneration - ARMD. In some embodiments, the protein construct or VEGF-PEDF construct is internalized in cells of the eye.

[0031] There are several anti-VEGF drugs currently available including AVASTIN product (bevacizumab), LUCENTIS product (ranibizumab), and EYLEA product (aflibercept) that have demonstrated excellent anti- angiogenic efficacy. However the effect of any conventional anti - VEGF product in clinical use loses efficacy after 4- 8 weeks; hence, patients undergo multiple intravitreal injections, some in excess of 50 - 100. It is contemplated that constructs in accordance with embodiments herein are useful in combination synergistic pharmacologic therapy that will result in more definitive and potent inhibition of angiogenesis. In some embodiments, a protein construct as described herein is administered to a patient no more than 10 times to inhibit, prevent, ameliorate, treat, or reduce the severity of a disease of the eye or a cancer, for example, no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 times, including ranges between any two of the listed values.

First Domains (e.g., antibodies and/or binding proteins to VEGF )

[0032] In some embodiments, a protein construct as described herein comprises a first domain. The first domain of some embodiments comprises, consists essentially of, or consists of an antibody (e.g., an anti-VEGF antibody) or binding domain (e.g., a VEGF binding domain) or fragment thereof. By way of example, the antibody can comprise, consist essentially of, or consist of an antibody or fragment thereof that binds specifically to VEGF. By way of example, the binding domain can comprise, consist essentially of, or consist of VEGFR or a fragment thereof. In some embodiments, the first domain comprises, consists essentially of, or consists of an antigen binding protein that binds specifically to VEGF. In some embodiments, the first domain comprises, consists essentially of, or consists of a binding domain that binds specifically to VEGF. In some embodiments, the first domain comprises, consists essentially of, or consists of an antigen-binding portion of an antigen binding domain that binds specifically to VEGF.

[0033] First domains of protein constructs of some embodiments can comprise an antibody, or an antigen-binding fragment thereof. As used herein, "antibody" refers to full- length antibodies, and unless explicitly stated otherwise, also encompasses antigen-binding fragments thereof. Accordingly,“antibody,” as used herein, includes binding fragments of antibodies such as Fabs and ScFvs, as well as full-length antibodies. Antigen-binding fragments of antibodies can be formatted into a variety of formats in accordance with embodiments herein. Antigen-binding fragments of antibodies, or "antibody fragments" include a portion of an intact antibody comprising the antigen binding site or variable region of the intact antibody. Some antibody fragments are free of the constant heavy chain domains (i.e. Cm, CH3, and/or CH4, depending on antibody isotype) of the Fc region of the intact antibody, or a portion thereof. Examples of antibody fragments include, but are not limited to Fab, Fab', Fab'-SH, F(ab')₂, and Fv fragments such as a single-chain Fv (ScFv); minibodies; diabodies; any antibody fragment that is a polypeptide having a primary structure consisting of one uninterrupted sequence of contiguous amino acid residues (referred to herein as a "single-chain antibody fragment" or "single chain polypeptide"), including without limitation (1) single-chain Fv (scFv) molecules (2) single chain polypeptides containing only one light chain variable domain, or a fragment thereof that contains the three CDRs of the light chain variable domain, without an associated heavy chain moiety and (3) single chain polypeptides containing only one heavy chain variable region, or a fragment thereof containing the three CDRs of the heavy chain variable region, without an associated light chain moiety; and multispecific or multivalent structures formed from antibody fragments, for example bispecific antibodies. In some embodiments, the antibody is monoclonal. In some embodiments, the antibody is chimeric. In some embodiments, the antibody is selected from the group consisting of murine, rat, goat, guinea pig, donkey, and human. In some embodiments, the antibody is selected from the group consisting of murine, rat, goat, guinea pig, and donkey. In some embodiments, the antibody is humanized. In some embodiments, the antibody is human.

[0034] An immunoglobulin light or heavy chain variable region consists of a "framework" region ("FR," which may also be referred to herein as "FWR") interrupted by three hypervariable regions, also called complementarity-determining regions or CDRs. The CDRs are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus. From N-terminal to C-terminal, both light and heavy chains include domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. Domains of the heavy chain may be referred to herein as HFR1, HCDR1, HFR2, HCDR2, HFR3, HCDR3, and HFR4. Domains of the light chain may be referred to herein as LFR1, LCDR1, LFR2, LCDR2, LHFR3, LCDR3, and LFR4. The extent of the framework region and CDRs have been defined, for example according the definitions of Rabat {See, e.g., Rabat et al. (1987), "Sequences of Proteins of Immunological Interest," U.S. Department of Health and Human Services, which is incorporated by reference herein for all purposes), Chothia (Chothia et al., J. Mol. Biol. 196:901-917 (1987), which is incorporated by reference herein for all purposes), ImMunoGeneTics (IMGT) (Lefranc et al.,“IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains” Dev. Comp. Immunol., 27, 55-77 (2003); see also Giudicelli et ah, “IMGT/LIGM-DB, the IMG® comprehensive database of immunoglobulin and T cell receptor nucleotide sequences,” Nucl. Acids Res., 34, D781-D784 (2006), each of which is incorporated by reference herein for all purposes), the AbM definition (Martin et ah, Proc. Natl. Acad. Sci. USA, 86:9268-9272 (1989), which is incorporated by reference herein for all purposes), the contact definition (MacCallum et ah, J. Mol. Biol., 262:732-745 (1996), which is incorporated by reference herein for all purposes), and/or the automatic modeling and analysis tool (Honegger et ah, accessible on the world wide web at bioc dot uzh dot ch/antibody/Numbering/index dot html, which is incorporated by reference herein for all purposes). Accordingly, in some embodiments, the CDRs of the antibody or fragment thereof are defined according to the definition of Rabat, Chothia, IMGT, the AbM definition, the contact definition, and/or the automatic modeling and analysis tool. In some embodiments, the CDRs are defined according to any one of the Rabat, Chothia, AbM, IMGT, and/or contact definitions. The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three dimensional space. The CDRs are primarily responsible for binding to an epitope of an antigen.

[0035] Suitable antibodies that bind specifically to VEGF that can be used in first domains of protein constructs of some embodiments include, but are not limited to, bevacizumab, ranibizumab, and fragments thereof, such as Fab’s or scFv’s. Example sequences of bevacizumab are shown in FIGs. 3A-3D (e.g., SEQ ID NOs: 6-9). Example sequences of ranibizumab are shown in FIGs. 3E-3H (e.g., SEQ ID NOs: 10-13). Suitable antibodies (to VEGF and/or PEDFR, as described herein) can also be produced using a variety of different techniques. In some embodiments, a non-human host, for example a mouse, rat, guinea pig, rabbit, goat, sheep, donkey, horse, or camel is immunized with antigen such as VEGF (or PEDFR) as described herein. In some embodiments, the host organism comprises its endogenous immunoglobulin genes. In some embodiments, the host organism is genetically modified so as to comprise one or more immunoglobulin genes of a different organism, for example a human. Antibody-producing cells can be harvested from the host organism and isolated, for example flow cytometry, or fluorescence activated cell sorting (FACS™) technique. In some embodiments, the antibody-producing host cells are immortalized using hybridoma technology. In some embodiments, DNA sequences or RNA sequences are isolated from an antibody-producing cell, expressed as antibody polypeptides, and then screened for an antibody having desired binding characteristics to VEGF (or PEDFR). In some embodiments, nucleic acids encoding antibodies are identified using phage display technology. By way of example, nucleic acids can contain random mutations in antibody variable region coding sequences, and be screened, using phage display, for variant antibodies having the desired binding characteristics to VEGF (or PEDFR).

[0036] Suitable binding domains that bind specifically to VEGF that can be used in first domains of protein constructs of some embodiments include the VEGFR fragment of aflibercept that binds specifically to VEGF. Example sequences of aflibercept are shown in FIGs. 3I-3J (e.g., SEQ ID NOs: 14-15). In some embodiments, the first domain comprises a binding domain that comprises, consists essentially of, or consists of a VEGF binding domain in SEQ ID NO: 14 or 15. In some embodiments, the first domain comprises a binding domain that comprises, consists essentially of, or consists of a VEGFR fragment in SEQ ID NO: 14 or 15. The VEGFR fragment can bind to VEGF.

[0037] In some embodiments, the first domain comprises, consists essentially of, or consists of an antibody or fragment thereof comprising (a) a heavy chain variable region comprising a HCDR1 that is a HCDR1 in SEQ ID NO: 6, a HCDR2 that is a HCDR2 in SEQ ID NO: 6, and a HCDR3 that is a HCDR3 in SEQ ID NO: 6; and a light chain variable region comprising a LCDR1 that is a LCDR1 in SEQ ID NO: 8, a LCDR2 that is a LCDR2 in SEQ ID NO: 8, and a LCDR3 that is a LCDR3 in SEQ ID NO: 8; or (b) a heavy chain variable region comprising a HCDR1 that is a HCDR1 in SEQ ID NO: 11, a HCDR2 that is a HCDR2 in SEQ ID NO: 11, and a HCDR3 that is a HCDR3 in SEQ ID NO: 11; and a light chain variable region comprising a LCDR1 that is a LCDR1 in SEQ ID NO: 13, a LCDR2 that is a LCDR2 in SEQ ID NO: 13, and a LCDR3 that is a LCDR3 in SEQ ID NO: 13. In some embodiments, the first domain comprises, consists essentially of, or consists of a binding domain comprising (c) a polypeptide of SEQ ID NO: 15 (for example, a VEGF binding domain of SEQ ID NO: 15). In some embodiments, the first domain comprises, consists essentially of, or consists of a binding domain comprising a VEGFR fragment of SEQ ID NO: 14 that binds specifically to human VEGF. In some embodiments, the first domain comprises, consists essentially of, or consists of (a), (b), or (c). In some embodiments, the first domain comprises, consists essentially of, or consists of (a), (b), or a VEGFR fragment of SEQ ID NO: 14 that binds specifically to human VEGF. In some embodiments, the first domain comprises, consists essentially of, or consists of an antibody or fragment thereof comprising, consisting essentially of, or consisting of (a) or (b) binds specifically to VEGF. In some embodiments, the first domain comprises, consists essentially of, or consists of an antibody or fragment thereof comprising, consisting essentially of, or consisting of (a), (b), or (c) binds specifically to VEGF. In some embodiments, the first domain comprises, consists essentially of, or consists of an antibody or fragment thereof comprising, consisting essentially of, or consisting of (a) or (b), or a VEGFR fragment of SEQ ID NO: 14 that binds specifically to human VEGF binds specifically to VEGF. The CDR’s can be defined according to definitions known to one skilled in the art, for example the Rabat, Chothia, AbM, IMGT, and/or contact definitions.

Second Domains (e.g., PEDF polypeptides or antibodies that bind specifically to PEDFR)

[0038] In some embodiments, a protein construct as described herein comprises a second domain. The second domain can comprise, consist essentially of, or consist of a PEDF polypeptide, such as a PEDF agonist fragment, or can comprise, consist essentially of, or consist of an antibody that binds specifically to PEDF receptor (PEDFR). A“PEDF agonist” as used herein (including variations of this root term, for example“PEDF agonist fragment”) has its ordinary and customary meaning as would be understood by one of ordinary skill in the art in view of this disclosure. It refers to a protein (such as a polypeptide or antibody) that has detectable PEDF activity. PEDF activity can be confirmed in vitro or in vivo. For example, in some embodiments, PEDF activity can be detected as induction of differentiation of retinal precursor cells (which may also be referred to as primitive retinal cells) into cells of a neuronal phenotype. For example, in some embodiments, PEDF activity can be detected as binding to PEDFR in vivo or in vitro. For example, in some embodiments, PEDF activity can be detected as cleavage of VEGF receptor 1 (VEGFR-l) in vivo or in vitro. It has been observed that antibodies that bind specifically to receptors can act as agonists that induce signaling via that receptor, for example by binding to and stimulating the receptor (See, e.g., Vitetta, et al.,“Monoclonal antibodies as agonists: an expanded role for their use in cancer therapy,” Cancer Research 54: 5301-5309 (1994)), which is hereby incorporated by reference in its entirety. Accordingly, it is contemplated that antibodies that bind specifically to PEDFR can be suitable PEDF agonists in accordance with some embodiments herein.

[0039] In some embodiments, the second domain comprises, consists essentially of, or consists of a PEDF polypeptide. An example sequence of PEDF is shown as SEQ ID NO: 1. In some embodiments, the protein construct comprises a second domain comprising, consisting essentially of, or consisting of PEDF or a fragment thereof, for example a PEDF agonist fragment of PEDF. In some embodiments, the second domain comprises a PEDF polypeptide comprising at least 20 consecutive amino acid residues of SEQ ID NO: 1, for example at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, or 80 consecutive amino acid residues, including ranges between any two of the listed values, for example 20 - 34, 20 - 35, 20 - 40, 20 - 60, 20 - 80, 30 - 34, 30 - 35, 30 - 40, 30 - 60, 30 - 80, 34 - 35, 34 - 40, 34 - 60, 34 - 80, 40 - 60, 40 - 80, or 60 - 80 consecutive amino acid residues of SEQ ID NO: 1. In some embodiments, the second domain comprises a PEDF polypeptide comprising at least 20 consecutive amino acid residues of SEQ ID NO: 2 for example at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34 consecutive amino acid residues of SEQ ID NO: 2, including ranges between any two of the listed values, for example 20 - 30, 20 - 34, 25 - 30, 25 - 34, or 30 - 34 consecutive amino acid residues of SEQ ID NO: 2. In some embodiments, the second domain comprises a PEDF polypeptide comprising at least 20 consecutive amino acid residues of SEQ ID NO: 3 for example at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34 consecutive amino acid residues of SEQ ID NO: 3, including ranges between any two of the listed values, for example 20 - 30, 20 - 34, 25 - 30, 25 - 34, or 30 - 34 consecutive amino acid residues of SEQ ID NO: 3. In some embodiments, the second domain comprises, consists essentially of, or consists of a PEDF polypeptide of SEQ ID NO: 2 or SEQ ID NO: 3, or at least 20 consecutive amino acid residues of SEQ ID NO: 2 or SEQ ID NO: 3. In some embodiments, the second domain comprises, consists essentially of, or consists of SEQ ID NO: 2 or SEQ ID NO: 3. In some embodiments, the second domain has PEDF agonist activity. Accordingly, in some embodiments, the second domain comprises, consists essentially of, or consists of a PEDF agonist fragment. In some embodiments, the second domain comprises, consists essentially of, or consists of a PEDF agonist fragment comprising, consisting essentially of, or consisting of SEQ ID NO: 2 or SEQ ID NO: 3. In some embodiments, the second domain comprises, consists essentially of, or consists of a PEDF agonist fragment comprising, consisting essentially of, or consisting of SEQ ID NO: 2. In some embodiments, the second domain comprises, consists essentially of, or consists of a PEDF agonist fragment comprising, consisting essentially of, or consisting of SEQ ID NO: 3.

[0040] In some embodiments, the second domain comprises, consists essentially of, or consists of an antibody that binds specifically to PEDFR. PEDFR (also known as PNPLA2) is annotated as Uniprot ID Q96AD5. An example sequence of H. sapiens PEDFR is provided in FIG. 2F (SEQ ID NO: 22). In some embodiments, the antibody binds specifically to PEDFR and is a PEDF agonist. In some embodiments, the antibody that binds to PEDFR is a PEDF agonist that is effective to induce cleavage of VEGFR- 1 upon binding to PEDFR. Example antibodies that bind specifically to PEDFR have been described in Subramanian, et al. Adv. Exp. Med. Biol. 723: 799-805 (2012), which is hereby incorporated by reference in its entirety. Additional example antibodies that bind specifically to PEDFR are available commercially (See, e.g., Novus Biologicals catalog no. NB 110-41536; R&D Systems catalog no. RB01; and monoclonal antibody K.751.7, available as Thermofisher catalog no MA5- 14990). Additionally, antibodies that bind specifically to PEDFR can be generated against PEDFR protein or PEDFR peptides, for example, using methods as described herein, or according to the methods of Subramanian, et al.

Protein constructs (e.g., VEGF-PEDF constructs )

[0041] In some embodiments, protein constructs are described. The protein construct can comprise a first domain and a second domain as described herein. The protein construct may also be referred to herein as a“VEGF-PEDF construct,” including variations of this root term. In some embodiments, the protein construct is a PEDF agonist and VEGF antagonist.

[0042] A protein construct that“binds specifically” to a target such as VEGF or PEDF receptor has its ordinary and customary meaning as would be understood by one of ordinary skill in the art in view of this disclosure. It refers to a protein construct that binds preferentially to the target (e.g., VEGF or PEDFR) over other targets. For example, a protein construct that binds specifically to VEGF according to some embodiments can bind to VEGF with a micromolar, nanomolar, or subnanomolar- scale KD to VEGF. In some embodiments, a protein construct that binds specifically to VEGF binds to VEGF with a KD that is numerically less than 10 ⁶, 10 ⁷, 10 ⁸, 10 ⁹, 10 ¹⁰, or 10 ¹¹ M. In some embodiments, a protein construct that binds specifically to PEDFR binds to PEDFR with a KD that is numerically less than 10 ⁶, 10 ⁷, 10 ⁸, 10 ⁹, 10 ¹⁰, or 10 ¹¹ M. In some embodiments, a protein construct that binds specifically to VEGF and binds specifically PEDFR binds to each of VEGF and PEDFR with a respective KD that is numerically less than 10 ⁶, 10 ⁷, 10 ⁸, 10 ⁹, 10 ¹⁰, or 10 ^{1 1} M. KD can be determined, for example, using surface plasmon resonance (e.g., on a BIACORE apparatus). It will be appreciated, of course, that a protein construct with dual specificities (for example a bivalent or bispecific protein) can bind specifically to each of it targets, for example by binding preferentially to each of these targets compared to other substances. Thus, a protein construct comprising a first domain that binds specifically to (or is specific for) VEGF, and a second domain that binds specifically to (or is specific for) PEDFR can bind preferentially to VEGF and PEDFR compared to other substances that are neither VEGF nor PEDFR.

[0043] It is contemplated that a protein construct comprising a first domain that binds specifically to VEGF and second domain that is a PEDF agonist fragment can inhibit vascularization in accordance with some embodiments. In some embodiments, the protein construct comprises, consists essentially of, or consists of a first domain as described herein and a second domain as described herein. In some embodiments, the protein construct comprises at least 1, 2, 3, 4, 5, or 6 second domains as described herein, including ranges between any two of the listed values, for example 1-2, 1-4, 1-6, 2-4, 2-6, or 4-6 second domains as described herein.

[0044] For example, the first domain of the protein construct can comprise, consist essentially of, or consist of bevacizumab, ranibizumab, or a binding fragment thereof as described herein. For example, the first domain can comprise a heavy chain variable region comprising a HCDR1 that is a HCDR1 in SEQ ID NO: 6, a HCDR2 that is a HCDR2 in SEQ ID NO: 6, and a HCDR3 that is a HCDR3 in SEQ ID NO: 6; and a light chain variable region comprising a LCDR1 that is a LCDR1 in SEQ ID NO: 8, a LCDR2 that is a LCDR2 in SEQ ID NO: 8, and a LCDR3 that is a LCDR3 in SEQ ID NO: 8; or a heavy chain variable region comprising a HCDR1 that is a HCDR1 in SEQ ID NO: 11, a HCDR2 that is a HCDR2 in SEQ ID NO: 11, and a HCDR3 that is a HCDR3 in SEQ ID NO: 11; and a light chain variable region comprising a LCDR1 that is a LCDR1 in SEQ ID NO: 13, a LCDR2 that is a LCDR2 in SEQ ID NO: 13, and a LCDR3 that is a LCDR3 in SEQ ID NO: 13. For example, the first domain of the protein construct can comprise a heavy chain variable region comprising a heavy chain variable region of SEQ ID NO: 6 and a light chain variable region comprising a light chain variable region of SEQ ID NO: 8. For example, the first domain of the protein construct can comprise a heavy chain variable region comprising a heavy chain variable region of SEQ ID NO: 11 and a light chain variable region comprising a light chain variable region of SEQ ID NO: 13. For example, the second domain of the protein construct can comprise, consist essentially of, or consist of an antibody that binds specifically to PEDFR, or a PEDF polypeptide comprising at least 20 consecutive amino acid residues of SEQ ID NO: 1 as described herein, for example a PEDF polypeptide comprising, consisting essentially of, or consisting of SEQ ID NO: 2 or 3. In some embodiments, the second domain comprises at least 1, 2, 3, 4, 5, or 6 PEDF polypeptides as described herein, including ranges between any two of the listed values. [0045] Example protein construct sequences of some embodiments are shown in FIGs 5A (SEQ ID NO: 18), FIG. 5B (SEQ ID NO: 19), and FIG. 6A (SEQ ID NO: 20). In some embodiments, the protein construct comprises a polypeptide of SEQ ID NO: 18 or 19 and a polypeptide of SEQ ID NO: 20. The first domain can comprise a heavy chain variable region of SEQ ID NO: 18 or SEQ ID NO: 19 and a light chain variable region of SEQ ID NO: 20. The second domain can comprise a PEDF polypeptide of SEQ ID NO: 18 or 19. In some embodiments, the protein construct comprises, consists essentially of, or consists of a polypeptide of SEQ ID NO: 18 and a polypeptide of SEQ ID NO: 20. The first domain can comprise a heavy chain variable region of SEQ ID NO: 18 and a light chain variable region of SEQ ID NO: 20. The second domain can comprise a PEDF polypeptide of SEQ ID NO: 18. In some embodiments, the protein construct comprises, consists essentially of, or consists of a polypeptide of SEQ ID NO: 19 and a polypeptide of SEQ ID NO: 20. The first domain can comprise a heavy chain variable region of SEQ ID NO: 19 and a light chain variable region of SEQ ID NO: 20. The second domain can comprise a PEDF polypeptide of SEQ ID NO: 19. In some embodiments, the first domain of the protein construct binds specifically to VEGF. In some embodiments, the second domain of the protein construct is a PEDF agonist fragment. In some embodiments, the first domain of the protein construct binds specifically to VEGF and the second domain of the protein construct is a PEDF agonist fragment.

[0046] Protein constructs of some embodiments can comprise, consist essentially of, or consist of antibody formats, for example bivalent formats. In some embodiments, the protein construct comprises, consists essentially of, or consists of at least one of: a bispecific antibody comprising the first domain, a Fab comprising the first domain, a bispecific construct comprising the first domain and a Knob in Hole, a bispecific construct comprising the first domain and a duobody, a tetravalent construct comprising the first domain, a tetravalent dual variable domain (DVD) construct, a tetravalent IgGScv construct, a tetravalent Mbatryn construct, or a composite antibody. The antibody of some embodiments can be at least bispecific, having a first specificity to VEGF provided by the first domain, and a second specificity to PEDFR provided by the second domain. [0047] In some embodiments, the protein construct or VEGF-PEDF construct comprises, consists essentially of, or consists of i) a first antigen-binding site specifically binding to VEGF, such as a first domain as described herein (for example, the antigen binding site can comprise the heavy chain variable domain or HCDR3 region as described herein, for example a heavy chain variable domain or HCDR3 in SEQ ID NO: 6 or 11); ii) a second anti- VEGF site which can be the same as or different from i) (for example a heavy chain variable domain or HCDR3 region as described herein, or a heavy chain variable domain or HCDR3 in SEQ ID NO: 6 or 11); and iii) two human constant heavy chain regions (e.g., of human IgGl, IgG2, IgG3, or IgG4 subclass, such as human IgGl or IgG4 subclass) and two human light chain constant region (e.g., of kappa, lambda, or sigma subclass). In some embodiments, the protein construct or VEGF-PEDF construct comprises, consists essentially of, or consists of a bispecific antibody comprising a first antigen-binding site that specifically binds to human VEGF as described herein (for example a first domain as described herein) and a second antigen-binding site that specifically binds to human VEGF as described herein (for example a first domain as described herein, which can be the same as or different from the first domain of the first antigen-binding site). In some embodiments, the protein construct or VEGF-PEDF construct comprises, consists essentially of, or consists of a bispecific antibody comprising a first antigen-binding site that specifically binds to human VEGF as described herein (for example a first domain as described herein) and a second antigen-binding site that specifically binds to human PEDFR as described herein (for example a second domain as described herein). Any of the protein constructs described herein can further comprise one or more second domains as described herein, for example PEDF polypeptides or PEDF agonist fragments as described herein. For example, the protein construct of some embodiments comprises 1, 2, 3, 4, 5, or 6 second domains (e.g., PEDF polypeptides), including ranges between any two of the listed values, for example 1-6, 1-4, 2- 6, or 2-4 second domains as described herein.

[0048] In some embodiments, the VEGF-PEDF construct comprises, consists essentially of, or consists of a bispecific, bivalent antibody comprising two second domains as described herein (e.g., agonist PDEF 34mer sites), each site linked to distal end of the Fc fragment of antibody. [0049] Some embodiments comprise, consist essentially of, or consist of a a protein construct as described herein (e.g., bispecific antibody). The protein construct can be for medical use, for example use in the treatment of ocular vascular diseases. Some embodiments comprise, consist essentially of, or consist of a pharmaceutical composition comprising a protein construct as described herein (e.g., bispecific antibody). The pharmaceutical composition can be for use in the treatment of ocular vascular diseases. In some embodiments, a protein construct as described herein (e.g., comprising a bispecific antibody) can be for use in the manufacture of a medicament for the treatment of ocular vascular diseases. In some embodiments, a method of treating a patient suffering from ocular vascular diseases comprises administering a protein construct as described herein (e.g., comprising a bispecific antibody) to a patient in the need of such treatment. In some embodiments, the protein construct (e.g., comprising a bispecific antibody) or the pharmaceutical composition comprising the protein construct is administered via intravitreal application. In some embodiments, a protein construct as described herein is for medical use.

[0050] The protein construct as described herein (e.g., bispecific/bivalent VEGF- PEDF construct) can be injected subconjunctivally, Sub- Tenon’s, intravitreally, intravenously or surgically injected into the sub-retinal space.

[0051] The protein construct as described herein (e.g., bispecific/bivalent VEGF- PEDF construct) can be administered as well utilizing one or more lipophilic carriers.

[0052] Some embodiments utilizes nanotechnology so that any protein construct or VEGF-PEDF construct as described herein can be structured in a nanotechnologic shell (which may also be referred to as a“nanocage”) for sustained long term delivery. As such, it is contemplated that the VEGF-PEDF construct can be administered in a single administration (such as subretinal injection in an operating room) for long term delivery.

[0053] In methods of some embodiments, the protein construct (e.g., bispecific/bivalent molecule) is administered in topical eye drop form. In uses of some embodiments, the protein construct (e.g., bispecific/bivalent molecule) is for administration in topical eye drop form. In pharmaceutical compositions of some embodiments, the bispecific/bivalent molecule is formulated for in topical eye drop form. [0054] In some embodiments, the protein construct or VEGF-PEDF construct can be injected intravenously in manner utilized as chemotherapy for solid tumors.

[0055] In some embodiments, protein construct or VEGF-PEDF construct comprises a bevacizumab Fab, with PEDF agonists bivalently linked thereto.

[0056] In some embodiments, the protein construct or VEGF-PEDF construct comprises, consists of, or consists essentially of a bispecific antibody utilizing a knob-hole technique. For example, a binding valence with a first VEGF specificity and a binding valence with a second VEGF specificity can be associated via knob-hole technique. In some embodiments, a second domain comprising a PEDF agonist as described herein is associated with an antibody or fragment thereof specific to VEGF using knob-hole technology (for example, the PEDF can be fused to an antibody constant region or fragment thereof, which is associated with a constant region of the antibody or binding protein specific for VEGF).

[0057] Protein constructs of some embodiments comprise a Fab fragment of bevacizumab linked to a PEDF agonist fragment comprising, consisting essentially of, or consisting of a 34mer of human PEDF residues 44 - 77. Protein constructs of some embodiments comprise a Fab fragment of bevacizumab linked to a PEDF agonist fragment comprising, consisting essentially of, or consisting of a 34mer of human PEDF residues 24 - 57.

[0058] An example protein construct and DNA sequences thereof comprising a Fab bi-specific antibody of some embodiments are shown in FIGs. 4A-B and 5A-B.

[0059] Protein constructs or VEGF-PEDF constructs of protein constructs, compositions, methods, and uses of some embodiments comprise a bispecific form comprising, consisting essentially of, or consisting of a Triomab utilizing heteromeric heavy chains of Anti- VEGF and PEDF agonist fragment.

[0060] Protein constructs or VEGF-PEDF constructs of protein constructs, compositions, methods, and uses of some embodiments comprise a bispecific form in which a Knob in Hole is created utilizing heteromeric heavy chains of Anti- VEGF and PEDF agonist fragment.

[0061] Protein constructs or VEGF-PEDF constructs of protein constructs, compositions, methods, and uses of some embodiments comprise a bispecific form in which a Duobody is created utilizing heteromeric heavy chains of Anti- VEGF and PEDF agonist fragment.

[0062] Protein constructs or VEGF-PEDF constructs of protein constructs, compositions, methods, and uses of some embodiments comprise an Anti -VEGF and PEDF agonist fragment tetravalent multispecific antibodies comprising IgGs with other binding domains fused to either the N or C termini of either the heavy or light chains in a dual variable domain (DVD) with a non antibody binding scaffold centryn fused to the c terminal end of the heavy chains - also known as DVD.

[0063] Protein constructs or VEGF-PEDF constructs of protein constructs, compositions, methods, and uses of some embodiments comprise an anti -VEGF and PEDF agonist fragment tetravalent multispecific antibodies comprising an IgG with other binding domains fused to either the N or C termini of either the heavy or light chains in a dual variable domain with a non antibody binding scaffold centryn fused to the c terminal end of the heavy chains - also known as IgGScv - fusion.

[0064] Protein constructs or VEGF-PEDF constructs of protein constructs, compositions, methods, and uses of some embodiments comprise Anti -VEGF and PEDF agonist fragment tetravalent multispecific antibodies comprising of IgGs with other binding domains fused to either the N or C termini of either the heavy or light chains in a dual variable domain with a non antibody binding scaffold centryn fused to the c terminal end of the heavy chains - also known as Mbatryn.

[0065] Protein constructs or VEGF-PEDF constructs of protein constructs, compositions, methods, and uses of some embodiments comprise a composite antibody structure of Anti - VEGF and PEDF agonist fragment are constructed utilizing IgGs into which additional antigen combining sites are added within the structure such as 2- in - 1 antibodies, MAT - Modular antibody technology.

[0066] Protein constructs or VEGF-PEDF constructs of protein constructs, compositions, methods, and uses of some embodiments comprise engineered antibody fragments of Anti - VEGF and PEDF agonist fragments linked by short peptide linkers can be made into bivalent, trivalent or tetravalent formats addressing two to three targets e.g. bispecific T cell engager (BiTE), nanobody platforms, and DART - dual affinity retargeting antibodies, and tandem antibody structures. In some embodiments, the VEGF-PEDF constructs comprises, consists essentially of, or consists of a triabody or tetrabody. In some embodiments, the protein construct (which may also be referred to as a VEGF-PEDF construct) comprises, consists essentially of, or consists of an antibody, bispecific antibody, nanobody, tandem antibody, triomab, kih IgG (common light chain), crossmab, ortho-Fab IgG, DVD-Ig, 2 in l-Ig, IgG-scFv, scFv₂-Fc, bi-Nanobody, BiTE, tandAbs, DART, DART- Fc, scFv-HSA-scFv, DNF-Fab3, minibody (eg., scFv-CH3), scFv-Fc, scFv-zipper, scFv, Fab, Fab₂ (bispecific), Fab₃ trispecific, scFab, Bis-scFv (bispecific), sdAb (VH/VHH), tetrabody (e.g., tetravalent), triabody (e.g., trivalent), diabody, diabody (e.g., bispecific), camel Ig, IgNAR, IgG, bispecific construct comprising a Knob in Hole, a bispecific construct comprising a duobody, a tetravalent multispecific antibody, a tetravalent construct, a tetravalent dual variable domain (DVD) construct, a tetravalent IgGScv construct, a tetravalent Mbatryn construct, and a composite antibody (e.g., comprising MAT as described herein), including a combination of two or more of the listed items. Example schematic structures of formats described herein are depicted in Figs. 1A-1BBB. In some embodiments, the protein construct is multivalent and multispecific (for example, a bivalent construct that is bispecific for VEGF and PEDFR, or a bivalent construct that is bispecific with a first specificity for a first epitope on VEGF and a second specificity for a second epitope on VEGF). It will be appreciated herein that a domain that binds to a target, for example a PEDF polypeptide that binds to PEDFR as described herein, or a VEGFR fragment that binds to VEGF, may provide valence and/or specificity to a protein construct, even if it is not a traditional immunoglobulin variable domain. Of course, traditional immunoglobulin variable domains, such as those of antibodies that bind specifically to VEGF or PEDFR can also provide the valences and specificities of the noted constructs. In some embodiments, the protein construct comprises, consists essentially of, or consists of a non human immunoglobulin, for example murine or porcine (see, e.g., Figs. IGG and 1JJ). In some embodiments, the protein construct comprises, consists essentially of, or consists of a chimeric construct (for example, human x mouse chimeric, or pig x mouse chimeric; See, e.g., Fig. 1HH). In some embodiments, the protein construct comprises, consists essentially of, or consists of a reformatted construct, such as humanized or porcinized (See, e.g., Fig. 1JJ). In some embodiments, the protein construct comprises, consists essentially of, or consists of a non-human host immunoglobulin, chimeric construct, humanized construct, or human construct. Protein constructs or VEGF-PEDF constructs of protein constructs, compositions, methods, and uses of some embodiments comprise anti-VEGF and PEDF agonist fragments constructed utilizing chemically coupled IgGs.

[0067] Some embodiments comprise a pharmaceutical composition comprising the protein construct or VEGF-PEDF construct as described herein. In some embodiment, the pharmaceutical composition is formulated for topical administration, intravitral administration, subretinal administration, sub-Tenon’s administration, or subconjuntival administration. In some embodiments, the pharmaceutical composition is formulated for a single sub-retinal injection. In some embodiments, the pharmaceutical composition is formulated for repeated topical administration. In some embodiments, the pharmaceutical composition comprises the VEGF-PEDF construct in a nanocage for extended release as described herein.

Methods

[0068] Some embodiments include methods of using any of the protein constructs described herein. The method can include administering the protein construct to a patient in need thereof, for example a patient suffering from a disease of the eye, or a cancer. The method can treat, ameliorate, reduce the risk of, delay the onset of, or prevent the disease of the eye and/or the cancer in the patient. In some embodiments, the protein construct is administered in an amount effective to induce proliferation of retinal pigment epithelial cells. In some embodiments, the protein construct is administered in an amount effective to inhibit growth or proliferation of cancer cells. It is contemplated herein that the anti-VEGF and PEDFR agonist activities of protein constructs of some embodiments can synergize to inhibit vascularization and/or growth or proliferation of cancer cells. Accordingly, in some embodiments, few administrations of the protein construct are required compared to conventional pharmacotherapy (which can require 50 to 100 administrations of a pharmaceutical composition to the eye of a patient). In the method of some embodiments, the protein construct is administered to the patient in no more than 10, 9, 8, 7, 6, 5, 4, 3, or 2 administrations. In some embodiments, the patient (who may also be referred to herein as a “subject”) is a human. In some embodiments, the patient (who may also be referred to herein as a“subject”) is a non-human mammal.

[0069] Wherever an in vivo method comprising a protein construct or VEGF- PEDF construct is described herein, the corresponding a protein construct or VEGF-PEDF construct for use in that in vivo contemplated is also expressly contemplated, as protein construct or VEGF-PEDF be for use in the manufacture of a medicament for the treatment of the relevant disease. Thus, wherever a method of treating, ameliorating, reducing the risk of, delaying the onset of, or preventing a disease of the eye or a cancer comprising administering a protein construct is described herein, the corresponding protein construct for use in treating, ameliorating, reducing the risk of, delaying the onset of, or preventing the disease of the eye or the cancer is also expressly contemplated. The protein construct for use in the manufacture of a medicament for use in treating, ameliorating, reducing the risk of, delaying the onset of, or preventing the disease of the eye or the cancer is also expressly contemplated. In some embodiments, a protein construct as described herein is for medical use.

[0070] In the method of some embodiments, a protein construct or VEGF-PEDF construct is administered intravitreally, subretinally, subconjuntivally, sub-Tenon’s, or topically to the eye of a patient. In the method of some embodiments, the protein construct or VEGF-PEDF construct is administered topically to the eye of a patient.

[0071] In the method of some embodiments, the disease of the eye is selected from the group consisting of: macular degeneration, diabetes, retinal vascular occlusion, proliferative diabetic retinopathy, diabetic macular edema, ischemic retinopathy secondary to CRVO - central retinal vein occlusion, BRVO - branch retinal vein occlusion, macular edema secondary to CRVO and BRVO, and/or choroidal neovascular diseases such as wet age related macular degeneration - ARMD.

[0072] The method of some embodiments comprises using the protein construct or VEGF-PEDF construct as described herein for treating, ameliorating, reducing the risk of, delaying the onset of, or preventing a solid cancer or solid tumor. The protein construct or VEGF-PEDF construct can be administered to a subject having a solid cancer or tumor, for example topically, intravenously, subcutaneously, intratumorally, or peritumorally. In some embodiments, the protein construct or VEGF-PEDF construct has anti-angiogenic effects, and thus can treat, ameliorate, reduce the risk of, delay the onset of, or prevent the solid tumor or cancer. Some embodiments include a composition comprising the protein construct or VEGF-PEDF construct as described herein for use in treating, ameliorating, reducing the risk of, delaying the onset of, or preventing a solid cancer or solid tumor. The composition can be formulated for administration topically, intravenously, subcutaneously, intratumorally, and/or peritumorally.

[0073] The method of some embodiments comprises using a protein construct or VEGF-PEDF construct as described herein for treating, ameliorating, reducing the risk of, delaying the onset of, or preventing a disease of the eye associated with angiogenesis, such as macular degeneration, diabetes, retinal vascular occlusion, proliferative diabetic retinopathy, diabetic macular edema, ischemic retinopathy secondary to CRVO - central retinal vein occlusion, BRVO - branch retinal vein occlusion, macular edema secondary to CRVO and BRVO, and/or choroidal neovascular diseases such as wet age related macular degeneration - ARMD. The protein construct or VEGF-PEDF construct can be administered to a subject having a disease of the eye associated with angiogenesis, such as macular degeneration, diabetes, or retinal vascular occlusion. In some embodiments, the protein construct or VEGF-PEDF construct is administered intravitreally, subretinally, subconjuntivally, sub- Tenon’s, or topically to the eye. In some embodiments, the protein construct or VEGF-PEDF construct has anti- angiogenic effects, and thus can treat, ameliorate, reduce the risk of, delay the onset of, or prevent the disease of the eye associated with angiogenesis. Some embodiments include a composition comprising the protein construct or VEGF-PEDF construct as described herein for use in treating, ameliorating, reducing the risk of, delaying the onset of, or preventing a disease of the eye, as macular degeneration, diabetes, retinal vascular occlusion, proliferative diabetic retinopathy, diabetic macular edema, ischemic retinopathy secondary to CRVO - central retinal vein occlusion, BRVO - branch retinal vein occlusion, macular edema secondary to CRVO and BRVO, and/or choroidal neovascular diseases such as wet age related macular degeneration - ARMD. The composition can be formulated for administration intravitreally, subretinally, subconjuntivally, sub-Tenon’s, or topically to the eye. In some embodiments, the method or use further comprises the protein construct or VEGF-PEDF construct to generate new rod and/or cone cells. Without being limited by theory, it is contemplated that the PEDF agonist fragments as described herein can retain pro-proliferative activity for retinal pigment epithelial cells, and thus can induce the production of new rod and/or cone cells in the eye of a patient in need thereof.

[0074] In the method of some embodiments, a separate protein comprising a VEGF binding domain (for example, an anti-VEGF antibody such as bevacizumab, ranibizumab, or a VEGF binding protein such as aflibercept, or a VEGF binding fragment of any of these) and a PEDF polypeptide (for example a peptide comprising, consisting essentially of, or consisting of a PEDF polypeptide as described herein) or antibody that binds specifically for PEDFR are administered instead of a single protein construct (such as a VEGF-PEDF construct) described herein. Thus, it is contemplated that for any method describe herein comprising administering a single protein construct (such as a VEGF-PEDF construct), a protein comprising a VEGF binding domain and a separate PEDF polypeptide (or antibody that binds specifically to PEDFR) can be administered instead, for example concurrently (e.g., separately, or in a single composition), or at separate times. In some embodiments, the protein comprising the VEGF binding domain and the PEDF polypeptide (or antibody that binds specifically to PEDFR) are administered in a single composition. In some embodiments, the protein comprising the VEGF binding domain and the PEDF polypeptide (or antibody that binds specifically to PEDFR) are administered in separate compositions. In some embodiments, the protein comprising the VEGF binding domain and the PEDF polypeptide (or antibody that binds specifically to PEDFR) are administered concurrently. In some embodiments, the protein comprising the VEGF binding domain and the PEDF polypeptide (or antibody that binds specifically to PEDFR) are administered separately. In some embodiments, the protein comprising the VEGF binding domain and the PEDF polypeptide (or antibody that binds specifically to PEDFR) are administered via the same route of administration. In some embodiments, the protein comprising the VEGF binding domain and the PEDF polypeptide (or antibody that binds specifically to PEDFR) are administered via the different routes of administration. In some embodiments, the protein comprising the VEGF binding domain and the PEDF polypeptide (or antibody that binds specifically to PEDFR) are administered two or more times, for example two, three, four, five, or six times. In some embodiments, the protein comprising the VEGF binding domain and the PEDF polypeptide (or antibody that binds specifically to PEDFR) are administered no more than twenty times, for example no more than twenty, nineteen, eighteen, seventeen, sixteen, fifteen, fourteen, thirteen, twelve, eleven, ten, nine, eight, seven, six, five, four, three, two, or one times. In some embodiments, the protein comprising the VEGF binding domain and the PEDF polypeptide (or antibody that binds specifically to PEDFR) are administered the same number of times each. In some embodiments, the protein comprising the VEGF binding domain and the PEDF polypeptide (or antibody that binds specifically to PEDFR) are each administered a different number of times (for example, the VEGF binding domain can be administered three times, and the PEDF polypeptide or antibody that binds specifically to PEDFR can be administered twice). In some embodiments, the protein comprising the VEGF binding domain comprises an antibody against VEGF or a VEGF binding fragment thereof. In some embodiments, the protein comprising the VEGF binding domain comprises bevacizumab, ranibizumab, or aflibercept, or a VEGF binding fragment thereof. In some embodiments, the protein comprising the VEGF binding domain comprises bevacizumab, ranibizumab, or aflibercept. In some embodiments, the PEDF polypeptide is administered and comprises, consists essentially of, or consists of at least 20, 25, 30, 31, 32, 33, 34, or, 35 consecutive amino acids of SEQ ID NO: 1. In some embodiments, the PEDF polypeptide is administered and comprises, consists essentially of, or consists of SEQ ID NO: 1. In some embodiments, the PEDF polypeptide comprises, consists essentially of, or consists of SEQ ID NO: 2 or SEQ ID NO: 3. In some embodiments, the PEDF polypeptide is a PEDF agonist. In some embodiments, the antibody that binds specifically to PEDFR is administered. In some embodiments, the antibody that binds specifically to PEDFR is administered and is a PEDF agonist. In some embodiments, the PEDF polypeptide or antibody that binds specifically to PEDFR is administered to the patient in an amount effective to induce proliferation of retinal pigment epithelial cells. In some embodiments, the protein comprising the VEGF binding domain is administered to the patient in an amount effective to inhibit growth or proliferation of cancer cells. In some embodiments, the PEDF polypeptide or antibody that binds specifically to PEDFR is administered to the patient in an amount effective to induce proliferation of retinal pigment epithelial cells, and the protein comprising the VEGF binding domain is administered to the patient in an amount effective to inhibit growth or proliferation of cancer cells. In the method of some embodiments, a separate protein comprising a VEGF binding domain (for example, an anti-VEGF antibody such as bevacizumab, ranibizumab, or a VEGF binding protein such as aflibercept, or a VEGF binding fragment of any of these) and a PEDF agonist (for example a peptide comprising, consisting essentially of, or consisting of a PEDF polypeptide as described herein or antibody that binds specifically to PEDFR) are administered instead of a single protein construct (such as a VEGF-PEDF construct) described herein. For any method comprising in vivo use of a separate protein comprising a VEGF binding domain (for example, an anti-VEGF antibody such as bevacizumab, ranibizumab, or a VEGF binding protein such as aflibercept) and a PEDF polypeptide as described herein or antibody that binds specifically to PEDFR, a corresponding composition, or combination of compositions (for example, a first composition comprising the protein comprising a VEGF binding domain and a second composition comprising a PEDF polypeptide as described herein or antibody that binds specifically to PEDFR) for the in vivo use is also expressly contemplated. The use of the a protein comprising a VEGF binding domain and PEDF polypeptide as described herein or antibody that binds specifically to PEDFR for the manufacture of a medicament for treatment of the relevant disease (e.g.,. a cancer or a disease of the eye as described herein) is also expressly contemplated.

[0075] Some embodiments include methods of making a protein construct or VEGF-PEDF construct as described herein. In some embodiments, the protein construct or VEGF-PEDF construct is produced in a cellular expression system, a cell-free expression system, or is chemically synthesized. Exemplary systems for producing protein constructs as described herein include, but are not limited to, cellular expression system (e.g., mammalian cells, E. coli, insect cells, Saccharomyces, Pichid) transformed with recombinant expression vectors containing the nucleotide sequences encoding antibodies; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing sequences encoding antibodies; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV, or tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing nucleotide sequences encoding antibodies; mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses. Exemplary cell free systems include E. coli extracts and yeast extracts. The extracts can be lysates. The extracts can be purified, for example, to enrich for ribosomes and/or to remove undesired materials such as debris or host genomic DNA. Nucleic acids encoding antibodies in cell-free systems can include plasmid DNA, linear DNA, or RNA. In some embodiments, a host cell comprising a nucleic acid (e.g., a single nucleic acid or multiple nucleic acids) encoding the polypeptides of a protein construct or VEGF-PEDF construct as described herein can express the polypeptides. In some embodiments, the protein construct or VEGF-PEDF construct is produced in Chinese Hamster Ovary (CHO) or Baby Hamster Kidney (BHK) cells.

Example 1: Administration of a protein construct in vivo

[0076] A protein construct comprising a polypeptide of SEQ ID NO: 18 and a polypeptide of SEQ ID NO: 20 is provided. The protein construct comprises a first domain comprising the heavy chain variable region of SEQ ID NO: 18 and the light chain variable region of SEQ ID NO: 20. The first domain binds to VEGF. The protein construct comprises a second domain comprising the PEDF polypeptide in SEQ ID NO: 18. The PEDF polypeptide comprises PEDF residues 44-77. The protein construct is administered parenterally to a B16 mouse model of human melanoma. The protein construct inhibits vascularization of the melanoma.

Example 2: Administration of a protein construct to a subject having macular degeneration:

[0077] A protein construct comprising a polypeptide of SEQ ID NO: 16 and a polypeptide of SEQ ID NO: 20 is provided. The protein construct comprises a first domain comprising the heavy chain variable region of SEQ ID NO: 16 and the light chain variable region of SEQ ID NO: 20. The first domain binds to VEGF. The protein construct comprises a second domain comprising the PEDF polypeptide in SEQ ID NO: 16. The PEDF polypeptide comprises PEDF residues 24-57. The protein construct is administered by intravitreal injection to the eye of a subject suffering from macular degeneration. Following the administrations of the protein construct, vascularization in the eye of the subject is inhibited.

Example 3: Administration of a protein construct to a subject having branch retinal vein occlusion

[0078] A protein construct comprising a polypeptide of SEQ ID NO: 18 and a polypeptide of SEQ ID NO: 20 is provided. The protein construct comprises a first domain comprising the heavy chain variable region of SEQ ID NO: 18 and the light chain variable region of SEQ ID NO: 20. The first domain binds to VEGF. The protein construct comprises a second domain comprising the PEDF polypeptide in SEQ ID NO: 18. The PEDF polypeptide comprises PEDF residues 44-77. The protein construct is administered topically to the eye of a subject suffering from branch retinal vein occlusion. Following a single administration of the protein construct, vascularization in the eye of the subject is inhibited.

Example 4: Administration of a protein construct to a subject having breast cancer

[0079] A protein construct comprising a polypeptide of SEQ ID NO: 16 and a polypeptide of SEQ ID NO: 20 is provided. The protein construct comprises a first domain comprising the heavy chain variable region of SEQ ID NO: 16 and the light chain variable region of SEQ ID NO: 20. The first domain binds to VEGF. The protein construct comprises a second domain comprising the PEDF polypeptide in SEQ ID NO: 16. The PEDF polypeptide comprises PEDF residues 24-57. The protein construct is administered intratumorally to a tumor of a subject suffering from breast cancer. Following administration of the protein construct, vascularization of the tumor is inhibited.

Example 5: Co-administration of an antibody that binds specifically to VEGF and an antibody that binds specifically to PEDFR in vivo

[0080] A first antibody that binds specifically to VEGF, and a second antibody that binds specifically to PEDFR are administered parenterally to a B16 mouse model of human melanoma. The antibodies inhibits vascularization of the melanoma to a greater degree than either antibody on its own (as determined in control B16 mice that received only one of the two antibodies).

[0081] In at least some of the previously described embodiments, one or more elements used in an embodiment can interchangeably be used in another embodiment unless such a replacement is not technically feasible. It will be appreciated by those skilled in the art that various other omissions, additions and modifications may be made to the methods, compositions, kits, and uses described herein without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter, as defined by the appended claims.

[0082] With respect to the use of substantially any plural and/or singular terms herein, those having skill in the art can translate from the plural to the singular and/or from the singular to the plural as is appropriate to the context and/or application. The various singular/plural permutations may be expressly set forth herein for sake of clarity.

[0083] It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims ( e.g ., bodies of the appended claims) are generally intended as“open” terms (e.g., the term“including” should be interpreted as “including but not limited to,” the term“having” should be interpreted as“having at least,” the term“includes” should be interpreted as“includes but is not limited to,” etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases "at least one" and "one or more" to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles "a" or "an" limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases "one or more" or "at least one" and indefinite articles such as "a" or "an" (e.g.,“a” and/or“an” should be interpreted to mean“at least one” or“one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number ( e.g ., the bare recitation of "two recitations," without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to“at least one of A, B, and C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g.,“ a protein construct having at least one of A, B, and C” would include but not be limited to protein constructs that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). In those instances where a convention analogous to“at least one of A, B, or C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g.,“ a protein construct having at least one of A, B, or C” would include but not be limited to protein constructs that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase“A or B” will be understood to include the possibilities of“A” or “B” or“A and B.”

[0084] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0085] As will be understood by one of skill in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as“up to,”“at least,”“greater than,”“less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

[0086] Wherever a method comprising the use of a composition is disclosed (e.g., a method of using a protein construct or VEGF-PEDF construct as described herein) is disclosed herein, the corresponding composition (e.g., a pharmaceutical composition comprising the protein construct or VEGF-PEDF construct) or product (e.g. protein construct or VEGF-PEDF construct) for use is also expressly contemplated.

[0087] While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those of skill in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims.

Claims

WHAT IS CLAIMED IS:

1. A protein construct comprising:

a first domain comprising an antibody or binding domain that binds specifically to human vascular endothelial growth factor (VEGF); and

a second domain comprising:

a PEDF polypeptide that is a PEDF agonist fragment; or

an antibody that binds specifically to PEDF receptor (PEDFR) that is a PEDF agonist.

2. The protein construct of claim 1, the first domain comprising:

(a) an antibody or fragment thereof comprising: a heavy chain variable region comprising a HCDR1 that is a HCDR1 in SEQ ID NO: 6, a HCDR2 that is a HCDR2 in SEQ ID NO: 6, and a HCDR3 that is a HCDR3 in SEQ ID NO: 6; and a light chain variable region comprising a LCDR1 that is a LCDR1 in SEQ ID NO: 8, a LCDR2 that is a LCDR2 in SEQ ID NO: 8, and a LCDR3 that is a LCDR3 in SEQ ID NO: 8; or

(b) an antibody or fragment thereof comprising: a heavy chain variable region comprising a HCDR1 that is a HCDR1 in SEQ ID NO: 11, a HCDR2 that is a HCDR2 in SEQ ID NO: 11, and a HCDR3 that is a HCDR3 in SEQ ID NO: 11; and a light chain variable region comprising a LCDR1 that is a LCDR1 in SEQ ID NO: 13, a LCDR2 that is a LCDR2 in SEQ ID NO: 13, and a LCDR3 that is a LCDR3 in SEQ ID NO: 13; or

(c) a binding domain comprising a polypeptide of SEQ ID NO: 15.

3. The protein construct of claim 2, wherein:

(a) the heavy chain variable region is a heavy chain variable region of SEQ ID NO: 6 and the light chain variable region is a light chain variable region of SEQ ID NO:8; or (b) the heavy chain variable region is a heavy chain variable region of SEQ ID NO: 11 and the light chain variable region is a light chain variable region of SEQ ID NO:l3.

4. The protein construct of claim 1, wherein the first domain comprises a VEGFR fragment of SEQ ID NO: 14 that binds specifically to human VEGF.

5. The protein construct of any one of claims 1 to 4, wherein the second domain comprises the PEDF polypeptide.

6. The protein construct of any one of claims 1 to 4, wherein the second domain comprises the PEDF polypeptide, comprising at least 20, 25, 30, 31, 32, 33, 34, or, 35 consecutive amino acids of SEQ ID NO: 1.

7. The protein construct of any one of claims 1 to 4, wherein the second domain comprises the the PEDF polypeptide, comprising SEQ ID NO: 2 or SEQ ID NO: 3.

8. The protein construct of any one of claims 1 to 4, wherein the second domain comprises the antibody that binds specifically to PEDFR.

9. The protein construct of claim 1, wherein the protein construct comprises:

(i) SEQ ID NO: 16 or 18; and

(ii) SEQ ID NO: 20.

10. The protein construct of claim 9,

wherein the first domain comprises a heavy chain variable region of SEQ ID NO: 16 or 18; and a light chain variable region of SEQ ID NO: 2, and

wherein the second domain comprises the PEDF polypeptide of SEQ ID NO: 16 or 18.

11. A protein construct comprising:

a first domain comprising:

(a) a heavy chain variable region comprising a HCDR1 that is a HCDR1 in SEQ ID NO: 6, a HCDR2 that is a HCDR2 in SEQ ID NO: 6, and a HCDR3 that is a HCDR3 in SEQ ID NO: 6 and

a light chain variable region comprising a LCDR1 that is a LCDR1 in SEQ ID NO: 8, a LCDR2 that is a LCDR2 in SEQ ID NO: 8, and a LCDR3 that is a LCDR3 in SEQ ID NO: 8; or (b) a heavy chain variable region comprising a HCDR1 that is a HCDR1 in SEQ ID NO: 11, a HCDR2 that is a HCDR2 in SEQ ID NO: 11, and a HCDR3 that is a HCDR3 in SEQ ID NO: 11; and

a light chain variable region comprising a LCDR1 that is a LCDR1 in SEQ ID NO: 13, a LCDR2 that is a LCDR2 in SEQ ID NO: 13, and a LCDR3 that is a LCDR3 in SEQ ID NO: 13; or

(c) a polypeptide of SEQ ID NO: 15; and

a second domain comprising a PEDF polypeptide or an antibody that binds specifically to PEDFR.

12. The protein construct of claim 11, wherein:

(a) the heavy chain variable region is a heavy chain variable region of SEQ ID NO: 6 and the light chain variable region is a light chain variable region of SEQ ID NO:8; or

(b) the heavy chain variable region is a heavy chain variable region of SEQ ID NO: 11 and the light chain variable region is a light chain variable region of SEQ ID NO:l3.

13. The protein construct of claim 11, wherein the first domain comprises a VEGF binding domain that is a VEGF binding domain of SEQ ID NO: 14.

14. The protein construct of any one of claims 11 to 13, wherein the second domain comprises the PEDF polypeptide.

15. The protein construct of any one of claims 11 to 13, wherein the second domain comprises the PEDF polypeptide, comprising at least 20, 25, 30, 31, 32, 33, 34, or, 35 consecutive amino acids of SEQ ID NO: 1.

16. The protein construct of any one of claims 11 to 13, wherein the second domain comprises the PEDF polypeptide, comprising SEQ ID NO: 2 or SEQ ID NO: 3.

17. The protein construct of any one of claims 10 to 13, wherein the second domain comprises the antibody that binds specifically to PEDFR.

18. The protein construct of claim 11, wherein the protein construct comprises:

(i) SEQ ID NO: 16 or 18; and

(ii) SEQ ID NO: 20.

19. The protein construct of claim 18, wherein the first domain comprises a heavy chain variable region of SEQ ID NO: 16 or 18; and a light chain variable region of SEQ ID NO: 20, and

wherein the second domain comprises a PEDF polypeptide that that is of SEQ ID NO: 16 or 18.

20. The protein construct of any one of claims 10 to 19, wherein the first domain binds specifically to human VEGF.

21. The protein construct of any one of claims 10 to 16, wherein the second domain comprises the PEDF polypeptide, and wherein the PEDF polypeptide is a PEDF agonist.

22. The protein construct of any one of claims 10 to 17, wherein the second domain comprises the antibody that binds specifically to PEDFR, and wherein the antibody that binds specifically to PEDFR is a PEDF agonist.

23. The protein construct of any one of claims 1 to 22, wherein the protein construct comprises an immunoglobulin.

24. The protein construct of any one of claims 1 to 23, wherein the protein construct comprises a bispecific antibody comprising the first domain, a Fab comprising the first domain, a bispecific construct comprising the first domain and a Knob in Hole, a bispecific construct comprising the first domain and a duobody, a tetravalent construct comprising the first domain, a tetravalent dual variable domain (DVD) construct, a tetravalent IgGScv construct, a tetravalent Mbatryn construct, or a composite antibody.

25. The protein construct of any one of claims 1 to 23, wherein the protein construct comprises an antibody, bispecific T cell engager (BiTE), nanobody, dual affinity retargeting antibody (DART), tandem antibody, triabody, and/or tetrabody.

26. The protein construct of any one of claims 1 to 18, wherein the protein construct comprises an antibody, bispecific antibody, nanobody, tandem antibody, triomab, kih IgG (common light chain), crossmab, ortho-Fab IgG, DVD-Ig, 2 in l-Ig, IgG-scFv, scFv₂- Fc, bi-Nanobody, BiTE, tandAbs, DART, DART-Fc, scFv-HSA-scFv, DNL-Fab3, minibody, scFv-Fc, scFv-zipper, scFv, Fab, Fab₂ (bispecific), Fab₃ (trispecific), scFab, Bis-scFv (bispecific), sdAb (VH/VHH), tetrabody, triabody, diabody, diabody, camel Ig, IgNAR, IgG, bispecific construct comprising a Knob in Hole, a bispecific construct comprising a duobody, a tetravalent multispecific antibody, tetravalent construct, tetravalent dual variable domain (DVD) construct, tetravalent IgGScv construct, tetravalent Mbatryn construct, or a composite antibody, or a combination of two or more of any of the listed items.

27. A method of treating a patient suffering from ocular vascular disease, the method comprising administering the protein construct of any one of claims 1 to 26 to the patient.

28. A method of treating, ameliorating, reducing the risk of, delaying the onset of, or preventing a disease of the eye in a patient, the method comprising administering the protein construct of any one of claims 1 to 26 to the patient.

29. The method of claim 28, wherein the disease of the eye is selected from the group consisting of: macular degeneration, diabetes, retinal vascular occlusion, proliferative diabetic retinopathy, diabetic macular edema, ischemic retinopathy secondary to CRVO - central retinal vein occlusion, BRVO - branch retinal vein occlusion, macular edema secondary to CRVO and BRVO, and/or choroidal neovascular diseases such as wet age related macular degeneration - ARMD.

30. A method of treating, ameliorating, reducing the risk of, delaying the onset of, or preventing a solid cancer or solid tumor, the method comprising administering the protein construct of any one of claims 1 to 26 to the patient.

31. A method of treating, ameliorating, reducing the risk of, delaying the onset of, or preventing a disease of the eye in a patient, the method comprising administering to the patient a protein construct comprising:

(a) SEQ ID NO: 16 or 18; and

(b) SEQ ID NO: 20.

32. The method of claim 31, wherein the first domain comprises a heavy chain variable region of SEQ ID NO: 16 or 18; and a light chain variable region of SEQ ID NO: 20, and

wherein the second domain comprises the PEDF polypeptide of SEQ ID NO: 16 or

18.

33. The method of claim 31 or 32, wherein the disease of the eye is selected from the group consisting of: macular degeneration, diabetes, retinal vascular occlusion, proliferative diabetic retinopathy, diabetic macular edema, ischemic retinopathy secondary to CRVO - central retinal vein occlusion, BRVO - branch retinal vein occlusion, macular edema secondary to CRVO and BRVO, and/or choroidal neovascular diseases such as wet age related macular degeneration - ARMD.

34. The method of any one of claim 27 to 33, wherein the protein construct is administered to the patient in an amount effective to induce proliferation of retinal pigment epithelial cells.

35. The method of any one of claim 27 to 34, wherein the protein construct is administered to the patient in an amount effective to induce proliferation of retinal pigment epithelial cells.

36. The method of any one of claims 27 to 35, wherein the protein construct is administered intravitreally, subretinally, subconjuntivally, sub-Tenon’s, or topically to an eye of the patient.

37. The method of any one of claims 27 to 35, wherein the protein construct is administered intravitreally to an eye of the patient.

38. The method of any one of claims 37 to 35, wherein the protein construct is administered subretinally to an eye of the patient.

39. The method of any one of claims 27 to 35, wherein the protein construct is administered subconjuntivally to an eye of the patient.

40. The method of any one of claims 27 to 35, wherein the protein construct is administered sub-Tenon’s, or topically to an eye of the patient.

41. The method of any one of claims 27 to 35, wherein the protein construct is administered topically to an eye of the patient.

42. The method of any one of claims 27-41, wherein the protein construct is administered to the patient in no more than 10, 9, 8, 7, 6, 5, 4, 3, or 2 administrations.

43. A nucleic acid encoding the protein construct of any one of claims 1 to 26.

44. The nucleic acid of claim 43, comprising two or more polynucleotides in trans, that together encode the protein construct of any one of claims 1 to 21.

45. The nucleic acid of claim 43, wherein the nucleic acid that encodes the protein construct is a single polynucleotide molecule.

46. A composition comprising:

a first nucleic acid comprising SEQ ID NO: 17 or SEQ ID NO: 19; and a second nucleic acid comprising SEQ ID NO: 21.

47. The composition of claim 46, wherein:

(a) the first and second nucleic acid are comprised by a single vector; or

(b) the first and second nucleic acid are comprised by separate vectors.

48. A method, comprising:

administering a protein comprising a VEGF binding domain to a patient who has or is at risk of having a solid cancer or solid tumor, or a disease of the eye; and

administering a PEDF polypeptide or an antibody that binds specifically to PEDFR to the patient.

49. The method of claim 48, wherein the patient has or is at risk of having the solid cancer or solid tumor, or wherein the patient has or is at risk of having the disease of the eye.

50. The method of claim 49, wherein the cancer or solid tumor is treated, ameliorated, reduced in risk of, delayed in onset, or prevented.

51. The method of any one of claims 48-50, wherein the patient has or is at risk of having the disease of the eye.

52. The method of claim 51, wherein the disease of the eye is selected from the group consisting of proliferative diabetic retinopathy, diabetic macular edema, ischemic retinopathy secondary to CRVO - central retinal vein occlusion, BRVO - branch retinal vein occlusion, macular edema secondary to CRVO and BRVO, and choroidal neovascular diseases such as wet age related macular degeneration - ARMD.

53. The method of any one of claims claim 51-52, wherein the disease of the eye is treated, ameliorated, reduced in risk of, delayed in onset, or prevented.

54. The method of any one of claims 48-52, wherein the protein comprising the VEGF binding domain comprises an antibody against VEGF or a VEGF binding fragment thereof.

55. The method of any one of claims 48-53, wherein the protein comprising the VEGF binding domain comprises bevacizumab, ranibizumab, or aflibercept, or a VEGF binding fragment thereof.

56. The method of any one of claims 48-53, wherein the protein comprising the VEGF binding domain comprises bevacizumab, ranibizumab, or aflibercept.

57. The method of any one of claims 48-56, wherein the PEDF polypeptide is administered.

58. The method of any one of claims 48-57, wherein the PEDF polypeptide is administered, and comprises at least 20, 25, 30, 31, 32, 33, 34, or, 35 consecutive amino acids of SEQ ID NO: 1.

59. The method of any one of claims 48-57, wherein the PEDF polypeptide is administered, and comprises SEQ ID NO: 2 or SEQ ID NO: 3.

60. The method of any one of claims 48-59, wherein the PEDF polypeptide is administered, and is a PEDF agonist.

61. The method of any one of claims 48-56, wherein the antibody that binds specifically to PEDFR is administered.

62. The method of any one of claims 48-56, wherein the antibody that binds specifically to PEDFR is administered, and is a PEDF agonist.

63. The method of any one of claims 48-62, wherein the protein comprising the VEGF binding domain and the PEDF polypeptide or antibody that binds specifically to PEDFR are part of a single molecule.

64. The method of any one of claims 48-59, wherein the protein comprising the VEGF binding domain and the PEDF polypeptide or antibody that binds specifically to PEDFR are in separate molecules.

65. The method of any one of claims 48-62 or 64, wherein the protein comprising the VEGF binding domain and the PEDF polypeptide are administered at the same time.

66. The method of any one of claims 48-62 or 64, wherein the protein comprising the VEGF binding domain and the PEDF polypeptide are administered at different times, for example separate and/or sequentially.

67. The method of any one of claims 48-58, wherein the PEDF polypeptide or antibody that binds specifically to PEDFR is administered to the patient in an amount effective to induce proliferation of retinal pigment epithelial cells.

68. The method of any one of claims 48-67, wherein the protein comprising the VEGF binding domain is administered intravitreally, subretinally, subconjuntivally, sub- Tenon’s, or topically to an eye of the patient.

69. The method of any one of claims 48-67, wherein the PEDF polypeptide or antibody that binds specifically to PEDFR is administered intravitreally, subretinally, subconjuntivally, sub-Tenon’s, or topically to an eye of the patient.

70. The method of any one of claims 48-62 or 64-67, wherein the protein comprising the VEGF binding domain and the PEDF polypeptide or antibody that binds specifically to PEDFR are administered via the same route of administration.

71. The method of any one of claims 48-62 or 64-67, wherein the protein comprising the VEGF binding domain and the PEDF polypeptide or antibody that binds specifically to PEDFR are administered via different routes of administration.