Fusion molecules targeting VEGF and angiopoietin and uses thereof

Abstract

Polypeptides comprising two or three VEGF binding domains; and a domain that binds to angiopoietin, and gene therapy methods for delivering such polypeptides.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of PCT application PCT / CN2021 / 084559, filed March 31, 2021, which is incorporated by reference in its entirety. (Reference to electronically submitted sequence listing) This application contains a Sequence Listing submitted electronically via EFS-Web as a file titled "14652-025-228_SEQ_LISTING.txt" and an ASCII formatted Sequence Listing having a size of 217,975 bytes with a creation date of March 18, 2022. The Sequence Listing submitted via EFS-Web is a part of the present specification and is incorporated herein by reference in its entirety. (1. Field) The present disclosure relates to fusion molecules (eg, polypeptides) that bind to both vascular endothelial growth factor (VEGF) and angiopoietins (eg, angiopoietin 1 and angiopoietin 2), gene therapy methods based on such fusion molecules, and methods of use thereof. [Background technology]

[0002] (2.Background) The VEGF / VEGFR and angiopoietin / Tie-2 signaling pathways are important in the process of vascular endothelial growth (angiogenesis) and the maintenance of angiogenesis-associated blood vessels. See Biela and Siemann, Cancer Lett. 380(2): 525-533 (2016). Abnormal angiogenesis has been implicated in many diseases, such as diabetic retinopathy, psoriasis, exudative or "wet" age-related macular degeneration ("wAMD"), rheumatoid arthritis and other inflammatory diseases, and most cancers. Diseased tissues or tumors associated with these diseases usually express abnormally high levels of VEGF and exhibit high levels of angiogenesis or vascular permeability. For example, wAMD is an angiogenic disease characterized by choroidal neovascularization in one or both eyes of aging individuals and is a leading cause of blindness. See Gehrs et al., Ann Med. 38(7): 450-471 (2006). Several therapeutic strategies exist to target VEGF or angiopoietin to inhibit abnormal angiogenesis. See Biela and Siemann, Cancer Lett. 380(2): 525-533 (2016). However, such treatments typically require repeated injections, which may increase the risk of inflammation, infection, and other adverse effects in some patients. Repeated injections also come with challenges in patient compliance and adherence, and non-compliance may lead to reduced vision and worsening of ocular or ocular diseases. Rates of non-compliance and non-adherence to treatment regimens that require repeated or frequent visits to a medical institution for administration are particularly high among elderly patients who are most affected by AMD. There is a need in the art for improved therapeutic molecules, particularly for use in gene therapy to treat diseases such as wAMD. Summary of the Invention

[0003] (3. Overview) In one aspect, provided herein is a polypeptide comprising: (i) a first domain that binds VEGF; (ii) a second domain that binds VEGF; and (iii) a third domain that binds angiopoietin (e.g., angiopoietin 1 and angiopoietin 2). In some embodiments, the third domain is optionally N-terminal to the first domain and the second domain.

[0004] In some embodiments, the first domain is derived from VEGF receptor-1 (VEGFR-1 or FLT-1). In some embodiments, the first domain comprises domain 2 of VEGFR-1 or a variant thereof. In some embodiments, the first domain comprises or consists of the amino acid sequence of SEQ ID NO:1. In some embodiments, the first domain comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:1.

[0005] In some embodiments, the second domain is derived from VEGF receptor-2 (VEGFR-2 or Flk-1). In some embodiments, the second domain comprises domain 3 of VEGFR-2 or a variant thereof. In some embodiments, the second domain comprises or consists of the amino acid sequence of SEQ ID NO:2. In some embodiments, the second domain comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:2.

[0006] In some embodiments, the third domain (ABD) comprises one or two repeats of the amino acid sequence of SEQ ID NO: 3. In some embodiments, the third domain comprises one or two repeats of the amino acid sequence of SEQ ID NO: 51. In some embodiments, the third domain comprises two repeats of the amino acid sequence of SEQ ID NO: 3. In some embodiments, the third domain comprises two repeats of the amino acid sequence of SEQ ID NO: 51. In some embodiments, the third domain comprises the amino acid sequence of SEQ ID NO: 3 and the amino acid sequence of SEQ ID NO: 51.

[0007] In some embodiments, the third domain comprises or consists of the amino acid sequence of SEQ ID NO:4, or the third domain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:4.

[0008] In some embodiments, the third domain comprises or consists of the amino acid sequence of SEQ ID NO:52, or the third domain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:52.

[0009] In some embodiments, the third domain comprises or consists of the amino acid sequence of SEQ ID NO:53, or the third domain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:53.

[0010] In some embodiments, the third domain comprises or consists of the amino acid sequence of SEQ ID NO:54, or the third domain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:54.

[0011] In another aspect, provided herein is a polypeptide comprising: (i) a first domain that binds VEGF, the first domain comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1; (ii) a second domain that binds VEGF, the second domain comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:2; and (iii) a third domain that binds angiopoietin, the third domain comprising two amino acid sequences each comprising an amino acid sequence having at least 80%, 85%, 90%, or 100% identity to SEQ ID NO:3.

[0012] In another aspect, provided herein is a polypeptide comprising: (i) a first domain that binds VEGF, the first domain comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1; (ii) a second domain that binds VEGF, the second domain comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:2; and (iii) a third domain that binds angiopoietin, the third domain comprising two amino acid sequences each comprising an amino acid sequence having at least 80%, 85%, 90%, or 100% identity to SEQ ID NO:51.

[0013] In another aspect, provided herein is (i) a first domain that binds VEGF, the first domain comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1; (ii) a second domain that binds VEGF, the first domain comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:2; a second domain comprising an amino acid sequence having 5%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:3; and (iii) a third domain that binds to angiopoietin, the third domain comprising one amino acid sequence having at least 80%, 85%, 90% or 100% identity to SEQ ID NO:3 and one amino acid sequence having at least 80%, 85%, 90% or 100% identity to SEQ ID NO:51.

[0014] In some embodiments, the polypeptide further comprises an Fc region of an antibody. In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO:5.

[0015] In some embodiments, the polypeptide further comprises a signal peptide. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO:6.

[0016] In some embodiments, the polypeptide further comprises one or more linkers.

[0017] In some embodiments, provided herein is a polypeptide comprising an amino acid sequence of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10.

[0018] In some embodiments, provided herein is a polypeptide comprising the amino acid sequence of SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, or SEQ ID NO:66, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, or SEQ ID NO:66.

[0019] In some embodiments, the polypeptide provided herein further comprises a VEGFC binding domain. In some embodiments, the VEGFC binding domain is derived from VEGFR-2. In other embodiments, the VEGFC binding domain is derived from VEGFR-3.

[0020] In some embodiments, the VEGFC binding domain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:55.

[0021] In some embodiments, the VEGFC binding domain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:56.

[0022] In some embodiments, the VEGFC binding domain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:57.

[0023] In some embodiments, the polypeptide is genetically fused or chemically conjugated to the agent.

[0024] In another aspect, provided herein is an isolated nucleic acid comprising a nucleic acid sequence encoding a polypeptide provided herein.

[0025] In yet another aspect, provided herein is a vector comprising the isolated nucleic acid provided herein. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is an adeno-associated viral (AAV) vector. In some embodiments, the AAV vector is derived from AAV1, AAV2, AAV2i8, AAV3, AAV3-B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV13, AAV-DJ, AAV LK03, AAVrh74, AAV44-9, or combinations or variants thereof. In some embodiments, the vector is a recombinant AAV2 (rAAV2) vector, a recombinant AAV8 (rAAV8) vector, or variants thereof.

[0026] In another aspect, provided herein is a recombinant AAV (rAAV) vector comprising a nucleic acid encoding a polypeptide, wherein the polypeptide comprises (i) a first domain derived from VEGFR-1; (ii) a second domain derived from VEGFR-2; and (iii) a third domain capable of binding to angiopoietin (e.g., angiopoietin 1 and angiopoietin 2), wherein the rAAV vector comprises an inverted terminal repeat (ITR) from AAV1, AAV2, AAV2i8, AAV3, AAV3-B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV13, AAV-DJ, AAV LK03, AAVrh74, AAV44-9, or a combination or variant thereof. In some embodiments, the ITR is from AAV2. In other embodiments, the ITRs are from AAV 8. In some embodiments, the third domain is N-terminal to the first domain and the second domain.

[0027] In yet another aspect, provided herein is a recombinant AAV (rAAV) vector comprising a nucleic acid encoding a polypeptide, wherein the polypeptide comprises (i) a first domain derived from VEGFR-1; (ii) a second domain derived from VEGFR-2; (iii) a third domain capable of binding to angiopoietin; and (iv) a fourth domain capable of binding to VEGFC, wherein the rAAV vector comprises an inverted terminal repeat (ITR) from AAV1, AAV2, AAV2i8, AAV3, AAV3-B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV13, AAV-DJ, AAV LK03, AAVrh74, or AAV44-9.In some embodiments, the ITR is derived from AAV2.In other embodiments, the ITR is derived from AAV8. In some embodiments, the third domain is N-terminal to the first domain and the second domain.

[0028] In some embodiments, provided herein is a vector comprising the nucleic acid sequence of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, or SEQ ID NO:23, or a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, or SEQ ID NO:23.

[0029] In some embodiments, provided herein is a vector comprising the nucleic acid sequence of SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, or SEQ ID NO:75, or a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, or SEQ ID NO:75.

[0030] In yet another aspect, provided herein is a recombinant AAV (rAAV) particle comprising: (a) a nucleic acid encoding a polypeptide, wherein the polypeptide comprises: (i) a first domain derived from VEGFR-1; (ii) a second domain derived from VEGFR-2; and (iii) a third domain capable of binding to angiopoietin (e.g., angiopoietin 1 and angiopoietin 2); and (b) a capsid protein of AAV1, AAV2, AAV2i8, AAV3, AAV3-B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV13, AAV-DJ, AAV LK03, AAVrh74, AAV44-9, or a variant thereof. In some embodiments, the third domain is N-terminal to the first domain and the second domain. In some embodiments, the capsid protein is an AAV2 capsid protein. In some embodiments, the capsid protein is an AAV8 capsid protein. In some embodiments, the capsid protein is a variant of the AAV2 capsid protein comprising the amino acid sequence of SEQ ID NO: 48, wherein the variant comprises the following amino acid substitutions of AAV2 capsid protein VP1: Y444F, R487G, T491V, Y500F, R585S, R588T, and Y730F.

[0031] In yet another aspect, provided herein are recombinant AAV (rAAV) particles comprising: (a) a nucleic acid encoding a polypeptide, wherein the polypeptide comprises (i) a first domain derived from VEGFR-1; (ii) a second domain derived from VEGFR-2; and (iii) a third domain capable of binding to angiopoietin; and (iv) a fourth domain capable of binding to VEGFC; and (b) a capsid protein of AAV1, AAV2, AAV2i8, AAV3, AAV3-B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV13, AAV-DJ, AAV LK03, AAVrh74, AAV44-9, or a variant thereof.

[0032] A pharmaceutical composition comprising a polypeptide, vector or rAAV vector, or rAAV particle provided herein, and a pharma- ceutical acceptable excipient.

[0033] In yet another aspect, provided herein is a method of treating a disease or disorder in a subject, comprising administering to the subject a polypeptide, vector, or pharmaceutical composition provided herein. In some embodiments, the disease or disorder is an angiogenic or neovascular disease or disorder. In some embodiments, the disease or disorder is an inflammatory disease, an ocular disease, an autoimmune disease, or cancer. In some embodiments, the disease or disorder is an ocular disease or disorder. In some embodiments, the ocular disease or disorder is selected from the group consisting of uveitis, retinitis pigmentosa, neovascular glaucoma, diabetic retinopathy (DR) (including proliferative diabetic retinopathy), ischemic retinopathy, intraocular neovascularization, age-related macular degeneration (AMD), retinal neovascularization, diabetic macular edema (DME), diabetic retinal ischemia, diabetic retinal edema, retinal vein occlusion (including central retinal vein occlusion and branch retinal vein occlusion), macular edema, and macular edema after retinal vein occlusion (RVO). In some embodiments, the disease or disorder is age-related macular degeneration (AMD). In some embodiments, the AMD is wet AMD (wAMD).

[0034] In some embodiments, the methods comprise administering by intravitreal or subretinal injection into the subject's eye. [Brief description of the drawings]

[0035] (4. BRIEF DESCRIPTION OF THE DRAWINGS) [Figure 1] FIG. 1 shows exemplary fusion protein constructs provided herein (Flt1 signal: signal peptide sequence of VEGFR1 (Flt1); VEGFR1 D2: IgG-like domain 2 of VEGF receptor 1; VEGFR2 D3: IgG-like domain 3 of VEGF receptor 2; Ang BD: angiopoietin binding domain; IgG Fc: Fc fragment of human IgG).

[0036] [Diagram 2]2 shows an exemplary rAAV vector provided herein, where TR represents AAV2 inverted terminal repeat, CBA represents 1.68 kb chicken β-actin promoter, CB represents 0.78 kb chicken small β-actin promoter, D2 represents IgG-like domain 2 of VEGFR-1, D3 represents IgG-like domain 3 of VEGFR-2, ABD (or Ang BD) represents angiopoietin binding domain, IgG Fc represents Fc fragment of human IgG, Fc-Hinger represents 21 amino acids at the N-terminus of Fc plus a 6 amino acid GS linker, WPRE represents woodchuck hepatitis virus posttranscriptional regulatory element (600 bp), mini-mWPRE is WPRE (240 bp), SV40pA is simian virus 40 polyadenylation signal, and bGHpA is bovine growth hormone polyadenylation signal.

[0037] [Diagram 3] 3A-3B show the experimental design and assay for testing the expression and binding affinity of the constructs provided herein.

[0038] [Figure 4] Figures 4A-4B show that positioning of the Ang BD at the C-terminus did not affect binding ability to rVEGF, but did reduce transgene expression levels.

[0039] [Diagram 5] 5A-5B show that positioning of the Ang BD between the VEGF binding domain and the Fc region did not affect binding capacity to rVEGF and transgene expression.

[0040] [Figure 6]Figures 6A-6B show that positioning the Ang BD between the VEGF binding domain and the Fc region inhibited its ability to bind to Ang2. In the top panel of Figure 6B, the right bar of each of the two pairs represents binding for EXG102-09, and the left bar of each of the two pairs represents binding for EXG102-04.

[0041] [Figure 7] Figures 7A-7D show that constructs with an N-terminal Ang BD strongly bind to both VEGF and Ang2 while maintaining high levels of transgene expression. In Figure 7D, for each EXG construct, the left bar represents binding to rVEGF, the center bar represents binding to Ang2, and the right bar represents protein expression levels.

[0042] [Figure 8] 8 shows a schematic design of additional exemplary fusion protein constructs comprising a VEGF-C binding domain (Trap-C1, C2, or C3) and an angiopoietin binding domain (ABD or ABD2) provided herein. TR, inverted terminal repeat; CBA, chimeric CMV-chicken β-actin promoter; ABD, angiopoietin binding domain; D2, IgG-like domain 2 of VEGF receptor 1; D3, IgG-like domain 3 of VEGF receptor 2; Trap C, VEGF F C binding domain; Fc, fragment crystallizable of IgG1; bGHpA, bovine growth hormone polyadenylation signal; VEGF, vascular endothelial growth factor; IgG1, immunoglobulin G1.

[0043] [Figure 9]Figures 9A-9I show EXG102-24, EXG102-25, EXG102-26, EXG102-27, EXG102-28, EXG102-29, EXG102-30, EXG102-31, and EXG102-32, respectively. Sequences are shown and numbered (the fusion proteins illustrated in Figures 9A-9I include SEQ ID NOs: 58-66, respectively). The signal peptide, ABD, D2, D3, Trap C, and Fc constructs are also shown in the figures. The GS linker is highlighted. TR, inverted terminal repeat; CBA, chimeric CMV-chicken β-actin promoter; ABD, angiopoietin binding domain; D2, IgG-like domain 2 of VEGF receptor 1; D3, IgG-like domain 3 of VEGF receptor 2; Trap C, VEGFC binding domain; Fc, fragment crystallizable of IgG1; bGHpA, bovine growth hormone polyadenylation signal; VEGF, vascular endothelial growth factor; IgG1, immunoglobulin G1.

[0044] [Figure 10] 10A-10F show that constructs containing ABD2 are comparable to constructs with the ABD domain in protein expression, VEGF-A binding, or Ang2 binding.

[0045] [Figure 11] Figure 11 shows the FFA mean score in a laser-induced choroidal neovascularization (CNV) mouse model. The FFA mean score was measured in eyes treated with AAV-GFP (negative control), EXG102-02 (positive control), EXG102-04, EXG102-09, EXG102-10, or EXG102-11 at 29 and 36 days after injection. The angiograms were scored as follows: score 0, no staining; score 1, slightly stained; score 2, moderately stained; and score 3, strongly stained. The two constructs with the greatest CNV inhibition are highlighted with arrows. Bars indicate mean values ​​with SD.

[0046] [Figure 12] Figure 12 shows the proportion of grade 3 CNV lesions in a laser-induced CNV mouse model. FFA mean scores were measured in eyes treated with AAV-GFP (negative control), EXG102-02 (positive control), EXG102-04, EXG102-09, EXG102-10, or EXG102-11. Angiograms were graded as follows: score 0, no staining; score 1, slightly stained; score 2, moderately stained; and score 3, strongly stained. Bars indicate mean values ​​with SD.

[0047] [Figure 13] 13A-13C show the in vitro VEGF-A, Ang2, or VEGF-C binding affinity of transgene products expressed from HEK293T cells transfected with plasmid DNA. HEK293T cells were transfected with pEXG102-02, pEXG102-30, or pEXG102-31 plasmid DNA, and expressed target proteins were affinity purified from cell lysates. The binding ability of EXG102-02, EXG102-30, or EXG102-31 to VEGF-A, Ang2, or VEGF-C, respectively, was measured by ELISA. Bars indicate mean values ​​with SD.

[0048] [Figure 14] FIG. 14 shows the FFA mean score in the laser-induced CNV mouse model. The FFA mean score was measured in eyes treated with vehicle control (black), EXG102-02 (medium gray), EXG102-30 (dark gray), or EXG102-31 (light gray). Prior to fluorescein angiography, animals were administered an intraperitoneal injection of sodium fluorescein (100 mg / ml, 30 μL / animal). FFA images were acquired 3 min after injection. Angiograms were scored as follows: score 0, no staining; score 1, slightly stained; score 2, moderately stained; and score 3, strongly stained. The percentage of lesions with score 3 and the mean score were calculated. Bars indicate the mean with SD.

[0049] [Figure 15] FIG. 15 shows the percentage of grade 3 CNV lesions in a laser-induced CNV mouse model. The percentage of grade 3 CNV lesions was measured in eyes treated with vehicle control, EXG102-02, EXG102-30, or EXG102-31. Prior to fluorescein angiography, animals were injected intraperitoneally with sodium fluorescein (100 mg / ml, 30 μL / animal). FFA images were acquired 3 min after injection. Angiograms were scored as follows: score 0, no staining; score 1, slightly stained; score 2, moderately stained; and score 3, strongly stained. The percentage of lesions with score 3 and the mean score were calculated. Bars indicate mean values ​​with SD. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0050] (5. Detailed Description) The present disclosure is based in part on novel fusion proteins comprising domains derived from VEGFR-1, VEGFR-2, and / or VEGFR-3 and an angiopoietin binding domain, AAV vectors comprising nucleic acids encoding such fusion proteins, and the improved properties thereof.

[0051] (5.1.Definition) The techniques and procedures described or referred to herein are, as a whole, well understood by those of skill in the art and / or commonly employed using conventional methods, such as the widely used methods described in Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd ed., 2001); Current Protocols in Molecular Biology (Ausubel et al., eds., 2003); Therapeutic Monoclonal Antibodies: From Bench to Clinic (An, eds., 2009); Monoclonal Antibodies: Methods and Protocols (Albitar, eds., 2010); and Antibody Engineering, vols. 1 and 2 (Kontermann and Dubel, eds., 2010).

[0052] Unless otherwise defined herein, technical and scientific terms used in this description have the meanings commonly understood by those skilled in the art. For the purposes of interpreting this specification, the following explanations of terms shall apply, and whenever appropriate, terms used in the singular shall also include the plural and vice versa. In the event that any explanation of a term given conflicts with any document incorporated herein by reference, the explanation of the term given below shall prevail.

[0053] The terms "polypeptide" and "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymers may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. These terms also encompass amino acid polymers that are modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification. Also included in this definition are polypeptides that contain one or more analogs of an amino acid, including, for example, but not limited to, unnatural amino acids, and other modifications known in the art.

[0054] The term "binds" or "binding" refers to interactions between molecules, including, for example, forming a complex. The interactions can be non-covalent interactions, including, for example, hydrogen bonds, ionic bonds, hydrophobic interactions, and / or van der Waals interactions. A complex can also include the binding of two or more molecules held together by covalent or non-covalent bonds, interactions, or forces. The strength of the overall non-covalent interactions between two molecules is the affinity of one molecule for another. The dissociation rate (k off ) and association rate (k on ) ratio (k off / k on ) is the dissociation constant K D which correlates inversely with affinity. D The lower the value, the higher the affinity. D The value of varies depending on the complex, and k on and k off The dissociation constant K D can be determined using any of the methods provided herein or any other method known to one of skill in the art.

[0055] In the context of the binding molecules described herein, terms such as "bind to," "specifically bind to," and similar terms are also used interchangeably herein to refer to a binding domain specifically binding to another molecule, e.g., a polypeptide. A binding molecule or binding domain that binds to or specifically binds to another molecule can be identified, for example, by immunoassay, Octet®, Biacore®, or other techniques known to those of skill in the art. In some embodiments, a binding molecule or binding domain binds to or specifically binds to a molecule if it binds to the molecule with higher affinity than to any cross-reactive molecules, as determined using experimental techniques such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). Typically, a specific or selective response is at least twice the background signal or noise, and may be more than 10 times the background. See, for example, Fundamental Immunology, 332-36 (Paul, ed., 2nd ed., 1989) for a discussion of binding specificity. In some embodiments, the degree of binding of a binding molecule or binding domain to a "non-target" protein is less than about 10% of the binding of the binding molecule or binding domain to its specific target, as determined, for example, by fluorescence-activated cell sorting (FACS) analysis or RIA. In this regard, terms such as "specific binding," "specifically binds to," or "specific for" refer to binding that is measurably different from non-specific interactions. Specific binding can be measured, for example, by determining the binding of a molecule compared to the binding of a control molecule, which is usually a molecule of similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target, for example, an excess of unlabeled target. In this case, specific binding is indicated when the binding of the labeled target to the probe is competitively inhibited by an excess of unlabeled target. Binding molecules or binding domains that bind to a molecule include those that can bind to a molecule with sufficient affinity such that the binding molecule is useful, for example, as a diagnostic or therapeutic agent in targeting the molecule.In certain embodiments, a binding molecule or binding domain that binds to a target molecule has a dissociation constant (K) of 1 μM, 800 nM, 600 nM, 550 nM, 500 nM, 300 nM, 250 nM, 100 nM, 50 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, or 0.1 nM or less. D ).

[0056] "Binding affinity" typically refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule and its binding partner. Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair. The affinity of a binding molecule X for its binding partner Y is typically measured by the dissociation constant (K D Affinity can be measured by common methods known in the art, including those described herein. A variety of methods for measuring binding affinity are known in the art, any of which may be used for purposes of the present disclosure. Specific exemplary embodiments include the following. In one embodiment, "K D " or "K D The "K value" can be measured by assays known in the art, for example, by binding assays. D Or K DValues ​​can also be measured using Biolayer Interferometry (BLI) or Surface Plasmon Resonance (SPR) assays, e.g., by Octet® using the Octet® Red96 system, or by Biacore® using, e.g., a Biacore® TM-2000 or Biacore® TM-3000. "On-rate" or "rate of association" or "association rate" or "kon" can also be determined using the same Biolayer Interferometry (BLI) or Surface Plasmon Resonance (SPR) methods described above, e.g., using the Octet® Red96, Biacore® TM-2000, or Biacore® TM-3000 systems.

[0057] The terms "antibody," "immunoglobulin," or "Ig" are used interchangeably herein and are used in the broadest sense to specifically cover monoclonal antibodies (including agonist, antagonist, neutralizing, full-length or intact monoclonal antibodies), antibody compositions with multi-epitope or single epitopic specificity, polyclonal or monovalent antibodies, multivalent antibodies, multispecific antibodies formed from at least two intact antibodies (e.g., bispecific antibodies, so long as they exhibit the desired biological activity), single chain antibodies, and fragments thereof, e.g., as described below. Antibodies can be human, humanized, chimeric, and / or affinity matured antibodies, as well as antibodies derived from other species, e.g., mouse, rabbit, llama, etc. The term "antibody" is intended to include polypeptide products of B cells within the immunoglobulin class of polypeptides that can bind to a specific molecular antigen and are composed of two identical pairs of polypeptide chains, where each pair has one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa), where the amino-terminal portion of each chain contains a variable region of about 100 to about 130 or more amino acids, and the carboxy-terminal portion of each chain contains a constant region. See, for example, Antibody Engineering (Borrebaeck, ed., 2nd ed., 1995); and Kuby, Immunology (3rd ed., 1997). In specific embodiments, specific molecular antigens, including polypeptides or epitopes, can be bound by the antibodies provided herein. Antibodies also include, but are not limited to, synthetic antibodies, recombinantly produced antibodies, single domain antibodies or humanized variants thereof, including those derived from Camelidae species (e.g., llama or alpaca), intrabodies, anti-idiotypic (anti-Id) antibodies, and functional fragments (e.g., antigen-binding fragments) of any of the above (which refers to a portion of an antibody heavy or light chain polypeptide that retains some or all of the binding activity of the antibody from which the fragment is derived).Non-limiting examples of functional fragments (e.g., antigen-binding fragments) include single chain Fvs (scFvs) (including, e.g., monospecific, bispecific, etc.), Fab fragments, F(ab') fragments, F(ab)2 fragments, F(ab')2 fragments, disulfide-linked Fvs (dsFvs), Fd fragments, Fv fragments, diabodies, triabodies, tetrabodies, and minibodies. In particular, antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, such as antigen-binding domains or molecules that contain an antigen-binding site that binds an antigen (e.g., one or more CDRs of an antibody). Such antibody fragments can be found, for example, in Harlow and Lane, Antibodies: A Laboratory Manual (1989); Mol. Biology and Biotechnology: A Comprehensive Desk Reference (Myers, ed., 1995); Huston et al., 1993, Cell Biophysics 22:189-224; Pluckthun and Skerra, 1989, Meth. Enzymol. 178:497-515; and Day, Advanced Immunochemistry (2nd ed., 1990). The antibodies provided herein can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) of immunoglobulin molecules. The antibody may be an agonistic or antagonistic antibody. The antibody may be neither agonistic nor antagonistic.

[0058] The term "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain, including, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of an immunoglobulin heavy chain Fc region can vary, the human IgG heavy chain Fc region is often defined as extending from an amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) can be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding the antibody heavy chain. Thus, a composition of intact antibodies can include antibody populations in which all K447 residues have been removed, antibody populations in which the K447 residue has not been removed, and antibody populations having a mixture of antibodies with and without the K447 residue. A "functional Fc region" possesses the "effector functions" of a native sequence Fc region. Exemplary "effector functions" include C1q binding; CDC; Fc receptor binding; ADCC; phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptors); and the like. Such effector functions typically require that the Fc region be combined with a binding region or domain (e.g., an antibody variable region or domain) and can be assessed using a variety of assays known to those of skill in the art. A "variant Fc region" comprises an amino acid sequence that differs from the amino acid sequence of a native sequence Fc region by at least one amino acid modification (e.g., substitution, addition, or deletion). In certain embodiments, the variant Fc region has at least one amino acid substitution, e.g., about 1 to about 10 amino acid substitutions, or about 1 to about 5 amino acid substitutions, in the native sequence Fc region or in the Fc region of the parent polypeptide compared to the native sequence Fc region or the Fc region of the parent polypeptide. The variant Fc region herein can retain at least about 80% homology to a native sequence Fc region and / or the Fc region of a parent polypeptide, or at least about 90% homology thereto, such as at least about 95% homology thereto.

[0059] "Polynucleotide" or "nucleic acid", as used interchangeably herein, refers to a polymer of nucleotides of any length, including DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. A polynucleotide can include modified nucleotides, such as methylated nucleotides and their analogs. An "oligonucleotide", as used herein, refers to a short, usually single-stranded, synthetic polynucleotide, which is usually, but not necessarily, less than about 200 nucleotides in length. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above description of polynucleotides is equally and fully applicable to oligonucleotides. Cells producing the binding molecules of the present disclosure can include parent hybridoma cells, as well as bacterial and eukaryotic host cells into which a nucleic acid encoding a polypeptide has been introduced. Unless otherwise specified, the left-hand end of any single-stranded polynucleotide sequence disclosed herein is the 5' end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The direction of 5' to 3' addition of nascent RNA transcripts is referred to as the transcription direction; the sequence region on the DNA strand that has the same sequence as the RNA transcript that is 5' to the 5' end of the RNA transcript is referred to as the "upstream sequence"; the sequence region on the DNA strand that has the same sequence as the RNA transcript that is 3' to the 3' end of the RNA transcript is referred to as the "downstream sequence".

[0060] An "isolated nucleic acid" is a nucleic acid, e.g., RNA, DNA, or mixed nucleic acid, that is usually substantially separated from other genomic DNA sequences, and proteins or complexes that naturally accompany the native sequence, e.g., ribosomes and polymerases. An "isolated" nucleic acid molecule is a nucleic acid molecule that is separated from other nucleic acid molecules present in the natural source of the nucleic acid molecule. Furthermore, an "isolated" nucleic acid molecule, e.g., a cDNA molecule, can be substantially free of other cellular material, or culture medium if produced by recombinant techniques, or substantially free of chemical precursors or other chemicals if chemically synthesized. In a specific embodiment, one or more nucleic acid molecules encoding the fusion proteins described herein are isolated or purified. This term encompasses a nucleic acid sequence that has been removed from its natural environment, including recombinant or cloned DNA isolates and chemically synthesized analogs or analogs biosynthesized by heterologous systems. A substantially pure molecule can include isolated forms of the molecule. Specifically, an "isolated" nucleic acid molecule encoding a polypeptide described herein is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the environment in which it is produced.

[0061] The term "variant" as used with respect to a polypeptide or protein refers to a polypeptide having a particular percent sequence identity with a reference polypeptide, e.g., at least about 80% amino acid sequence identity with the reference polypeptide, e.g., the corresponding full-length native sequence. Such polypeptide variants include, for example, polypeptides in which one or more amino acid residues have been added or deleted. In embodiments, a variant has at least about 80% amino acid sequence identity, at least about 81% amino acid sequence identity, at least about 82% amino acid sequence identity, at least about 83% amino acid sequence identity, at least about 84% amino acid sequence identity, at least about 85% amino acid sequence identity, at least about 86% amino acid sequence identity, at least about 87% amino acid sequence identity, at least about 88% amino acid sequence identity, at least about 89% amino acid sequence identity, at least about 90% amino acid sequence identity, or at least about 91% amino acid sequence identity, at least about 92% amino acid sequence identity, at least about 93% amino acid sequence identity, at least about 94% amino acid sequence identity, at least about 95% amino acid sequence identity, at least about 96% amino acid sequence identity, at least about 97% amino acid sequence identity, at least about 98% amino acid sequence identity, or at least about 99% amino acid sequence identity to a reference polypeptide, e.g., a corresponding full-length native sequence. In embodiments, variant polypeptides are at least about 10 amino acids in length, at least about 20 amino acids in length, at least about 30 amino acids in length, at least about 40 amino acids in length, at least about 50 amino acids in length, at least about 60 amino acids in length, at least about 70 amino acids in length, at least about 80 amino acids in length, at least about 90 amino acids in length, at least about 100 amino acids in length, at least about 150 amino acids in length, at least about 200 amino acids in length, at least about 300 amino acids in length, or longer. Variants include substitutions that are conservative or non-conservative in nature.For example, a polypeptide of interest can include up to about 5-10 conservative or non-conservative amino acid substitutions, or even up to about 15-25 or 50 conservative or non-conservative amino acid substitutions, or any number between 5-50.

[0062] The term "homology" as used herein refers to the percent identity between two polynucleotides or two polypeptide moieties. Two DNA or two polypeptide sequences are "substantially homologous" to each other if the sequences exhibit at least about 50%, at least about 75%, at least about 80%-85%, at least about 90%, at least about 95%-98% sequence identity, at least about 99%, or any percentage therebetween, over a defined length of the molecule. As used herein, substantially homologous also refers to a sequence that exhibits complete identity with a particular DNA or polypeptide sequence.

[0063] The term "identity" as used herein refers to an exact nucleotide-to-nucleotide or amino acid-to-amino acid match of two polynucleotide or polypeptide sequences, respectively. Methods for determining percent identity are well known in the art. For example, percent identity can be determined by a direct comparison of sequence information between two molecules by aligning the sequences, counting the number of exact matches between the two aligned sequences, dividing by the length of the shorter sequence, and multiplying the result by 100. Readily available computer programs such as ALIGN can be used to aid in the analysis, for example, Dayhoff, MO, in Atlas of Protein Sequence and Structure, MO Dayhoff, ed., 5th Supplement, 3:353-358, National Biomedical Research Foundation, Washington, DC, which applies the local homology algorithm of Smith and Waterman, Advances in Appl. Math. 2:482-489, 1981, to peptide analysis. Programs for determining nucleotide sequence identity, such as the BESTFIT, FASTA, and GAP programs, are available in the Wisconsin Sequence Analysis Package, version 8 (available from Genetics Computer Group, Madison, Wis.), which also rely on the Smith and Waterman algorithm. These programs are readily used with the default parameters recommended by the manufacturer and described in the Wisconsin Sequence Analysis Package referenced above. For example, the percent identity between a particular nucleotide sequence and a reference sequence can be determined using the Smith and Waterman homology algorithm with a default scoring table and a gap penalty of six nucleotide positions.Another method for establishing percent identity in the context of the present invention is to use the MPSRCH package of programs copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S. Sturrok and distributed by IntelliGenetics, Inc. (Mountain View, Calif.). From this suite of packages, the Smith-Waterman algorithm can be utilized, with default parameters (e.g., gap opening penalty 12, gap extension penalty 1, gap 6) used in the scoring table. From the data generated, the "match" value reflects the "sequence identity". Other suitable programs for calculating percent identity or similarity between sequences are generally known in the art, for example, another alignment program is BLAST, used with default parameters. For example, BLASTN and BLASTP can be used with the following default parameters: genetic code=standard; filter=none; strand=both; cutoff=60; expectation=10; matrix=BLOSUM62; description=50 sequences; sorting method=high score; database=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translation+Swiss-protein+Spupdate+PIR. Details of these programs are well known in the art. Alternatively, homology can be determined by hybridization of polynucleotides under conditions that form stable duplexes between homologous regions, followed by digestion with a single-strand specific nuclease and sizing of the digested fragments. DNA sequences that are substantially homologous can be identified in Southern hybridization experiments under, for example, stringent conditions defined for that particular system. It is within the skill of the art to define appropriate hybridization conditions. See, for example, Sambrook et al., supra, DNA Cloning, supra, and Nucleic Acid Hybridization, supra.

[0064] The term "vector" as used herein refers to a material used to carry or contain a nucleic acid sequence, for example, to introduce the nucleic acid sequence into a host cell. Vectors applicable for use include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which can include a selection sequence or marker operable for stable integration into a host cell chromosome. In addition, the vector can include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes that can be included, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply essential nutrients not present in the culture medium. Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like, which are well known in the art. When two or more nucleic acid molecules are to be co-expressed, both nucleic acid molecules can be inserted, for example, into a single expression vector or into separate expression vectors. Introduction of the nucleic acid molecules into the host cell can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis, such as Northern blot or polymerase chain reaction (PCR) amplification of mRNA, immunoblotting of gene product expression, or other suitable analytical methods for testing the expression of introduced nucleic acid sequence or its corresponding gene product.The term "vector" includes cloning and expression vehicles, and viral vectors.In some embodiments, the vector provided herein is a recombinant AAV vector.

[0065] As used herein, the term "recombinant AAV vector (rAAV vector)" refers to a polynucleotide vector that includes a nucleic acid sequence derived from AAV and one or more heterologous sequences (i.e., a nucleic acid sequence that is not of AAV origin). In some embodiments, the one or more heterologous sequences are flanked by at least one, and in some embodiments, two, AAV inverted terminal repeats (ITRs). In some embodiments, such rAAV vectors can be replicated and packaged into infectious viral capsid particles, for example, when present in a host cell that is infected with a suitable helper virus (or expresses suitable helper functions) and expresses the AAV rep and cap gene products (i.e., AAV Rep and Cap proteins). rAAV vectors can be incorporated into a larger polynucleotide (e.g., in a chromosome or in another vector, such as a plasmid used for cloning or transfection) and can be "rescued" by replication and encapsidation in the presence of AAV packaging functions and suitable helper functions. rAAV vectors can be in any of a number of forms, including, but not limited to, plasmids, linear artificial chromosomes, complexed with lipids, encapsulated within liposomes, and encapsidated within viral capsid particles, particularly AAV particles. rAAV vectors can be packaged within AAV capsids to generate "recombinant adeno-associated virus capsid particles (rAAV particles)."

[0066] The term "heterologous" as used herein with respect to nucleic acid sequences, such as coding sequences and control sequences, refers to sequences that are not normally linked together and / or are not normally associated with a particular cell. Thus, a "heterologous" region of a nucleic acid construct or vector is a segment of nucleic acid within or attached to another nucleic acid molecule that is not found in association with the other molecule in nature. For example, a heterologous region of a nucleic acid construct may include a coding sequence that is flanked by sequences that are not found in association with the coding sequence in nature. Another example of a heterologous coding sequence is a construct in which the coding sequence itself is not found in nature (e.g., a synthetic sequence with different codons than the native gene).

[0067] The term "adjacent" as used herein with respect to a sequence flanked by other elements indicates that there are one or more adjacent elements upstream and / or downstream, i.e., 5' and / or 3', relative to the sequence. The term "adjacent" does not indicate that the sequences are necessarily contiguous. For example, there may be an intervening sequence between the nucleic acid encoding the transgene and the flanking element. A sequence (e.g., a transgene) "adjacent" to two other elements (e.g., TRs) indicates that one element is located 5' and the other is located 3' of the sequence; however, there may be an intervening sequence between them.

[0068] The term "inverted terminal repeat" or "ITR" sequence as used herein refers to a relatively short sequence found at the end of a viral genome that is inverted. "AAV inverted terminal repeat (ITR)" sequences are well known in the art and are typically sequences of about 145 nucleotides present at both ends of a native single-stranded AAV genome. The outermost 125 nucleotides of the ITR can be in either of two alternative orientations, resulting in heterogeneity between different AAV genomes and between the two ends of a single AAV genome. The outermost 125 nucleotides also contain several shorter regions (called A, A', B, B', C, C', and D regions) that are self-complementary, allowing intrastrand base pairing to occur within this portion of the ITR.

[0069] A "coding sequence" or a sequence "encoding" a selected polypeptide is a nucleic acid molecule that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A transcription termination sequence may be located 3' to the coding sequence.

[0070] The term "control sequence" refers to a DNA sequence necessary for the expression of an operably linked coding sequence in a particular host organism. Control sequences suitable for prokaryotes include, for example, a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0071] As used herein, the term "operably linked" and similar phrases (e.g., genetically fused), when used in reference to nucleic acids or amino acids, refer to the functional linkage of nucleic acid sequences or amino acid sequences, respectively, placed in a functional relationship with each other. For example, operably linked promoters, enhancer elements, open reading frames, 5' and 3' UTRs, and terminator sequences result in the correct production of nucleic acid molecules (e.g., RNA). In some embodiments, operably linked nucleic acid elements result in the transcription of the open reading frame, ultimately resulting in the production of a polypeptide (i.e., expression of the open reading frame). As another example, an operably linked peptide is one in which functional domains are positioned at an appropriate distance from each other to confer the intended function of each domain.

[0072] The term "promoter," as used herein in its ordinary sense, refers to a nucleotide region that contains DNA regulatory sequences derived from a gene that are capable of binding RNA polymerase and initiating transcription of a downstream (3' direction) coding sequence. Transcriptional promoters can include "inducible promoters" (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), "repressible promoters" (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), and "constitutive promoters."

[0073] The term "transgene" as used broadly herein means any heterologous nucleotide sequence incorporated into a viral vector for expression in, for example, a target cell, which may be associated with an expression control sequence, such as a promoter. It will be understood by those skilled in the art that the expression control sequence is selected based on its ability to promote transgene expression in the target cell. Examples of transgenes are nucleic acids encoding therapeutic polypeptides or detectable markers.

[0074] As used herein, the term "AAV capsid" or "AAV capsid protein" or "AAV cap" refers to the proteins encoded by the AAV capsid genes (e.g., VPI, VP2, and VP3) or variants thereof. For example, the term includes, but is not limited to, capsid proteins from any AAV serotype, such as AAV1, AAV2, AAV2i8, AAV3, AAV3-B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV13, AAV-DJ, AAV-2 / 1, AAV2 / 6, AAV2 / 7, AAV2 / 8, AAV2 / 9, AAVLK03, AAVrh10, AAVrh74, AAV44-9, or variants thereof. The term also includes capsid proteins expressed by or derived from recombinant AAV, such as chimeric AAV.

[0075] As used herein, the term "AAV capsid particle" or "AAV particle" includes at least one AAV capsid protein (e.g., a VP1 protein, a VP2 protein, a VP3 protein, or a variant thereof) and optionally encapsulates a nucleic acid derived from the AAV genome or a nucleic acid derived from the AAV genome.

[0076] The term "serotype" as used in reference to a vector or viral capsid is defined by a distinct immunological profile based on capsid protein sequence and capsid structure.

[0077] As used herein, the term "chimeric," with respect to a viral capsid or particle, means that the capsid or particle contains sequences from different parvoviruses, preferably different AAV serotypes, as described in Rabinowitz et al., U.S. Pat. No. 6,491,907, the disclosure of which is incorporated herein by reference in its entirety.

[0078] The term "recombinant" refers to a genetic entity that differs from that normally found in nature. When applied to a polynucleotide or gene, it means that the polynucleotide is the product of various combinations of cloning, restriction, and / or ligation steps, as well as other procedures, that result in the production of constructs that differ from polynucleotides found in nature.

[0079] As used herein, the term "recombinant virus" refers to a virus that has been genetically modified, for example, by the addition or insertion of a heterologous nucleic acid construct into the particle. For example, as used herein, the term "recombinant AAV particle" or "rAAV" refers to an AAV that has been genetically modified, for example, by deletion or other mutation of an endogenous AAV gene and / or the addition or insertion of a heterologous nucleic acid construct into the polynucleotide of the AAV particle.

[0080] The terms "transfected" or "transformed" or "transduced" as used herein refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed or transduced with exogenous nucleic acid. For example, the term "transfection" is used to refer to the uptake of foreign DNA by a cell, and a cell is "transfected" when the exogenous DNA has been introduced inside the cell membrane. Several transfection techniques are commonly known in the art. See, e.g., Graham et al. (1973), Virology, 52:456; Sambrook et al. (1989), Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York; Davis et al. (1986), Basic Methods in Molecular Biology, Elsevier; and Chu et al. (1981), Gene 13:197. Using such techniques, one or more exogenous molecules can be introduced into a suitable host cell. "Transduction" of a cell by a virus means that there is the transfer of nucleic acid, e.g., DNA or RNA, from a viral particle to the cell.

[0081] The term "host cell," as used herein, refers to a particular cell that can be transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. A host cell can be a bacterial cell, a yeast cell, an insect cell, or a mammalian cell.

[0082] The term "purified" refers to the isolation of a substance (compound, polynucleotide, protein, polypeptide, polypeptide composition) such that the substance of interest constitutes the majority percentage of the sample in which it is present. Typically, in a sample, a substantially purified component constitutes 50%, 80%-85%, 90-99%, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the sample. Techniques for purifying polynucleotides and polypeptides of interest are well known in the art and include, for example, ion exchange chromatography, affinity chromatography, and sedimentation by density.

[0083] As used herein, the term "pharmaceutical acceptable" means approved by a regulatory agency of the Federal or state government, or listed in the United States Pharmacopoeia, the European Pharmacopoeia, or other generally recognized pharmacopoeias, for use in animals, and more specifically, in humans.

[0084] In one embodiment, each component is "pharmaceutical acceptable" in the sense that it is compatible with other components of pharmaceutical preparations, and is suitable for use in contact with human and animal tissues or organs without undue toxicity, irritation, allergic response, immunogenicity or other problems or complications, and is commensurate with a reasonable benefit / risk ratio.See, for example, Lippincott Williams & Wilkins: Philadelphia, PA, 2005; Handbook of Pharmaceutical Excipients, 6th Edition; Rowe et al., eds.; The Pharmaceutical Press and the American Pharmaceutical Association: 2009; Handbook of Pharmaceutical Additives, 3rd Edition; Ash and Ash, eds.; Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, 2nd Edition; Gibson, ed.; CRC Press LLC: Boca Raton, FL, 2009. In some embodiments, the pharma- ceutically acceptable excipient is non-toxic to cells or mammals exposed thereto at the dosages and concentrations employed, hi some embodiments, the pharma-ceutically acceptable excipient is an aqueous pH buffered solution.

[0085] As used herein, the terms "treat", "treatment" and "treating" refer to a reduction or amelioration of the progression, severity and / or duration of a disease or condition resulting from the administration of one or more therapies. Treating can be determined by assessing whether there is a decrease, alleviation and / or reduction in one or more symptoms associated with the underlying disorder such that an improvement is observed in a patient, even though the patient may still suffer from the underlying disorder. The term "treating" includes both managing and ameliorating a disease. The terms "manage", "managing" and "management" refer to the beneficial effects a subject derives from a therapy, which does not necessarily result in a cure of the disease. "Treatment" and "treating" include: (1) preventing a disease, i.e., preventing the onset of a disease or causing a disease to occur less severely in a subject who may be exposed to or susceptible to a disease, but who has not yet experienced or displayed symptoms of the disease; (2) inhibiting a disease, i.e., halting the onset, preventing or slowing progression, or reversing the disease state; (3) alleviating symptoms of a disease, i.e., reducing the number of symptoms experienced by a subject; and (4) reducing, preventing, or slowing the progression of a disease or its symptoms. The terms "prevent," "preventing," and "prevention" refer to reducing the likelihood of the onset (or recurrence) of a disease, disorder, condition, or related condition.

[0086] As used herein, "administer", "administration", or "administering" refers to the act of injecting or otherwise physically delivering a substance (e.g., a conjugate or pharmaceutical composition provided herein) to a subject or patient (e.g., a human), for example, by oral, mucosal, topical, intradermal, parenteral, intravenous, intravitreal, intraarticular, subretinal, intramuscular, intrathecal delivery, and / or by any other physical delivery method described herein or known in the art. In certain embodiments, administration is by intravenous injection. The conjugates or compositions provided herein can be delivered systemically or to a specific tissue.

[0087] As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of a therapeutic agent (e.g., a conjugate or pharmaceutical composition provided herein) that is sufficient to treat, diagnose, prevent, delay the onset, alleviate, and / or ameliorate the severity and / or duration of a given disease, disorder, or condition and / or symptoms associated therewith. These terms also encompass the amount necessary to alleviate, slow, or ameliorate the development or progression of a given disease, alleviate, slow, or ameliorate the recurrence, occurrence, or onset of a given disease, and / or to improve or enhance the prophylactic or therapeutic effect of another therapy, or to act as a bridge to another therapy. In some embodiments, as used herein, "effective amount" also refers to the amount of a conjugate described herein to achieve a specified result. As used herein, the terms "subject" and "patient" are used interchangeably.

[0088] As used herein, a subject is a mammal, such as a non-primate (e.g., cows, pigs, horses, cats, dogs, goats, rabbits, rats, mice, etc.) or a primate (e.g., monkeys and humans), such as a human. In some embodiments, the subject is a mammal, such as a human, diagnosed with a disease or disorder provided herein. In another embodiment, the subject is a mammal, such as a human, at risk of developing a disease or disorder provided herein. In a specific embodiment, the subject is a human.

[0089] As used herein, the terms "therapies" and "therapy" can refer to any protocol, method, composition, formulation, and / or agent that can be used in the prevention, treatment, management, or amelioration of a disease or disorder or a symptom thereof (e.g., a disease or disorder provided herein or one or more symptoms or conditions associated therewith). In certain embodiments, the terms "therapies" and "therapy" refer to drug therapy, adjuvant therapy, radiation, surgery, biological therapy, supportive therapy, and / or other therapies useful in the treatment, management, prevention, or amelioration of a disease or disorder or one or more symptoms thereof. In certain embodiments, the term "therapy" refers to a therapy other than a conjugate or pharmaceutical composition thereof described herein.

[0090] As used herein, the term "angiogenesis-associated disease or disorder" refers to a disease or disorder in which angiogenesis (including aberrant angiogenesis) is implicated, for example, as either a symptom or a direct or indirect cause. The term includes diseases or disorders in which angiogenesis is implicated in their development, including, for example, cancer and ocular diseases or disorders.

[0091] The terms "about" and "approximately" mean within 20%, within 15%, within 10%, within 9%, within 8%, within 7%, within 6%, within 5%, within 4%, within 3%, within 2%, within 1%, or less of a given value or range.

[0092] As used in this disclosure and claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise.

[0093] Whenever an embodiment is described herein using the term "comprising," it is understood that other similar embodiments described with the terms "consisting of" and / or "consisting essentially of" are also provided. Whenever an embodiment is described herein using the phrase "consisting essentially of," it is also understood that other similar embodiments described with the term "consisting of" are also provided.

[0094] The term "between" when used in phrases such as "between A and B" or "between A and B" refers to a range that includes both A and B.

[0095] The term "and / or" as used herein in phrases such as "A and / or B" is intended to include both A and B; A or B; A (only); and B (only). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (only); B (only); and C (only).

[0096] 5.2. Fusion Proteins or Variants Thereof 5.2.1. Multispecific Fusion Proteins In one aspect, provided herein is a multispecific fusion protein (or polypeptide) comprising multiple VEGF binding domains (e.g., two or three VEGF binding domains) and a domain that binds to angiopoietin (e.g., angiopoietin 1 and angiopoietin 2). In some embodiments, the fusion protein provided herein comprises at least three binding domains - a first domain that binds to VEGF, a second domain that binds to VEGF, and a third domain that binds to angiopoietin (e.g., angiopoietin 1 and angiopoietin 2). In some embodiments, the multispecific fusion protein further comprises a fourth domain that binds to VEGF, and thus, in some embodiments, provided herein is a fusion protein comprising at least four binding domains - a first domain that binds to VEGF, a second domain that binds to VEGF, a third domain that binds to angiopoietin (e.g., angiopoietin 1 and angiopoietin 2), and a fourth domain that binds to VEGF (more specifically, VEGFC).

[0097] Vascular endothelial growth factor (VEGF) belongs to the PDGF supergene family characterized by eight conserved cysteines, functions as a homodimeric structure, and is produced by many cell types, including tumor cells, macrophages, platelets, keratinocytes, and renal mesangial cells. VEGF-A regulates angiogenesis and vascular permeability by activating two receptors, VEGFR-1 (Flt-1) and VEGFR-2 (KDR / Flk1). In addition to its function in the vasculature, VEGF plays a role in normal physiological functions such as bone formation, hematopoiesis, wound healing, and development. For example, the formation of new blood vessels caused by the overproduction of growth factors such as VEGF is a critical component of diseases such as tumor growth, age-related macular degeneration (AMD), and proliferative diabetic retinopathy (PDR). The VEGF-VEGFR system is both an important target for antiangiogenic therapy in cancer and an attractive system for proangiogenic therapy in the treatment of neurodegenerative and ischemic diseases. Shibuya, Genes Cancer, 2(12): 1097-1105 (2011). Binding of VEGF-C to VEGFR-3 is involved in and / or responsible for most of the biological actions of VEGFR-3. The discovery of a soluble form of VEGFR-3 (sVEGFR-3) and experiments with transgenic mice expressing this gene led to the conclusion that sVEGFR-3 inhibits signals mediated by VEGF-C and VEGF-D, inhibiting lymphatic vessel development and inducing edema.

[0098] Angiopoietins are a family of growth factors that include the glycoproteins angiopoietin 1 (ANGPT1 or Ang1) and angiopoietin 2 (ANGPT2 or Ang2) and orthologues 3 (mouse) and 4 (human). Angiopoietins are involved in embryonic vascular development. Angiopoietin 1 is expressed by numerous cell types, while angiopoietin 2 is almost exclusively restricted to endothelial cells. Both act on the TIE2 receptor tyrosine kinase, which is found primarily on endothelial cells and hematopoietic stem cells. These proteins are key regulators of angiogenesis and maintenance of vascular integrity. As used herein, "angiopoietin" refers to any angiopoietin peptide, including angiopoietin 1 and angiopoietin 2; "angiopoietin-binding domain" refers to a domain that binds to angiopoietin 1 and / or angiopoietin 2.

[0099] In some embodiments, the first and second domains provided herein bind to human VEGF. In some embodiments, the third domain provided herein binds to human angiopoietin (e.g., angiopoietin 1 and angiopoietin 2). In some embodiments, the fourth domain provided herein binds to human VEGF (e.g., VEGFC).

[0100] In some embodiments, the fusion proteins provided herein modulate one or more VEGF activities. In some embodiments, the fusion proteins provided herein modulate one or more angiopoietin (e.g., angiopoietin 1 and angiopoietin 2) activities.

[0101] In some embodiments, the first domain provided herein has a cytotoxicity of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g., 10 -8 M~10 -13 M, for example, 10 -9 M~10 -13 Dissociation constant (KD ) and binds to VEGF (e.g., human VEGF).

[0102] In some embodiments, the second domain provided herein has a cytotoxicity of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g., 10 -8 M~10 -13 M, for example, 10 -9 M~10 -13 Dissociation constant (K D ) and binds to VEGF (e.g., human VEGF).

[0103] In some embodiments, the third domain provided herein has a cytotoxicity of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g., 10 -8 M~10 -13 M, for example, 10 -9 M~10 -13 Dissociation constant (K D ) and binds to angiopoietins (e.g., angiopoietin 1 and angiopoietin 2).

[0104] In some embodiments, the fourth domain provided herein has a cytotoxicity of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g., 10 -8 M~10 -13 M, for example, 10 -9 M~10 -13 Dissociation constant (K D ) and binds to VEGF (e.g., human VEGF).

[0105] K D can be measured using techniques known in the art and described in Section 5.1 above.

[0106] In some embodiments, the first domain is derived from VEGF receptor-1 (VEGFR-1 or FLT-1). In some embodiments, the first domain comprises IgG-like domain 2 (or domain 2 or D2) of VEGFR-1 or a variant thereof. This domain is therefore referred to as D2 in the figures, e.g., in FIG. 1, FIG. 2, FIG. 8, and FIG. 9A-9I. In some embodiments, the first domain comprises or consists of the amino acid sequence of SEQ ID NO: 1. In some embodiments, the first domain comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO: 1.

[0107] In some embodiments, the second domain is derived from VEGF receptor-2 (VEGFR-2 or Flk-1). In some embodiments, the second domain comprises IgG-like domain 3 (or domain 3 or D3) of VEGFR-2 or a variant thereof. This domain is therefore referred to as D3 in the figures, e.g., in FIG. 1, FIG. 2, FIG. 8, and FIG. 9A-9I. In some embodiments, the second domain comprises or consists of the amino acid sequence of SEQ ID NO:2. In some embodiments, the second domain comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to an SEQ ID NO:2.

[0108] In some embodiments, the third domain (ABD) comprises one or more repeats of the amino acid sequence of SEQ ID NO:3. In some embodiments, the third domain comprises one or more repeats of the amino acid sequence of SEQ ID NO:51. In some embodiments, the third domain comprises two repeats of the amino acid sequence of SEQ ID NO:3. In some embodiments, the third domain comprises two repeats of the amino acid sequence of SEQ ID NO:51. In other embodiments, the third domain comprises the amino acid sequence of SEQ ID NO:3 and the amino acid sequence of SEQ ID NO:51. When the ABD in the fusion protein comprises the amino acid sequence of SEQ ID NO:3 and the amino acid sequence of SEQ ID NO:51, these two sequences can be in any order, for example, the amino acid sequence of SEQ ID NO:3 can be N-terminal or C-terminal to the amino acid sequence of SEQ ID NO:51.

[0109] In some specific embodiments, the third domain (ABD) comprises or consists of the amino acid sequence of SEQ ID NO: 4. In some embodiments, the third domain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO: 4 and capable of binding to angiopoietin (e.g., angiopoietin 2 or Ang2).

[0110] In some embodiments, the third domain (ABD) comprises or consists of the amino acid sequence of SEQ ID NO: 52. In some embodiments, the third domain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO: 52 and capable of binding to angiopoietin (e.g., angiopoietin 2 or Ang2).

[0111] In some embodiments, the third domain (ABD) comprises or consists of the amino acid sequence of SEQ ID NO: 53. In some embodiments, the third domain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO: 53 and capable of binding to angiopoietin (e.g., angiopoietin 2 or Ang2).

[0112] In some embodiments, the third domain (ABD) comprises or consists of the amino acid sequence of SEQ ID NO: 54. In some embodiments, the third domain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO: 54 and capable of binding to angiopoietin (e.g., angiopoietin 2 or Ang2).

[0113] In certain embodiments, the third domain that binds to angiopoietin (e.g., angiopoietin 1 and angiopoietin 2) is N-terminal to the first domain or the second domain. In a specific embodiment, the polypeptide comprises, from N-terminus to C-terminus, the third domain, the first domain, and the second domain. In another specific embodiment, the polypeptide comprises, from N-terminus to C-terminus, the third domain, the second domain, and the first domain.

[0114] When a fourth domain, i.e., a VEGFC binding domain (also called Trap C), is present in the fusion protein, in some embodiments, the VEGFC binding domain is derived from VEGFR-2 (e.g., domain 2). In other embodiments, the VEGFC binding domain is derived from VEGFR-3 (e.g., domain 1 and / or domain 2).

[0115] In some embodiments, the VEGFC binding domain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:55.

[0116] In some embodiments, the VEGFC binding domain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:56.

[0117] In some embodiments, the VEGFC binding domain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:57.

[0118] In some embodiments, the polypeptide further comprises an Fc region of an antibody. In some embodiments, the Fc region is derived from human IgG. In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO:5. In some embodiments, the Fc region is at the C-terminus of the polypeptide. In other embodiments, the Fc region is not at the C-terminus of the polypeptide.

[0119] The various domains in the fusion protein may be in any order. In some embodiments, D2, D3, ABD, Fc region, and / or Trap C are in the order as shown in Figure 1, Figure 2, Figure 8, and Figures 9A-9I. In some more specific embodiments, D2, D3, ABD, Fc region, and / or Trap C are in the order as shown in Figure 9A. In some more specific embodiments, D2, D3, ABD, Fc region, and / or Trap C are in the order as shown in Figure 9B. In some more specific embodiments, D2, D3, ABD, Fc region, and / or Trap C are in the order as shown in Figure 9C. In some more specific embodiments, D2, D3, ABD, Fc region, and / or Trap C are in the order as shown in Figure 9D. In some more specific embodiments, D2, D3, ABD, Fc region, and / or Trap C are in the order as shown in Figure 9E. In some more specific embodiments, D2, D3, ABD, Fc region, and / or Trap C are in the order as shown in Figure 9F. In some more specific embodiments, D2, D3, ABD, Fc region, and / or Trap C are in the order as shown in Figure 9G. In some more specific embodiments, D2, D3, ABD, Fc region, and / or Trap C are in the order as shown in Figure 9H. In some more specific embodiments, D2, D3, ABD, Fc region, and / or Trap C are in the order as shown in Figure 9I.

[0120] In some embodiments, the polypeptide further comprises a signal peptide at the N-terminus of the polypeptide. In general, a signal peptide is a peptide sequence that targets the polypeptide to a desired site in a cell. In some embodiments, the signal peptide targets the effector molecule to the secretory pathway of the cell and allows the effector molecule to be incorporated and anchored in the lipid bilayer. Signal peptides, including natural protein signal sequences or synthetic non-natural signal sequences, that are suitable for use in the fusion proteins described herein will be clear to those skilled in the art. In some specific embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO:6.

[0121] In some embodiments, the polypeptide further comprises one or more linkers between the various domains described above. The various domains described herein can be fused to each other via peptide linkers. In some embodiments, certain domains are directly fused to each other without any peptide linker. The peptide linkers connecting different domains can be the same or different. In some embodiments, the polypeptide provided herein comprises peptide linkers between certain domains but not between other domains therein.

[0122] Each peptide linker in the polypeptides provided herein may comprise the same or different length and / or sequence. Each peptide linker may be independently selected and optimized. The length, degree of flexibility, and / or other properties of the peptide linker used in the fusion protein may have some effect on properties including, but not limited to, affinity, specificity, or avidity for one or more specific target molecules. In some embodiments, the peptide linker comprises flexible residues (e.g., glycine and serine) so that adjacent domains can move freely relative to each other. For example, a glycine-serine doublet can be a suitable peptide linker.

[0123] The peptide linker can be of any suitable length. In some embodiments, the peptide linker is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, or more amino acids in length. In some embodiments, the peptide linker is about 100, 75, 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or less amino acids in length. In some embodiments, the length of the peptide linker is any of about 1 amino acid to about 10 amino acids, about 1 amino acid to about 20 amino acids, about 1 amino acid to about 30 amino acids, about 5 amino acids to about 15 amino acids, about 10 amino acids to about 25 amino acids, about 5 amino acids to about 30 amino acids, about 10 amino acids to about 30 amino acids in length, or about 30 amino acids to about 50 amino acids.

[0124] The peptide linker can have a natural sequence or a non-natural sequence. For example, a sequence derived from the hinge region of a heavy chain-only antibody can be used as a linker. See, for example, WO1996 / 34103. In some embodiments, the peptide linker is a flexible linker. In some embodiments, the peptide linker provided herein is a (GxS)n linker (wherein x and n can be, independently, an integer between 3 and 12, including 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or greater). Exemplary flexible linkers include glycine polymers (G) n , glycine-serine polymers (e.g., (GS) n , (GSGGS) n , GGGS) n , and (GGGGS) n (wherein n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Exemplary peptide linkers are listed in the table below. Table 1. Exemplary peptide linkers [Table 1]

[0125] The fusion proteins of the present disclosure may include a hinge domain located between the above domains. A hinge domain is an amino acid segment usually found between two domains of a protein that may allow protein flexibility and movement of one or both of the domains relative to one another.

[0126] The hinge domain may contain about 10 to 100 amino acids, e.g., about any one of 15 to 75 amino acids, 20 to 50 amino acids, or 30 to 60 amino acids. In some embodiments, the hinge domain is at least about any one of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 amino acids in length.

[0127] In some embodiments, the hinge domain is a hinge domain of a natural protein. In some embodiments, the hinge domain is at least a portion of a hinge domain of a natural protein. The hinge domain of an antibody, for example, an IgG, IgA, IgM, IgE, or IgD antibody, is also adapted for use in the fusion proteins described herein. In some embodiments, the hinge domain is a hinge domain that connects the constant domains CH1 and CH2 of the antibody. In some embodiments, the hinge domain is of an antibody and comprises the hinge domain of the antibody and one or more constant regions of the antibody. In some embodiments, the hinge domain comprises the hinge domain of the antibody and the CH3 constant region of the antibody. In some embodiments, the hinge domain comprises the hinge domain of the antibody and the CH2 and CH3 constant regions of the antibody. In some embodiments, the antibody is an IgG, IgA, IgM, IgE, or IgD antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the hinge region comprises the hinge region and the CH2 and CH3 constant regions of an IgG1 antibody. In some embodiments, the hinge region comprises the hinge region and the CH3 constant region of an IgG1 antibody.

[0128] Non-naturally occurring peptides may also be used as hinge domains of the fusion proteins described herein. In some embodiments, the linker is a self-cleaving linker.

[0129] Other linkers known in the art and described, for example, in WO2016014789, WO2015158671, WO2016102965, US20150299317, WO2018067992, US7741465, Colcher et al., J. Nat. Cancer Inst. 82:1191-1197 (1990), and Bird et al., Science 242:423-426 (1988), the disclosures of each of which are incorporated herein by reference, can also be included in the fusion proteins provided herein.

[0130] In certain embodiments, the polypeptides provided herein are illustrated in FIG.

[0131] In some specific embodiments, provided herein is a polypeptide comprising the amino acid sequence of SEQ ID NO:7.

[0132] In some specific embodiments, provided herein is a polypeptide comprising the amino acid sequence of SEQ ID NO:8.

[0133] In some specific embodiments, provided herein is a polypeptide comprising the amino acid sequence of SEQ ID NO:9.

[0134] In some specific embodiments, provided herein is a polypeptide comprising the amino acid sequence of SEQ ID NO:10.

[0135] In certain embodiments, the polypeptides provided herein are illustrated in FIG.

[0136] In some specific embodiments, provided herein is a polypeptide comprising the amino acid sequence of SEQ ID NO:58.

[0137] In some specific embodiments, provided herein is a polypeptide comprising the amino acid sequence of SEQ ID NO:59.

[0138] In some specific embodiments, provided herein is a polypeptide comprising the amino acid sequence of SEQ ID NO:60.

[0139] In some specific embodiments, provided herein is a polypeptide comprising the amino acid sequence of SEQ ID NO:61.

[0140] In some specific embodiments, provided herein is a polypeptide comprising the amino acid sequence of SEQ ID NO:62.

[0141] In some specific embodiments, provided herein is a polypeptide comprising the amino acid sequence of SEQ ID NO:63.

[0142] In some specific embodiments, provided herein is a polypeptide comprising the amino acid sequence of SEQ ID NO:64.

[0143] In some specific embodiments, provided herein is a polypeptide comprising the amino acid sequence of SEQ ID NO:65.

[0144] In some specific embodiments, provided herein is a polypeptide comprising the amino acid sequence of SEQ ID NO:66.

[0145] In certain embodiments, the fusion proteins described herein contain an amino acid sequence having a particular percent identity to any one of the polypeptides described in Section 6 below.

[0146] The determination of percent identity between two sequences (e.g., amino acid sequences or nucleic acid sequences) can be accomplished using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm utilized for comparing two sequences is the algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264 2268 (1990), modified as found in Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873 5877 (1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., J. Mol. Biol. 215:403 (1990). BLAST nucleotide searches can be performed, for example, with the NBLAST nucleotide program parameters set to score=100, word length=12 to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed, for example, with XBLAST program parameters set at score=50, word length=3 to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25:3389 3402 (1997). Alternatively, PSI BLAST can be used to perform an iterated search that detects distant relationships between molecules (ibid.). When utilizing BLAST, Gapped BLAST, and PSI Blast programs, the default parameters of the respective programs (e.g., those of XBLAST and NBLAST) can be used (see, e.g., the National Center for Biotechnology Information (NCBI) site at http: / / www.ncbi.nlm.nih.gov). Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS 4:11-17 (1998).Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

[0147] The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. Typically, only exact matches are counted when calculating percent identity.

[0148] In some embodiments, a polypeptide is provided having at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 7-10 and 58-66. In some embodiments, a polypeptide sequence having at least about any one of 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to a reference sequence, but two domains or three domains within the polypeptide comprising that sequence retain the ability to bind to VEGF and one domain within the polypeptide retains the ability to bind to angiopoietin (e.g., angiopoietin 1 and angiopoietin 2).

[0149] In some embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in an amino acid sequence selected from SEQ ID NOs: 7-10 and 58-66.

[0150] In certain embodiments, the fusion protein described herein comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:7, wherein the first domain and the second domain retain the ability to bind to VEGF and the third domain retains the ability to bind to angiopoietin (e.g., angiopoietin 1 and angiopoietin 2).

[0151] In certain embodiments, the fusion protein described herein comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:8, wherein the first domain and the second domain retain the ability to bind to VEGF and the third domain retains the ability to bind to angiopoietin (e.g., angiopoietin 1 and angiopoietin 2).

[0152] In certain embodiments, the fusion protein described herein comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:9, wherein the first domain and the second domain retain the ability to bind to VEGF and the third domain retains the ability to bind to angiopoietin.

[0153] In certain embodiments, the fusion protein described herein comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 10, wherein the first domain and the second domain retain the ability to bind to VEGF and the third domain retains the ability to bind to angiopoietin (e.g., angiopoietin 1 and angiopoietin 2).

[0154] In certain embodiments, the fusion protein described herein comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:58, wherein the first domain, the second domain, and the fourth domain retain the ability to bind to VEGF, and the third domain retains the ability to bind to angiopoietin (e.g., angiopoietin 1 and angiopoietin 2).

[0155] In certain embodiments, the fusion protein described herein comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:59, wherein the first domain, the second domain, and the fourth domain retain the ability to bind to VEGF, and the third domain retains the ability to bind to angiopoietin (e.g., angiopoietin 1 and angiopoietin 2).

[0156] In certain embodiments, the fusion protein described herein comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 60, wherein the first domain, the second domain, and the fourth domain retain the ability to bind to VEGF, and the third domain retains the ability to bind to angiopoietin (e.g., angiopoietin 1 and angiopoietin 2).

[0157] In certain embodiments, the fusion protein described herein comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:61, wherein the first domain, the second domain, and the fourth domain retain the ability to bind to VEGF, and the third domain retains the ability to bind to angiopoietin (e.g., angiopoietin 1 and angiopoietin 2).

[0158] In certain embodiments, the fusion protein described herein comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:62, wherein the first domain, the second domain, and the fourth domain retain the ability to bind to VEGF, and the third domain retains the ability to bind to angiopoietin (e.g., angiopoietin 1 and angiopoietin 2).

[0159] In certain embodiments, the fusion protein described herein comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:63, wherein the first domain, the second domain, and the fourth domain retain the ability to bind to VEGF, and the third domain retains the ability to bind to angiopoietin (e.g., angiopoietin 1 and angiopoietin 2).

[0160] In certain embodiments, the fusion protein described herein comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:64, wherein the first domain, the second domain, and the fourth domain retain the ability to bind to VEGF, and the third domain retains the ability to bind to angiopoietin (e.g., angiopoietin 1 and angiopoietin 2).

[0161] In certain embodiments, the fusion protein described herein comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:65, wherein the first domain, the second domain, and the fourth domain retain the ability to bind to VEGF, and the third domain retains the ability to bind to angiopoietin (e.g., angiopoietin 1 and angiopoietin 2).

[0162] In certain embodiments, the fusion protein described herein comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:66, wherein the first domain, the second domain, and the fourth domain retain the ability to bind to VEGF, and the third domain retains the ability to bind to angiopoietin (e.g., angiopoietin 1 and angiopoietin 2).

[0163] Other exemplary fusion proteins provided herein are described in more detail in the following sections: In some embodiments, a fusion protein according to any of the above embodiments may incorporate any of the features described in Sections 5.2.2-5.2.4 below, either alone or in combination.

[0164] 5.2.2. Polypeptide Variants and Modifications In some embodiments, amino acid sequence modification of the fusion proteins described herein is contemplated. For example, it may be desirable to optimize the binding affinity and / or other biological characteristics of the fusion protein, including, but not limited to, specificity, thermostability, expression level, glycosylation, reduced immunogenicity, or solubility. Thus, in addition to the fusion proteins described herein, it is contemplated that variants of the fusion proteins described herein can be prepared. For example, peptide variants can be prepared by introducing appropriate nucleotide changes into the coding DNA and / or by synthesis of the desired polypeptide. Those skilled in the art who understand amino acid changes can modify post-translational processes of peptides.

[0165] A mutation can be a substitution, deletion or insertion of one or more codons encoding the polypeptide, which results in a change in the amino acid sequence when compared to the original polypeptide.

[0166] Amino acid substitutions can be, for example, conservative amino acid substitutions that result from replacing one amino acid with another amino acid having similar structural and / or chemical properties, such as a leucine with a serine. Standard techniques known to those of skill in the art, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis that result in amino acid substitutions, can be used to introduce mutations into the nucleotide sequence encoding the molecules provided herein. Insertions or deletions can optionally range from about 1 to 5 amino acids. In certain embodiments, the substitutions, deletions, or insertions include fewer than 25 amino acid substitutions, fewer than 20 amino acid substitutions, fewer than 15 amino acid substitutions, fewer than 10 amino acid substitutions, fewer than 5 amino acid substitutions, fewer than 4 amino acid substitutions, fewer than 3 amino acid substitutions, or fewer than 2 amino acid substitutions compared to the original molecule. In specific embodiments, the substitutions are conservative amino acid substitutions made at one or more predicted non-essential amino acid residues. Permitted mutations can be determined by systematically making amino acid insertions, deletions, or substitutions in a sequence and testing the resulting variants for activity exhibited by the parent peptide.

[0167] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing multiple residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is a polypeptide with an N-terminal methionyl residue.

[0168] Fusion proteins made by conservative amino acid substitution are included in the present disclosure. In conservative amino acid substitution, an amino acid residue is replaced with an amino acid residue having a side chain with a similar charge. As mentioned above, families of amino acid residues with side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be introduced randomly along all or part of the coding sequence, for example by saturation mutagenesis, and the resulting mutants can be screened for biological activity to identify mutants that retain activity. After mutagenesis, the encoded protein can be expressed and the activity of the protein can be determined. Conservative (e.g., within a group of amino acids with similar properties and / or side chains) substitutions can be made to maintain or not significantly change the properties. Exemplary substitutions are shown in the table below. Table 2. Amino acid substitutions [Table 2]

[0169] Amino acids can be grouped according to the similarity of their side chain properties: (1) nonpolar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); and (4) basic: Lys (K), Arg (R), His (H) (see, e.g., Lehninger, Biochemistry 73-75 (2d ed., 1975)). Alternatively, natural residues can be divided into groups based on common side chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that affect chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

[0170] For example, any cysteine ​​residue not involved in maintaining the proper conformation of the polypeptides provided herein can also be substituted with another amino acid, such as, for example, alanine or serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking.

[0171] Non-conservative substitutions involve exchanging a member of one of these classes for another class.

[0172] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Exemplary terminal insertions include polypeptides with an N-terminal methionyl residue.

[0173] Mutations can be made using methods known in the art, such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis (see, e.g., Carter, Biochem J. 237:1-7 (1986), and Zoller et al., Nucl. Acids Res. 10:6487-500 (1982)), cassette mutagenesis (see, e.g., Wells et al., Gene 34:315-23 (1985)), or other known techniques can be performed on cloned DNA to produce polypeptide variant DNA.

[0174] Covalent modifications of the fusion proteins provided herein are included within the scope of this disclosure. Covalent modifications include reacting targeted amino acid residues of the polypeptides provided herein with organic derivatizing agents capable of reacting with selected side chains or N- or C-terminal residues of the polypeptides. Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of the hydroxyl groups of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine, and histidine side chains (see, e.g., Creighton, Proteins: Structure and Molecular Properties, 79-86 (1983)), acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

[0175] In some embodiments, the fusion protein provided herein is chemically modified, for example, by covalently binding any type of molecule to the fusion protein.Polypeptide derivatives can include polypeptides that are chemically modified, for example, by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, binding to cellular ligands or other proteins, or conjugation to one or more immunoglobulin domains (e.g., Fc or part of Fc).Any of the numerous chemical modifications can be performed by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formulation, metabolic synthesis of tunicamycin, etc.In addition, polypeptides can contain one or more non-typical amino acids.

[0176] In some embodiments, the fusion proteins provided herein are modified to increase or decrease the degree to which the fusion protein is glycosylated. Addition or deletion of glycosylation sites to a polypeptide can be conveniently accomplished by modifying the amino acid sequence such that one or more glycosylation sites are created or removed.

[0177] When the fusions provided herein are fused to an Fc region, the carbohydrate attached thereto can be modified. Native antibodies produced by mammals usually contain biantennary branched oligosaccharides that are usually attached by N-linkage to Asn297 of the CH2 domain of the Fc region. See, for example, Wright et al., TIBTECH 15:26-32 (1997). The oligosaccharides can include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to the GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modification of the oligosaccharides in the binding molecules provided herein can be performed to generate variants with specific improved properties.

[0178] In molecules that contain an Fc region, one or more amino acid modifications can be introduced into the Fc region, thereby creating an Fc region variant. The Fc region variant can include a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) that contains an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0179] In some embodiments, the present application contemplates variants that retain some, but not all, effector functions, making them desirable candidates for applications where the half-life of the binding molecule in vivo is important, but certain effector functions (e.g., complement and ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / depleted CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to confirm that the binding molecule lacks FcγR binding (and thus likely lacks ADCC activity) but retains FcRn binding activity. Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); U.S. Pat. No. 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods can be utilized (see, e.g., the ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Mountain View, CA; and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest can be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays can also be performed to confirm that the binding molecule is unable to bind C1q and therefore lacks CDC activity.See, e.g., the C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, CDC assays can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0180] Binding molecules with reduced effector function include those with substitutions of one or more of residues 238, 265, 269, 270, 297, 327, and 329 of the Fc region (U.S. Patent No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant with substitutions of residues 265 and 297 to alanine (U.S. Patent No. 7,332,581).

[0181] Certain variants have been described with improved or reduced binding to FcR (see, e.g., U.S. Pat. No. 6,737,056; WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001)).

[0182] In some embodiments, the variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, e.g., substitutions at positions 298, 333, and / or 334 (EU residue numbering) of the Fc region.

[0183] In some embodiments, modifications are made in the Fc region that result in altered (i.e., either improved or reduced) C1q binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol. 164: 4178-4184 (2000).

[0184] Binding molecules with increased half-life and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), have been described in US2005 / 0014934A1 (Hinton et al.). These molecules comprise an Fc region having one or more substitutions therein which improve binding of the Fc region to FcRn. Such Fc variants include those having a substitution at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, e.g., a substitution at Fc region residue 434 (U.S. Patent No. 7,371,826). See also Duncan and Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351 for other examples of Fc region variants.

[0185] In some embodiments, it may be desirable to generate cysteine ​​modified polypeptides, in which one or more residues of polypeptide are replaced with cysteine ​​residues.In some embodiments, the replaced residues occur at accessible sites of peptide.By replacing these residues with cysteine, reactive thiol groups are thereby placed at accessible sites of peptide, which can be used to conjugate peptides to other moieties, such as drug moieties or linker-drug moieties, to generate immunoconjugates, as further described herein.

[0186] 5.2.3. Preparation of Fusion Proteins Also provided herein are methods of making the various fusion proteins provided herein.

[0187] In a specific embodiment, the fusion proteins provided herein are recombinantly expressed. Recombinant expression of the fusion proteins provided herein may require the construction of an expression vector containing a polynucleotide encoding the protein or a fragment thereof. Once a polynucleotide encoding the protein or a fragment thereof provided herein is obtained, a vector for the production of the molecule can be produced by recombinant DNA technology using techniques well known in the art. Thus, methods for preparing a protein by expressing a polynucleotide containing a coding nucleotide sequence are described herein. Methods well known to those skilled in the art can be used to construct an expression vector containing a coding sequence and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Also provided is a replicable vector containing a nucleotide sequence encoding the fusion protein or a fragment thereof provided herein, operably linked to a promoter.

[0188] The expression vector can be transferred into a host cell by conventional techniques, and the transfected cells are then cultured by conventional techniques to produce the fusion proteins provided herein. Thus, also provided herein is a host cell containing a polynucleotide encoding a fusion protein provided herein, or a fragment thereof, operably linked to a heterologous promoter.

[0189] A variety of host-expression vector systems can be utilized to express the fusion proteins provided herein. Such host-expression systems represent vehicles in which a coding sequence of interest may be produced and subsequently purified, but also represent cells that, when transformed or transfected with the appropriate nucleotide coding sequence, can express the fusion proteins provided herein in situ. These include, but are not limited to, microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with a recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vector containing the coding sequence; yeast (e.g., Saccharomyces Pichia) transformed with a recombinant yeast expression vector containing the coding sequence; insect cell systems infected with a recombinant viral expression vector (e.g., baculovirus) containing the coding sequence; plant cell systems infected with a recombinant viral expression vector (e.g., Cauliflower Mosaic Virus, CaMV, Tobacco Mosaic Virus, TMV) or transformed with a recombinant plasmid expression vector (e.g., Ti plasmid) containing the coding sequence; or mammalian cell systems (e.g., COS, CHO, BHK, 293, NS0, and 3T3 cells) harboring a recombinant expression construct containing a promoter derived from the genome of a mammalian cell (e.g., metallothionein promoter) or a promoter derived from a mammalian virus (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter). In particular, bacterial cells such as E. coli for the expression of whole recombinant molecules, or eukaryotic cells can be used for the expression of recombinant fusion proteins. For example, mammalian cells such as Chinese hamster ovary cells (CHO) in conjunction with vectors such as the major immediate early gene promoter element from human cytomegalovirus are effective expression systems for antibodies or variants thereof. In a specific embodiment, the expression of the nucleotide sequence encoding the fusion protein provided herein is regulated by a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

[0190] In bacterial systems, several expression vectors can be advantageously selected depending on the intended use of the fusion protein being expressed. When large quantities of such fusion proteins are to be produced, for example for the creation of pharmaceutical compositions of the fusion proteins, vectors directing high-level expression of fusion protein products that are easily purified may be desirable. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al., EMBO 12:1791 (1983)), in which coding sequences can be individually ligated into the vector in frame with the lac Z coding region to produce a fusion protein; pIN vectors (Inouye and Inouye, Nucleic Acids Res. 13:3101-3109 (1985); Van Heeke and Schuster, J. Biol. Chem. 24:5503-5509 (1989)); and the like. pGEX vectors can also be used to express foreign polypeptides as fusion proteins with glutathione 5-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to matrix glutathione agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to contain thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

[0191] In mammalian host cells, several virus-based expression systems are available. When adenovirus is used as an expression vector, the coding sequence of interest can be ligated to the adenovirus transcription / translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) results in a recombinant virus that is viable in the infected host and capable of expressing the fusion protein (see, e.g., Logan and Shenk, Proc. Natl. Acad. Sci. USA 8 1:355-359 (1984)). Specific initiation signals may be required for efficient translation of the inserted coding sequence. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see, eg, Bittner et al., Methods in Enzymol. 153:51-544 (1987)).

[0192] Furthermore, a host cell line can be selected that modulates the expression of the inserted sequences or modifies and processes the gene product in a specific manner desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products can be important for the function of the protein. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. To ensure correct modification and processing of the expressed foreign protein, an appropriate cell line or host system can be selected. To this end, eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used. Such mammalian host cells include, but are not limited to, CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT2O, and T47D, NS0 (a mouse myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O, and HsS78Bst cells.

[0193] For long-term, high-yield production of recombinant proteins, stable expression can be utilized. For example, cell lines can be engineered that stably express the fusion protein. Rather than using expression vectors containing viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) and a selection marker. After introduction of the foreign DNA, engineered cells can be grown in rich medium for 1-2 days and then transferred to selective medium. The selection marker in the recombinant plasmid confers resistance to selection, allowing the cells to stably integrate the plasmid into their chromosomes and grow to form foci, which can then be cloned and expanded into cell lines. This method can be advantageously used to engineer cell lines that express the fusion protein. Such engineered cell lines can be particularly useful in screening and evaluating compositions that interact directly or indirectly with the binding molecule.

[0194] Several selection systems may be used, including, but not limited to, the herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223 (1977)), hypoxanthine guanine phosphoribosyltransferase (Szybalska and Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adenine phosphoribosyltransferase (Lowy et al., Cell 22:8-17 (1980)) genes, which may be utilized in tk- cells, hgprt- cells, or aprt- cells, respectively. Anti-metabolite resistance can also be used as the basis for selection of the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance to mycophenolic acid (Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78:2072 (1981)); neo, which confers resistance to the aminoglycoside G-418 (Wu and Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May, TIB TECH 11(5):155-215 (1993); and hygro, which confers resistance to hygromycin (Santerre et al., 1984, Gene 30:147).The desired recombinant clones can be selected by routine application of methods generally known in the art of recombinant DNA technology, such as those described in, for example, Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and Dracopoli et al. (eds.), Current Protocols in Human Genetics, Chapters 12 and 13, John Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), which are incorporated herein by reference in their entireties.

[0195] The expression level of the fusion protein can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3, Academic Press, New York, 1987). If the vector-based marker expressing the fusion protein is amplifiable, increasing the level of inhibitor present in the host cell culture will increase the number of copies of the marker gene. Since the amplified region is associated with the fusion protein gene, production of the fusion protein will also increase (Crouse et al., Mol. Cell. Biol. 3:257 (1983)).

[0196] A host cell can be co-transfected with multiple expression vectors provided herein. The vectors can contain the same selectable markers that allow equal expression of each encoded polypeptide. Alternatively, a single vector can be used that encodes and expresses multiple polypeptides. The coding sequence can include cDNA or genomic DNA.

[0197] Once a fusion protein provided herein has been produced by recombinant expression, it can be purified by any method known in the art for the purification of polypeptides (e.g., immunoglobulin molecules), such as, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for a specific antigen followed by Protein A, sizing column chromatography, and Kappa Select affinity chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. Additionally, the fusion protein molecules provided herein can be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification.

[0198] Various aspects of the recombinant production of the fusion proteins provided herein are described in more detail below in the context of prokaryotic or eukaryotic cells.

[0199] (Recombinant production in prokaryotic cells) Polynucleic acid sequences encoding the fusion proteins of the present disclosure can be obtained using standard recombinant techniques. For example, polynucleotides can be synthesized using a nucleotide synthesizer or PCR techniques. Once obtained, the sequences encoding the polypeptides are inserted into a recombinant vector capable of replicating and expressing heterologous polynucleotides in a prokaryotic host. Many vectors available and known in the art can be used for the purposes of the present disclosure. The selection of an appropriate vector depends mainly on the size of the nucleic acid to be inserted into the vector and the specific host cell to be transformed with the vector. Each vector contains various components depending on its function (amplification or expression of heterologous polynucleotides, or both) and its compatibility with the specific host cell in which it resides. Vector components usually include, but are not limited to, an origin of replication, a selectable marker gene, a promoter, a ribosome binding site (RBS), a signal sequence, the heterologous nucleic acid insert, and a transcription termination sequence.

[0200] Generally, plasmid vectors containing replicon and control sequences derived from species compatible with the host cell are used in connection with these hosts. The vector usually carries a replication site and a marking sequence capable of providing phenotypic selection in transformed cells. For example, E. coli is usually transformed with pBR322, a plasmid derived from E. coli species. pBR322 contains genes encoding ampicillin (Amp) and tetracycline (Tet) resistance, thus providing a simple means for identifying transformed cells. pBR322, its derivatives, or other microbial plasmids or bacteriophages may also contain, or be modified to contain, promoters that can be used by the microorganism for expression of endogenous proteins. Examples of pBR322 derivatives used for expression of specific antibodies are described in detail in Carter et al., U.S. Pat. No. 5,648,237.

[0201] Additionally, phage vectors containing replicon and control sequences compatible with the host microorganism can be used as transforming vectors in the context of these hosts. For example, bacteriophages such as GEM™-11 can be utilized to generate recombinant vectors that can be used to transform susceptible host cells, such as E. coli LE392.

[0202] The expression vector of the present application may contain two or more promoter-cistron pairs, each encoding a polypeptide component. A promoter is a non-translated regulatory sequence located upstream (5') of a cistron that regulates its expression. Prokaryotic promoters are usually divided into two classes: inducible and constitutive. An inducible promoter is a promoter that causes an increased level of transcription of the cistron under its control in response to a change in culture conditions, e.g., the presence or absence of a nutrient or a change in temperature.

[0203] Numerous promoters recognized by various potential host cells are well known. The selected promoter can be operably linked to the cistron DNA encoding the present fusion protein by removing the promoter from the source DNA by restriction enzyme digestion and inserting the isolated promoter sequence into the vector of the present application. Both native promoter sequences and many heterologous promoters can be used to direct the amplification and / or expression of the target gene. In some embodiments, heterologous promoters are utilized because they usually allow more transcription and higher yields of the expressed target gene compared to the native target polypeptide promoter.

[0204] Suitable promoters for use with prokaryotic hosts include the PhoA promoter, the galactamase and lactose promoter systems, the tryptophan (trp) promoter system, and hybrid promoters such as the tac or trc promoter. However, other promoters that are functional in bacteria (e.g., other known bacterial or phage promoters) are suitable as well. These nucleic acid sequences have been published, allowing one skilled in the art to operably link them to a cistron encoding a target peptide (Siebenlist, Cell 20:269-281 (1980)) using linkers or adapters to provide any required restriction sites.

[0205] In one embodiment, each cistron in the recombinant vector contains a secretory signal sequence component that directs translocation of the expressed polypeptide across the membrane. In general, the signal sequence may be a component of the vector or it may be part of the target polypeptide DNA inserted into the vector. The signal sequence selected for the present invention should be one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. For prokaryotic host cells that do not recognize and process the signal sequence native to the heterologous polypeptide, the signal sequence is replaced by a prokaryotic signal sequence selected from the group consisting of, for example, alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II (STII) leader, LamB, PhoE, PelB, OmpA, and MBP. In some embodiments of the present disclosure, the signal sequence used in both cistrons of the expression system is a STII signal sequence or a variant thereof.

[0206] In some embodiments, production of the fusion proteins according to the present disclosure can occur in the cytoplasm of the host cell and therefore does not require the presence of a secretion signal sequence within each cistron. -The recombinant yeast strain (S. cerevisiae) provides cytoplasmic conditions that are favorable for disulfide bond formation, thereby allowing proper folding and association of the expressed protein subunits.

[0207] Suitable prokaryotic host cells for expressing the fusion proteins of the present disclosure include Archaebacteria and Eubacteria, such as Gram-negative or Gram-positive organisms. Examples of useful bacteria include Escherichia (e.g., E. coli), Bacilli (e.g., Bacillus subtilis), Enterobacteria, Pseudomonas species (e.g., P. aeruginosa), Salmonella typhimurium, Serratia marcescans, Klebsiella, Proteus, Shigella, Rhizobia, Vitreoscilla, or Paracoccus. In some embodiments, Gram-negative cells are used. In one embodiment, E. coli is used as a host. Exemplary E. coli strains include those of the genotype W3110 AfhuA(AtonA) ptr3 lac Iq lacL8 AompT A(nmpc-fepE) degP41 kan RExamples of suitable strains include strain W3110 (Bachmann, Cellular and Molecular Biology, Vol. 2 (Washington, DC: American Society for Microbiology, 1987), pp. 1190-1219; ATCC Accession No. 27,325), including strain 33D3 (U.S. Pat. No. 5,639,635) having the genotype 1110. Other strains and derivatives thereof, such as E. coli 294 (ATCC 31,446), E. coli B, E. coli 1776 (ATCC 31,537), and E. coli RV308 (ATCC 31,608), are also suitable. These examples are illustrative rather than limiting. Methods for constructing derivatives of any of the above-mentioned bacteria having defined genotypes are known in the art and are described, for example, in Bass et al., Proteins, 8:309-314 (1990). Generally, it is necessary to select an appropriate bacterium taking into consideration the replicability of the replicon in the bacterial cell. For example, when the replicon is supplied using a well-known plasmid such as pBR322, pBR325, pACYC177, or pKN410, Escherichia coli, Serratia sp., or Salmonella sp. can be suitably used as the host.

[0208] Generally, the host cell should secrete minimal amounts of proteolytic enzymes and it may be desirable to incorporate additional protease inhibitors into the cell culture.

[0209] Host cells are transformed with the above expression vectors and cultured in conventional nutrient media modified as appropriate to induce promoters, select transformants, or amplify genes encoding the desired sequences. Transformation means introducing DNA into a prokaryotic host so that the DNA is replicable either as an extrachromosomal element or by a chromosomal integrant. Depending on the host cells used, transformation is carried out using standard techniques appropriate for such cells. The calcium treatment, which utilizes calcium chloride, is usually used for bacterial cells that contain substantial cell wall barriers. Another transformation method utilizes polyethylene glycol / DMSO. Yet another technique used is electroporation.

[0210] The prokaryotic cells used to produce the fusion proteins of the present application are grown in a medium known in the art and suitable for culturing the selected host cells. An example of a suitable medium includes Luria Broth (LB) + necessary nutritional supplements. In some embodiments, the medium may contain a selection agent selected based on the structure of the expression vector to selectively allow the growth of prokaryotic cells containing the expression vector. For example, ampicillin is added to the medium for the growth of cells expressing an ampicillin resistance gene.

[0211] Any necessary supplements other than carbon, nitrogen, and inorganic phosphate sources can also be included at appropriate concentrations introduced alone or in mixtures with other supplements or media, e.g., complex nitrogen sources. Optionally, the culture medium may contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thioglycolate, dithioerythritol, and dithiothreitol.

[0212] Prokaryotic host cells are cultured at a suitable temperature. For growth of E. coli, for example, preferred temperatures range from about 20° C. to about 39° C., more preferably from about 25° C. to about 37° C., and even more preferably about 30° C. The pH of the medium depends primarily on the host organism, but can be any pH in the range of about 5 to about 9. For E. coli, the pH is preferably about 6.8 to about 7.4, more preferably about 7.0.

[0213] When an inducible promoter is used in the expression vector of the present application, protein expression is induced under conditions suitable for the activation of the promoter. In one embodiment of the present application, the PhoA promoter is used to control the transcription of the polypeptide. Thus, the transformed host cell is cultured in a phosphate-limited medium for induction. Preferably, the phosphate-limited medium is CRAP medium (see, for example, Simmons et al., J. Immunol. Methods 263:133-147 (2002)). As known in the art, a variety of other inducers can be used depending on the vector construct utilized.

[0214] The expressed fusion protein of the present disclosure is secreted into the periplasm of the host cell and recovered therefrom. Protein recovery typically involves disrupting the microorganism, usually by means such as osmotic shock, sonication, or lysis. Once the cells are disrupted, cell debris or whole cells can be removed by centrifugation or filtration. The protein can be further purified, for example, by affinity resin chromatography. Alternatively, the protein can be carried into the culture medium and isolated there. The cells can be removed from the culture and culture supernatant, filtered and centrifuged for further purification of the produced protein. The expressed polypeptide can be further isolated and identified using commonly known methods such as polyacrylamide gel electrophoresis (PAGE) and Western blot assay.

[0215] Alternatively, protein production is carried out in large quantities by fermentation processes. A variety of large-scale fed-batch fermentation procedures are available for the production of recombinant proteins. Large-scale fermentations have a volume of at least 1000 liters, and preferably from about 1,000 to 100,000 liters. These fermenters use impellers to distribute oxygen and nutrients, especially glucose (the preferred carbon / energy source). Small-scale fermentation usually refers to fermentation in culture tanks that are no larger than about 100 liters in volume and can range from about 1 liter to about 100 liters.

[0216] In a fermentation process, induction of protein expression usually occurs when cells reach a desired density, e.g., an OD of about 180-220, under suitable conditions. 550 The induction process begins after proliferation to 300 s, at which stage the cells are in early stationary phase. As known in the art and as described above, a variety of inducers can be used depending on the vector construct utilized. The cells may be grown for a shorter period before induction. The cells are typically induced for about 12-50 hours, although longer or shorter induction times may be used.

[0217] Various fermentation conditions can be modified to improve the production yield and quality of the fusion proteins of the present disclosure. For example, to improve the proper assembly and folding of secreted polypeptides, the host prokaryotic cells can be co-transformed with additional vectors that overexpress Dsb proteins (DsbA, DsbB, DsbC, DsbD, and / or DsbG) or chaperone proteins such as FkpA (peptidyl prolyl cis,trans-isomerase with chaperone activity). Chaperone proteins have been shown to promote proper folding and solubility of heterologous proteins produced in bacterial host cells. Chen et al., J Bio Chem 274:19601-19605 (1999); U.S. Patent No. 6,083,715; U.S. Patent No. 6,027,888; Bothmann and Pluckthun, J. Biol. Chem. 275:17100-17105 (2000); Ramm and Pluckthun, J. Biol. Chem. 275:17106-17113 (2000); Arie et al., Mol. Microbiol. 39:199-210 (2001).

[0218] In order to minimize proteolysis of expressed heterologous proteins (especially those that are sensitive to proteolysis), certain host strains that are deficient in proteolytic enzymes can be used in the present invention. For example, the host cell strain can be modified to achieve genetic mutations in the genes encoding known bacterial proteases, such as protease III, OmpT, DegP, Tsp, protease I, protease Mi, protease V, protease VI, and combinations thereof. Several E. coli protease-deficient strains are available and are described, for example, in U.S. Patent No. 5,264,365; U.S. Patent No. 5,508,192; Hara et al., Microbial Drug Resistance, 2:63-72 (1996).

[0219] E. coli strains that are deficient in proteolytic enzymes and transformed with plasmids overexpressing one or more chaperone proteins can be used as host cells for expression systems encoding the antibodies of the present application.

[0220] The fusion proteins produced herein can be further purified to obtain preparations that are substantially homogeneous for further assays and uses. Standard protein purification methods known in the art can be utilized. The following procedures are examples of suitable purification procedures: fractionation on immunoaffinity or ion exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica or on cation exchange resins such as DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and gel filtration, for example, using Sephadex G-75.

[0221] For example, in some embodiments, Protein A immobilized on a solid phase can be used for immunoaffinity purification of the binding molecules of the present disclosure. The solid phase on which Protein A is immobilized is preferably a column comprising a glass or silica surface, more preferably a controlled pore glass column or a silicic acid column. In some embodiments, the column is coated with a reagent such as glycerol to prevent non-specific adhesion of contaminants. The solid phase is then washed to remove contaminants non-specifically bound to the solid phase. Finally, the antibody of interest is recovered from the solid phase by elution.

[0222] (Recombinant production in eukaryotic cells) For eukaryotic expression, the vector components usually include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, and an enhancer element, a promoter, and a transcription termination sequence.

[0223] Vectors for use with eukaryotic hosts may contain an insert encoding a signal sequence or other polypeptide with a specific cleavage site at the N-terminus of the mature protein or polypeptide. The heterologous signal sequence selected preferably is one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. In mammalian expression, mammalian signal sequences and viral secretory leaders, for example, the herpes simplex gD signal, are available. The DNA of such precursor regions can be ligated in reading frame to DNA encoding the antibody of the present application.

[0224] Generally, the origin of replication component is not needed for mammalian expression vectors (the SV40 origin can usually be used only because it contains the early promoter).

[0225] Expression and cloning vectors may contain a selection gene, also called a selectable marker, that confers resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, or tetracycline; complements an auxotrophic deficiency; or encodes a protein that supplies a vital nutrient not available from complex media.

[0226] One example of a selection scheme utilizes drugs to arrest the growth of host cells. Cells that have been successfully transformed with a heterologous gene produce a protein that confers drug resistance and therefore survive the selection regime. Examples of such dominant selection use the drugs neomycin, mycophenolic acid, and hygromycin.

[0227] Another example of a suitable selectable marker for mammalian cells is one that allows the identification of cells capable of incorporating nucleic acid encoding the antibody of the present application. For example, cells transformed with a DHFR selection gene are first identified by culturing all transformants in a culture medium containing methotrexate (Mtx), a competitive antagonist of DHFR. An exemplary suitable host cell when wild-type DHFR is utilized is a Chinese Hamster Ovary (CHO) cell line that is deficient in DHFR activity. Alternatively, host cells transformed or co-transformed with a DNA sequence encoding a polypeptide, a wild-type DHFR protein, and another selectable marker, such as aminoglycoside 3'-phosphotransferase (APH), (particularly wild-type hosts containing endogenous DHFR), can be selected by growing the cells in a medium containing a selection agent for the selectable marker, such as an aminoglycoside antibiotic.

[0228] Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to a nucleic acid encoding a desired polypeptide sequence. Eukaryotic genes have an AT-rich region located approximately 25-30 bases upstream from the site where transcription is initiated. Additional sequences may be included that are found 70-80 bases upstream from the transcription start point of many genes. The 3' end of most eukaryotic genes may be a signal for addition of a polyA tail to the 3' end of the coding sequence. All of these sequences can be inserted into eukaryotic expression vectors.

[0229] Polypeptide transcription from the vector in mammalian host cells can be controlled by a promoter derived from the genome of a virus, such as polyoma virus, fowlpox virus, adenovirus (e.g., adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, and simian virus 40 (SV40), from a heterologous mammalian promoter, such as the actin promoter or an immunoglobulin promoter, from a heat shock promoter, provided that such promoter is compatible with the host cell system.

[0230] Transcription of DNA encoding the fusion proteins of the present disclosure by higher eukaryotes is often increased by inserting an enhancer sequence into the vector. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin). Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late replication origin, and adenovirus enhancers. See also Yaniv, Nature 297:17-18 (1982) for enhancing elements for activation of eukaryotic promoters. The enhancer can be spliced ​​into the vector at a position 5' or 3' to the polypeptide coding sequence, but is preferably located at a site 5' from the promoter.

[0231] Expression vectors used in eukaryotic host cells (yeast, fungi, insects, plants, animals, humans, or nucleated cells from other multicellular organisms) also contain sequences necessary for the termination of transcription and stabilization of the mRNA. Such sequences are commonly available from the 5' and, occasionally, 3' untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding a polypeptide. One useful transcription termination component is the bovine growth hormone polyadenylation region.

[0232] Suitable host cells for cloning or expressing the DNA in the vectors herein include the higher eukaryotic cells described herein, including vertebrate host cells. Propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are the SV40 transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham, J. Gen. Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub, Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TR1 cells (Mather, Annals. NY Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).

[0233] Host cells can be transformed with the above-described expression or cloning vectors for protein production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying genes encoding the desired sequences.

[0234] The host cells used to produce the fusion proteins of the present application can be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimum Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells. Further, see Ham et al., Meth. Enz. 58:44 (1979); Barnes et al., Anal. Biochem. 102:255 (1980); U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90 / 03430; WO 99 / 023631; WO 99 / 023632; WO 99 / 023633; WO 99 / 023634; WO 99 / 023635; WO 99 / 023636; WO 99 / 023637; WO 99 / 023638; WO 99 / 023639 ... Any of the media described in US Patent Application Publication No. 87 / 00195; or US Patent Application Rep. No. 30,985 can be used as the culture medium for the host cells. Any of these media can be supplemented with hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, and phosphate), buffers (e.g., HEPES), nucleotides (e.g., adenosine and thymidine), antibiotics (e.g., GENTAMYCIN™ drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source, as necessary. Any other necessary supplements can also be included at appropriate concentrations known to those skilled in the art. Culture conditions, such as temperature, pH, etc., will be those previously used with the host cells selected for expression and will be apparent to those skilled in the art.

[0235] When using recombinant techniques, the fusion protein can be produced intracellularly in the periplasmic space or directly secreted into the medium. If the fusion protein is produced intracellularly, as a first step, particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. If the fusion protein is secreted into the medium, the supernatant from such an expression system can usually first be concentrated using a commercially available protein concentration filter, for example, an ultrafiltration unit from Amicon or Millipore Pellicon. A protease inhibitor, such as PMSF, can be included in any of the aforementioned steps to inhibit proteolysis, and an antibiotic can be included to prevent the buildup of adventitious contaminants.

[0236] The antibody composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique. The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrene-divinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Other techniques for protein purification, such as fractionation on ion exchange columns, ethanol precipitation, reversed-phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™, chromatography on anion or cation exchange resins (e.g., polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation, are also available depending on the polypeptide to be recovered. After any preliminary purification steps, the mixture containing the polypeptide of interest and contaminants can be subjected to low pH hydrophobic interaction chromatography.

[0237] 5.2.4. Other Binding Molecules Including Fusion Proteins In another aspect, provided herein are binding molecules comprising the fusion proteins provided herein. In some embodiments, the fusion proteins provided herein are portions of other binding molecules. Exemplary binding molecules of the present disclosure are described herein.

[0238] (Other fusion proteins) In various embodiments, the fusion proteins provided herein can be genetically fused or chemically conjugated with another agent, such as a protein-based entity. The fusion proteins can be chemically conjugated with the agent or non-covalently conjugated with the agent in other ways. The agent can be a peptide or an antibody (or a fragment thereof).

[0239] Thus, in some embodiments, provided herein are the above fusion proteins recombinantly fused or chemically conjugated (covalently or non-covalently conjugated) to a heterologous protein or polypeptide (or a fragment thereof, e.g., about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, or about 500 amino acids, or a polypeptide of more than 500 amino acids) to generate a fusion protein, and uses thereof.

[0240] Additionally, the fusion proteins provided herein can be fused to a marker or "tag" sequence, such as a peptide, to facilitate purification. In specific embodiments, the marker or tag amino acid sequences are a hexahistidine peptide, a hemagglutinin ("HA") tag, and a "FLAG" tag.

[0241] The fusion proteins provided herein can be fused or conjugated to an antibody. Methods for fusing or conjugating moieties (including polypeptides) to antibodies are known (see, e.g., Arnon et al., Monoclonal Antibodies for Immunotargeting of Drugs in Cancer Therapy, Monoclonal Antibodies and Cancer Therapy 243-56 (Reisfeld et al., eds., 1985); Hellstrom et al., Antibodies for Drug Delivery, Controlled Drug Delivery 623-53 (Robinson et al., eds., 2nd ed., 1987); Thorpe, Antibody Carriers of Cytotoxic Agents in Cancer Therapy: A Review, Monoclonal Antibodies: Biological and Clinical Applications). 475-506 (Pinchera et al., eds., 1985); Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody in Cancer Therapy, Monoclonal Antibodies for Cancer Detection and Therapy, 303-16 (Baldwin et al., eds., 1985); Thorpe et al., Immunol. Rev.62:119-58 (1982); U.S. Patent Nos. 5,336,603; 5,622,929; 5,359,046; 5,349,053; 5,447,851; 5,723,125; 5,783,181; 5,908,626; 5,844,095; and 5,112,946; EP 307,434; EP 367,166; EP 394,827; PCT Publication Nos. WO 91 / 06570, WO 96 / 04388, WO 96 / 22024, WO 97 / 34631, and WO 99 / 04813; See Ashkenazi et al., Proc. Natl. Acad. Sci. USA, 88: 10535-39 (1991); Traunecker et al., Nature, 331:84-86 (1988); Zheng et al., J. Immunol. 154:5590-600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA 89:11337-41 (1992).

[0242] Fusion proteins can be produced, for example, by the techniques of gene shuffling, motif shuffling, exon shuffling, and / or codon shuffling (collectively referred to as "DNA shuffling").

[0243] In various embodiments, the fusion protein can be genetically fused to the agent. Genetic fusion can be achieved by placing a linker (e.g., a polypeptide) between the fusion protein and the agent. The linker can be a flexible linker.

[0244] In various embodiments, the fusion protein can be genetically conjugated to the therapeutic molecule by means of a hinge region that allows the fusion protein to be linked to the therapeutic molecule.

[0245] In some embodiments, the linker is a peptide linker as described above in Section 5.2.1. These other fusion proteins can be made according to methods well known in the art and described above in Section 5.2.3.

[0246] (Immunoconjugates) In some embodiments, the present disclosure also provides immunoconjugates comprising any of the fusion proteins described herein conjugated to one or more cytotoxic agents, e.g., chemotherapeutic agents or drugs, growth inhibitors, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioisotopes.

[0247] Drugs include maytansinoids (see U.S. Pat. Nos. 5,208,020, 5,416,064, and European Patent No. 0 425 235 B1); auristatins, e.g., monomethylauristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483, 5,780,588, and 7,498,298); dolastatins; calicheamicin or derivatives thereof (see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode et al., Cancer Res. 58:2925-2928 (1998)); anthracyclines, such as daunomycin or doxorubicin (Kratz et al., Current Med. Chem. 13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters 16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); See King et al., J. Med. Chem. 45:4336-4343 (2002); and U.S. Patent No. 6,630,579; methotrexate; vindesine; taxanes, such as docetaxel, paclitaxel, larotaxel, tacetaxel, and ortataxel; trichothecenes; and CC1065.

[0248] In some embodiments, the immunoconjugate comprises a fusion protein as described herein conjugated to an enzymatically active toxin or fragment thereof, including, but not limited to, diphtheria A chain, nonbinding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, diansin protein, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogenin, restrictocin, phenomycin, enomycin, and the trichothecenes.

[0249] In some embodiments, the immunoconjugate comprises a fusion protein as described herein that is conjugated to a radioactive atom to form a radioconjugate. A variety of radioisotopes are available for the production of radioconjugates. Examples include At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 and radioactive isotopes of Lu. When a radioactive conjugate is used for detection, it may contain a radioactive atom for scintigraphic examination, such as tc99m or I123, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as iodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

[0250] Conjugates of fusion proteins and cytotoxic agents are made using a variety of bifunctional protein-coupling agents, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., triene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science, 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotides to polypeptides. See WO94 / 11026.

[0251] The linker is a "cleavable linker" that facilitates the release of the conjugated drug in cells, although non-cleavable linkers are also contemplated herein. Linkers for use in the conjugates of the present disclosure include, but are not limited to, acid-labile linkers (e.g., hydrazone linkers), disulfide-containing linkers, peptidase-sensitive linkers (e.g., peptide linkers containing amino acids such as valine and / or citrulline, e.g., citrulline-valine, or phenylalanine-lysine), photolabile linkers, dimethyl linkers, thioether linkers, or hydrophilic linkers designed to avoid multidrug transporter-mediated resistance.

[0252] The immunoconjugates provided herein contemplate, but are not limited to, such conjugates prepared with cross-linker reagents, including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, as well as SVSB (succinimidyl-(4-vinylsulfone)benzoate) (e.g., from Pierce Biotechnology, Rockford, Ill., USA), which are commercially available.

[0253] 5.3. Recombinant Viral Vectors and Viral Particles for Gene Therapy Also provided herein is a virus-based gene therapy for delivering the fusion protein provided herein.Thus, in another aspect, provided herein is a virus vector (e.g., rAAV vector) that comprises (as a transgene) a nucleic acid that encodes the fusion protein provided herein.In yet another aspect, provided herein is a virus particle (e.g., rAAV or rAAV particle) that comprises (as a transgene) a nucleic acid that encodes the fusion protein provided herein.

[0254] 5.3.1. Viral-Based Gene Delivery Systems Many virus-based systems have been developed for gene transfer into mammalian cells. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, lentivirus vectors, retrovirus vectors, vaccinia vectors, herpes simplex virus vectors, and their derivatives. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals.

[0255] In some embodiments, the viral vector or viral particle provided herein is derived from adenovirus. Exemplary vectors are based on or derived from HAd5, ChAd3, HAd26, HAd6, AdCH3NSmut, HAd35, ChAd63, HAd4, rcAd26. Recombinant adenovirus vectors can be constructed according to methods known in the art. See, for example, O'Connor et al., Virology, 217(1):11-22(1996); Hardy et al., Journal of Virology, 73(9):7835-7841(1999); Hardy et al., Journal of Virology, 71(3):1842-1849(1997). In some embodiments, third generation adenoviral vectors (also called "high-capacity adenoviral vectors" (HCAd), helper-dependent or "gutless" adenoviral vectors) can be used herein to deliver longer sequences. In some embodiments, the polynucleotide of interest, e.g., transgene, is cloned into an adenoviral vector that contains only ITRs and packaging signals. The helper adenoviral vector can be co-transfected into HEK cells to generate adenoviral particles. See Lee et al., Genes and Diseases, 4(2):43-63 (2007).

[0256] In some embodiments, the viral vector or viral particle provided herein is derived from lentivirus.Exemplary vectors are based on or derived from HIV-1, HIV-2, SIVSM, SIVAGM, EIAV, FIV, VNV, CAEV, or BIV.Lentiviral vectors can be produced according to methods known in the art, for example, as described in Cribbs et al., BMC Biotechnology, 13:98 (2003); Merten et al., Mol Ther Methods Clin Dev., 13(3):16017 (2016); Durand and Cimarelli, Viruses, 3:132-159 (2011).In some embodiments, third generation self-inactivating lentiviral vectors are used herein.

[0257] In some embodiments, the viral vector or viral particle provided herein is derived from herpes simplex virus (HSV). In some embodiments, the herpes simplex virus is herpes simplex type 1 virus (HSV-1), herpes simplex type 2 virus (HSV-2), or any derivative thereof. Exemplary vectors are based on or derived from HSV-1, HSV-2, CMV, VZV, EBV, and KSHV. HSV-based vectors can be constructed according to methods known in the art, for example, as described in U.S. Pat. Nos. 7,078,029, 6,261,552, 5,998,174, 5,879,934, 5,849,572, 5,849,571, 5,837,532, 5,804,413, and 5,658,724, and International Patent Applications WO 91 / 02788, WO 96 / 04394, WO 98 / 15637, and WO 99 / 06583, which are incorporated by reference in their entireties.

[0258] In some embodiments, the HSV-based vector provided herein is an amplicon vector.In other embodiments, the HSV-based vector provided herein is a replication-deficient vector.In other embodiments, the HSV-based vector provided herein is a replication-competent vector.

[0259] Amplicons are plasmid-derived vectors that have been modified to contain both the HSV DNA origin of replication (ori) and the HSV cleavage-packaging recognition sequence (pac). When amplicons are transfected into mammalian cells with HSV helper function, they are replicated to form head-to-tail linked concatemers, which are then packaged into viral particles. Two main methods are currently used to produce amplicon particles, one based on infection with defective helper HSV, and the other based on transfection of HSV-1 genes, such as a set of pac-deleted overlapping cosmids or pac-deleted and ICP27-deleted BAC-HSV-1. In some embodiments, the amplicons used herein can accommodate large fragments of foreign DNA (e.g., up to 152 kb), including multiple copies (e.g., up to 15 copies) of the transgene, and are non-toxic.

[0260] In some embodiments, the HSV-based vector used herein is deficient in at least one essential HSV gene, and the HSV-based vector may also include one or more deletions of non-essential genes. In some embodiments, the HSV-based vector is replication-deficient. Most replication-deficient HSV-based vectors contain deletions that remove one or more immediate early, early, or late HSV genes to prevent replication. In other embodiments, the HSV-based vector is deficient in an immediate early gene selected from the group consisting of ICP0, ICP4, ICP22, ICP27, ICP47, and combinations thereof. In a specific embodiment, the HSV-based vector is deficient in all of ICP0, ICP4, ICP22, ICP27, and ICP47. Exemplary replication-competent vectors include NV-1020 (HSV-1), RAV9395 (HSV-2), AD-472 (HSV-2), NS-gEnull (HSV-1), and ImmunoVEX (HSV2). Exemplary replication-deficient vectors include dl5-29 (HSV-2), dl5-29-41L (HSV-1), DISC-dH (HSV-1 and HSV-2), CJ9gD (HSV-1), TOH-OVA (HSV-1), d106 (HSV-1), d81 (HSV-1), HSV-SIV d106 (HSV-1), and d106 (HSV-1).

[0261] Replication-deficient HSV-based vectors are usually produced in complementing cell lines that provide the gene functions necessary for viral growth that are not present in the replication-deficient HSV-based vectors, but at appropriate levels to generate high-titer viral vector stocks.Exemplary cell lines complement at least one, and in some embodiments, all, replication-essential gene functions that are not present in the replication-deficient HSV-based vectors.For example, HSV-based vectors that are defective in ICP0, ICP4, ICP22, ICP27, and ICP47 can be complemented by human osteosarcoma line U2OS.The cell line can also complement non-essential genes (e.g., UL55) that reduce growth or replication efficiency when missing. The complementing cell line can complement the deficiency of at least one replication-essential gene function encoded by early region, immediate early region, late region, viral packaging region, viral associated region, or combinations thereof, including all HSV functions (e.g., allowing the propagation of HSV amplicons containing minimal HSV sequences, such as only inverted terminal repeats and packaging signals, or only ITRs and HSV promoters).In some embodiments, the cell line is further characterized by containing the complementing gene in a manner that does not overlap with the HSV-based vector, minimizing and substantially eliminating the possibility that the genome of the HSV-based vector recombines with the DNA of the cell.Therefore, the presence of replicable HSV is minimized, if not avoided, in the vector stock, which is therefore suitable for certain therapeutic purposes, particularly gene therapy purposes.The construction of the complementing cell line involves standard molecular biology and cell culture techniques well known in the art.

[0262] In certain embodiments, the viral vectors or viral particles provided herein are derived from adeno-associated viruses (AAV). A more detailed description of AAV is provided below in Sections 5.3.2-5.3.4.

[0263] The nucleic acid of interest can be cloned into the vector using any known molecular cloning method in the art, including, for example, the use of a restriction endonuclease site and one or more selectable markers. In some embodiments, the nucleic acid is operably linked to a promoter. A variety of promoters have been explored for gene expression in mammalian cells, and any promoter known in the art can be used in the present disclosure. Promoters can be broadly classified as constitutive promoters or regulated promoters, such as inducible promoters.

[0264] In some embodiments, the nucleic acid encoding the fusion protein is functionally linked to a constitutive promoter. A constitutive promoter allows heterologous genes (also called transgenes) to be constitutively expressed in host cells. Exemplary constitutive promoters contemplated herein include, but are not limited to, cytomegalovirus (CMV) promoter, human elongation factor-1 alpha (hEF1α), ubiquitin C promoter (UbiC), phosphoglycerokinase promoter (PGK), simian virus 40 early promoter (SV40), and chicken β-actin promoter (CAGG) in combination with CMV early enhancer. The efficiency of such constitutive promoters in driving transgene expression has been widely compared in a large number of studies.

[0265] In some embodiments, the nucleic acid encoding the fusion protein is functionally linked to an inducible promoter.The inducible promoter belongs to the category of regulated promoter.The inducible promoter can be induced by one or more conditions, such as physical conditions, the microenvironment of modified immune effector cells, or the physiological state of modified immune effector cells, inducer (i.e., inducer), or a combination thereof.

[0266] In some embodiments, the inducing conditions do not induce expression of endogenous genes in the modified mammalian cell and / or in the subject receiving the pharmaceutical composition. In some embodiments, the inducing conditions are selected from the group consisting of an inducer, irradiation (e.g., ionizing radiation, light), temperature (e.g., heat), redox state, tumor environment, and activation state of the modified mammalian cell.

[0267] In some embodiments, the vector also contains a selectable marker gene or a reporter gene for selecting cells expressing the fusion protein from a population of host cells transfected via the vector. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences to allow expression in the host cell. For example, the vector may include transcription and translation terminators, initiation sequences, and promoters useful for regulating the expression of the nucleic acid sequence.

[0268] 5.3.2. Recombinant AAV Vectors In certain more specific embodiments, the fusion proteins provided herein are delivered by an AAV-based system and thus are contained in a recombinant AAV vector.

[0269] Any AAV serotype or variant thereof can be used in the present disclosure, including AAV1 (Genbank Accession Nos. NC_002077.1; HC000057.1), AAV2 (Genbank Accession Nos. NC_001401.2, JC527779.1), AAV2i8 (Asokan, A., 2010, Discov. Med. 9:399), AAV3 (Genbank Accession No. NC_001729.1), AAV3-B (Genbank Accession No. AF028705.1), AAV4 (Genbank Accession No. NC_001829.1), AAV5 (Genbank Accession Nos. NC_006152.1; JC527780.1), AAV6 (Genbank Accession Nos. AF028704.1; JC527781.1), AAV7 (Genbank accession nos. NC_006260.1; JC527782.1), AAV8 (Genbank accession nos. NC_006261.1; JC527783.1), AAV9 (Genbank accession nos. AX753250.1; JC527784.1), AAV10 (Genbank accession no. AY631965.1), AAVrh10 (Genbank accession no. AY243015.1), AAV11 (Genbank accession no. AY631966.1), AAV12 (Genbank accession no. DQ813647.1), AAV13 (Genbank accession no. EU285562.1), AAV LK03, AAVrh74, and AAV DJ (Wu Z et al., J Virol. 80:11393-7(2006)), AAVAnc81, Anc82, Anc83, Anc84, Anc110, Anc113, Anc126, or Anc127 (Zin, E. et al., Cell. Rep. 12:1056(2016)), AAV_go.1 (Arbetum, A. E. et al., J. Virol. 79:15238(2005)), AAVhu.37, AAVrh8, AAVrh8R, and AAV rh.8 (Wang et al., Mol. Ther. 18:119-125(2010), or variants thereof.

[0270] AAV variants include AAV1 variants (e.g., AAVs comprising an AAV1 variant capsid protein), AAV2 variants (e.g., AAVs comprising an AAV2 variant capsid protein), AAV3 variants (e.g., AAVs comprising an AAV3 variant capsid protein), AAV3-B variants (e.g., AAVs comprising an AAV3-B variant capsid protein), AAV4 variants (e.g., AAVs comprising an AAV4 variant capsid protein), AAV5 variants (e.g., AAVs comprising an AAV5 variant capsid protein), AAV6 variants (e.g., AAVs comprising an AAV6 variant capsid protein), AAV7 variants (e.g., AAVs comprising an AAV7 variant capsid protein), and AAV8 variants. (e.g., an AAV comprising an AAV8 variant capsid protein), AAVrh8, AAVrh8R (e.g., an AAV comprising an AAVrh8 or AAVrh8R variant capsid protein), AAV9 variant (e.g., an AAV comprising an AAV9 variant capsid protein), AAV10 variant (e.g., an AAV comprising an AAV10 variant capsid protein), AAVrh10 variant (e.g., an AAV comprising an AAVrh10 variant capsid protein), AAV11 variant (e.g., an AAV comprising an AAV11 variant capsid protein), AAV12 variant (e.g., an AAV comprising an AAV12 variant capsid protein), AAV13 variant (e.g., an AAV comprising an AAV13 variant capsid protein), AAV Examples of AAV variants include, but are not limited to, an AAV LK03 variant (e.g., an AAV comprising an AAV LK03 variant capsid protein), or an AAVrh74 variant (e.g., an AAV comprising an AAVrh74 variant capsid protein).

[0271] The recombinant AAV (rAAV) vector used in the present disclosure can be constructed according to known techniques. In some embodiments, the rAAV vector is constructed to include the transcriptionally operably linked components, control elements including a transcription initiation region, a polynucleotide encoding the fusion protein provided herein, and a transcription termination region. The control elements can be selected based on the cell of interest. In some embodiments, the resulting rAAV vector construct that includes the operably linked components is flanked (5' and 3') by functional AAV ITR sequences.

[0272] In some embodiments, the polypeptide encoding the fusion protein is functionally linked to at least one regulatory sequence.In some embodiments, the regulatory sequence may include, for example, a promoter sequence, an enhancer sequence such as an upstream enhancer sequence (USE), an RNA processing signal such as a splicing signal, a polyadenylation signal sequence, a sequence that stabilizes cytoplasmic mRNA, a post-transcriptional regulatory element (PRE), and / or a microRNA (miRNA) target sequence.In some embodiments, the regulatory sequence may include a sequence that enhances translation efficiency (e.g., Kozak sequence), a sequence that enhances protein stability, and / or a sequence that enhances protein processing and / or secretion.In some embodiments, the polynucleotide may code for a regulatory miRNA.

[0273] In some embodiments, the regulatory sequence comprises a constitutive promoter and / or a regulatory control element. In some embodiments, the regulatory sequence comprises a regulatable promoter and / or a regulatory control element. In some embodiments, the regulatory sequence comprises a ubiquitous promoter and / or a regulatory control element. In some embodiments, the regulatory sequence comprises a cell- or tissue-specific promoter and / or a regulatory control element. In some embodiments, the regulatory control element is 5' of the coding sequence of the fusion protein (i.e., in the 5' untranslated region (5'UTR)). In other embodiments, the regulatory control element is 3' of the coding sequence of the fusion protein (i.e., in the 3' untranslated region (3'UTR)). In some embodiments, the polynucleotide may comprise multiple regulatory control elements, for example, two, three, four, or five regulatory elements. When the polynucleotide comprises multiple regulatory elements, each regulatory element may be independently located 5' of the coding sequence of the fusion protein, 3' of the coding sequence, adjacent to the coding sequence, or within the coding sequence.

[0274] In some embodiments, the control element is an enhancer. In some embodiments, the included control element directs transcription or expression of the polynucleotide of the fusion protein in a subject in vivo. The control element can include control sequences normally associated with the selected polynucleotide of interest, or alternatively, heterologous control sequences.

[0275] Exemplary regulatory sequences include those derived from sequences encoding mammalian or viral genes, such as the neuron-specific enolase promoter, the GFAP promoter, the SV40 early promoter, the mouse mammary tumor virus LTR promoter, the adenovirus major late promoter (Ad MLP); herpes simplex virus (HSV) promoter, the cytomegalovirus (CMV) promoter, such as the CMV immediate early promoter region (CMVIE), the Rous sarcoma virus (RSV) promoter, synthetic promoters, and hybrid promoters.

[0276] In some embodiments, the promoter is not cell-specific or tissue-specific.For example, the promoter is considered to be a ubiquitous promoter.Examples of promoter sequences that can promote expression in multiple cell types or tissue types include, for example, human elongation factor 1a-subunit (EFla), cytomegalovirus (CMV) immediate early enhancer and / or promoter, chicken β-actin (CBA) and its derivatives, such as CAG, for example, CBA promoter with S40 intron, β-glucuronidase (GUSB), or ubiquitin C (UBC).

[0277] In some embodiments, the promoter sequence can promote expression in a particular cell type or tissue. For example, in some embodiments, the promoter can be a muscle-specific promoter, such as a mammalian muscle creatine kinase (MCK) promoter, a mammalian desmin (DES) promoter, a mammalian troponin I (TNNI2) promoter, or a mammalian skeletal alpha-actin (ASKA) promoter. In other embodiments, the promoter sequence can promote expression in a neuronal cell or neuronal cell type, such as a neuron-specific enolase (NSE), synapsin (Syn), methyl-CpG binding protein 2 (MeCP2), Ca2+ / calmodulin-dependent protein kinase II (CaMKII), metabotropic glutamate receptor 2 (mGluR2), neurofilament light chain (NFL) or heavy chain (NFH), β-globin minigene hb2, preproenkephalin (PPE), enkephalin (Enk), or excitatory amino acid transporter 2 (EAAT2) promoter. In other embodiments, the promoter sequence can promote expression in the liver, such as the alpha-1-antitrypsin (hAAT) or thyroxine-binding globulin (TBG) promoters. In yet other embodiments, the promoter sequence can promote expression in cardiac tissue, such as a cardiomyocyte-specific promoter, such as the MHC, cTnT, or CMV-MUC2k promoters.

[0278] In some embodiments, the polynucleotide can include at least one polyadenylation (polyA) signal sequence, which is well known in the art. When the polyadenylation sequence is present, it is usually located between the 3' end of the transgene coding sequence and the 5' end of the 3'ITR. In some embodiments, the polynucleotide further includes a polyA upstream enhancer sequence 5' of the polyA signal sequence. In some instances, the regulatory sequence is a sequence that enhances translation efficiency, such as a Kozak sequence.

[0279] In some embodiments, the polynucleotide comprises an intron. In some embodiments, the intron is present within the coding sequence of the fusion protein provided herein. In some embodiments, the intron is 5' or 3' of the coding sequence of the fusion protein. In some embodiments, the intron is adjacent to the 5' or 3' end of the coding sequence of the fusion protein. In some embodiments, the polynucleotide comprises two introns. In some embodiments, one intron is 5' and one intron is 3' of the coding sequence of the fusion protein. In some embodiments, one intron is adjacent to the 5' end of the coding sequence of the fusion protein and a second intron is adjacent to the 3' end of the coding sequence of the fusion protein. In some embodiments, the intron is an SV40 intron, for example, a 5'UTR SV40 intron.

[0280] Any AAV ITR sequence known in the art can be used in the rAAV vector. In some embodiments, the AAV ITR used in the vector has a wild-type nucleotide sequence. In other embodiments, the AAV ITR sequence used in the vector is not a wild-type sequence, but instead contains, for example, an insertion, deletion, or substitution of nucleotides. The AAV ITRs provided herein can be derived from any AAV serotype, including, but not limited to, AAV1, AAV2, AAV2i8, AAV3, AAV3-B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV13, AAV-DJ, AAVLK03, AAVrh74, AAV44-9, or variants thereof.

[0281] In some embodiments, the 5' and 3' ITRs flanking the nucleotide sequences in the rAAV vectors provided herein are identical and derived from the same AAV serotype. In other embodiments, the 5' and 3' ITRs flanking the nucleotide sequences in the rAAV vectors provided herein are different and / or derived from different AAV serotypes.

[0282] In some embodiments, a rAAV vector containing a polynucleotide of a fusion protein flanked by AAV ITRs can be constructed by directly inserting a polynucleotide of interest into the AAV genome, e.g., into an excised AAV open reading frame, and specific portions of the AAV genome can be optionally deleted, e.g., as described in WO 1993 / 003769; Kotin (1994), Human Gene Therapy 5:793-801; Shelling and Smith (1994), Gene Therapy 1:165-169; and Zhou et al. (1994), J. Exp. Med. 179:1867-1875.

[0283] In other embodiments, the AAV ITRs are excised from the AAV genome, or from an AAV vector containing such ITRs, and then fused 5' and 3' to the polynucleotide sequence of a fusion protein present in another vector using standard ligation techniques.

[0284] In some embodiments, the rAAV vectors provided herein comprise a recombinant self-complementary genome. rAAVs that comprise a self-complementary genome can usually rapidly form double-stranded DNA molecules due to their partially complementary (e.g., transgene coding and non-coding) sequences. More specifically, in some embodiments, the rAAV vectors provided herein comprise a rAAV genome that comprises a first heterologous polynucleotide sequence (e.g., a therapeutic transgene coding strand) and a second heterologous polynucleotide sequence (e.g., a therapeutic transgene non-coding or antisense strand), where the first heterologous polynucleotide sequence can form intrastrand base pairs with the second polynucleotide sequence. In some embodiments, the first heterologous polynucleotide sequence and the second heterologous polynucleotide sequence are linked by a sequence that promotes intrastrand base pairing, e.g., a hairpin DNA structure. In some embodiments, the first heterologous polynucleotide sequence and the second heterologous polynucleotide sequence are linked by a mutated ITR, such that the rep protein does not cleave the viral genome at the mutated ITR. rAAV vectors containing self-complementary genomes can be produced using methods known in the art, for example, as described in U.S. Pat. Nos. 7,125,717; 7,785,888; 7,790,154; 7,846,729; 8,093,054; and 8,361,457.

[0285] In some embodiments, the polynucleotide molecule in the rAAV vector provided herein is less than about 5 kilobases (kb) in size. In some embodiments, the polynucleotide molecule in the rAAV vector provided herein is less than about 4.5 kb in size. In some embodiments, the polynucleotide molecule in the rAAV vector provided herein is less than about 4.0 kb in size. In some embodiments, the polynucleotide molecule in the rAAV vector provided herein is less than about 3.5 kb in size. In some embodiments, the polynucleotide molecule in the rAAV vector provided herein is less than about 3.0 kb in size. In some embodiments, the polynucleotide molecule in the rAAV vector provided herein is less than about 2.5 kb in size.

[0286] In certain embodiments, provided herein is a recombinant AAV (rAAV) vector comprising a nucleic acid encoding a polypeptide, wherein the polypeptide comprises (i) a first domain derived from VEGFR-1; (ii) a second domain derived from VEGFR-2; and (iii) a third domain capable of binding to angiopoietin (e.g., angiopoietin 1 and angiopoietin 2), wherein the rAAV vector comprises inverted terminal repeats (ITRs) from AAV1, AAV2, AAV2i8, AAV3, AAV3-B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV13, AAV-DJ, AAV LK03, AAVrh74, AAV44-9, or a combination or variant thereof. In some embodiments, the third domain is N-terminal to the first domain and the second domain.

[0287] In certain embodiments, provided herein is a recombinant AAV (rAAV) vector comprising a nucleic acid encoding a polypeptide, wherein the polypeptide comprises (i) a first domain derived from VEGFR-1; (ii) a second domain derived from VEGFR-2; (iii) a third domain capable of binding to angiopoietin (e.g., angiopoietin 1 and angiopoietin 2), and (iv) a fourth domain capable of binding to VEGFC, wherein the rAAV vector comprises inverted terminal repeats (ITRs) from AAV1, AAV2, AAV2i8, AAV3, AAV3-B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV13, AAV-DJ, AAV LK03, AAVrh74, AAV44-9, or a combination or variant thereof. In some embodiments, the third domain is N-terminal to the first domain and the second domain.

[0288] In some specific embodiments, the rAAV vector comprises a nucleic acid encoding a polypeptide having SEQ ID NO:7 or a polypeptide having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:7. In some embodiments, the rAAV vector comprises an ITR from AAV1. In some embodiments, the rAAV vector comprises an ITR from AAV2. In some embodiments, the rAAV vector comprises an ITR from AAV2i8. In some embodiments, the rAAV vector comprises an ITR from AAV3. In some embodiments, the rAAV vector comprises an ITR from AAV3-B. In some embodiments, the rAAV vector comprises an ITR from AAV4. In some embodiments, the rAAV vector comprises an ITR from AAV5. In some embodiments, the rAAV vector comprises an ITR from AAV6. In some embodiments, the rAAV vector comprises an ITR from AAV7. In some embodiments, the rAAV vector comprises an ITR from AAV8. In some embodiments, the rAAV vector comprises an ITR from AAVrh8. In some embodiments, the rAAV vector comprises an ITR from AAVrh8R. In some embodiments, the rAAV vector comprises an ITR from AAV9. In some embodiments, the rAAV vector comprises an ITR from AAV10. In some embodiments, the rAAV vector comprises an ITR from AAVrh10. In some embodiments, the rAAV vector comprises an ITR from AAV11. In some embodiments, the rAAV vector comprises an ITR from AAV12. In some embodiments, the rAAV vector comprises an ITR from AAV13. In some embodiments, the rAAV vector comprises an ITR from AAV-DJ. In some embodiments, the rAAV vector comprises an ITR from AAV LK03. In some embodiments, the rAAV vector comprises an ITR from AAVrh74.

[0289] In some specific embodiments, the rAAV vector comprises a nucleic acid encoding a polypeptide having SEQ ID NO:8 or a polypeptide having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:8. In some embodiments, the rAAV vector comprises an ITR from AAV1. In some embodiments, the rAAV vector comprises an ITR from AAV2. In some embodiments, the rAAV vector comprises an ITR from AAV2i8. In some embodiments, the rAAV vector comprises an ITR from AAV3. In some embodiments, the rAAV vector comprises an ITR from AAV3-B. In some embodiments, the rAAV vector comprises an ITR from AAV4. In some embodiments, the rAAV vector comprises an ITR from AAV5. In some embodiments, the rAAV vector comprises an ITR from AAV6. In some embodiments, the rAAV vector comprises an ITR from AAV7. In some embodiments, the rAAV vector comprises an ITR from AAV8. In some embodiments, the rAAV vector comprises an ITR from AAVrh8. In some embodiments, the rAAV vector comprises an ITR from AAVrh8R. In some embodiments, the rAAV vector comprises an ITR from AAV9. In some embodiments, the rAAV vector comprises an ITR from AAV10. In some embodiments, the rAAV vector comprises an ITR from AAVrh10. In some embodiments, the rAAV vector comprises an ITR from AAV11. In some embodiments, the rAAV vector comprises an ITR from AAV12. In some embodiments, the rAAV vector comprises an ITR from AAV13. In some embodiments, the rAAV vector comprises an ITR from AAV-DJ. In some embodiments, the rAAV vector comprises an ITR from AAV LK03. In some embodiments, the rAAV vector comprises an ITR from AAVrh74.

[0290] In some specific embodiments, the rAAV vector comprises a nucleic acid encoding a polypeptide having SEQ ID NO:9 or a polypeptide having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:9. In some embodiments, the rAAV vector comprises an ITR from AAV1. In some embodiments, the rAAV vector comprises an ITR from AAV2. In some embodiments, the rAAV vector comprises an ITR from AAV2i8. In some embodiments, the rAAV vector comprises an ITR from AAV3. In some embodiments, the rAAV vector comprises an ITR from AAV3-B. In some embodiments, the rAAV vector comprises an ITR from AAV4. In some embodiments, the rAAV vector comprises an ITR from AAV5. In some embodiments, the rAAV vector comprises an ITR from AAV6. In some embodiments, the rAAV vector comprises an ITR from AAV7. In some embodiments, the rAAV vector comprises an ITR from AAV8. In some embodiments, the rAAV vector comprises an ITR from AAVrh8. In some embodiments, the rAAV vector comprises an ITR from AAVrh8R. In some embodiments, the rAAV vector comprises an ITR from AAV9. In some embodiments, the rAAV vector comprises an ITR from AAV10. In some embodiments, the rAAV vector comprises an ITR from AAVrh10. In some embodiments, the rAAV vector comprises an ITR from AAV11. In some embodiments, the rAAV vector comprises an ITR from AAV12. In some embodiments, the rAAV vector comprises an ITR from AAV13. In some embodiments, the rAAV vector comprises an ITR from AAV-DJ. In some embodiments, the rAAV vector comprises an ITR from AAV LK03. In some embodiments, the rAAV vector comprises an ITR from AAVrh74.

[0291] In some specific embodiments, the rAAV vector comprises a nucleic acid encoding a polypeptide having SEQ ID NO: 10 or a polypeptide having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 10. In some embodiments, the rAAV vector comprises an ITR from AAV1. In some embodiments, the rAAV vector comprises an ITR from AAV2. In some embodiments, the rAAV vector comprises an ITR from AAV2i8. In some embodiments, the rAAV vector comprises an ITR from AAV3. In some embodiments, the rAAV vector comprises an ITR from AAV3-B. In some embodiments, the rAAV vector comprises an ITR from AAV4. In some embodiments, the rAAV vector comprises an ITR from AAV5. In some embodiments, the rAAV vector comprises an ITR from AAV6. In some embodiments, the rAAV vector comprises an ITR from AAV7. In some embodiments, the rAAV vector comprises an ITR from AAV8. In some embodiments, the rAAV vector comprises an ITR from AAVrh8. In some embodiments, the rAAV vector comprises an ITR from AAVrh8R. In some embodiments, the rAAV vector comprises an ITR from AAV9. In some embodiments, the rAAV vector comprises an ITR from AAV10. In some embodiments, the rAAV vector comprises an ITR from AAVrh10. In some embodiments, the rAAV vector comprises an ITR from AAV11. In some embodiments, the rAAV vector comprises an ITR from AAV12. In some embodiments, the rAAV vector comprises an ITR from AAV13. In some embodiments, the rAAV vector comprises an ITR from AAV-DJ. In some embodiments, the rAAV vector comprises an ITR from AAV LK03. In some embodiments, the rAAV vector comprises an ITR from AAVrh74.

[0292] In some specific embodiments, the rAAV vector comprises a nucleic acid encoding a polypeptide having SEQ ID NO:58 or a polypeptide having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:58. In some embodiments, the rAAV vector comprises an ITR from AAV1. In some embodiments, the rAAV vector comprises an ITR from AAV2. In some embodiments, the rAAV vector comprises an ITR from AAV2i8. In some embodiments, the rAAV vector comprises an ITR from AAV3. In some embodiments, the rAAV vector comprises an ITR from AAV3-B. In some embodiments, the rAAV vector comprises an ITR from AAV4. In some embodiments, the rAAV vector comprises an ITR from AAV5. In some embodiments, the rAAV vector comprises an ITR from AAV6. In some embodiments, the rAAV vector comprises an ITR from AAV7. In some embodiments, the rAAV vector comprises an ITR from AAV8. In some embodiments, the rAAV vector comprises an ITR from AAVrh8. In some embodiments, the rAAV vector comprises an ITR from AAVrh8R. In some embodiments, the rAAV vector comprises an ITR from AAV9. In some embodiments, the rAAV vector comprises an ITR from AAV10. In some embodiments, the rAAV vector comprises an ITR from AAVrh10. In some embodiments, the rAAV vector comprises an ITR from AAV11. In some embodiments, the rAAV vector comprises an ITR from AAV12. In some embodiments, the rAAV vector comprises an ITR from AAV13. In some embodiments, the rAAV vector comprises an ITR from AAV-DJ. In some embodiments, the rAAV vector comprises an ITR from AAV LK03. In some embodiments, the rAAV vector comprises an ITR from AAVrh74.

[0293] In some specific embodiments, the rAAV vector comprises a nucleic acid encoding a polypeptide having SEQ ID NO:59 or a polypeptide having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:59. In some embodiments, the rAAV vector comprises an ITR from AAV1. In some embodiments, the rAAV vector comprises an ITR from AAV2. In some embodiments, the rAAV vector comprises an ITR from AAV2i8. In some embodiments, the rAAV vector comprises an ITR from AAV3. In some embodiments, the rAAV vector comprises an ITR from AAV3-B. In some embodiments, the rAAV vector comprises an ITR from AAV4. In some embodiments, the rAAV vector comprises an ITR from AAV5. In some embodiments, the rAAV vector comprises an ITR from AAV6. In some embodiments, the rAAV vector comprises an ITR from AAV7. In some embodiments, the rAAV vector comprises an ITR from AAV8. In some embodiments, the rAAV vector comprises an ITR from AAVrh8. In some embodiments, the rAAV vector comprises an ITR from AAVrh8R. In some embodiments, the rAAV vector comprises an ITR from AAV9. In some embodiments, the rAAV vector comprises an ITR from AAV10. In some embodiments, the rAAV vector comprises an ITR from AAVrh10. In some embodiments, the rAAV vector comprises an ITR from AAV11. In some embodiments, the rAAV vector comprises an ITR from AAV12. In some embodiments, the rAAV vector comprises an ITR from AAV13. In some embodiments, the rAAV vector comprises an ITR from AAV-DJ. In some embodiments, the rAAV vector comprises an ITR from AAV LK03. In some embodiments, the rAAV vector comprises an ITR from AAVrh74.

[0294] In some specific embodiments, the rAAV vector comprises a nucleic acid encoding a polypeptide having SEQ ID NO:60 or a polypeptide having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:60. In some embodiments, the rAAV vector comprises an ITR from AAV1. In some embodiments, the rAAV vector comprises an ITR from AAV2. In some embodiments, the rAAV vector comprises an ITR from AAV2i8. In some embodiments, the rAAV vector comprises an ITR from AAV3. In some embodiments, the rAAV vector comprises an ITR from AAV3-B. In some embodiments, the rAAV vector comprises an ITR from AAV4. In some embodiments, the rAAV vector comprises an ITR from AAV5. In some embodiments, the rAAV vector comprises an ITR from AAV6. In some embodiments, the rAAV vector comprises an ITR from AAV7. In some embodiments, the rAAV vector comprises an ITR from AAV8. In some embodiments, the rAAV vector comprises an ITR from AAVrh8. In some embodiments, the rAAV vector comprises an ITR from AAVrh8R. In some embodiments, the rAAV vector comprises an ITR from AAV9. In some embodiments, the rAAV vector comprises an ITR from AAV10. In some embodiments, the rAAV vector comprises an ITR from AAVrh10. In some embodiments, the rAAV vector comprises an ITR from AAV11. In some embodiments, the rAAV vector comprises an ITR from AAV12. In some embodiments, the rAAV vector comprises an ITR from AAV13. In some embodiments, the rAAV vector comprises an ITR from AAV-DJ. In some embodiments, the rAAV vector comprises an ITR from AAV LK03. In some embodiments, the rAAV vector comprises an ITR from AAVrh74.

[0295] In some specific embodiments, the rAAV vector comprises a nucleic acid encoding a polypeptide having SEQ ID NO:61 or a polypeptide having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:61. In some embodiments, the rAAV vector comprises an ITR from AAV1. In some embodiments, the rAAV vector comprises an ITR from AAV2. In some embodiments, the rAAV vector comprises an ITR from AAV2i8. In some embodiments, the rAAV vector comprises an ITR from AAV3. In some embodiments, the rAAV vector comprises an ITR from AAV3-B. In some embodiments, the rAAV vector comprises an ITR from AAV4. In some embodiments, the rAAV vector comprises an ITR from AAV5. In some embodiments, the rAAV vector comprises an ITR from AAV6. In some embodiments, the rAAV vector comprises an ITR from AAV7. In some embodiments, the rAAV vector comprises an ITR from AAV8. In some embodiments, the rAAV vector comprises an ITR from AAVrh8. In some embodiments, the rAAV vector comprises an ITR from AAVrh8R. In some embodiments, the rAAV vector comprises an ITR from AAV9. In some embodiments, the rAAV vector comprises an ITR from AAV10. In some embodiments, the rAAV vector comprises an ITR from AAVrh10. In some embodiments, the rAAV vector comprises an ITR from AAV11. In some embodiments, the rAAV vector comprises an ITR from AAV12. In some embodiments, the rAAV vector comprises an ITR from AAV13. In some embodiments, the rAAV vector comprises an ITR from AAV-DJ. In some embodiments, the rAAV vector comprises an ITR from AAV LK03. In some embodiments, the rAAV vector comprises an ITR from AAVrh74.

[0296] In some specific embodiments, the rAAV vector comprises a nucleic acid encoding a polypeptide having SEQ ID NO:62 or a polypeptide having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:62. In some embodiments, the rAAV vector comprises an ITR from AAV1. In some embodiments, the rAAV vector comprises an ITR from AAV2. In some embodiments, the rAAV vector comprises an ITR from AAV2i8. In some embodiments, the rAAV vector comprises an ITR from AAV3. In some embodiments, the rAAV vector comprises an ITR from AAV3-B. In some embodiments, the rAAV vector comprises an ITR from AAV4. In some embodiments, the rAAV vector comprises an ITR from AAV5. In some embodiments, the rAAV vector comprises an ITR from AAV6. In some embodiments, the rAAV vector comprises an ITR from AAV7. In some embodiments, the rAAV vector comprises an ITR from AAV8. In some embodiments, the rAAV vector comprises an ITR from AAVrh8. In some embodiments, the rAAV vector comprises an ITR from AAVrh8R. In some embodiments, the rAAV vector comprises an ITR from AAV9. In some embodiments, the rAAV vector comprises an ITR from AAV10. In some embodiments, the rAAV vector comprises an ITR from AAVrh10. In some embodiments, the rAAV vector comprises an ITR from AAV11. In some embodiments, the rAAV vector comprises an ITR from AAV12. In some embodiments, the rAAV vector comprises an ITR from AAV13. In some embodiments, the rAAV vector comprises an ITR from AAV-DJ. In some embodiments, the rAAV vector comprises an ITR from AAV LK03. In some embodiments, the rAAV vector comprises an ITR from AAVrh74.

[0297] In some specific embodiments, the rAAV vector comprises a nucleic acid encoding a polypeptide having SEQ ID NO:63 or a polypeptide having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:63. In some embodiments, the rAAV vector comprises an ITR from AAV1. In some embodiments, the rAAV vector comprises an ITR from AAV2. In some embodiments, the rAAV vector comprises an ITR from AAV2i8. In some embodiments, the rAAV vector comprises an ITR from AAV3. In some embodiments, the rAAV vector comprises an ITR from AAV3-B. In some embodiments, the rAAV vector comprises an ITR from AAV4. In some embodiments, the rAAV vector comprises an ITR from AAV5. In some embodiments, the rAAV vector comprises an ITR from AAV6. In some embodiments, the rAAV vector comprises an ITR from AAV7. In some embodiments, the rAAV vector comprises an ITR from AAV8. In some embodiments, the rAAV vector comprises an ITR from AAVrh8. In some embodiments, the rAAV vector comprises an ITR from AAVrh8R. In some embodiments, the rAAV vector comprises an ITR from AAV9. In some embodiments, the rAAV vector comprises an ITR from AAV10. In some embodiments, the rAAV vector comprises an ITR from AAVrh10. In some embodiments, the rAAV vector comprises an ITR from AAV11. In some embodiments, the rAAV vector comprises an ITR from AAV12. In some embodiments, the rAAV vector comprises an ITR from AAV13. In some embodiments, the rAAV vector comprises an ITR from AAV-DJ. In some embodiments, the rAAV vector comprises an ITR from AAV LK03. In some embodiments, the rAAV vector comprises an ITR from AAVrh74.

[0298] In some specific embodiments, the rAAV vector comprises a nucleic acid encoding a polypeptide having SEQ ID NO:64 or a polypeptide having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:64. In some embodiments, the rAAV vector comprises an ITR from AAV1. In some embodiments, the rAAV vector comprises an ITR from AAV2. In some embodiments, the rAAV vector comprises an ITR from AAV2i8. In some embodiments, the rAAV vector comprises an ITR from AAV3. In some embodiments, the rAAV vector comprises an ITR from AAV3-B. In some embodiments, the rAAV vector comprises an ITR from AAV4. In some embodiments, the rAAV vector comprises an ITR from AAV5. In some embodiments, the rAAV vector comprises an ITR from AAV6. In some embodiments, the rAAV vector comprises an ITR from AAV7. In some embodiments, the rAAV vector comprises an ITR from AAV8. In some embodiments, the rAAV vector comprises an ITR from AAVrh8. In some embodiments, the rAAV vector comprises an ITR from AAVrh8R. In some embodiments, the rAAV vector comprises an ITR from AAV9. In some embodiments, the rAAV vector comprises an ITR from AAV10. In some embodiments, the rAAV vector comprises an ITR from AAVrh10. In some embodiments, the rAAV vector comprises an ITR from AAV11. In some embodiments, the rAAV vector comprises an ITR from AAV12. In some embodiments, the rAAV vector comprises an ITR from AAV13. In some embodiments, the rAAV vector comprises an ITR from AAV-DJ. In some embodiments, the rAAV vector comprises an ITR from AAV LK03. In some embodiments, the rAAV vector comprises an ITR from AAVrh74.

[0299] In some specific embodiments, the rAAV vector comprises a nucleic acid encoding a polypeptide having SEQ ID NO:65 or a polypeptide having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:65. In some embodiments, the rAAV vector comprises an ITR from AAV1. In some embodiments, the rAAV vector comprises an ITR from AAV2. In some embodiments, the rAAV vector comprises an ITR from AAV2i8. In some embodiments, the rAAV vector comprises an ITR from AAV3. In some embodiments, the rAAV vector comprises an ITR from AAV3-B. In some embodiments, the rAAV vector comprises an ITR from AAV4. In some embodiments, the rAAV vector comprises an ITR from AAV5. In some embodiments, the rAAV vector comprises an ITR from AAV6. In some embodiments, the rAAV vector comprises an ITR from AAV7. In some embodiments, the rAAV vector comprises an ITR from AAV8. In some embodiments, the rAAV vector comprises an ITR from AAVrh8. In some embodiments, the rAAV vector comprises an ITR from AAVrh8R. In some embodiments, the rAAV vector comprises an ITR from AAV9. In some embodiments, the rAAV vector comprises an ITR from AAV10. In some embodiments, the rAAV vector comprises an ITR from AAVrh10. In some embodiments, the rAAV vector comprises an ITR from AAV11. In some embodiments, the rAAV vector comprises an ITR from AAV12. In some embodiments, the rAAV vector comprises an ITR from AAV13. In some embodiments, the rAAV vector comprises an ITR from AAV-DJ. In some embodiments, the rAAV vector comprises an ITR from AAV LK03. In some embodiments, the rAAV vector comprises an ITR from AAVrh74.

[0300] In some specific embodiments, the rAAV vector comprises a nucleic acid encoding a polypeptide having SEQ ID NO:66 or a polypeptide having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:66. In some embodiments, the rAAV vector comprises an ITR from AAV1. In some embodiments, the rAAV vector comprises an ITR from AAV2. In some embodiments, the rAAV vector comprises an ITR from AAV2i8. In some embodiments, the rAAV vector comprises an ITR from AAV3. In some embodiments, the rAAV vector comprises an ITR from AAV3-B. In some embodiments, the rAAV vector comprises an ITR from AAV4. In some embodiments, the rAAV vector comprises an ITR from AAV5. In some embodiments, the rAAV vector comprises an ITR from AAV6. In some embodiments, the rAAV vector comprises an ITR from AAV7. In some embodiments, the rAAV vector comprises an ITR from AAV8. In some embodiments, the rAAV vector comprises an ITR from AAVrh8. In some embodiments, the rAAV vector comprises an ITR from AAVrh8R. In some embodiments, the rAAV vector comprises an ITR from AAV9. In some embodiments, the rAAV vector comprises an ITR from AAV10. In some embodiments, the rAAV vector comprises an ITR from AAVrh10. In some embodiments, the rAAV vector comprises an ITR from AAV11. In some embodiments, the rAAV vector comprises an ITR from AAV12. In some embodiments, the rAAV vector comprises an ITR from AAV13. In some embodiments, the rAAV vector comprises an ITR from AAV-DJ. In some embodiments, the rAAV vector comprises an ITR from AAV LK03. In some embodiments, the rAAV vector comprises an ITR from AAVrh74.

[0301] In some more specific embodiments, provided herein is a vector comprising the nucleic acid sequence of SEQ ID NO:11 or a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:11.

[0302] In some more specific embodiments, provided herein is a vector comprising the nucleic acid sequence of SEQ ID NO:12 or a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:12.

[0303] In some more specific embodiments, provided herein is a vector comprising the nucleic acid sequence of SEQ ID NO:13 or a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:13.

[0304] In some more specific embodiments, provided herein is a vector comprising the nucleic acid sequence of SEQ ID NO:14 or a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:14.

[0305] In some more specific embodiments, provided herein is a vector comprising the nucleic acid sequence of SEQ ID NO:15 or a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:15.

[0306] In some more specific embodiments, provided herein is a vector comprising the nucleic acid sequence of SEQ ID NO:16 or a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:16.

[0307] In some more specific embodiments, provided herein is a vector comprising the nucleic acid sequence of SEQ ID NO:17 or a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:17.

[0308] In some more specific embodiments, provided herein is a vector comprising the nucleic acid sequence of SEQ ID NO:18 or a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:18.

[0309] In some more specific embodiments, provided herein is a vector comprising the nucleic acid sequence of SEQ ID NO:19 or a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:19.

[0310] In some more specific embodiments, provided herein is a vector comprising the nucleic acid sequence of SEQ ID NO:20 or a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:20.

[0311] In some more specific embodiments, provided herein is a vector comprising the nucleic acid sequence of SEQ ID NO:21 or a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:21.

[0312] In some more specific embodiments, provided herein is a vector comprising the nucleic acid sequence of SEQ ID NO:22 or a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:22.

[0313] In some more specific embodiments, provided herein is a vector comprising the nucleic acid sequence of SEQ ID NO:23 or a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:23.

[0314] In some more specific embodiments, provided herein is a vector comprising a nucleic acid sequence of any one of SEQ ID NOs: 67-75, or a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to any one of SEQ ID NOs: 67-75.

[0315] In yet another aspect, the rAAV vector provided herein comprises a nucleic acid encoding a first domain derived from VEGFR-1 provided herein, a second domain derived from VEGFR-2 provided herein, and a third domain capable of binding to angiopoietin provided herein, each of which is described in more detail above; and the rAAV vector is constructed in such a way that two or more peptides are expressed instead of a single fusion protein. For example, in some embodiments, the first domain, the second domain, and the third domain are each expressed as separate proteins. In other embodiments, the first domain and the second domain are expressed as a single polypeptide, and the third domain is expressed as a second polypeptide.

[0316] Methods for generating such separately expressed polypeptides via one vector, for example, via IRES-based and 2A peptide-based vector systems or intein-mediated protein splicing systems, are known in the art. In some embodiments, an internal ribosome entry site (IRES) is used herein to express multiple genes from one promoter. In other embodiments, 2A self-cleaving peptides are used herein. Members of the 2A peptides are named after the viruses in which they were first described. For example, F2A, the first described 2A peptide, is derived from the foot and mouth disease virus. The self-cleaving 18-22 amino acid long 2A peptides mediate "ribosome skipping" between proline and glycine residues, inhibiting peptide bond formation without affecting downstream translation. These peptides allow multiple proteins to be encoded as polyproteins that dissociate into component proteins upon translation. Self-cleaving peptides are found in members of the Picornaviridae virus family, including aphthoviruses, such as foot and mouth disease virus (FMDV), equine rhinitis A virus (ERAV), thosea asigna virus (TaV), and porcine teschovirus-1 (PTV-1) (see Donnelly et al., J. Gen. Virol., 82: 1027-101 (2001); Ryan et al., J. Gen. Virol., 72: 2727-2732 (2001)), and cardioviruses, such as theloviruses (e.g., Theiler's murine encephalomyelitis) and encephalomyocarditis virus.The 2A peptides derived from FMDV, ERAV, PTV-1, and TaV may also be referred to as "F2A," "E2A," "P2A," and "T2A," respectively, and are included in the present disclosure, as described, for example, in Donnelly et al., J. Gen. Virol., 78: 13-21 (1997); Ryan and Drew, EMBO J., 13: 928-933 (1994); Szymczak et al., Nature Biotech., 5: 589-594 (2004); Hasegawa et al., Stem Cells, 25(7): 1707-12 (2007). In yet other embodiments, an intein-mediated protein splicing system is used herein, e.g., as described in Shah and Muir, Chem Sci., 5(1): 446-461 (2014) and Topilina and Mills, Mobile DNA, 5(5) (2014). Other methods known in the art can also be used with the present constructs.

[0317] 5.3.3. Recombinant AAV Particles In another aspect, provided herein is a recombinant AAV (rAAV) or rAAV particle comprising a nucleic acid encoding a fusion protein provided herein, including any of the rAAV vectors described above in Section 5.3.2, and at least an AAV capsid protein.

[0318] The capsid protein may be from the same serotype as the ITR or a derivative thereof. The capsid may be of a different serotype than the ITR. For example, in certain embodiments, the AAV particle comprises an AAV2 ITR and an AAV6 capsid (AAV2 / 6), an AAV2 ITR and an AAV7 capsid (AAV2 / 7), an AAV2 ITR and an AAV8 capsid (AAV2 / 8), or an AAV2 ITR and an AAV9 capsid (AAV2 / 9).

[0319] Natural AAV capsid comprises AAV VP1, VP2 and VP3 capsid proteins, each encoded by a splice variant of the AAV cap gene. In general, AAV particles comprise three proteins, VP1, VP2 and VP3, where VP2 and VP3 are truncated versions of VP1 and therefore have sequences also contained in VP1. The amino acid sequence of VP1 usually defines the serotype of the capsid. Thus, for example, if the VP1 capsid protein encodes the AAV2 VP1 protein, the AAV is of the AAV2 serotype, whereas if the VP1 capsid protein encodes the AAV8 VP1 protein, the AAV is of the AAV8 serotype.

[0320] In some embodiments, the AAV capsid proteins (e.g., VP1, VP2, and / or VP3) in the rAAV particles are not native capsid proteins. In some embodiments, the AAV capsid proteins (e.g., VP1, VP2, and / or VP3) are derived from native capsid proteins.

[0321] In some embodiments, the AAV capsid protein is VP1 capsid protein. In other embodiments, the AAV capsid protein is VP2 capsid protein. In other embodiments, the AAV capsid protein is VP3 capsid protein. In some embodiments, the rAAV particle comprises VP1 capsid protein, VP2 capsid protein, and / or VP3 capsid protein. In other embodiments, the rAAV particle comprises VP1 capsid protein, VP2 capsid protein, and VP3 capsid protein. In some embodiments, the rAAV particle comprises VP1 capsid protein, VP2 capsid protein, and / or VP3 capsid protein, wherein the capsid proteins of the rAAV particle are of the same serotype. In other embodiments, the rAAV particle comprises a VP1 capsid protein, a VP2 capsid protein, and a VP3 capsid protein, wherein the capsid proteins of the AAV particle are of the same serotype.

[0322] In some embodiments, the capsid protein is a variant capsid protein. A variant capsid protein can include one or more mutations, such as amino acid substitutions, amino acid deletions, and heterologous peptide insertions, compared to a corresponding reference capsid protein, such as a naturally occurring parent capsid protein, i.e., the capsid protein from which it is derived. In some embodiments, the amino acid sequence of the AAV capsid protein is identical to the amino acid sequence of a wild-type, reference, or parent AAV capsid protein, except for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid residues, for example, except for a substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid residues. In some embodiments, the capsid proteins or AAV particles described herein may be chimeric capsid proteins or AAV particles, respectively, that comprise protein sequences of two or more AAV serotype capsid proteins or particles, respectively.

[0323] In some embodiments, the capsid protein in the rAAV particles provided herein is derived from AAV1, AAV2, AAV2i8, AAV3, AAV3-B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV13, AAV-DJ, AAV LK03, AAVrh74, AAV44-9 capsid protein. In specific embodiments, the capsid protein in the rAAV particles provided herein has an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 100% identical to the amino acid sequence of an AAV1, AAV2, AAV2i8, AAV3, AAV3-B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV13, AAV-DJ, AAV LK03, AAVrh74, AAV44-9 capsid protein.

[0324] In certain embodiments, the AAV particles provided herein comprise VP1, VP2, and / or VP3 capsid proteins comprising VP1, VP2, and / or VP3 capsid protein sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% sequence identity to any of the VP1, VP2, or VP3 amino acid sequences of AAV1.

[0325] In certain embodiments, the AAV particles provided herein comprise VP1, VP2, and / or VP3 capsid proteins comprising VP1, VP2, and / or VP3 capsid protein sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% sequence identity to any of the VP1, VP2, or VP3 amino acid sequences of AAV2.

[0326] In certain embodiments, the AAV particles provided herein comprise VP1, VP2, and / or VP3 capsid proteins comprising VP1, VP2, and / or VP3 capsid protein sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% sequence identity to any of the VP1, VP2, or VP3 amino acid sequences of AAV2i8.

[0327] In certain embodiments, the AAV particles provided herein comprise VP1, VP2, and / or VP3 capsid proteins comprising VP1, VP2, and / or VP3 capsid protein sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% sequence identity to any of the VP1, VP2, or VP3 amino acid sequences of AAV3.

[0328] In certain embodiments, the AAV particles provided herein comprise VP1, VP2, and / or VP3 capsid proteins comprising VP1, VP2, and / or VP3 capsid protein sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% sequence identity to any of the VP1, VP2, or VP3 amino acid sequences of AAV3-B.

[0329] In certain embodiments, the AAV particles provided herein comprise VP1, VP2, and / or VP3 capsid proteins comprising VP1, VP2, and / or VP3 capsid protein sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% sequence identity to any of the VP1, VP2, or VP3 amino acid sequences of AAV4.

[0330] In certain embodiments, the AAV particles provided herein comprise VP1, VP2, and / or VP3 capsid proteins comprising VP1, VP2, and / or VP3 capsid protein sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% sequence identity to any of the VP1, VP2, or VP3 amino acid sequences of AAV5.

[0331] In certain embodiments, the AAV particles provided herein comprise VP1, VP2, and / or VP3 capsid proteins comprising VP1, VP2, and / or VP3 capsid protein sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% sequence identity to any of the VP1, VP2, or VP3 amino acid sequences of AAV6.

[0332] In certain embodiments, the AAV particles provided herein comprise VP1, VP2, and / or VP3 capsid proteins comprising VP1, VP2, and / or VP3 capsid protein sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% sequence identity to any of the VP1, VP2, or VP3 amino acid sequences of AAV7.

[0333] In certain embodiments, the AAV particles provided herein comprise VP1, VP2, and / or VP3 capsid proteins comprising VP1, VP2, and / or VP3 capsid protein sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% sequence identity to any of the VP1, VP2, or VP3 amino acid sequences of AAV8.

[0334] In certain embodiments, the AAV particles provided herein comprise VP1, VP2, and / or VP3 capsid proteins comprising VP1, VP2, and / or VP3 capsid protein sequences that have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% sequence identity to any of the VP1, VP2, or VP3 amino acid sequences of AAVrh8.

[0335] In certain embodiments, the AAV particles provided herein comprise VP1, VP2, and / or VP3 capsid proteins comprising VP1, VP2, and / or VP3 capsid protein sequences that have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% sequence identity to any of the VP1, VP2, or VP3 amino acid sequences of AAVrh8R.

[0336] In certain embodiments, the AAV particles provided herein comprise VP1, VP2, and / or VP3 capsid proteins comprising VP1, VP2, and / or VP3 capsid protein sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% sequence identity to any of the VP1, VP2, or VP3 amino acid sequences of AAV9.

[0337] In certain embodiments, the AAV particles provided herein comprise VP1, VP2, and / or VP3 capsid proteins comprising VP1, VP2, and / or VP3 capsid protein sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% sequence identity to any of the VP1, VP2, or VP3 amino acid sequences of AAV10.

[0338] In certain embodiments, the AAV particles provided herein comprise VP1, VP2, and / or VP3 capsid proteins comprising VP1, VP2, and / or VP3 capsid protein sequences that have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% sequence identity to any of the VP1, VP2, or VP3 amino acid sequences of AAVrh10.

[0339] In certain embodiments, the AAV particles provided herein comprise VP1, VP2, and / or VP3 capsid proteins comprising VP1, VP2, and / or VP3 capsid protein sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% sequence identity to any of the VP1, VP2, or VP3 amino acid sequences of AAV11.

[0340] In certain embodiments, the AAV particles provided herein comprise VP1, VP2, and / or VP3 capsid proteins comprising VP1, VP2, and / or VP3 capsid protein sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% sequence identity to any of the VP1, VP2, or VP3 amino acid sequences of AAV12.

[0341] In certain embodiments, the AAV particles provided herein comprise VP1, VP2, and / or VP3 capsid proteins comprising VP1, VP2, and / or VP3 capsid protein sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% sequence identity to any of the VP1, VP2, or VP3 amino acid sequences of AAV13.

[0342] In certain embodiments, the AAV particles provided herein comprise VP1, VP2, and / or VP3 capsid proteins comprising VP1, VP2, and / or VP3 capsid protein sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% sequence identity to any of the VP1, VP2, or VP3 amino acid sequences of AAV-DJ.

[0343] In certain embodiments, the AAV particles provided herein comprise VP1, VP2, and / or VP3 capsid proteins comprising VP1, VP2, and / or VP3 capsid protein sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% sequence identity to any of the VP1, VP2, or VP3 amino acid sequences of AAV LK03.

[0344] In certain embodiments, the AAV particles provided herein comprise VP1, VP2, and / or VP3 capsid proteins comprising VP1, VP2, and / or VP3 capsid protein sequences that have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% sequence identity to any of the VP1, VP2, or VP3 amino acid sequences of AAVrh74.

[0345] In certain embodiments, the AAV particles provided herein comprise VP1, VP2, and / or VP3 capsid proteins comprising VP1, VP2, and / or VP3 capsid protein sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% sequence identity to any of the VP1, VP2, or VP3 amino acid sequences of AAV44-9.

[0346] In some specific embodiments, the rAAV particles provided herein comprise a nucleic acid encoding a fusion protein provided herein and a VP1 of AAV comprising the amino acid sequence of SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, or SEQ ID NO: 49. In a specific embodiment, VP1 comprises the amino acid sequence of SEQ ID NO: 43. In a specific embodiment, VP1 comprises the amino acid sequence of SEQ ID NO: 44. In a specific embodiment, VP1 comprises the amino acid sequence of SEQ ID NO: 48, which is a variant VP1 of AAV2 comprising the amino acid substitutions Y444F, R487G, T491V, Y500F, R585S, R588T, and Y730F. In yet another specific embodiment, VP1 comprises the amino acid sequence of SEQ ID NO: 49. Table 3. Exemplary VP1, VP2, and VP3 proteins [Table 3] TIFF2024514072000004.tif243170TIFF2024514072000005.tif237170TIFF2024514072 000006.tif242170TIFF2024514072000007.tif242170TIFF2024514072000008.tif61170

[0347] The rAAV particles described herein can be produced using any suitable method known in the art. For example, a host cell (e.g., a mammalian cell) can be modified to stably express the necessary components for AAV particle production. This can be accomplished by integrating a plasmid (or multiple plasmids) containing the AAV rep and cap genes and a selectable marker, such as an antibiotic (e.g., neomycin or ampicillin) resistance gene, into the genome of the cell. The cell can be, for example, an insect or mammalian cell, which can then be co-infected with a helper virus (e.g., an adenovirus or baculovirus that provides helper functions) and a rAAV vector containing the 5' and 3' AAV ITRs. The use of a selectable marker allows for large-scale production of rAAV. As another non-limiting example, the rep and cap genes can be introduced into the packaging cell using an adenovirus or baculovirus, rather than a plasmid. As another non-limiting example, both viral vectors containing the 5' and 3' AAV ITRs and the rep and cap genes can be stably integrated into the DNA of a producer cell, and helper functions can be provided by wild-type adenovirus to produce rAAV.

[0348] A helper virus for AAV refers to a virus that allows AAV to replicate and be packaged by a host cell. Helper viruses provide helper functions that allow AAV to replicate. Several such helper viruses have been identified, including adenoviruses, herpesviruses, and poxviruses, such as vaccinia. Adenoviruses encompass several different subgroups, with adenovirus type 5 (Ad5) of subgroup C being the most commonly used. Numerous adenoviruses of human, non-human mammalian, and avian origin are known and available from depositories such as the ATCC. Viruses of the Herpes family (also available from depositories such as the ATCC) include, for example, herpes simplex virus (HSV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), and pseudorabies virus (PRV). Examples of adenoviral helper functions for AAV replication include E1A, E1B, E2A, VA, and E4orf6 functions.

[0349] A preparation of AAV is said to be substantially free of helper virus when the ratio of infectious AAV particles to infectious helper virus particles is at least about 102:1, at least about 104:1, at least about 106:1, or at least about 108:1. A preparation may also be free of equivalent amounts of helper virus proteins (i.e., proteins present as a result of such levels of helper virus if the above-mentioned helper virus particle impurities are present in disrupted form). Contamination with viral and / or cellular proteins can usually be observed as the presence of Coomassie-stained bands on SDS gels (e.g., the appearance of bands other than those corresponding to the AAV capsid proteins VP1, VP2, and VP3).

[0350] In some embodiments, the host cell containing the above rAAV vector is adapted to provide AAV helper functions to replicate and package the polynucleotide encoding the fusion protein provided herein flanked by AAV ITRs to produce rAAV particles. AAV helper functions are usually coding sequences derived from AAV that can be expressed to provide AAV gene products that then function in trans for productive AAV replication. AAV helper functions are used herein to complement essential AAV functions that are missing from rAAV vectors. In some embodiments, AAV helper functions include the main AAV ORFs, i.e., rep and cap coding regions, or one or both of their functional homologs.

[0351] AAV helper functions can be introduced into host cells by transfecting the host cells with AAV helper constructs either before or at the same time as transfection of rAAV vectors.For example, AAV helper constructs can be used to provide at least transient expression of AAV rep and / or cap genes to complement the missing AAV functions required for productive AAV infection.Usually, AAV helper constructs lack AAV ITRs and cannot replicate or package themselves.AAV helper constructs can be, for example, in the form of plasmids, phages, transposons, cosmids, viruses, or virions.

[0352] In certain embodiments, the host cell can also provide or is provided with non-AAV derived or "accessory functions" for producing rAAV particles. Accessory functions are non-AAV derived viral and / or cellular functions on which AAV depends for its replication, such as non-AAV proteins and RNAs required for AAV replication, including those involved in AAV gene transcription, stage-specific AAV mRNA splicing, AAV DNA replication, synthesis of Cap expression products, and activation of AAV capsid assembly. In some embodiments, viral-based accessory functions can be derived from known helper viruses.

[0353] In some embodiments, recombinant AAV particles are produced as a result of infection of host cells with the helper virus and / or accessory function vectors, and the rAAV particles produced are infectious, replication-defective viruses and contain an AAV protein shell encapsulating a heterologous nucleotide sequence of interest flanked on both sides by AAV ITRs.

[0354] The rAAV particles can be purified from the host cells using purification methods known in the art, such as chromatography, CsCl gradients, and other methods described, for example, in U.S. Patent Nos. 6,989,264 and 8,137,948, and WO 2010 / 148143. In some embodiments, residual helper virus can be inactivated using known methods, such as by heating.

[0355] (5.3.4.Cell) Various host cells can be used to produce the rAAV particles described herein.Suitable host cells for producing AAV particles from the polynucleotides and AAV vectors provided herein include microorganisms, yeast cells, insect cells, and mammalian cells.Typically, such cells can be or have been used as recipients of heterologous nucleic acid molecules, and can be grown, for example, in suspension culture and bioreactor.

[0356] In some embodiments, the cell is a mammalian host cell, such as HEK293, HEK293-T, A549, WEHI, 10T1 / 2, BHK, MDCK, COS1, COS7, BSC 1, BSC 40, BMT 10, VERO, W138, HeLa, 293, Jurkat, 2V6.11, Saos, C2C12, L, HT1080, HepG2, primary fibroblasts, hepatocytes, and myoblasts.

[0357] In other embodiments, the cells are insect cells, such as Sf9, SF21, SF900+, or Drosophila cell lines, mosquito cell lines, such as Aedes albopictus cell lines, silkworm cell lines, such as Bombyx mori cell lines, Trichoplusia ni cell lines, such as High Five cells, or Lepidoptera cell lines, such as Ascalapha odorata cell lines. In some embodiments, the insect cells are cells from insect species susceptible to baculovirus infection, including High Five, Sf9, Se301, SeIZD2109, SeUCR1, Sf900+, Sf21, BTI-TN-5B1-4, MG-1, Tn368, HzAm1, BM-N, Ha2302, Hz2E5, and Ao38. For example, large-scale production of recombinant AAV in cells, including Sf9 insect cells, has been described by Kotin RM. Hum Mol Genet. 20(R1):R2-R6 (2011) doi:10.1093 / hmg / ddr141.Methods for molecular engineering and expression of polypeptides in insect cells are described, for example, in Summers and Smith, A Manual of Methods for Baculovirus Vectors and Insect Culture Procedures, Texas Agricultural Experimental Station Bull. No. 7555, College Station, Tex. (1986); King, LA and RD Possee, The baculovirus expression system, Chapman and Hall, United Kingdom (1992); O'Reilly, DR, LK Miller, VA Luckow, Baculovirus Expression Vectors: A Laboratory Manual, New York (1992); WH Freeman and Richardson, CD, Baculovirus Expression Protocols, Methods in Molecular Biology, volume 39 (1995).

[0358] 5.4. Polynucleotides In certain embodiments, the present disclosure provides polynucleotides encoding various fusion proteins provided herein. In an exemplary embodiment, the nucleic acid molecule provided herein comprises a sequence encoding a fusion protein having the sequence of SEQ ID NO:7. In an exemplary embodiment, the nucleic acid molecule provided herein comprises a sequence encoding a fusion protein having the sequence of SEQ ID NO:8. In an exemplary embodiment, the nucleic acid molecule provided herein comprises a sequence encoding a fusion protein having the sequence of SEQ ID NO:9. In an exemplary embodiment, the nucleic acid molecule provided herein comprises a sequence encoding a fusion protein having the sequence of SEQ ID NO:10. In an exemplary embodiment, the nucleic acid molecule provided herein comprises a sequence encoding a fusion protein having the sequence of SEQ ID NO:58. In an exemplary embodiment, the nucleic acid molecule provided herein comprises a sequence encoding a fusion protein having the sequence of SEQ ID NO:59. In an exemplary embodiment, the nucleic acid molecule provided herein comprises a sequence encoding a fusion protein having the sequence of SEQ ID NO:60. In an exemplary embodiment, the nucleic acid molecule provided herein comprises a sequence encoding a fusion protein having the sequence of SEQ ID NO:61. In an exemplary embodiment, the nucleic acid molecule provided herein comprises a sequence encoding a fusion protein having the sequence of SEQ ID NO:62. In an exemplary embodiment, the nucleic acid molecule provided herein comprises a sequence encoding a fusion protein having the sequence of SEQ ID NO:63. In exemplary embodiments, the nucleic acid molecules provided herein comprise a sequence that encodes a fusion protein having the sequence of SEQ ID NO: 64. In exemplary embodiments, the nucleic acid molecules provided herein comprise a sequence that encodes a fusion protein having the sequence of SEQ ID NO: 65. In exemplary embodiments, the nucleic acid molecules provided herein comprise a sequence that encodes a fusion protein having the sequence of SEQ ID NO: 66.

[0359] In some embodiments, the nucleic acid molecule provided herein comprises SEQ ID NO: 67. In some embodiments, the nucleic acid molecule provided herein comprises SEQ ID NO: 68. In some embodiments, the nucleic acid molecule provided herein comprises SEQ ID NO: 69. In some embodiments, the nucleic acid molecule provided herein comprises SEQ ID NO: 70. In some embodiments, the nucleic acid molecule provided herein comprises SEQ ID NO: 71. In some embodiments, the nucleic acid molecule provided herein comprises SEQ ID NO: 72. In some embodiments, the nucleic acid molecule provided herein comprises SEQ ID NO: 73. In some embodiments, the nucleic acid molecule provided herein comprises SEQ ID NO: 74. In some embodiments, the nucleic acid molecule provided herein comprises SEQ ID NO: 75.

[0360] In certain embodiments, the disclosure provides a polynucleotide of any of the recombinant vectors provided herein, including, for example, SEQ ID NOs: 11-23.

[0361] The polynucleotide of the present disclosure can be in the form of RNA or in the form of DNA.DNA includes cDNA, genomic DNA, and synthetic DNA; it can be double-stranded or single-stranded, and if single-stranded, it can be coding strand or non-coding (antisense) strand.In some embodiments, the polynucleotide is in the form of cDNA.In some embodiments, the polynucleotide is a synthetic polynucleotide.

[0362] The present disclosure further relates to variants of the polynucleotides described herein, where the variants encode, for example, fragments, analogs, and / or derivatives of the fusion proteins of the present disclosure. In certain embodiments, the present disclosure provides polynucleotides, including polynucleotides having a nucleotide sequence at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, and in some embodiments, at least about 96%, 97%, 98%, or 99% identical to a polynucleotide encoding a fusion protein of the present disclosure.

[0363] In certain embodiments, the disclosure provides polynucleotides comprising a polynucleotide having a nucleotide sequence at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, and in some embodiments, at least about 96%, 97%, 98%, or 99% identical to the polynucleotides of the vectors provided herein.

[0364] As used herein, the phrase "a polynucleotide having a nucleotide sequence that is at least, for example, 95% "identical" to a reference nucleotide sequence" is intended to mean that the nucleotide sequence of the polynucleotide is identical to the reference sequence, except that the polynucleotide sequence may contain up to 5 point mutations per each 100 nucleotides of the reference nucleotide sequence. That is, to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to the reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or replaced with another nucleotide, or several nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may be present at the 5' or 3' terminal position of the reference nucleotide sequence, or anywhere between these terminal positions, either individually dispersed among the nucleotides in the reference sequence or dispersed in one or more consecutive groups within the reference sequence.

[0365] Polynucleotide variants can contain changes in coding regions, non-coding regions, or both. In some embodiments, polynucleotide variants can contain changes that produce silent substitutions, additions, or deletions, but do not change the properties or activity of the encoded polypeptide. In some embodiments, polynucleotide variants contain silent substitutions that do not cause changes in the amino acid sequence of a polypeptide (due to the degeneracy of the genetic code). Polynucleotide variants can be produced for various reasons, such as to optimize codon expression for a particular host (i.e., to change the codons in human mRNA to those preferred by bacterial hosts, such as E. coli). In some embodiments, polynucleotide variants contain at least one silent mutation in the non-coding or coding regions of the sequence.

[0366] In some embodiments, polynucleotide variants are produced to modulate or alter expression (or expression levels) of an encoded polypeptide. In some embodiments, polynucleotide variants are produced to increase expression of an encoded polypeptide. In some embodiments, polynucleotide variants are produced to decrease expression of an encoded polypeptide. In some embodiments, polynucleotide variants increase expression of an encoded polypeptide compared to the parent polynucleotide sequence. In some embodiments, polynucleotide variants decrease expression of an encoded polypeptide compared to the parent polynucleotide sequence.

[0367] In certain embodiments, the polynucleotide is isolated, hi certain embodiments, the polynucleotide is substantially pure.

[0368] 5.5. Pharmaceutical Compositions In one aspect, the present disclosure further provides a pharmaceutical composition comprising the fusion protein, vector, or viral particle of the present disclosure. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the fusion protein, vector, or viral particle provided herein, and a pharma- ceutical acceptable excipient.

[0369] In some embodiments, provided herein is a pharmaceutical composition comprising a therapeutically effective amount of a fusion protein provided herein and a pharma- ceutical acceptable excipient.

[0370] In some embodiments, provided herein is a pharmaceutical composition comprising a therapeutically effective amount of a rAAV vector provided herein and a pharma- ceutical acceptable excipient.

[0371] In other embodiments, provided herein is a pharmaceutical composition comprising a therapeutically effective amount of the rAAV particles provided herein and a pharma- ceutical acceptable excipient.

[0372] In specific embodiments, the term "excipient" can also refer to a diluent, adjuvant (e.g., Freund's adjuvant (complete or incomplete), carrier, or vehicle. Pharmaceutical excipients can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, mineral oil, sesame oil, and the like. Saline solutions and aqueous glucose and glycerol solutions can also be employed as liquid excipients. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, glycerol ... Examples of suitable pharmaceutical excipients include malt, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, nonfat dry milk, glycerol, propylene, glycol, water, ethanol, etc. The compositions can also contain small amounts of wetting or emulsifying agents, or pH buffering agents, if desired. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like. Examples of suitable pharmaceutical excipients are described in "Remington's Pharmaceutical Sciences" (1990) Mack Publishing Co., Easton, PA. Such compositions contain a prophylactically or therapeutically effective amount of the active ingredient provided herein, e.g., in purified form, together with a suitable amount of excipients, so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

[0373] In some embodiments, the choice of excipient will be determined, in part, by the particular cells, binding molecules, viral particles, and / or method of administration. Accordingly, a variety of suitable formulations exist.

[0374] Typically, acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include buffers, antioxidants including ascorbic acid, methionine, vitamin E, sodium metabisulfite; preservatives, isotonicity agents, stabilizers, metal complexes (e.g., Zn-protein complexes); chelating agents such as EDTA, and / or non-ionic surfactants.

[0375] In particular, when stability is pH-dependent, buffering agents can be used to adjust pH in a range that optimizes therapeutic effectiveness.Suitable buffering agents for use with the present disclosure include both organic and inorganic acids and their salts.For example, citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, acetate.Furthermore, buffering agents can include histidine and trimethylamine salts, such as Tris.

[0376] Preservatives can be added to retard microbial growth.Suitable preservatives for use with the present disclosure include octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium halides (e.g., benzalkonium chloride, benzalkonium bromide, benzalkonium iodide), benzethonium chloride; thimerosal, phenol, butyl alcohol or benzyl alcohol; alkyl parabens, e.g., methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol, 3-pentanol, and m-cresol.

[0377] Tonicity agents, sometimes known as "stabilizers", can be present to adjust or maintain the tonicity of the liquid in the composition.When used with large charged biomolecules such as proteins and antibodies, they are often referred to as "stabilizers" because they can interact with the charged groups of amino acid side chains, thereby reducing the possibility of inter- and intra-molecular interactions.Exemplary tonicity agents include polyhydric sugar alcohols, trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, and mannitol.

[0378] Further exemplary excipients include: (1) bulking agents, (2) solubility enhancers, (3) stabilizers, and (4) agents that prevent denaturation or adhesion to container walls. Such excipients include: polyhydric sugar alcohols (listed above); amino acids such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, threonine, and the like; organic acids or sugar alcohols such as sucrose, lactose, lactitol, trehalose, stachyose, mannose, sorbose, xylose, ribose, ribitol, myoinisitose ... Examples of suitable sugars include myoinisitol, galactose, galactitol, glycerol, cyclitols (e.g., inositol), polyethylene glycol; sulfur-containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol, and sodium thiosulfate; low molecular weight proteins, such as human serum albumin, bovine serum albumin, gelatin, or other immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; monosaccharides (e.g., xylose, mannose, fructose, glucose; disaccharides (e.g., lactose, maltose, sucrose); trisaccharides, such as raffinose; and polysaccharides, such as dextrin or dextran.

[0379] Non-ionic surfactants or detergents (also known as "wetting agents") can be present to aid in solubilization of the therapeutic agent and to protect the therapeutic protein from agitation-induced aggregation, which also allows the formulation to be exposed to shear surface stresses without causing denaturation of the active therapeutic protein. Suitable non-ionic surfactants include, for example, polysorbates (20, 40, 60, 65, 80, etc.), poloxamers (184, 188, etc.), PLURONIC® polyols, TRITON®, polyoxyethylene sorbitan monoethers (TWEEN®-20, TWEEN®-80, etc.), lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50, and 60, glycerol monostearate, sucrose fatty acid esters, methylcellulose, and carboxymethylcellulose. Anionic detergents that can be used include sodium lauryl sulfate, dioctyl sodium sulfosuccinate, and dioctyl sodium sulfonate. Cationic detergents include benzalkonium chloride or benzethonium chloride.

[0380] For use in in vivo administration, the pharmaceutical composition is preferably sterilized.The pharmaceutical composition may be sterilized by filtration through a sterile filtration membrane.The pharmaceutical composition herein can be usually placed in a container with a sterile access port, for example, an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic injection needle.

[0381] Routes of administration are in accordance with known and accepted methods, such as by single or multiple boluses or infusions over an extended period of time in a suitable manner, such as injection or infusion by subcutaneous, intravenous, intraperitoneal, intramuscular, intraarterial, intralesional, or intraarticular routes, intravitreal, subretinal injection, topical administration, inhalation, or by sustained or sustained release means.

[0382] In another embodiment, pharmaceutical composition can be provided as a controlled release or sustained release system.In one embodiment, controlled or sustained release can be achieved by using pump (see, for example, Sefton, Crit. Ref. Biomed. Eng. 14:201-40(1987); Buchwald et al., Surgery, 88:507-16(1980); and Saudek et al., N. Engl. J. Med. 321:569-74(1989)). In another embodiment, polymeric materials can be used to achieve controlled or sustained release of a prophylactic or therapeutic agent (e.g., a fusion protein described herein) or composition provided herein (see, e.g., Medical Applications of Controlled Release (Langer and Wise, eds., 1974); Controlled Drug Bioavailability, Drug Product Design and Performance (Smolen and Ball, eds., 1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61-126 (1983); Levy et al., Science 228:190-92 (1985); During et al., Ann. Neurol. 25:351-56 (1989); Howard et al., J. Neurosurg. 71:105-12 (1989); U.S. Patent Nos. 5,679,377; 5,916,597; 5,912,015; 5,989,463; and 5,128,326; see PCT Publication Nos. WO 99 / 15154 and WO 99 / 20253).Examples of polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolide (PLG), polyanhydride, poly(N-vinylpyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactide (PLA), poly(lactide-co-glycolide) (PLGA), and polyorthoester.In one embodiment, the polymers used in sustained release formulations are inert, free of eluting impurities, stable during storage, sterile, and biodegradable.

[0383] In yet another embodiment, a controlled or sustained release system can be placed in close proximity to a specific target tissue, such as the nasal cavity or lungs, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, Medical Applications of Controlled Release Vol. 2, 115-38 (1984)). Controlled release systems are discussed, for example, by Langer, Science 249:1527-33 (1990). Any technique known to one of skill in the art can be used to make sustained release formulations containing one or more of the agents described herein (see, e.g., U.S. Pat. No. 4,526,938; PCT Publication Nos. WO 91 / 05548 and WO 96 / 20698; Ning et al., Radiotherapy & Oncology 39:179-89 (1996); Song et al., PDA J. of Pharma. Sci. & Tech. 50:372-97 (1995); Cleek et al., Pro. Int'l. Symp. Control. Rel. Bioact. Mater. 24:853-54 (1997); and Lam et al., Proc. Int'l. Symp. Control Rel. Bioact. Mater. 24:759-60 (1997)).

[0384] The pharmaceutical compositions described herein may also contain multiple active compounds or agents as needed to treat a particular indication. Alternatively, or in addition, the composition may include a cytotoxic agent, a chemotherapeutic agent, a cytokine, an immunosuppressant, or an antiproliferative agent. Such molecules are preferably present in combination in amounts that are effective for the intended purpose.

[0385] The active ingredient can also be encapsulated in microcapsules, such as hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, respectively, colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or macroemulsions, prepared, for example, by coacervation techniques or by interfacial polymerization. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 18th Edition.

[0386] A variety of compositions and delivery systems are known and can be used with the therapeutic agents provided herein, including, but not limited to, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the therapeutic molecules provided herein, construction of nucleic acids as part of viral or other vectors.

[0387] In some embodiments, the pharmaceutical composition provided herein contains the binding molecule and / or the viral particle in an amount effective for treating or preventing disease or disorder, for example, a therapeutically effective amount or a prophylactically effective amount.The therapeutic or prophylactic efficacy in some embodiments is monitored by periodic evaluation of the treated subject.For repeated administration over several days or longer, depending on the condition, the treatment is repeated until the desired suppression of disease symptoms occurs.However, other dosing regimes may be useful and may be determined.

[0388] In specific embodiments, the pharmaceutical compositions provided herein are suitable for intravitreal or subretinal injection into the eye of a subject.

[0389] 5.6. Methods and Uses In another aspect, provided herein are methods and uses of the fusion proteins, vectors, or viral particles (rAAV) provided herein.

[0390] Such methods and uses include, for example, therapeutic methods and uses that include administration of the molecule, rAAV, or a composition comprising the same to a subject having a disease or disorder. In some embodiments, the molecule, viral particle, and / or composition is administered in an effective amount to achieve treatment of the disease or disorder. Uses include the use of the fusion protein or viral particle in such methods and treatments, and in the preparation of a medicament for carrying out such therapeutic methods. In some embodiments, the method is carried out by administering the fusion protein or viral particle, or a composition comprising the same, to a subject having or suspected of having a disease or disorder. In some embodiments, the method thereby treats the disease or disorder in the subject.

[0391] In some embodiments, the treatment provided herein causes complete or partial improvement or alleviation of a disease or disorder, or symptoms, adverse effects or outcomes, or phenotypes associated therewith. The desired effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological consequences of disease, prevention of metastasis, reduction of the rate of disease progression, improvement or mitigation of disease state, and remission or improved prognosis. These terms include, but do not necessarily include, complete cure of disease or complete elimination of any symptoms or effects on all symptoms or outcomes.

[0392] As used herein, in some embodiments, the treatments provided herein delay the onset of a disease or disorder, for example, suspend, prevent, slow, delay, stabilize, inhibit, and / or postpone the onset of a disease (e.g., cancer or eye disease). This delay can be of varying lengths, depending on the history of the disease and / or individual being treated. As will be apparent to one of skill in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease or disorder.

[0393] In other embodiments, the methods or uses provided herein prevent a disease or disorder. In some embodiments, the disease or disorder is associated with VEGF and / or angiopoietin. In some embodiments, the disease or disorder is associated with angiogenesis.

[0394] The disease or disorder herein includes, but is not limited to, inflammatory disease, eye disease, autoimmune disease, or cancer.In some embodiments, the disease or disorder is selected from the group consisting of rheumatoid arthritis, inflammatory arthritis, osteoarthritis, cancer, age-related macular degeneration (AMD) (e.g., wet AMD or dry AMD), eye disease characterized by neovascularization (e.g., choroidal neovascularization), uveitis (e.g., anterior uveitis or posterior uveitis), retinitis pigmentosa, and diabetic retinopathy.In some embodiments, the disease or disorder is age-related macular degeneration (AMD) (e.g., wet AMD or dry AMD).

[0395] In some embodiments, treatment with a rAAV comprising a fusion protein or a nucleic acid encoding a fusion protein results in lower lesion levels in a CNV animal model than treatment with no treatment or a negative control. In some embodiments, the CNV animal model is a mouse model. In some embodiments, the lesions are grade 3 lesions, whereas the angiogram is scored as follows: score 0, no staining; score 1, slightly staining; score 2, moderately staining; and score 3, strongly staining. In some embodiments, treatment with a rAAV comprising a fusion protein or a nucleic acid encoding a fusion protein results in lower lesion levels in a CNV animal model than treatment with a reference agent. In some embodiments, the reference agent is an agent that treats AMD. In some embodiments, the reference agent is a known drug that treats AMD. In some embodiments, treatment with a rAAV comprising a fusion protein or a nucleic acid encoding a fusion protein inhibits late CNV lesions.

[0396] In some embodiments, the fusion proteins provided herein bind to VEGF-A. In some embodiments, the fusion proteins provided herein bind to Ang2. In some embodiments, the fusion proteins provided herein bind to VEGF-C. In some embodiments, the fusion proteins provided herein bind to any two of VEGF-A, Ang2, and VEGF-C. In some embodiments, the fusion proteins provided herein bind to all three of VEGF-A, Ang2, and VEGF-C.

[0397] In some embodiments, the disease or disorder is an autoimmune disease, e.g., rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, vasculitis (inflammation of the blood vessels), polyneuropathy, skin ulceration, visceral infarction, pleuritis, interstitial fibrosis, Kaplan's syndrome, pleuropulmonary nodules, pneumonitis, rheumatic lung disease, or arteritis.

[0398] In certain embodiments, the disease or disorder is an inflammatory disease, such as inflammatory arthritis, osteoarthritis, psoriasis, chronic inflammation, irritable bowel disease, pulmonary inflammation, or asthma.

[0399] In some embodiments, the disease or disorder is cancer, including hematological cancer and solid tumor cancer.In some embodiments, the cancer is prostate cancer, breast cancer, lung cancer, esophageal cancer, colon cancer, rectal cancer, liver cancer, urinary tract cancer (e.g., bladder cancer), kidney cancer, lung cancer (e.g., non-small cell lung cancer), ovarian cancer, cervical cancer, endometrial cancer, pancreatic cancer, gastric cancer, thyroid cancer, skin cancer (e.g., melanoma), hematopoietic cancer of lymphoid or myeloid lineage, head and neck cancer, nasopharyngeal carcinoma (NPC), glioblastoma, teratoma, neuroblastoma, adenocarcinoma, cancer of mesenchymal origin, such as fibrosarcoma or rhabdomyosarcoma, soft tissue sarcoma and carcinoma, choriomas, hepatoblastoma, Kaposi's sarcoma, or Wilms' tumor.

[0400] Other diseases associated with angiogenesis can be treated with the methods and compositions disclosed herein, including, but not limited to, atherosclerosis, retrolental fibroplasia, thyroid hyperplasia (including Grave's disease), nephrotic syndrome, preeclampsia, ascites, pericardial effusion (e.g., associated with pericarditis), and pleural effusion.

[0401] In some embodiments, the methods and compositions provided herein can be used to treat ocular or ocular diseases or disorders.In some embodiments, the ocular disease is uveitis, retinitis pigmentosa, neovascular glaucoma, diabetic retinopathy (DR) (including proliferative diabetic retinopathy), ischemic retinopathy, intraocular neovascularization, age-related macular degeneration (AMD) including wet AMD and dry AMD, retinal neovascularization, diabetic macular edema (DME), diabetic retinal ischemia, diabetic retinal edema, retinal vein occlusion (including central retinal vein occlusion and branch retinal vein occlusion), or macular edema, macular edema after retinal vein occlusion (RVO).In some embodiments, the compositions or methods provided herein treat or prevent one or more symptoms of ocular disease, including but not limited to the formation of intraocular drusen, inflammation of eye or ocular tissue, and vision loss.

[0402] In a specific embodiment, the methods and compositions provided herein are used to treat AMD. AMD is characterized by the progressive loss of central vision resulting from damage to photoreceptor cells in an area of ​​the retina called the macula. AMD is broadly divided into two clinical conditions: wet and dry. It is generally accepted that wet AMD precedes and develops from dry AMD. Dry AMD is characterized by the formation of macular drusen, which are small yellow or white deposits of extracellular material that accumulate between the Bruch's membrane and the retinal pigment epithelium of the eye. Wet AMD, which accounts for the majority of severe vision loss, is associated with neovascularization, where blood vessels grow from the choroid under the retina, and the leakage of these new vessels. The accumulation of blood and fluid leads to retinal detachment, which then leads to rapid photoreceptor degeneration and vision loss in both forms of AMD.

[0403] The fusion protein can be delivered to a subject in a composition. The fusion protein can also be delivered to a subject by a rAAV that contains a nucleic acid that encodes the fusion protein. Pharmaceutical compositions that include the fusion protein or a rAAV that contains a nucleic acid that encodes the fusion protein are provided herein and described in more detail above.

[0404] The pharmaceutical compositions described herein can be administered to an individual by any route, for example, intravascularly (e.g., intravenously (IV) or intraarterially), directly intraarterially, systemically (e.g., by intravenous injection), or locally (e.g., by intraarterial or intraocular injection). Non-limiting exemplary methods of administration include intravenous (e.g., by infusion pump), intraperitoneally, intraocular, intraarterial, intrapulmonary, oral, inhalation, intravesicular, intramuscular, intratracheal, subcutaneous, intraocular, intrathecal, transdermal, transpleural, intraarterial, topical, inhalation (e.g., as a spray mist), mucosal (via nasal mucosa), subcutaneous, transdermal, gastrointestinal, intraarticular, intracapsular, intraventricular, intracranial, intraurethral, ​​intrahepatic, intratumoral, intravitreal, and subretinal injection.

[0405] In some embodiments, the composition is administered directly to the eye or ocular tissue, for example, via intravitreal or subretinal injection. In some embodiments, the composition is administered locally to the eye, for example, via eye drops. In some embodiments, the composition is administered by injection into the eye (intraocular injection) or into tissues related to the eye. The composition can be administered, for example, by intraocular injection, periocular injection, subretinal injection, intravitreal injection, transseptal injection, subscleral injection, intrachoroidal injection, intracameral injection, subconjunctival injection, subtenon injection, retrobulbar injection, peribulbar injection, or posterior juxtascleral delivery.

[0406] The composition can be administered, for example, to the vitreous, aqueous humor, sclera, conjunctiva, the area between the sclera and conjunctiva, retinal choroidal tissue, macula, or other area in or adjacent to the eye of an individual. The composition can also be administered to an individual as an implant, such as a biocompatible and / or biodegradable sustained release formulation that slowly releases the compound over a period of time. The composition can also be administered to an individual using iontophoresis.

[0407] The effective amount of the composition can be determined empirically by one of skill in the art and depends on the type and severity of the disease, the route of administration, disease progression and health status, etc. For example, when administered intraocularly, an rAAV comprising a nucleic acid encoding a fusion protein described herein can be administered in an amount of 1×10 8 ~1×10 15 Vector genome (vg), e.g., 1 × 10 9 ~1×10 11 Vector genome (vg) or 1×10 11 ~1×10 13 Vector genome (vg), including, for example, 1×10 10 , 2×10 10 , 3×10 10 , 4×10 10 , 5×10 10 , 6×10 10 , 7×10 10 , 8×10 10 , 9×10 10 , 1×10 11 , 2×10 11 , 3×1011 , 4×10 11 , 5×10 11 , 6×10 11 , 7×10 11 , 8×10 11 , 9×10 11 , 1×10 12 , 2×10 12 , 3×10 12 , 4×10 12 , 5×10 12 , 6×10 12 , 7×10 12 , 8×10 12 , 9×10 12 , 1×10 13 , 2×10 13 , 3×10 13 , 4×10 13 , 5×10 13 , 6×10 13 , 7×10 13 , 8×10 13 , 9×10 13 , 1×10 14 , 2×10 14 , 3×10 14 , 4×10 14 , 5×10 14 , 6×10 14 , 7×10 14 , 8×10 14 , 9×10 14 A dose of vector genome (vg) can be administered to the subject.

[0408] In some embodiments, the unit dose comprises a volume not exceeding 1 mL. In some embodiments, the unit dose comprises a volume not exceeding 0.5-1.0 mL. In some embodiments, the unit dose comprises a volume not exceeding 0.5 mL. In some embodiments, the unit dose comprises a volume not exceeding 500 μL.

[0409] A pharmaceutical composition comprising a fusion protein may be administered in a single daily dose, or the daily dose may be administered in divided dosages two, three, or four times daily. A composition comprising a fusion protein may be administered multiple times (e.g., two, three, four, or five times) within a period of time (e.g., one week, two weeks, three weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, one year, two years, or three years).

[0410] A pharmaceutical composition comprising a rAAV comprising a nucleic acid encoding a fusion protein can be administered less frequently, for example, once every 3 months, once every 4 months, once every 5 months, once every 6 months, once every 7 months, once every 8 months, once every 9 months, once every 10 months, once every 11 months, once every year, once every 2 years, once every 3 years, once every 4 years, once every 5 years, or even less frequently. In some embodiments, a single dose of a composition comprising a rAAV comprising a nucleic acid encoding a fusion protein as described herein is administered.

[0411] In some embodiments, the pharmaceutical composition provided herein is a suspension, for example, a refrigerated suspension.In some embodiments, the method further comprises stirring the suspension before the administration step to ensure uniform distribution of the suspension.In some embodiments, the method further comprises warming the pharmaceutical composition to room temperature before the administration step.

[0412] The compositions can also be administered in sustained release formulations. Sustained release devices (pellets, nanoparticles, microparticles, nanospheres, microspheres, etc.) can be administered by injection or surgical implantation into the eye or eye-related tissues, for example, intraocular, intravitreal, subretinal, periocular, subconjunctival, or subtenon's capsule at various locations.

[0413] The rAAV comprising the fusion protein or the nucleic acid encoding the fusion protein or the pharmaceutical composition comprising the same can be used alone or in combination with one or more additional therapeutic agents or other therapies. Exemplary additional therapeutic agents include complement inhibitors, antiangiogenic agents, and anti-VEGF agents, such as Lucentis®, Avastin®, Trebananib, and Conbercept. In some embodiments, the combination is provided as a simultaneous administration, in which the fusion protein or the rAAV comprising the nucleic acid encoding the fusion protein and at least one therapeutic agent are administered together in the same composition or simultaneously in different compositions. In some embodiments, the combination is provided as a separate administration, in which the administration of the fusion protein or the rAAV comprising the nucleic acid encoding the fusion protein can occur before, simultaneously, and / or after the administration of at least one therapeutic agent.

[0414] 5.8. Kits and Articles of Manufacture Also provided are kits, unit doses, and articles of manufacture that contain any of the compositions described herein. In some embodiments, kits are provided that contain any of the pharmaceutical compositions described herein, and preferably provide instructions for use thereof.

[0415] The kits of the present application are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar bags or plastic), and the like. The kits can optionally be provided with additional components such as cushioning and instructional information. Thus, the present application also provides articles of manufacture that include vials (e.g., sealed vials), bottles, jars, flexible packaging, and the like.

[0416] The article of manufacture may include a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The containers may be formed from a variety of materials, such as glass or plastic. Typically, the container holds a composition that is effective for treating a disease or disorder described herein (e.g., an ocular disease or disorder) and may have a sterile access port (e.g., the container may be an intravenous fluid bag or vial with a stopper pierceable by a hypodermic needle). The label or package insert indicates that the composition is used to treat a particular disease in an individual. The label or package insert further includes instructions for administering the composition to an individual. The label may indicate reconstitution and / or use instructions. The container holding the pharmaceutical composition may be a multi-use vial that allows for repeated administration (e.g., 2-6 administrations) of the reconstituted formulation. Package insert refers to instructions customarily included in commercial packaging for therapeutic products, which contain information regarding the indications, usage, dosage, administration, contraindications, and / or warnings for the use of such therapeutic products. In addition, the article of manufacture may further include a second container containing a pharma- ceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0417] The kit or article of manufacture may include multiple unit doses of the pharmaceutical composition and instructions for use, packaged in an amount sufficient for storage and use in a pharmacy, such as a hospital pharmacy or compounding pharmacy.

[0418] For the sake of brevity, certain abbreviations are used herein. One example is the one-letter abbreviations that represent amino acid residues. The amino acids and their corresponding three-letter and one-letter abbreviations are as follows: [Table 4]

[0419] The present disclosure is generally disclosed herein using definitive language to describe many embodiments. The present disclosure also specifically includes embodiments in which certain subject matter, such as substances or materials, method steps and conditions, protocols, procedures, assays, or analyses, is entirely or partially excluded. Thus, even if the present disclosure is not generally expressed herein with respect to what the disclosure does not include, aspects that are not explicitly included in the present disclosure are nevertheless disclosed herein.

[0420] Some embodiments of the present disclosure have been described. Nevertheless, it will be understood that various modifications can be made without departing from the spirit and scope of the present disclosure. Thus, the following examples are intended to illustrate, but are not intended to limit the scope of the disclosure described in the claims. EXAMPLES

[0421] 6. Working Examples The following is a description of various methods and materials used in the testing, presented to provide those skilled in the art with a complete disclosure and description of how to make and use the present disclosure, and is not intended to limit the scope of what the inventors regard as their disclosure, nor is it intended to represent all of the experiments that have been and may be performed. It should be understood that the exemplary descriptions written in the present tense have not necessarily been performed, but rather, the descriptions may be performed to generate data, etc., relevant to the teachings of the present disclosure. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, percentages, etc.), but some experimental error and deviation should be accounted for.

[0422] 6.1. Example 1 - Construction of a Fusion Protein Capable of Binding to VEGF and Angiopoietin Exemplary fusion proteins according to the present disclosure have been constructed, and Figure 1 shows these exemplary constructs, from top to bottom: Exemplary Polypeptide 1, Exemplary Polypeptide 2, Exemplary Polypeptide 3, and Exemplary Polypeptide 4.

[0423] Specifically, these fusion proteins contain three binding domains - one derived from VEGFR-1 (i.e., IgG-like domain 2 of VEGFR-1), one derived from VEGFR-2 (i.e., IgG-like domain 3 of VEGFR-2), and one domain capable of binding to angiopoietin (e.g., angiopoietin 1 and angiopoietin 2). A Flt-1 signal domain was also included at the N-terminus of these exemplary constructs. In some constructs, the fusion protein further comprises a human IgG Fc fragment. These exemplary domains and their sequences for the exemplary fusion proteins are provided in the table below. Table 4. Exemplary fusion proteins and their domains [Table 5] TIFF2024514072000011.tif241170TIFF2024514072000012.tif249170

[0424] 6.2. Example 2 - Construction of AAV Vectors Containing Nucleic Acids Encoding Fusion Proteins Nucleic acid sequences encoding the exemplary fusion proteins described in Section 6.1 were introduced into exemplary rAAV vectors derived from rAAV2 in this example to generate rAAV vectors EXG102-02, EXG102-03-01, EXG102-03-02, EXG102-04, EXG102-05, EXG102-06, EXG102-07, EXG102-08, EXG102-09, EXG102-10, EXG102-11, EXG102-12, and EXG102-13.

[0425] Specifically, exemplary polypeptide 1 is the gene of interest in EXG102-04, EXG102-07, and EXG102-08; exemplary polypeptide 2 is the gene of interest in EXG102-09, EXG102-10; exemplary polypeptide 3 is the gene of interest in EXG102-11, EXG102-12. Exemplary polypeptide 4 is the gene of interest in EXG102-13; and control polypeptides without Ang BD were constructed in EXG102-02, EXG102-03-1, EXG102-03-2, EXG102-05, and EXG102-06.

[0426] These vectors are illustrated in Figure 2, where TR represents the AAV2 inverted terminal repeat, CBA represents the 1.68 kb chicken β-actin promoter, CB represents the 0.78 kb small chicken β-actin promoter, D2 represents the IgG-like domain 2 of VEGFR-1, D3 represents the IgG-like domain 3 of VEGFR-2, ABD (or Ang BD) represents the angiopoietin binding domain, IgG Fc represents the Fc fragment of human IgG, Fc-Hinger represents the 21 amino acids at the N-terminus of Fc plus a 6 amino acid GS linker, WPRE represents the woodchuck hepatitis virus posttranscriptional regulatory element (600 bp), mWPRE is the miniWPRE (240 bp), SV40pA is the simian virus 40 polyadenylation signal, and bGHpA is the bovine growth hormone polyadenylation signal. In these constructs, the small CB promoter is only associated with self-complementary AAV (scAAV).

[0427] The nucleic acid sequences of these exemplary rAAV vectors are shown in the table below. Table 5. Exemplary rAAV Vectors [Table 6] TIFF2024514072000014.tif244170TIFF2024514072000015.tif241170TIFF2024514072000016.tif242170TIFF2024514072000017.tif242170TIFF2024514072000018.tif244170TIFF2024514072000019.tif240170TIFF2024514072000020.tif239170TIFF2024514072000021.tif242170TIFF2024514072000022.tif243170TIFF2024514072000023.tif240170TIFF2024514072000024.tif240170TIFF2024514072000025.tif243170TIFF2024514072000026.tif241170TIFF2024514072000027.tif241170TIFF2024514072000028.tif243170TIFF2024514072000029.tif239170TIFF2024514072000030.tif243170TIFF2024514072000031.tif240170TIFF2024514072000032.tif240170TIFF2024514072000033.tif243170TIFF2024514072000034.tif238170TIFF2024514072000035.tif239170TIFF2024514072000036.tif240170TIFF2024514072000037.tif239170TIFF2024514072000038.tif239170TIFF2024514072000039.tif240170TIFF2024514072000040.tif238170TIFF2024514072000041.tif239170TIFF2024514072000042.tif241170TIFF2024514072000043.tif242170TIFF2024514072000044.tif240170TIFF2024514072000045.tif240170TIFF2024514072000046.tif243170TIFF2024514072000047.tif240170TIFF2024514072000048.tif239170 TIFF2024514072000049.tif242170TIFF2024514072000050.tif242170TIFF202451 4072000051.tif238170TIFF2024514072000052.tif241170TIFF2024514072000053 .tif239170TIFF2024514072000054.tif240170TIFF2024514072000055.tif81170.

[0428] 6.3. Example 3 - Protein Expression and Ligand Binding Assays HEK293 cells were transiently transfected with plasmid constructs or infected with AAV vectors expressing D2D3 or D2D3 / ABD (see FIG. 3A). Culture medium containing D2D3 or D2D3 / ABD was added to wells coated with VEGF or Ang, which can capture D2D3 or ABD, respectively. The captured D2D3 or D2D3 / ABD was then incubated with anti-Fc-HRP and quantified by reading OD450 (see FIG. 3B).

[0429] These results indicate that transgene expression from the three codon-optimized versions, EXG102-02, EXG102-03-01, and EXG102-03-02, was comparable to that of scAAV102-06. As shown in Figures 4A-4B and 5A-5B, the position of the Ang BD at the C-terminus did not affect the binding ability of D2D3 to rVEGF, but it significantly reduced the transgene expression level (see EXG102-04, EXG102-07, and EXG102-08).

[0430] In contrast, positioning the Ang BD intermediate D2D3 and Fc did not affect rVEGF binding or transgene expression (see EXG102-09), as shown in Figures 5A-5B. However, positioning the Ang BD intermediate inhibited Ang2 binding, as shown in Figures 6A-6B.

[0431] As shown in Figures 7A-7C, constructs with an Ang BD at the N-terminus (e.g., EXG102-11) can bind to VEGF similarly to construct EXG102-09 in the same CM from transient transfection; moreover, EXG102-04 and EXG102-11 showed the strongest binding to Ang2 in the same CM from transient transfection. Furthermore, EXG102-11 did not reduce transgene expression (see Figure 7D). In summary, EXG102-11 was shown to bind strongly to both VEGF and Ang2 while maintaining high transgene expression levels.

[0432] 6.4. Example 4 - Construction of Fusion Proteins Containing Additional VEGFC Binding Domains (Trap C) Further exemplary fusion proteins according to the present disclosure were constructed as shown in Figure 8. These further constructs contain an additional VEGF binding domain, i.e., a VEGFC binding domain (Trap C). Figures 9A-9I show exemplary polypeptide constructs provided herein encoded by the nucleic acids (transgenes) in EXG102-24, EXG102-25, EXG102-26, EXG102-27, EXG102-28, and EXG102-29, respectively. The signal peptide, ABD, D2, D3, Trap C, and Fc constructs are shown in these figures. Specifically, these exemplary fusion proteins contain four binding domains - one derived from VEGFR-1 (i.e., IgG-like domain 2 of VEGFR-1), one derived from VEGFR-2 (i.e., IgG-like domain 3 of VEGFR-2), one VEGFC binding domain, and one domain capable of binding to angiopoietin (e.g., angiopoietin 1 and angiopoietin 2). These fusion proteins further contain an Fc fragment of human IgG. A Flt-1 signal domain was also included at the N-terminus of these exemplary constructs.

[0433] In some of the present constructs, the ABD comprises one or more repeats of an amino acid sequence having SEQ ID NO:3 or SEQ ID NO:51. In some constructs, the ABD provided herein comprises one repeat of SEQ ID NO:3. In some constructs, the ABD provided herein comprises two repeats of SEQ ID NO:3. In some constructs, the ABD provided herein comprises one repeat of SEQ ID NO:51. In some constructs, the ABD provided herein comprises two repeats of SEQ ID NO:51. In some constructs, the ABD provided herein comprises SEQ ID NO:4. In some constructs, the ABD provided herein comprises SEQ ID NO:52. In some constructs, the ABD provided herein comprises SEQ ID NO:53. In some constructs, the ABD provided herein comprises SEQ ID NO:54.

[0434] Exemplary Trap C sequences that can be included in the constructs are provided below. In some constructs, Trap C comprises the amino acid sequence of SEQ ID NO: 55. In some constructs, Trap C comprises the amino acid sequence of SEQ ID NO: 56. In some constructs, Trap C comprises the amino acid sequence of SEQ ID NO: 57.

[0435] These sequences of these exemplary domains and exemplary fusion proteins are provided in the table below. Table 6. Sequences of exemplary domains and exemplary fusion proteins containing Trap C [Table 7] TIFF2024514072000057.tif238170TIFF2024514072000058.tif241170TIFF2024514072000059.tif239170TIFF2024514072000060.tif236170TIFF2024514072000061.tif242170TIFF2024514072000062.tif241170TIFF2024514072000063.tif140170Table 7. Nucleic acid sequences of exemplary fusion proteins [Table 8] TIFF2024514072000065.tif241170TIFF2024514072000066.tif242170TIFF202 4514072000067.tif238170TIFF2024514072000068.tif238170TIFF20245140720 00069.tif239170TIFF2024514072000070.tif240170TIFF2024514072000071.t if239170TIFF2024514072000072.tif239170TIFF2024514072000073.tif239170 TIFF2024514072000074.tif242170TIFF2024514072000075.tif242170TIFF202 4514072000076.tif239170TIFF2024514072000077.tif239170TIFF20245140720 00078.tif242170TIFF2024514072000079.tif241170TIFF2024514072000080.t if241170TIFF2024514072000081.tif239170TIFF2024514072000082.tif104170

[0436] 6.5. Example 5 - Construction of AAV vectors containing nucleic acids encoding fusion proteins containing additional Trap C domains Nucleic acid sequences encoding the exemplary fusion proteins described in Section 6.4 were introduced into exemplary rAAV vectors in this example, derived from, for example, rAAV2 and rAAV8, to generate rAAV vectors as shown in Figure 8 and Figures 9A-9I.

[0437] 6.6. Example 6 - Protein Expression, Ligand Binding, and Other Functional Assays HEK293 cells are transiently transfected with the plasmid constructs or infected with the AAV vectors described above. Transgene expression is analyzed and compared. Various in vitro and in vivo assays are performed with these constructs.

[0438] As shown in Figures 10A-10F, constructs containing ABD2 are comparable to constructs with the ABD domain in terms of protein expression, VEGF-A binding, or Ang2 binding.

[0439] 6.7. Example 7 - Effect of ANG-2 Binding Domain on Inhibition of CNV Lesions in a CNV Mouse Model FFA mean scores were measured in eyes treated with AAV-GFP (negative control), EXG102-02 (positive control), EXG102-04, EXG102-09, EXG102-10, or EXG102-11 using the CNV mouse model at 29 and 36 days post-injection. Angiograms were scored as follows: score 0, no staining; score 1, slightly stained; score 2, moderately stained; and score 3, strongly stained.

[0440] Figure 11 shows the effect of ANG-2 binding domain compounds on inhibition of CNV lesions compared to a positive control with only a VEGF inhibitor. EXG102-04 and EXG102-11 showed the most effective inhibition of CNV lesions (four wide arrows).

[0441] 6.8. Example 8 - No Grade 3 CNV Lesions in Eyes Treated with EXG102-04, EXG102-09, EXG102-10, or EXG102-11 in a CNV Mouse Model FFA mean scores were measured in eyes treated with AAV-GFP (negative control), EXG102-02 (positive control), EXG102-04, EXG102-09, EXG102-10, or EXG102-11 on days 29 and 36 after injection. Angiograms were scored as follows: score 0, no staining; score 1, slightly stained; score 2, moderately stained; and score 3, strongly stained.

[0442] As shown in FIG. 12, no grade 3 CNV lesions were seen in any eyes treated with EXG102-04, EXG102-09, EXG102-10, or EXG102-11, whereas 40-50% grade 3 lesions were seen in eyes that received AAV2-GFP (negative control).

[0443] 6.9. Example 9 - EXG102-31 binds to VEGF-A, Ang2, and VEGF-C The in vitro VEGF-A, Ang2, or VEGF-C binding affinity of the transgene products expressed from HEK293T cells was measured. HEK293T cells were transfected with plasmid DNA pEXG102-02, pEXG102-30, or pEXG102-31. The expressed target proteins were affinity purified from cell lysates. The binding ability of EXG102-02, EXG102-30, and EXG102-31 to VEGF-A, Ang2, and VEGF-C, respectively, was measured by ELISA.

[0444] 12A-12C show that EXG102-31 was able to bind to each of VEGF-A, Ang2, and VEGF-C. Surprisingly, the VEGF-A binding affinity of EXG102-31 is similar to that of the positive control EXG102-02.

[0445] 6.10. Example 10 - CNV lesion scores were reduced in treated groups in a CNV mouse model FFA mean scores were measured in eyes treated with vehicle control, EXG102-02, EXG102-30, or EXG102-31. Prior to fluorescein angiography, animals received an intraperitoneal injection of sodium fluorescein (100 mg / ml, 30 μL / animal). FFA images were taken 3 minutes after injection. Angiograms were scored as follows: score 0, no staining; score 1, slightly stained; score 2, moderately stained; and score 3, strongly stained. The percentage of score 3 lesions and the mean score were calculated.

[0446] Figure 14 shows the FFA mean score in the laser-induced CNV mouse model. Compared with the vehicle-treated group, the CNV lesion scores in all treatment groups were significantly reduced on days 29 and 36, respectively (p≦0.001). The CNV lesion scores in the EXG102-31-treated group were higher than those in the EXG102-02 and EXG102-30-treated groups on day 29, but there was no significant difference between the groups treated with the test article on day 36 (p>0.05). This is likely due to the large variability between individual mice. Notably, compared with day 29, the mean volume of CNV lesions in the EXG102-31-treated group was significantly reduced on day 36 (p≦0.05), suggesting the compound effect of the VEGF-C binding domain in EXG102-31 on inhibiting late CNV lesions. Figure 14 suggests that EXG102-31 is more advantageous than EXG102-30 for long-term CNV inhibition because the CNV lesion score improved significantly from 29 to 36 days when compared with that of EXG102-30. According to Cabral, T. et al., Ophthalmol Retina, 2018. 2(1): p. 31-37, the levels of VEGF-C and Ang-2 increased significantly after VEGF-A was inhibited. EXG102-31 can neutralize almost all VEGF subtypes, including VEGF-C, and Ang-2.

[0447] 6.11. Example 11 - No Grade 3 CNV Lesions Observed in Eyes Treated with EXG102-02, EXG102-30, or EXG102-31 The percentage of grade 3 CNV lesions was measured in eyes treated with vehicle control, EXG102-02, EXG102-30, or EXG102-31. Prior to fluorescein angiography, animals received an intraperitoneal injection of sodium fluorescein (100 mg / ml, 30 μL / animal). FFA images were taken 3 minutes after injection. Angiograms were scored as follows: score 0, no staining; score 1, slightly stained; score 2, moderately stained; and score 3, strongly stained. The percentage of score 3 lesions and the mean score were calculated.

[0448] As shown in FIG. 15, no grade 3 CNV lesions were seen in any eyes treated with EXG102-02, EXG102-30, or EXG102-31, whereas 29 and 21 of 47 CNV grade 3 lesions were seen in eyes receiving the vehicle control.

[0449] The teachings of all patents, published applications, and references cited herein are incorporated by reference in their entirety.

[0450] Although example embodiments have been specifically shown and described, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the embodiments encompassed by the appended claims.

[0451] From the foregoing, it will be understood that, although specific embodiments have been described herein for purposes of illustration, various modifications may be made without departing from the spirit and scope of what is provided herein. All references mentioned above are incorporated herein by reference in their entirety.

Claims

1. (i) the first domain that binds VEGF; (ii) a second domain that binds to VEGF; and (iii) a third domain that binds to angiopoietin; A polypeptide comprising:

2. The polypeptide of claim 1 , wherein the third domain is N-terminal to the first domain and the second domain.

3. The first domain is (i) derived from VEGF receptor-1 (VEGFR-1 or FLT-1); (ii) contains domain 2 of VEGFR-1 or a variant thereof; (iii) comprises or consists of the amino acid sequence of SEQ ID NO:1; and / or (iv) comprising or consisting of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:1; The polypeptide of claim 1.

4. The second domain is (i) derived from VEGF receptor-2 (VEGFR-2 or Flk-1); (ii) contains domain 3 of VEGFR-2 or a variant thereof; (iii) comprises or consists of the amino acid sequence of SEQ ID NO:2; or (iv) comprising or consisting of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:2; A polypeptide according to any one of claims 1 to 3.

5. The third domain is (i) one or two repeats of the amino acid sequence of SEQ ID NO:3; and / or one or two repeats of the amino acid sequence of SEQ ID NO:51, optionally, wherein (ia) the third domain comprises two repeats of the amino acid sequence of SEQ ID NO:3, (ib) the third domain comprises two repeats of the amino acid sequence of SEQ ID NO:51; or (ic) the third domain comprises the amino acid sequence of SEQ ID NO:3 and the amino acid sequence of SEQ ID NO:51; (ii) comprises or consists of the amino acid sequence of SEQ ID NO:4, or the third domain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:4; (iii) comprising or consisting of the amino acid sequence of SEQ ID NO:52, or the third domain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:52; (iv) comprises or consists of the amino acid sequence of SEQ ID NO:53, or the third domain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:53; (v) comprises or consists of the amino acid sequence of SEQ ID NO:54, or the third domain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:54; or (vi) at the N-terminus of the first domain and the second domain, A polypeptide according to any one of claims 1 to 4.

6. (i) a first domain that binds VEGF, said first domain comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1; (ii) a second domain that binds VEGF, the second domain comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:2; and (iii) a third domain that binds to angiopoietin, the third domain comprising two amino acid sequences each comprising an amino acid sequence having at least 80%, 85%, 90%, or 100% identity to SEQ ID NO:3; two amino acid sequences each comprising an amino acid sequence having at least 80%, 85%, 90%, or 100% identity to SEQ ID NO:51; or one amino acid sequence having at least 80%, 85%, 90%, or 100% identity to SEQ ID NO:3 and one amino acid sequence having at least 80%, 85%, 90%, or 100% identity to SEQ ID NO:51; A polypeptide comprising:

7. (i) an Fc region of an antibody or variant thereof, optionally comprising the amino acid sequence of SEQ ID NO:5; (ii) a signal peptide, optionally comprising the amino acid sequence of SEQ ID NO: 6; and / or (iii) one or more linkers The polypeptide of any one of claims 1 to 6, further comprising:

8. (i) an amino acid sequence of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10; or (ii) a polypeptide comprising the amino acid sequence of SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, or SEQ ID NO:66, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, or SEQ ID NO:

66.

9. the polypeptide further comprises a VEGFC binding domain, optionally wherein the VEGFC binding domain (i) derived from VEGFR-2; (ii) derived from VEGF receptor-3 (VEGFR-3); (iii) comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:55; (iv) comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 56; or (v) comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 57; A polypeptide according to any one of claims 1 to 8.

10. The polypeptide of any one of claims 1 to 9, wherein the polypeptide is genetically fused or chemically conjugated to the agent.

11. 10. An isolated nucleic acid comprising a nucleic acid sequence encoding the polypeptide of any one of claims 1 to 9.

12. 12. A vector comprising the isolated nucleic acid of claim 11, wherein optionally the vector is a viral vector, wherein optionally the viral vector is an adeno-associated viral (AAV) vector, wherein optionally the AAV vector is derived from AAV1, AAV2, AAV2i8, AAV3, AAV3-B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV13, AAV-DJ, AAV LK03, AAVrh74, AAV44-9, or a combination or variant thereof, and optionally the vector is a recombinant AAV2 (rAAV2) vector, a recombinant AAV8 (rAAV8) vector, or a variant thereof.

13. A recombinant AAV (rAAV) vector comprising: (i) a nucleic acid encoding a polypeptide, the polypeptide comprising: (ia) a first domain derived from VEGFR-1; (ib) a second domain derived from VEGFR-2; and (ic) a third domain capable of binding to angiopoietin, wherein the rAAV vector comprises inverted terminal repeats (ITRs) from AAV1, AAV2, AAV2i8, AAV3, AAV3-B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV13, AAV-DJ, AAV LK03, AAVrh74, or AAV44-9; or (ii) a nucleic acid encoding a polypeptide, the polypeptide comprising: (ii-a) a first domain derived from VEGFR-1; (ii-b) a second domain derived from VEGFR-2; (ii-c) a third domain capable of binding to angiopoietin; and (ii-d) a fourth domain capable of binding to VEGFC, wherein the rAAV vector comprises inverted terminal repeats (ITRs) from AAV1, AAV2, AAV2i8, AAV3, AAV3-B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV13, AAV-DJ, AAV LK03, AAVrh74, or AAV44-9.

14. The rAAV vector of claim 13, wherein the ITRs are derived from AAV2 or AAV8, and optionally the third domain is N-terminal to the first domain and the second domain.

15. A vector comprising: (i) the nucleic acid sequence of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, or SEQ ID NO:23, or a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, or SEQ ID NO:23; or (ii) a nucleic acid sequence of SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, or SEQ ID NO:75, or a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, or SEQ ID NO:

75.

16. A recombinant AAV (rAAV) particle comprising: (a) a nucleic acid encoding a polypeptide, the polypeptide comprising: (i) a first domain derived from VEGFR-1; (ii) a second domain derived from VEGFR-2; and (iii) a third domain capable of binding to angiopoietin; or A nucleic acid encoding a polypeptide, the polypeptide comprising: (i) a first domain derived from VEGFR-1; (ii) a second domain derived from VEGFR-2; (iii) a third domain capable of binding to angiopoietin; and (iv) a fourth domain capable of binding to VEGFC. and, (b) The capsid protein of AAV1, AAV2, AAV2i8, AAV3, AAV3-B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV13, AAV-DJ, AAV LK03, AAVrh74, AAV44-9, or a variant thereof.

17. the third domain is N-terminal to the first domain and the second domain, and optionally the capsid protein (i) the AAV2 capsid protein; (ii) is an AAV8 capsid protein; or 17. The rAAV particle of claim 16, wherein (iii) a variant of an AAV2 capsid protein comprising the amino acid sequence of SEQ ID NO: 48, wherein the variant of the AAV2 capsid protein comprises the following amino acid substitutions in AAV2 capsid protein VP1: Y444F, R487G, T491V, Y500F, R585S, R588T, and Y730F.

18. A pharmaceutical composition comprising a polypeptide according to any one of claims 1 to 10, a vector or rAAV vector according to any one of claims 12 to 15, or an rAAV particle according to claim 16 or 17, and a pharma- ceutical acceptable excipient.

19. 19. A composition comprising a polypeptide according to any one of claims 1 to 10, a vector or an rAAV vector according to any one of claims 12 to 15, an rAAV particle according to claim 16 or 17, or a pharmaceutical composition according to claim 18, for use in a method of treating a disease or disorder in a subject by administering the composition to the subject.

20. The disease or disorder (i) an angiogenic or neovascular disease or disorder, (ii) the disease is an inflammatory disease, an eye disease, an autoimmune disease, or cancer; (iii) an ocular disease or disorder, optionally wherein the ocular disease or disorder is selected from the group consisting of uveitis, retinitis pigmentosa, neovascular glaucoma, diabetic retinopathy (DR) (including proliferative diabetic retinopathy), ischemic retinopathy, intraocular neovascularization, age-related macular degeneration (AMD), retinal neovascularization, diabetic macular edema (DME), diabetic retinal ischemia, diabetic retinal edema, retinal vein occlusion (including central retinal vein occlusion and branch retinal vein occlusion), macular edema, and macular edema following retinal vein occlusion (RVO); or (iv) age-related macular degeneration (AMD), optionally wherein the AMD is wet AMD (wAMD); 20. The composition of claim 19, wherein the method optionally comprises administering by intravitreal or subretinal injection into the eye of the subject.

21. A polypeptide according to any one of claims 1 to 10, a vector or rAAV vector according to any one of claims 12 to 15, an rAAV particle according to claim 16 or 17, or a pharmaceutical composition according to claim 18, for use in therapy.

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