Tie2 binding agents and methods of use thereof

Abstract

The invention provides Tie-2 antibodies and fragments and conjugates thereof and methods of using the same.

Description

[0001] This application is a divisional application of the application filed on March 22, 2021, Chinese Application No. 202180023203.0, entitled “TIE2 BINDING AGENTS AND METHODS OF USING THE SAME.”

[0002] Cross Reference to Related Applications

[0003] This application claims priority to U.S. Provisional Patent Application No. 62 / 993,930, filed March 24, 2020, and U.S. Provisional Patent Application No. 63 / 046,318, filed June 30, 2020, which are incorporated by reference in their entirety.

[0004] SEQUENCE LISTING

[0005] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and which is hereby incorporated by reference in its entirety. The ASCII copy, named P35891-WO_SeqList.txt was created on March 17, 2021, and is 107,354 bytes in size. TECHNICAL FIELD

[0006] The presently disclosed subject matter relates to Tie2 binding agents, including anti-Tie2 antibodies, as well as conjugates and methods of using the binding agents. BACKGROUND

[0007] Tie2 is a promising therapeutic target for the treatment of various ocular diseases (see, e.g., Campochiaro and Peters, 2016, Curr Diab Rep, 16:126, Whitehead et al., 2019, J Diabetes Res, 2019:5140521, Hussain et al., 2019, Expert Opin Investig Drug, 28:861-869). Tie2 is a receptor tyrosine kinase that is specifically expressed by endothelial cells and has been shown to promote endothelial stabilization and reduce vascular permeability. Vascular leakage is known to contribute to visual impairment in several common ocular diseases, including but not limited to diabetic macular edema (DME), diabetic retinopathy (DR), and age-related macular degeneration (AMD).

[0008] The most extensively studied Tie2 ligands are Angiopoietin 1 (Angl) and Angiopoietin 2 (Ang2). Angl is a strong agonist of Tie2 and has been shown to inhibit ocular neovascularization and breakdown of the blood-retinal barrier (see, e.g., Nambu et al., 2004, Gene Therapy, 11 :865-873). Ang2 is a context-dependent antagonist of Tie2 and its increased expression is associated with multiple ocular diseases, including DME, wet AMD, DR, metastasis, sepsis, and inflammation. In addition, Ang2 competitively binds to Tie2 and inhibits Angl signaling, leading to endothelial and vascular instability, breakdown of the blood-retinal barrier, and inflammation (Klaassen et al., 2013, Prog Retin Eye Res, 34: 19-48, Saharinen et al., 2017, Nat Rev Drug Discov, 16:635-661).

[0009] Tie receptors, including Tie 1 and Tie 2, are type 1 transmembrane protein receptor tyrosine kinases (RTKs) (Ramsauer, M. & D’Amore, P. A. J. Clin. Invest. (2002); 110: 1615-1617). Tie represents a tyrosine kinase receptor with immunoglobulin and EGF homology domains. Tie 2 is located in all endothelial cells that form blood vessels and in the endocardium of mouse embryos (Korhonen et al., Blood (1992); 80: 2548-2555). The extracellular domain or extracellular domain (“ECD”) of Tie 2 includes three immunoglobulin (Ig) domains (Igl, Ig2, and Ig3), three epidermal growth factor (EGF) domains, and a type III fibronectin domain (FNIII). The Ig-EGF region of Tie 2 mediates angiopoietin recognition and binding (Fiedler, U. et al., J. Biol. Chem. (2003); 278: 1721-1727; Barton, W. A., et al., Structure (2005); 13: 825-832).

[0010] Two ligands for the Tie2 receptor have been identified: Angiopoietin-1 (Angl) and Angiopoietin-2 (Ang2). Ang-1 is a Tie2 agonist that binds to and induces tyrosine phosphorylation of Tie2, and its expression in vivo is closely associated with vascular development (Davis et al., Cell (1996); 87: 1161-1169). Mice lacking Ang-1 exhibit defects in angiogenesis reminiscent of those observed in mice lacking Tie2, supporting the notion that Ang-1 is the primary physiological ligand for Tie2 and that Tie2 has a critical role in angiogenesis in vivo (Suri et al., Cell (1996); 87: 1171-1180). Ang-1 is anti-inflammatory, promotes vascular integrity, and decreases vascular permeability. Ang2 is recognized as a naturally occurring antagonist of Tie2. Overexpression of Ang2 by gene transfer disrupts vascular formation in mouse embryos (Maisonpierre et al., Science 277:55-60, 1997). Ang2 can be pro-inflammatory, disrupts EC quiescence, and increases vascular permeability. Collectively, studies support a critical role for the Angl / Ang2 / Tie2 system in angiogenesis. Given the important role of Tie2 in angiogenesis, there is a need to identify agents for Tie2 and methods of using these agents. In addition, compositions that act as Tie2 agonists and can decrease vascular permeability or increase vascular integrity have great potential as therapeutic agents, particularly for treating ocular diseases. SUMMARY

[0011] The present invention provides anti-Tie2 antibodies, compositions (e.g., conjugates) comprising the anti-Tie2 antibodies or fragments thereof, and methods of using the same.

[0012] The presently disclosed subject matter provides isolated antibodies, or antigen binding fragments thereof, that specifically bind Tie2, compositions comprising at least one or more anti-Tie2 antibodies or antigen binding fragments thereof, and methods of using the same. In one exemplary embodiment, the anti-Tie2 antibody is a Fab.

[0013] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds Tie2 is provided, wherein the anti-Tie2 antibody comprises a heavy chain (HC) variable domain (VH domain) and a light chain (LC) variable domain (VL domain), wherein the VH domain comprises: a CDR-H1 comprising the amino acid sequence of NTDIS (SEQ ID NO: 3), a CDR-H2 comprising the amino acid sequence of RISPSDGNTYYADSVKG (SEQ ID NO: 4), and a CDR-H3 comprising the amino acid sequence of (a) RTRWASXiAX2DY (SEQ ID NO: 5, wherein Xi is M, L, K, F, Y, R, N, Q, H, or W, and / or X2 is F, Y, L, Q, I, K, or H), (b) RTRWASWAMDY (SEQ ID NO: 6), or (c) RTRWASWAFDY (SEQ ID NO: 7); and the VL domain comprises: a CDR-L1 comprising the amino acid sequence of RASQDVSTAVA (SEQ ID NO: 8), a CDR-L2 comprising the amino acid sequence of SASFLYS (SEQ ID NO: 9), and a CDR-L3 comprising the amino acid sequence of QQSYTTPPT (SEQ ID NO: 10). In some embodiments, the CDR-H3 comprises SEQ ID NO: 6 or SEQ ID NO: 7. In particular embodiments, the CDR-H3 comprises SEQ ID NO: 7.

[0014] In some embodiments, the anti-Tie2 antibody comprises a VH framework FR1 sequence of SEQ ID NO: 11, a VH framework FR2 sequence of SEQ ID NO: 12, a VH framework FR3 sequence of SEQ ID NO: 13, and / or a VH framework FR4 sequence of SEQ ID NO: 14. In other embodiments, the anti-Tie2 antibody comprises a VL framework FR1 sequence of SEQ ID NO: 15, a VL framework FR2 sequence of SEQ ID NO: 16, a VL framework FR3 sequence of SEQ ID NO: 17, and / or a VL framework FR3 sequence of SEQ ID NO: 18.

[0015] In some embodiments, the anti-Tie2 antibody comprises a VH domain and a VL domain, wherein the VH domain comprises: a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 3, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 4, and, a CDR-H3 comprising the amino acid sequence of (a) SEQ ID NO: 5 (wherein X1is M, L, K, F, Y, R, N, Q, H, or W, and / or X2is F, Y, L, Q, I, K, or H), (b) SEQ ID NO: 6, or (c) SEQ ID NO: 7; and the VL domain comprises: a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, and, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10. In other embodiments, the CDR-H3 comprises SEQ ID NO: 6 or SEQ ID NO: 7. In particular embodiments, the CDR-H3 comprises SEQ ID NO: 7. In other embodiments, the VH domain has at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 20, and the VL domain has at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 21.

[0016] In some embodiments, the anti-Tie2 antibody or antigen-binding fragment thereof comprises a VH domain sequence of SEQ ID NO: 19 or SEQ ID NO: 22 and a VL domain sequence of SEQ ID NO: 21.

[0017] In some embodiments, the anti-Tie2 antibody comprises a VH domain sequence of SEQ ID NO: 20. In other embodiments, the anti-Tie2 antibody comprises a VL domain sequence of SEQ ID NO: 21. In other embodiments, the anti-Tie2 antibody comprises a VH domain sequence of SEQ ID NO: 20 and a VL domain sequence of SEQ ID NO: 21.

[0018] In some embodiments, the anti-Tie2 antibody or fragment thereof comprises a heavy chain (HC) domain sequence of SEQ ID NO: 55 and a light chain (LC) domain sequence of SEQ ID NO: 25. In other embodiments, the anti-Tie2 antibody or fragment thereof comprises a heavy chain (HC) domain sequence of SEQ ID NO: 23 and a light chain (LC) domain sequence of SEQ ID NO: 56.

[0019] In some aspects, there is provided an antibody or antigen-binding fragment thereof that specifically binds Tie2, comprising a VH domain and a VL domain, the VL domain comprising: a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 28, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 29, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 30; the VL domain comprising: a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10. In some embodiments, the VH domain comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 31, and the VL domain comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 21. In other embodiments, the VH domain comprises SEQ ID NO: 31, and the VL domain comprises SEQ ID NO: 21. In other embodiments, the HC comprises SEQ ID NO: 32, and the LC comprises SEQ ID NO: 25.

[0020] In some aspects, there is provided an antibody or antigen-binding fragment thereof that specifically binds Tie2, comprising a VH domain and a VL domain, the VH domain comprising: a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 33, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 34, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 35; the VL domain comprising: a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10. In some embodiments, the VH domain comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 36, and the VL domain comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 21. In other embodiments, the VH domain comprises SEQ ID NO: 36, and the VL domain comprises SEQ ID NO: 21. In other embodiments, the HC comprises SEQ ID NO: 37, and the LC comprises SEQ ID NO: 25.

[0021] In some aspects, an antibody or antigen-binding fragment thereof that specifically binds Tie2 is provided, comprising a VL domain and a VH domain, the VH domain comprising: a CDR-L1 comprising SEQ ID NO:8, a CDR-L2 comprising SEQ ID NO:9, and a CDR-L3 comprising SEQ ID NO: 10; the VH domain comprising: (a) a CDR-H1 comprising SEQ ID NO:38, a CDR-H2 comprising SEQ ID NO:39, and a CDR-H3 comprising SEQ ID NO:40; (b) a CDR-H1 comprising SEQ ID NO:43, a CDR-H2 comprising SEQ ID NO:44, and a CDR-H3 comprising SEQ ID NO:45; or (c) a CDR-H1 comprising SEQ ID NO:48, a CDR-H2 comprising SEQ ID NO:49, and a CDR-H3 comprising SEQ ID NO:50. In other embodiments, the VL domain comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:21, and the VH domain comprises: (a), (b), or (c) comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to (d) SEQ ID NO:41, (e) SEQ ID NO:46, or (f) SEQ ID NO:51, respectively.

[0022] In some embodiments, the antibody or antigen-binding fragment thereof comprises a LC of SEQ ID NO:21 and a HC comprising SEQ ID NO:42, SEQ ID NO:46, or SEQ ID NO:52.

[0023] In some aspects, an antibody or antigen-binding fragment thereof that specifically binds to Tie2 is provided, comprising 2 VH domains and 2 VL domains, the VH domain comprising, in N-terminal to C-terminal direction, a CDR-H1 comprising SEQ ID NO: 6, a CDR-H2 comprising SEQ ID NO: 8, a CDR-H3 comprising SEQ ID NO: 9, a CDR-H1 comprising SEQ ID NO: 48, a CDR-H2 comprising SEQ ID NO: 49, and a CDR-H3 comprising SEQ ID NO: 50; the VL domain comprising, in N-terminal to C-terminal direction, a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10, a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10. In some embodiments, the HC comprises SEQ ID NO: 54, and the LC comprises SEQ ID NO: 53.

[0024] In some embodiments, the anti-Tie2 antibody or antigen-binding fragment thereof comprises an engineered cysteine, wherein the engineered cysteine is in the HC constant domain and / or in the LC constant domain. In other embodiments, the engineered cysteine is selected from T120C, G166C, G178C, T187C, and T209C in the heavy chain, and Q124C, R142C, Q155C, L201C, T206C, K107C, K126C, and K149C in the light chain, wherein the residue numbers are according to EU numbering. In other embodiments, the anti-Tie2 antibody or antigen-binding fragment thereof is a Fab, wherein the HC of the Fab terminates at amino acids CDKTHTSPPC (SEQ ID NO: 83). In some embodiments, the Fab terminates at the amino acid sequence of SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 85, or SEQ ID NO: 86.

[0025] In some aspects, the anti-Tie2 antagonist antibody is a monoclonal antibody.

[0026] In some embodiments, the anti-Tie2 antibody is a humanized antibody, a chimeric antibody, or a human antibody.

[0027] In some embodiments, the anti-Tie2 antibody is a full-length IgGl or a full-length IgM antibody.

[0028] In some embodiments, the anti-Tie2 antibody or fragment thereof binds to Tie2, wherein Tie2 is a protein that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1.

[0029] In preferred embodiments, the anti-Tie2 antibody or fragment thereof is a Fab.

[0030] In some embodiments, the anti-Tie2 antibody is an antibody or Fab that competes with an anti-Tie2 antibody comprising a VH sequence of SEQ ID NO: 22 and a VL sequence of SEQ ID NO: 21.

[0031] In some embodiments, the anti-Tie2 antibody or fragment thereof does not bind to the Ig1 domain of Tie2. In other embodiments, the anti-Tie2 antibody or fragment thereof does not bind to the EGF domain of Tie2. In other embodiments, the anti-Tie2 antibody or fragment thereof does not bind to the Ig3 domain of Tie2. In other embodiments, the anti-Tie2 antibody or fragment thereof does not bind to the FNIII domain of the Tie2 protein.

[0032] In some embodiments, the antibody or antigen binding fragment thereof binds to cynomolgus Tie2. In other embodiments, the cynomolgus Tie2 comprises the amino acid sequence of SEQ ID NO: 2 or a variant thereof.

[0033] In some embodiments, the antibody or antigen binding fragment thereof that binds to Tie2 is a multispecific antibody. In other embodiments, the multispecific antibody binds to Tie2 and VEGF. In other embodiments, the multispecific antibody binds to Tie2 and Factor D. In other embodiments, the multispecific antibody binds to Tie2 and Ang2.

[0034] In some embodiments, the anti-Tie2 antibody is a multispecific antibody that activates Tie2. In other embodiments, the multispecific antibody binds to Tie2 and VEGF, wherein the multispecific antibody can activate Tie2. In other embodiments, the multispecific antibody binds to Tie2 and Factor D, wherein the multispecific antibody can activate Tie2. In other embodiments, the multispecific antibody binds to Tie2 and Ang2, wherein the multispecific antibody can activate Tie2.

[0035] In one aspect, an isolated nucleic acid encoding an anti-Tie2 antibody or antigen binding fragment thereof is provided.

[0036] In one aspect, a host cell comprising an isolated nucleic acid encoding an anti-Tie2 antibody or antigen binding fragment thereof is provided.

[0037] In one aspect, a method of making an antibody or antigen binding fragment thereof that binds Tie2 is provided. In some embodiments, the method comprises culturing a host cell comprising a nucleic acid encoding the anti-Tie2 antibody under conditions suitable for expression of the anti-Tie2 antibody. In other embodiments, the method further comprises recovering the anti-Tie2 antibody from the host cell.

[0038] In one aspect, an anti-Tie2 antibody made by the above method is provided.

[0039] In one aspect, a conjugate comprising at least two antibodies or antigen binding fragments thereof that specifically bind Tie2 according to the embodiments disclosed herein, and a multi-armed moiety is provided. In preferred embodiments, the at least two anti-Tie2 antibodies are each a Fab.

[0040] In some embodiments, the conjugate comprises an anti-Tie2 antibody that binds Tie2, and a multi-armed moiety, wherein the multi-armed moiety is linked to at least 2 anti-Tie2 Fabs. In some embodiments, the multi-armed moiety is linked to at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 anti-Tie2 Fabs. In other embodiments, the multi-armed moiety is linked to 2, 3, 4, 5, 6, 7, 8, 9, or 10 anti-Tie2 Fabs. In other embodiments, the multi-armed moiety is linked to 6 or 8 anti-Tie2 Fabs. In other embodiments, the multi-armed moiety is linked to 8 anti-Tie2 Fabs. In preferred embodiments, the multi-armed moiety is linked to 6 anti-Tie2 Fabs.

[0041] In one aspect, the conjugate binds and activates Tie2 activity.

[0042] In some embodiments, the conjugate that binds Tie2 activates AKT phosphorylation. In other embodiments, the activation of AKT phosphorylation is evidenced by an increase in phosphorylated AKT protein in an in vitro assay. In other embodiments, the Tie2 binding agent activates phosphorylation of Tie2. In other embodiments, the activation of Tie2 phosphorylation is measured in vitro.

[0043] In some embodiments, exposure of a Tie2-expressing cell to the conjugate does not decrease Tie2 protein levels in the cell. In other embodiments, the exposure does not decrease Tie2 protein levels in the cell by more than 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, or 75%. In other embodiments, the exposure decreases Tie2 protein levels in the cell by less than 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, or 75%. In other embodiments, the exposure decreases Tie2 protein levels in the cell by more than about 25% but less than about 75%, more than about 50% but less than about 75%, or more than about 60% but less than about 80%. In some embodiments, Tie2 protein levels are measured by Western blotting before and after in vitro incubation of the Tie2-binding agent with a Tie2-expressing cell. In other embodiments, the incubation is for 10 to 36 hours or 12 to 24 hours at 37°C.

[0044] In some embodiments, the anti-Tie2 antibody or antigen-binding fragment thereof has an equilibrium dissociation constant (Kd) ranging from 0.1 uM to 10 uM, from 0.01 uM to 10 uM, or from 0.1 uM to 100 uM.

[0045] In some embodiments, the conjugate comprising the multi-arm moiety and the anti-Tie2 antibody or antigen-binding fragment thereof increases translocation of Tie2 to cell-cell junctions.

[0046] In some embodiments, the conjugate decreases vascular permeability.

[0047] In some embodiments, the conjugate promotes vascular stability and / or increases vascular integrity.

[0048] In some embodiments, the conjugate does not inhibit or decrease binding of Angl to Tie2.

[0049] In some embodiments, the conjugate inhibits or decreases binding of Ang2 to Tie2.

[0050] In some embodiments, the conjugate activity is measured using an in vitro assay.

[0051] In some embodiments, the multi-arm polyol is selected from the group consisting of a dimer, a tetramer, a hexamer, and an octamer. In preferred embodiments, the multi-arm polyol is a hexamer or an octamer. In more preferred embodiments, the multi-arm polyol is a hexamer.

[0052] In some embodiments, the polyol is a poly(alkylene oxide) polymer. In other embodiments, the polyol is a poly(alkylene glycol) polymer. In other embodiments, the polyol is a polyethylene glycol (PEG). In some embodiments, the PEG is a functionalized multi-armed PEG.

[0053] In some embodiments, the PEG has the structure of Formula (la):

[0054]

[0055] wherein each m represents the length or size of a particular arm of the polyol (PEG) and is independently an integer from about 45 to about 1000, from about 20 to about 1000, from about 10 to about 1000, from about 3 to about 250, from about 3 to about 200, from about 3 to about 100, from about 10 to about 50, from about 10 to about 30, from about 20 to about 30, from about 50 to about 200, or from about 100 to about 150; and n is an integer from about 1 to about 10; each R 1 is independently or absent or a linking group; and each R 2 is independently or hydrogen or a terminal reactive group; wherein at least one R 2 is a terminal reactive group. In some embodiments, R 2 is independently selected from the group consisting of a thiol reactive group, an amino reactive group, and combinations thereof.

[0056] In some embodiments, the PEG has the structure of Formula (la) wherein n is an integer from 1 to 3.

[0057] In some embodiments, the PEG has the structure of Formula (la) wherein n is 1 and the multi-armed PEG is a tetramer.

[0058] In some embodiments, the PEG has the structure of Formula (la) wherein n is 2 and the multi-armed PEG is a hexamer. In such embodiments, the hexamer has the structure of Formula (lb):

[0059]

[0060] wherein each m is independently an integer from about 45 to about 1000, from about 20 to about 1000, from about 10 to about 1000, from about 3 to about 250, from about 3 to about 200, from about 3 to about 100, from about 10 to about 50, from about 10 to about 30, from about 20 to about 30, from about 50 to about 200, or from about 100 to about 150; each R 1 is independently or absent or a linking group; and each R 2 is independently or hydrogen or a terminal reactive group; wherein at least one R 2are terminal reactive groups and are covalently linked to an anti-Tie2 antibody fragment or Fab as described above. In some embodiments, each m is independently an integer from about 15 to 35 or from about 20 to 30. In other embodiments, each m is independently an integer of about 22.

[0061] In some embodiments, R 1 together have the structure 2 together have the structure wherein R 2 is maleic acid diamine;

[0062] In some embodiments, the PEG has the structure of Formula (la) wherein n is 3 and the multi-armed PEG is an octamer. In such embodiments, the octamer has the structure of Formula (lc):

[0063]

[0064] wherein each m is independently an integer from 3 to 250; each R 1 is independently or absent or a linking group; and each R 2 is independently or hydrogen or a terminal reactive group; wherein at least one R 2 is a terminal reactive group and is covalently linked to an anti-Tie2 Fab as described above. In some embodiments, each m is independently an integer from 15 to 35. In other embodiments, each m is independently an integer of about 22.

[0065] In some embodiments, at least two anti-Tie2 antibody fragments or Fabs described herein are covalently linked to the multi-armed polyol. In other embodiments, the multi-armed polyol of the conjugate is covalently linked to the at least two anti-Tie2 antibody fragments or Fabs via a free thiol group of a cysteine amino acid. In other embodiments, the cysteine amino acid is an engineered cysteine. In other embodiments, the cysteine amino acid is located in the anti-Tie2 constant domain. In other embodiments, the cysteine amino acid is located at the C-terminus of the heavy chain (HC) or light chain (LC) of the anti-Tie2 Fab. In preferred embodiments, the cysteine amino acid is not located at the N-terminus or C-terminus of the HC or LC.

[0066] In some embodiments, the conjugate comprising an anti-Tie2 antibody or antigen-binding fragment thereof comprises an engineered cysteine in the HC and / or LC thereof. In other embodiments, the engineered cysteine is selected from T120C, G166C, G178C, T187C, and T209C in the HC; or the engineered cysteine is selected from Q124C, R142C, Q155C, L201C, T206C, K107C, K126C, and K149C in the LC, wherein the residue numbers are according to EU numbering.

[0067] In some embodiments, the conjugate comprises a multi-armed polyol covalently linked to the at least two anti-Tie2 antibody fragments or Fabs via a free amino group of a lysine amino acid. In other embodiments, the lysine amino acid is in the constant region of the anti-Tie2 antibody fragment or Fab. In other embodiments, the lysine amino acid is at the C-terminus of the heavy chain or light chain of the anti-Tie2 antibody fragment or Fab. In alternative embodiments, the Tie2 binding agent is not covalently linked to the at least two anti-Tie2 Fabs via a free amino group of a lysine.

[0068] In some embodiments, less than 20%, less than 15%, or less than 10% of the conjugate is deconjugated per month under in vitro physiological conditions. In other embodiments, less than 20%, less than 15%, or less than 10% of the conjugate is deconjugated per month under in vivo physiological conditions.

[0069] In some embodiments, the conjugate is stable over time, losing less than 20%, less than 15%, or less than 10% of its Tie2 binding ability per month under physiological conditions.

[0070] In one aspect, a conjugate comprising an anti-Tie2 antibody or antigen-binding fragment thereof and a multi-armed moiety is provided, wherein the multi-armed moiety comprises an IgM molecule. In other embodiments, the IgM molecule comprises a J chain and the multi-armed moiety comprises 5 anti-Tie2 Fabs, wherein about each of the 5 anti-Tie2 Fabs is linked to the IgM molecule. In other embodiments, the IgM molecule does not comprise a J chain and the multi-armed moiety comprises 6 anti-Tie2 Fabs, wherein each of the 6 anti-Tie2 Fabs is linked to the IgM molecule.

[0071] In some embodiments, the conjugate comprises an IgM molecule that is an IgM variant having an amino acid substitution, whereby the IgM variant reduces or eliminates complement-dependent cytotoxicity (CDC) activity. In preferred embodiments, the IgM variant comprises a P436G substitution based on EU numbering.

[0072] In one aspect, conjugates comprising an anti-Tie2 antibody or antigen-binding fragment thereof and a multi-arm moiety are provided, wherein the multi-arm moiety comprises at least 2, 4, 6, 8, or 10 peptides, wherein about each of the peptides is covalently linked to an anti-Tie2 Fab. In some embodiments, the anti-Tie2 Fab sequence terminates at residue 221, 222, 223, 224, or 225 (EU numbering). In other embodiments, each of the peptides is a nucleoside diphosphokinase (NDK) peptide. In other embodiments, each of the NDK peptides comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 71.

[0073] In some embodiments, each of the NDK peptides is linked at its N-terminus to the C-terminus of the anti-Tie2 Fab heavy chain or light chain. In other embodiments, a linker is present between the Fab heavy chain or light chain and the peptide. In other embodiments, the linker is an amino acid linker. In other embodiments, the amino acid linker comprises 2, 3, 4, 5, 6, 7, 8, 2 to 20, 5 to 10, or 4 to 10 amino acids. In other embodiments, the linker comprises glycine.

[0074] In some embodiments, the multi-arm moiety comprises 6 or 8 peptides. In other embodiments, the multi-arm moiety comprises 6 peptides. In preferred embodiments, the multi-arm moiety comprises 6 NDK peptides.

[0075] In one aspect, conjugates comprising an antibody or antigen-binding fragment thereof and a multi-arm moiety are provided, wherein the multi-arm moiety comprises an IgM molecule. In other embodiments, the IgM molecule comprises a J chain, and the multi-arm moiety comprises 5 antibody fragments, Fabs, or antigen-binding fragments thereof, wherein about each of the 5 antibody fragments, Fabs, or antigen-binding fragments thereof is linked to the IgM molecule. In other embodiments, the IgM molecule does not comprise a J chain, and the multi-arm moiety comprises 6 antibody fragments, Fabs, or antigen-binding fragments thereof, wherein each of the 6 antibody fragments, Fabs, or antigen-binding fragments thereof is linked to the IgM molecule.

[0076] In some embodiments, the conjugate comprises an IgM molecule that is an IgM variant having an amino acid substitution, whereby the IgM variant reduces or eliminates complement-dependent cytotoxicity (CDC) activity. In preferred embodiments, the IgM variant comprises a P436G substitution based on EU numbering.

[0077] In one aspect, there is provided a conjugate comprising an antibody, antibody fragment, Fab, or antigen binding fragment thereof, and a multi-arm moiety, wherein the multi-arm moiety comprises at least 2, 4, 6, 8, or 10 peptides, wherein about each of the peptides is covalently linked to the antibody, antibody fragment, Fab, or antigen binding fragment thereof. In some embodiments, the antibody, antibody fragment, Fab, or antigen binding fragment thereof sequence terminates at residue 221, 222, 223, 224, or 225 (EU numbering). In other embodiments, each of the peptides is a nucleoside diphosphate kinase (NDK) peptide. In other embodiments, each of the NDK peptides comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 71.

[0078] In some embodiments, each of the NDK peptides is linked at its N-terminus to the C-terminus of a heavy chain or light chain of the antibody, antibody fragment, Fab, or antigen binding fragment thereof. In other embodiments, a linker is present between the Fab heavy chain or light chain and the peptide. In other embodiments, the linker is an amino acid linker. In other embodiments, the amino acid linker comprises 2, 3, 4, 5, 6, 7, 8, 2 to 20, 5 to 10, or 4 to 10 amino acids. In other embodiments, the linker comprises glycine.

[0079] In some embodiments, the multi-arm moiety comprises 6 or 8 peptides. In other embodiments, the multi-arm moiety comprises 6 peptides. In preferred embodiments, the multi-arm moiety comprises 6 NDK peptides.

[0080] In one aspect, there is provided a pharmaceutical composition comprising an anti-Tie2 antibody or fragment thereof according to the present disclosure and a pharmaceutically acceptable carrier, excipient, or diluent. In some embodiments, the pharmaceutical composition comprises an anti-Tie2 binding agent as described herein comprising a plurality of anti-Tie2 Fabs linked to a multi-arm moiety and a pharmaceutically acceptable carrier.

[0081] In some embodiments, the pharmaceutical composition comprising an anti-Tie2 antibody or Tie2 binding agent further comprises an additional therapeutic agent. In other embodiments, the additional therapeutic agent is selected from the group consisting of an anti-VEGF antibody, an anti-Tie2 antibody, and an anti-Ang2 antibody. In some embodiments, the additional therapeutic agent is selected from an Ang2 antagonist, a VEGF antagonist, a VEGF trap, an anti-VEGF antibody, an anti-Ang2 antibody, and a complement component antagonist.

[0082] In some embodiments, any of the foregoing pharmaceutical compositions can be used as a medicament.

[0083] In some embodiments, any of the foregoing pharmaceutical compositions can be used in the manufacture of a medicament for treating an ocular disease in a subject.

[0084] In some embodiments, any of the foregoing pharmaceutical compositions can be used to reduce or inhibit pathological vascular permeability in a subject having an ocular disease.

[0085] In another aspect, any of the foregoing pharmaceutical compositions can be used to treat an ocular disease in a subject.

[0086] In one aspect, methods for treating an individual in need of treatment are provided, comprising administering to the patient an anti-Tie2 antibody and / or Tie2 binding agent as described herein. In some embodiments, the methods comprise administering to the individual a pharmaceutical composition as described herein comprising an anti-Tie2 conjugate as described herein.

[0087] In some embodiments, the individual has been diagnosed with a vascular disease. In other embodiments, the individual has been diagnosed with an ocular vascular disease.

[0088] In another aspect, the application features a method of inhibiting vascular permeability in a patient having a disease associated with undesirable vascular permeability, the method comprising administering to the subject an effective amount of any of the foregoing antibodies or conjugates, thereby inhibiting vascular permeability in the subject.

[0089] In another aspect, the application features a method of treating a disease associated with undesirable vascular permeability, the method comprising administering to a subject in need of such treatment an effective amount of any of the foregoing antibodies or conjugates.

[0090] In some embodiments, the individual has been diagnosed with a disease associated with the Tie2 pathway.

[0091] In some embodiments, the individual has been diagnosed with an ocular disease selected from the group consisting of diabetic macular edema (DME), age-related macular degeneration (AMD) (including dry and wet (non-exudative and exudative) forms), choroidal neovascularization (CNV), uveitis, diabetic retinopathy, ischemia-related retinopathy, pathologic myopia, von Hippel-Lindau disease, ocular histoplasmosis, central retinal vein occlusion (CRVO), corneal neovascularization, glaucoma, non-edemic retinopathy, and retinal neovascularization.

[0092] In some embodiments, the individual has received treatment with an anti-VEGF antibody. In other embodiments, the individual has not experienced, has experienced reduced, and / or has ceased to experience therapeutic efficacy of an anti-VEGF antibody.

[0093] In some embodiments, the method further comprises administering to the individual a second therapeutic agent. In other embodiments, the second therapeutic agent is selected from the group consisting of an anti-VEGF antibody, an anti-Ang2 antibody, an anti-VEGF / anti-Ang2 bispecific antibody, a VEGF antagonist, and an Ang2 antagonist. BRIEF DESCRIPTION OF DRAWINGS

[0094] Figure 1 Schematic of Tie2 domains provided for use in the translational VH library.

[0095] Figures 2A to 2B Assay results showing evaluation of anti-Tie2 antibodies Tie2.1, Tie2.10, Tie2.11, and Tie2.12 binding to mouse Tie2 ECD5 domain ( Figure 2A ) and to human Tie2 ECD5 domain ( Figure 2B ). The ECD5 domain comprises the Ig1, Ig2, EGF, and Ig3 domains of Tie2.

[0096] Figures 3A to 3B Assay results showing evaluation of anti-Tie2 antibodies Tie2.1, Tie2.10, Tie2.11, and Tie2.12 ( Figure 3A ) and Tie2.2, Tie2.3, Tie2.4, Tie2.5, Tie2.7, Tie2.9, Tie2.15, Tie2.16, Tie2.17, and Tie2.20 ( Figure 3B ) binding to human Tie1.

[0097] Figures 4A to 4B Assay results showing evaluation of blocking of the interaction between Tie2 and Ang1 ( Figure 4A ) and Tie2 and Ang2 ( Figure 4B ) by anti-Tie2 antibodies Tie2.1, Tie2.12, and Tie2.20.

[0098] Figures 5A to 5B Assay results showing evaluation of anti-Tie2 antibodies Tie2.1, Tie2.10, Tie2.11, and Tie2.12 binding to HUVEC ( Figure 5A ) and to RAEC ( Figure 5B ).

[0099] Figures 6A to 6B Assay results showing evaluation of anti-Tie2 antibodies Tie2.1, Tie2.4, Tie2.5, and Tie2.20 activating AKT phosphorylation in RAEC ( Figure 6A ) and evaluation of the effect of anti-IgG cross-linking of the anti-Tie2 antibodies on AKT phosphorylation ( Figure 6BResults of assays to assess the ability of anti-Tie2 antibodies to activate AKT phosphorylation are shown. The level of phosphorylated AKT was determined by Western blot analysis.

[0100] 7A to 7B Results of assays to assess the ability of anti-Tie2 antibodies Tie2.1, Tie2.22, Tie2.23, Tie2.24, Tie2.27, Tie2.28, Tie2.31, Tie2.33, Tie2.34, Tie2.34, Tie2.38, and Tie2.1 to activate AKT phosphorylation are shown. Figure 7A Figure 7B The effect of anti-Tie2 antibody clustering or anti-IgG cross-linking on the activity of anti-Tie2 antibody Tie2.1 to induce AKT phosphorylation is shown. The level of phosphorylated AKT was determined by FRET analysis.

[0101] Figures 8A to 8B Methods (left) and results (right) of a classification study of anti-Tie2 antibodies using ELISA are shown. Figure 8A Figure 8B

[0102] Figure 9 This is a schematic showing the set of epitopes on Tie2 to which the anti-Tie2 antibodies of the present disclosure bind.

[0103] Figure 10A and Figure 10B A sequence alignment of the amino acid sequences of the heavy chain variable region (VH) of anti-Tie2 antibodies Tie2.1 (SEQ ID NO: 22), Tie2.1.M100cF (SEQ ID NO: 20), Tie2.12 (SEQ ID NO: 31), Tie2.24 (SEQ ID NO: 36), Tie2.33 (SEQ ID NO: 41), and Tie2.38 (SEQ ID NO: 51) Figure 10A Figure 10B

[0104] Figure 11 consists of Figure 11A (top) and Figure 11B (bottom). Figures 11A to 11B Results of classification assays using various anti-Tie2.1 antibodies generated via phage display or animal immunization are shown.​​​​​ Figure 11A A list of covalently immobilized antibodies and several of these antibodies in solution is provided, while Figure 11B A list of the remaining of this series of antibodies in solution is provided.

[0105] FIG. 12A to FIG. 12B Assays to evaluate agonist activity of multimeric anti-Tie2 antibodies are shown. Figure 12A AKT phosphorylation activity of Tie2.1 as a PEG hexamer and as a biparatopic Fab IgG (1.38) is compared. Figure 12B Multimeric formats of anti-Tie2 antibodies are compared.

[0106] 13A to 13B The effect of various ratios of huIgM heavy chains to light chains (with or without J chains) on total protein yield isolated on affinity columns is shown, as measured by total protein A280( Figure 13A ) and SEC profiles ( Figure 13B ).

[0107] Figure 13C Rheology measurements of IgM are shown, compared to ocular multivalent PEG formats targeting Factor D (fD).

[0108] Figure 13D Vitreous pharmacokinetic analysis of non-binding IgM and non-binding Fab is shown.

[0109] Figure 13E Systemic pharmacokinetic analysis of IgM is shown, comparing non-binding recombinant hIgM pentamer and recombinant hIgM hexamer to IgM isolated from human serum, with drug injected intravenously into female SCID mice. Figure 13E Serum IgM levels are shown.

[0110] Figure 13F LC-MS analysis of total N-linked glycan from serum samples is shown.

[0111] Figures 13G to 13H Assay results of anti-Tie2 antibodies characterized by the featured IgM multimeric formats are shown. Figure 13G Complement assay results to evaluate mutations in the IgM constant domain are shown. Figure 13H Agonist activity of anti-Tie antibodies in IgM hexamer formats is shown.

[0112] FIG. 14A to FIG. 14B Design and analysis of anti-Tie2 antibodies in hexamer formats linked via peptide moieties is shown. Figure 14A Schematic of multimer design. Figure 14BAKT phosphorylation assay results are shown comparing anti-Tie2 antibodies in a hexamer format linked via NDK peptide, IgM, and multi-armed PEG.

[0113] FIG. 15A to FIG. 15B AKT phosphorylation assay results are shown evaluating agonist activity of various anti-Tie2 antibodies in a hexamer format. Results are shown for Tie2.1, Tie2.38, Figure 15A Tie2.33 Figure 15B and Tie2.1, Tie2.1.M100cF, Tie2.12, Tie2.24

[0114] 16A to 16C Assay results are shown evaluating the effect of anti-Tie2 antibodies on the cellular level of Tie2 protein in in vitro assays. Figure 16A Figure 16B and Figure 16C Comparisons of Tie2 levels when HUVECs are exposed to anti-Tie2 antibodies generated via phage display and animal immunization are shown in Figure 16A Figure 16B and Figure 16C All assays in

[0115] Figure 17 In vivo assay results are shown evaluating the effect of anti-Tie2 antibodies on the cellular level of this Tie2 protein level.

[0116] 18A to 18B Assay methods Figure 18A and results Figure 18B are shown for in vitro endothelial cell assays investigating the effect of anti-Tie2.1 antibodies on endothelial cell barrier permeability.

[0117] Figures 19A to 19B Assay methods Figure 19A and results Figure 19B are shown for in vivo vascular permeability assays investigating the effect of anti-Tie2.1 antibodies on VEGF-induced vascular leakage.

[0118] FIG. 20A to FIG. 20B Assay methods Figure 20A and results Figure 20B are shown for in vivo vascular permeability assays investigating the effect of anti-Tie2.1 antibodies or anti-VEGF antibodies on VEGF-induced vascular leakage.

[0119] Figures 21A to 21B In vivo effects of a biparatopic anti-Tie2 agonist (anti-Tie2 Fab-IgF 1.38) on the protein level of Tie2 as measured by Western blot analysis are shown.​​ Figure 21A ), and in vivo vascular permeability assay results investigating the effect of anti-Tie2 Fab-IgG 1.38 on VEGF-induced vascular leakage ( Figure 21B ).

[0120] Figure 22 The effect of anti-Tie2 agonist on VE-cadherin (top panel) and F-actin (bottom panel) cytoarchitecture in cultured HUVECs is shown.

[0121] Figure 23 AKT phosphorylation assay results evaluating agonist activity of Tie2.1 anti-Tie2 antibody variants in IG1 format are shown.

[0122] Figure 24 AKT phosphorylation assay results evaluating agonist activity of Tie2.1 anti-Tie2 conjugate variants in hexamer format compared to non-PEG conjugated Tie2.1 Fab (no hexamer) are shown.

[0123] Figure 25 Pharmacokinetics of anti-Tie2 Fab hexamer conjugate after ocular injection in cynomolgus monkeys is shown.

[0124] Figures 26 to 27 Electropherogram analysis of anti-Tie2.1 PEG conjugate is shown, with monomer peaks included in relative quantification ( Figure 26 ) or not ( Figure 27 ).

[0125] Figure 28 Electropherogram analysis of anti-Tie2.1 PEG conjugate in samples taken from vitreous humor of cynomolgus monkeys is shown.

[0126] Figure 29 The relative amount of anti-Tie2 PEG conjugate hexamer to pentamer is shown.

[0127] FIG. 30A to FIG. 30B Data showing M100c oxidation ( Figure 30B ) in samples taken from the eyes of cynomolgus monkeys over 21 days is provided. Figure 30A

[0128] The effect of oxidation on pAKT activity is shown. Figure 31

[0129] The effect of conjugation on pAKT activity is shown ( Figures 32 to 33 ), with normalized values shown in Figure 32 . Figure 33

[0130] Figure 34 ​PEG conjugation site position effects on deconjugation are shown.

[0131] Figure 35A PEG conjugation site position effects on activity are shown.

[0132] Figure 35B PEG conjugation site position effects on activity are shown.

[0133] Figure 36 Pharmacokinetics of different Tie2.1M100cF PEG conjugates are shown.

[0134] Figure 37 In vivo stability of different Tie2.1M100cF PEG conjugates are compared.

[0135] Figures 38A to 38B Activity of different Tie2.1M100cF PEG conjugates are compared. Figure 38A pAKT activity is shown, while Figure 38B Total conjugate levels as determined via ELISA are shown.

[0136] Figure 39 Structure of PEG conjugate hexamer core molecule is shown, which is conjugated to an anti-Tie2 antibody according to the present disclosure. DETAILED DESCRIPTION

[0137] I. DEFINITIONS

[0138] Unless otherwise defined herein, the term "comprising" encompasses the term "consisting of."

[0139] The term "about" as used herein in connection with a particular value (e.g., temperature, concentration, time, etc.) shall mean + / - 1% variation of the particular value to which the term "about" refers.

[0140] For purposes herein, an "acceptor human framework" is a framework comprising an amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework can comprise the same amino acid sequence thereof, or it can contain amino acid sequence changes. In some embodiments, the number of amino acid changes is 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, or 2 or fewer. In some embodiments, the VL acceptor human framework is sequence-identical to a VL human immunoglobulin framework sequence or a human consensus framework sequence.

[0141] "Affinity" refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., antigen). Unless indicated otherwise, "binding affinity," as used herein, refers to the intrinsic binding affinity reflecting the 1 : 1 interaction between binding partners (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can be expressed by the dissociation constant (Kd). Affinity can be measured by routine methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

[0142] The term "affinity matured" antibody refers to an antibody with one or more alterations in one or more hypervariable regions (HVRs) that result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody that does not possess such alteration(s).

[0143] The terms "anti-Tie2 antibody," "antibody that binds to Tie2," and "antibody that specifically binds to Tie2" refer to an antibody that is capable of binding Tie2 with sufficient affinity to be useful as a therapeutic and / or diagnostic agent targeting Tie2. In one embodiment, the extent of binding of an anti-Tie2 antagonist antibody to an unrelated, non-Tie2 protein is less than about 10% of the binding of the antibody to Tie2 as measured, e.g., by a radioimmunoassay (RIA). In certain embodiments, an antibody that binds to Tie2 has a dissociation constant (Kd) of < 1 μΜ, < 100 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM (e.g., 10 D M or less, e.g., 10 -8 M to 10 -8 M, e.g., 10 -13 M, e.g., 10 -9 M to 10 -13 M). In certain embodiments, an anti-Tie2 antagonist antibody binds to an epitope of Tie2 that is conserved among Tie2 of different species.

[0144] The term "antibody," as used herein, is used in the broadest sense and includes various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.

[0145] "Antibody fragments" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibodies molecules (e.g., scFv), and multispecific antibodies formed from antibody fragments.

[0146] The terms "full-length antibody," "intact antibody," and "whole antibody" are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.

[0147] As used herein, "Fab" refers to an antibody comprising a heavy chain constant region that includes a CH1 domain or a sufficient portion of a CH1 domain to form a disulfide bond with a light chain constant region, but does not include a CH2 domain or a CH3 domain. As used herein, a Fab can include one or more amino acids of the hinge region. Thus, as used herein, the term "Fab" encompasses Fab' antibodies. A Fab can include additional non-native amino acids, such as a C-terminal cysteine, in which case it can refer to a Fab-C. As described below, the term Fab-C also encompasses Fabs that include native amino acids of the hinge region, including the native cysteine at the C-terminus. In some embodiments, the Fab includes an engineered cysteine (i.e., the Fab can be a THIOMAB). In some embodiments, the engineered cysteine is a cysteine amino acid residue in the Fab HC and / or LC polypeptide sequence that is substituted for a non-cysteine amino acid residue.

[0148] "Fab-C" refers to a Fab with a C-terminal cysteine, which can be a native cysteine that occurs at that residue position (e.g., a cysteine from the hinge region), or can be a cysteine added to the C-terminus that does not correspond to a native cysteine.

[0149] "Fab-SH" refers to a Fab with a free sulfhydryl group. In some embodiments, the free thiol group is positioned at the last 10 amino acids of the C-terminus of the Fab. A Fab-C antibody is typically also a Fab-SH antibody.

[0150] An antibody that "binds to the same epitope" as a reference antibody is one that blocks binding of the reference antibody to its antigen in a competitive assay by 50% or more, and vice versa. An exemplary competitive assay is provided herein.

[0151] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0152] The "class" of an antibody refers to the type of constant domains, or constant regions, possessed by its heavy chains. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of those can be further divided into subclasses (isotypes), e.g., IgGi, IgG2, IgG3, IgG4, IgAi, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, d, e, g, and m, respectively.

[0153] The term "conjugate" as used herein, according to its broadest definition, means joined or linked together. Molecules are "conjugated" when they act or operate as if they are joined together. A "conjugate" is an antibody (e.g., Fab) conjugated to one or more heterologous molecules, including but not limited to a polyol. In particular embodiments, a "conjugate" refers to an antibody (e.g., an antibody fragment, as detailed herein) covalently bound to a multi-armed moiety. In particular embodiments, the multi-armed moiety is a polyol, an IgM molecule, or a peptide in a multimeric (e.g., hexameric) form.

[0154] The term "Tie2" as used herein, unless otherwise indicated, refers to any native Tie2 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses "full-length," unprocessed Tie2 as well as any form of Tie2 that results from cellular processing. The term also encompasses naturally occurring variants of Tie2, e.g., splice variants or allelic variants. An exemplary human Tie2 protein has the amino acid sequence of NCBI Reference Number NP_000450 (SEQ ID NO: 1).

[0155] The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and / or causes destruction or death of cells. Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212and Lu's radioisotopes); chemotherapeutic agents or drugs (e.g., methotrexate, doxorubicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof, such as nucleolytic enzymes; antibiotics; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof, and various anti-tumor or anticancer agents disclosed below.

[0156] "Effector function" refers to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor); and B cell activation.

[0157] A "therapeutically effective amount" of an agent, such as a pharmaceutical formulation, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0158] The term "Fc region" herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226, or Pro230, to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region can or can not be present. Unless otherwise specified, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system (also referred to as the EU index), as set forth by Kabat et al. (Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991) (see also above).

[0159] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR residues are typically conserved within a species and only slightly variant between species. Thus, the HVR and FR sequences typically occur in the order: FR1-H1 (L1)-FR2-H2 (L2)-FR3-H3 (L3)-FR4 in a VHand (or VL).

[0160] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which an exogenous nucleic acid has been introduced, and include the progeny of the parent cell, which progeny will carry the exogenous nucleic acid. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. The nucleic acid content of the progeny cell may not be identical to that of the parent cell from which it originated, but may contain mutations. Mutant progeny that express the same functional or biological activity as screened or selected for in the originally transformed cell are included herein.

[0161] A "human antibody" is an antibody that possesses an amino acid sequence that corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes a human antibody repertoire or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.

[0162] A "human consensus framework" is a framework which represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Typically, the subgroup of sequences is a subgroup as in Kabat et al. Sequences of Proteins of Immunological Interest (5th ed., NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3). In one embodiment, for the VL, the subgroup is subgroup kappa I as in Kabat et al. supra. In one embodiment, for the VH, the subgroup is subgroup III as in Kabat et al. supra.

[0163] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally can comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0164] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains (VH and VL, respectively) of a native antibody generally have similar structures and each domain comprises four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindt et al. Kuby Immunology, 6th Ed., W.H. Freeman and Co., page 91 (2007).) A single VH or VL domain can be sufficient to confer antigen-binding specificity. Furthermore, VH or VL domains can be used to isolate antibodies that bind a particular antigen from libraries of antibodies that bind the antigen, to screen libraries of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0165] The term "hypervariable region," "HVR," or "HV," when used herein refers to the regions of an antibody variable domain that are hypervariable in sequence (also referred to herein as "complementarity determining regions" or "CDRs") and / or form structurally defined loops. In general, antibodies comprise six HVRs; three in the VH (H1, H2, H3), and three in the VL (L1, L2, L3). Of the six HVRs, H3 and L3 exhibit the most diversity, and, in particular, H3 is believed to play a unique role in conferring fine specificity to an antibody. See, e.g., Xu et al., Immunity 13:37-45 (2000); Johnson and Wu, in Methods in Molecular Biology 248:1-25 (Lo, ed., Human Press, Totowa, N.J., 2003). Indeed, naturally occurring camelid antibodies consisting of a heavy chain only are functional and stable in the absence of light chains. See, e.g., Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).

[0166] A number of descriptions of HVRs are in use, and are encompassed herein. The Kabat Complementarity Determining Regions (CDRs) are based on sequence variability, and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia refers instead to the location of the structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). The AbM HVRs represent an intermediate between the Kabat HVRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software. The "contact" HVRs are based on an analysis of the available complex crystal structures. The residues from each of these HVRs are noted below.

[0167]

[0168] HVRs can comprise "extended HVRs" as follows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in the VL, and 26-35 (H1), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) in the VH. With respect to each of these definitions, the variable domain residues are numbered according to Kabat et al. ( supra ).

[0169] The terms "variable domain residue numbering as in Kabat" or "amino acid position numbering as in Kabat" and variants thereof, refer to the numbering system used in the antibody compilation of Kabat et al. ( supra ) for the heavy chain variable domain or light chain variable domain. Using this numbering system, the actual linear amino acid sequence can include fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or HVR of the variable domain. For example, a heavy chain variable domain can include a single amino acid insertions (residue 52a according to Kabat) after residue 52 of H2 and insertions of residues (e.g., residues 82a, 82b, and 82c, etc. according to Kabat) after residue 82 of heavy chain FR. The Kabat numbering of residues in an antibody can be determined by alignment of the sequence of the given antibody with the "standard" Kabat numbered sequence.

[0170] When referring to residues in a variable domain, the Kabat numbering system is generally used (approximately residues 1-107 of a light chain and residues 1-113 of a heavy chain) (e.g., Kabat et al., supra). When referring to residues in an immunoglobulin heavy chain constant region, the "EU numbering system" or "EU index" is generally used (e.g., the EU index reported in Kabat et al., supra). "EU index as in Kabat" refers to the residue numbering of the human IgGl EU antibody. Unless otherwise specified herein, reference to residue numbering in an antibody variable domain refers to the residue numbering of the Kabat numbering system. Unless otherwise specified herein, reference to residue numbering in an antibody constant domain refers to the residue numbering of the EU numbering system (see, e.g., U.S. Patent Application Publication No. 2008 / 0181888, Figure for EU numbering).

[0171] An "immunoconjugate" is an antibody conjugated with one or more heterologous molecule(s), including but not limited to a cytotoxic agent.

[0172] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0173] An "isolated" antibody is one which has been separated from a component of its natural environment. In some embodiments, an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC) methods. See, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007) for a review of methods for assessing antibody purity.

[0174] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that are typically present in a population of cells, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from that at which it naturally occurs.

[0175] An "isolated nucleic acid encoding an antibody, e.g., an anti-Tie2 antibody" refers to one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of the antibody, including such nucleic acid molecule(s) in a single recombinant vector or separate vectors, and such nucleic acid molecule(s) present at one or more locations in a host cell.

[0176] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind the same epitope, except for possible variants that can arise during production of the monoclonal antibody, such variants are typically present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present application can be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and transgenic animals containing all or part of the human immunoglobulin loci, as described herein and as described generally in U.S. Patent No. 7,896,743.

[0177] A "naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The naked antibody can be present in a pharmaceutical formulation.

[0178] A "natural antibody" refers to a naturally generated immunoglobulin molecule having a different structure. For example, an Ig natural IgG antibody is a heterotetrameric glycoprotein of about 150,000 daltons, composed of two identical light chains and two identical heavy chains that are linked via disulfide bonds. From N- to C-terminus, each heavy chain has a variable region (VH), also called a variable heavy chain domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from N- to C-terminus, each light chain has a variable region (VL), also called a variable light chain domain or a light chain variable domain, followed by a light chain constant (CL) domain. The light chains of antibodies can be assigned to one of two types, called kappa (K) and lambda (l), based on the amino acid sequence of their constant domains.

[0179] The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and / or warnings concerning the use of such therapeutic products.

[0180] "Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within routine competence of those skilled in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or can be compiled from the source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and no variation can be made.

[0181] Where amino acid sequence comparisons are done using ALIGN-2, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which is alternatively expressed as a given amino acid sequence A which either has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows:

[0182] 100 times the fraction X / Y

[0183] where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in a program-generated alignment of A and B, and Y is the total number of amino acid residues in B. It will be appreciated that where amino acid sequence A is not the same length as amino acid sequence B, % amino acid sequence identity of A to B will not equal % amino acid sequence identity of B to A. All % amino acid sequence identity values used herein were obtained using the ALIGN-2 computer program, as set forth in the immediately preceding paragraph, unless otherwise specifically indicated.

[0184] The term "pharmaceutical formulation" refers to a preparation which is in a form to be used in the treatment of a subject and which is made from a pharmaceutical agent. The preparation takes a form that allows the biological activity of the active ingredient contained therein to be effective, and does not contain other ingredients that are unacceptable with respect to toxicity to the subject to which the formulation will be administered.

[0185] A "pharmaceutically acceptable carrier" refers to a component of a pharmaceutical formulation other than the active ingredient which is not toxic to the subject to which the formulation will be administered. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0186] The term "polyol" refers broadly to a polyhydroxy alcohol compound. For example, a polyol can be any water-soluble poly(alkylene oxide) polymer and can have a linear chain or a branched chain. Preferred polyols include those which are substituted at one or more hydroxyl positions with a chemical group such as an alkyl group having between one and four carbons. Typically, the polyol is a poly(alkylene glycol), preferably a polyethylene glycol (PEG). However, one skilled in the art will recognize that other polyols such as polypropylene glycol and polyethylene glycol-polypropylene glycol copolymers can be employed using the techniques described herein for conjugation to PEG. The polyols of the present disclosure include those which are well known in the art and those which are available, for example, from commercially available sources.

[0187] "Treatment" (and grammatical variations thereof such as "treat" or "treating"), as used herein, refers to clinical intervention in an attempt to alter the natural course of the disease of a subject being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or reoccurrence of the disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, antibodies of the application are used to delay development of a disease or to slow the progression of a disease.

[0188] The term "vector," as used herein, refers to a nucleic acid molecule capable of transmitting another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0189] A "port delivery system" or "PDS" is an implantable device for the eye that has a refillable reservoir allowing for delivery of a therapeutic agent over an extended period of time. The implant is configured to have a refill port in communication with the reservoir and a release control element that determines the rate of drug release into the eye. See, e.g., US20100174272, 8,277,830, 8,399,006, 8,795,712, and 8,808,727.

[0190] A "small gauge needle" refers to a needle for injection of a fluid composition that is about a 30, 29, 28, 27, 26, 25, 24, 23, or 22 gauge or higher, e.g., a 30 gauge needle. In some embodiments, the small gauge needle has a standard size needle wall. In another embodiment, the small gauge needle has a thin needle wall, which can be preferred for viscous solutions.

[0191] II. Compositions and Methods

[0192] Provided herein are novel Tie2 agonists that activate Tie2 function, as evidenced by, e.g., phosphorylation of Tie2. Direct agonism more effectively activates Tie2 signaling in the absence or deficiency of endogenous agonist Angl, driving vision improvement that can be therapeutically advantageous. Because activators of Tie2 can both block binding of Ang2 (block Ang2 antagonist function) and directly bind to Tie2 to activate Tie2 activity, e.g., increase phosphorylation of Tie2 and / or Akt, they can be more advantageous than inhibitors of Ang2. Activation of Tie2 has been shown to require clustering of Tie2 upon ligand binding, and as such, the Tie2 agonists provided herein are multimeric, preferably hexameric or octameric. The multimeric Tie2 agonists described herein can have the additional unexpected advantage of not significantly reducing cellular Tie2 levels in vitro or in vivo. Furthermore, the multimeric Tie2 agonists described herein can be formulated for intravitreal injection and possess a molecular size that confers advantageous pharmacokinetics and thus pharmacodynamics. Data are provided to show that the multimeric Tie2 agonists reduce endothelial cell membrane permeability and strengthen cell-cell junctions (see, e.g., Example 10).

[0193] In one aspect, the present application is based in part on antibodies that bind to Tie2. In particular, provided herein are binding agents (also referred to herein as conjugates) comprising more than one anti-Tie2 antibody or antigen binding fragment thereof, e.g., but not limited to, anti-Tie2 Fabs. It can be preferred that the binding agent comprises more than 4, e.g., comprises 5, 6, 7, 8, 9, or 10, anti-Tie2 antibodies, e.g., more than 4 anti-Tie2 Fabs, such that binding of the Tie2 binding agent to Tie2 located on the surface of a cell is associated with activation of Tie2. Tie2 clustering can also occur upon binding of the binding agent to Tie2.

[0194] In some embodiments, it is advantageous to have a Tie2 binding agent that, when it binds to Tie2 on the surface of a cell, does not cause a significant reduction in the level of Tie2 protein on the surface of the cell. In some embodiments, a Tie2 binding agent that activates Tie2 comprises an anti-Tie2 antibody (Fab) that has an affinity for Tie2 in the range of about 0.1 μΜ to 10 μΜ.

[0195] Activation of the Tie2 protein can be measured in vitro by measuring an increase in phosphorylation of the Tie2 protein (e.g., by Western blotting) or measuring an increase in phosphorylation of the associated Akt protein (AKT serine / threonine kinase 1, e.g., GenBank Accession No. NP_001014431) using materials and methods known to those of ordinary skill. In certain embodiments, antibodies that bind to Tie2 are provided, as well as compositions comprising more than one antibody that binds to Tie2. The antibodies and compositions comprising antibodies of the present application can be used, for example, to diagnose or treat vascular permeability disorders associated with Tie2 function, particularly ocular disorders.

[0196] A. Exemplary Anti-Tie2 Binding Agents

[0197] In one aspect, the present application provides a Tie2 binding agent comprising an isolated antibody that binds to Tie2. In some embodiments, the Tie2 binding agent comprises a multi-armed moiety, wherein each of the arms is conjugated or linked to an anti-Tie2 antibody or fragment thereof. In preferred embodiments, the anti-Tie2 antibody or fragment thereof is an anti-Tie2 Fab. Examples of multi-armed moieties include, but are not limited to, multi-armed polyols (e.g., polyethylene glycol (PEG)), IgM, and multimers such as hexameric peptides. In certain embodiments, a Tie2 binding agent is provided comprising more than one anti-Tie2 antibody or fragment thereof, wherein the anti-Tie2 antibody (when not part of the Tie2 binding agent, but optionally formalized as a full-length antibody) binds to Tie2 with a binding affinity of, for example, less than 100 μM, or less than 50 μM, or less than 10 μM, and / or a K D and / or more than 1 μM K DIt is understood that affinity is measured using an antibody rather than a Tie2 binding agent comprising more than one anti-Tie2 antibody. In some embodiments, affinity is monovalent affinity. Further, provided are Tie2 binding agents and Tie2 antibodies that activate Tie2 activity (e.g., function as Tie2 agonists). For example, activation of Tie2 is measured by measuring an increase in phosphorylation of Tie2 and / or an increase in phosphorylation of AKT in an in vitro assay. In some embodiments, the Tie2 binding agent does not down-regulate (decrease) Tie2 protein levels in a cell by more than about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, or 85%. In alternative embodiments, the Tie2 binding agent decreases Tie2 protein levels in a cell by less than about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, or 75%. In some embodiments, the Tie2 binding agent decreases vascular permeability. Decrease in vascular endothelial cell permeability can be measured in vivo or in vitro. In some embodiments, the Tie2 binding agent can increase translocation of Tie2 to cell-cell junctions and / or contribute to structural organization of actin and / or cadherins. In preferred embodiments, the Tie2 binding agent comprises a hexameric PEG molecule, wherein each of the 6 arms is bound to an anti-Tie2 Fab. Alternatively, the PEG molecule comprises 8 arms. In some embodiments, the Tie2 binding agent comprises more than 1 anti-Tie2 antibody or fragment thereof described herein. The present disclosure also provides anti-Tie2 antibodies or fragments thereof that bind to Tie2. In some embodiments, the anti-Tie2 antibody or fragment thereof binds to the Ig2 domain of Tie2 (e.g., binds to an epitope that exists at least partially within amino acid residues 23 to 120 of SEQ ID NO: 1).

[0198] In one aspect, the present application provides an antibody or antigen-binding fragment thereof that specifically binds to Tie2, comprising at least one, two, three, four, five, or six CDRs selected from the group consisting of: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:3; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:4; (c) CDR-H3 comprising the amino acid sequence of (a) SEQ ID NO:5 (wherein X1is M, L, K, F, Y, R, N, Q, H, or W, and / or X2is F, Y, L, Q, I, K, or H), SEQ ID NO:6, or SEQ ID NO:7; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:8; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:9, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 10. In a particular embodiment, the CDR-H3 comprises SEQ ID NO:7.

[0199] In one aspect, the present application provides an antibody comprising at least one, at least two, or all three VH domain CDR sequences selected from the group consisting of: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:3; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:4; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:5, wherein X1is M, L, K, F, Y, R, N, Q, H, or W and / or X2is F, Y, L, Q, I, K, or H. All possible combinations of the above substitutions are encompassed by the consensus sequence of SEQ ID NO:5. In one embodiment, the CDR-H3 comprises the amino acid sequence of SEQ ID NO:6. In another embodiment, the CDR-H3 comprises the amino acid sequence of SEQ ID NO:7

[0200] In another aspect, the present application provides an antibody comprising at least one, at least two, or all three VL domain CDR sequences selected from the group consisting of: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:8; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO:9; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10. In one embodiment, the VL domain comprises (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO:8; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO:9; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10.

[0201] In another aspect, an antibody of the application comprises: (a) a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:3; (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:4; and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO:5 (wherein X1is M, L, K, F, Y, R, N, Q, H, or W and / or X2is F, Y, L, Q, I, K, or H), SEQ ID NO:6, and SEQ ID NO:7; and (b) a VL domain comprising at least one, at least two, or all three VL CDR sequences selected from: (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO:8; (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO:9; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10.

[0202] In another aspect, the application provides an antibody comprising a VH domain and a VL domain, wherein the VH domain comprises: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:3; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:4; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:5 (wherein X1is M, L, K, F, Y, R, N, Q, H, or W and / or X2is F, Y, L, Q, I, K, or H), SEQ ID NO:6, or SEQ ID NO:7; and the VL domain comprises: (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:8; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:9, and (f) CDR-L3 comprising the amino acid sequence selected from SEQ ID NO: 10. In some embodiments, the VH domain comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:20, and the VL domain comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:21.

[0203] In any of the above embodiments, the anti-Tie2 antibody can be humanized. In one embodiment, the anti-Tie2 antibody comprises the CDRs as described in any of the above embodiments, and further comprises a human acceptor framework, e.g., a human immunoglobulin framework or a human consensus framework. In another embodiment, the anti-Tie2 antibody comprises the CDRs as described in any of the above embodiments, and further comprises a VH framework FR1 sequence of SEQ ID NO: 11, a VH framework FR2 sequence of SEQ ID NO: 12, a VH framework FR3 sequence of SEQ ID NO: 13, and / or a VH framework FR4 sequence of SEQ ID NO: 14. In other embodiments, the anti-Tie2 antibody comprises a VL framework FR1 sequence of SEQ ID NO: 15, a VL framework FR2 sequence of SEQ ID NO: 16, a VL framework FR3 sequence of SEQ ID NO: 17, and / or a VL framework FR3 sequence of SEQ ID NO: 18.

[0204] In another aspect, an anti-Tie2 antibody or antigen-binding fragment thereof comprises a heavy chain variable domain (VH) sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 20. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-Tie2 antibody comprising that sequence retains the ability to bind to Tie2. In certain embodiments, a total of 1 to 10 amino acids in SEQ ID NO: 20 are substituted, inserted and / or deleted. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the anti-Tie2 antibody comprises a VH sequence in SEQ ID NO: 20, including post-translational modifications of that sequence. In a particular embodiment, the VH comprises one, two, or three CDRs selected from: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 3; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 4; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 7.

[0205] In another aspect, an anti-Tie2 antibody is provided, wherein the antibody comprises a heavy chain variable domain (VH) sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 20. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-Tie2 antibody comprising that sequence retains the ability to bind to Tie2. In certain embodiments, a total of 1 to 10 amino acids in SEQ ID NO: 20 are substituted, inserted and / or deleted. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-Tie2 antibody comprises the VH sequence in SEQ ID NO: 20, including post-translational modifications of that sequence. In a particular embodiment, the VH comprises one, two or three HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 6; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 7; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 8.

[0206] In another aspect, an anti-Tie2 antibody is provided, wherein the antibody comprises a VH of any one of the embodiments provided above and a VL of any one of the embodiments provided above. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 20 and SEQ ID NO: 21, respectively, including post-translational modifications of those sequences.

[0207] In yet another aspect, the present application provides an antibody that binds to the same epitope as an anti-Tie2 antibody provided herein. For example, in certain embodiments, an antibody that binds to the same epitope as an anti-Tie2 antibody is provided, comprising the VH sequence of SEQ ID NO: 20 and the VL sequence of SEQ ID NO: 21. In certain embodiments, an antibody that binds to an epitope within a Tie2 fragment, e.g., the Ig1 domain of Tie2, is provided, wherein the Ig1 domain comprises amino acids 23-120 of SEQ ID NO: 1.

[0208] In yet another aspect of the application, the anti-Tie2 antibody according to any of the above embodiments is a monoclonal antibody, including a chimeric antibody, a humanized antibody, or a human antibody. In one embodiment, the anti-Tie2 antibody is an antibody fragment, such as a Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment. In another embodiment, the antibody is a full-length antibody, such as a whole IgGl antibody or other antibody class or isotype as defined herein.

[0209] In yet another aspect, the anti-Tie2 antibody according to any of the above embodiments can alone or in combination bind any of the features as described in Sections 1-7 below:

[0210] 1. Antibody Affinity

[0211] In certain embodiments, the dissociation constant (Kd) of an antibody provided herein is < 10 μΜ, < 100 μΜ, < 10 μΜ, < 1 μΜ, < 100 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM and / or > 0.01 μΜ, 0.1 μΜ, or 1 μM (e.g., 10 -5 M or less, 10 -6 M or more, 10 -8 M or less, e.g., 1 μΜ to 10 μΜ, e.g., 0.1 μΜ to 10 μΜ, e.g., 10 -6 M to 10 -9 M, e.g., 10 -8 M to 10 -13 M, e.g., 10 -9 M to 10 -13 M).

[0212] In one embodiment, the Kd is measured by a radioiodinated antigen binding assay (RIA). In one aspect, the RIA is performed with the Fab form of the antibody of interest and its antigen. For example, the solution binding affinity of the Fab for antigen is measured by equilibrating the Fab with a minimal concentration of 125 I) labeled antigen in the presence of a titration series of unlabeled antigen, then capturing the antigen-bound Fab with an anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). To determine the conditions for the assay, a capture anti-Fab antibody (Cappel Labs) is dissolved in 50 mM sodium carbonate (pH 9.6) at 5 μg / ml and coated on plates for 24 hours. Nonspecific protein binding is blocked with a solution of 1% bovine serum albumin (BSA) in PBS (pH 7.4). The Fab is then added to the plates at 10 nM, 5 nM, 2.5 nM, 1.25 nM, 0.625 nM, and 0.3125 nM in PBS containing 0.1% BSA with a final Multiwell plates (ThermoScientific) were coated overnight and then blocked with 2% (w / v) bovine serum albumin in PBS for two to five hours at room temperature (approximately 23°C). In non-adsorbent plates (Nunc #269620), 100 pM or 26 pM [ 125 The antigen is mixed with a serial dilution of the Fab of interest (e.g., consistent with the evaluation of the anti-VEGF antibody Fab-12 described by Presta et al. in Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then incubated overnight; however, the incubation may be continued for a longer period (e.g., about 65 hours) to ensure that equilibrium is reached. Thereafter, the mixture is transferred to a capture plate and incubated at room temperature (e.g., for 1 hour). The solution is then removed and the plate is washed with 0.1% polysorbate 20 (TWEEN-20) in PBS. ) The plate was washed eight times. When the plate was dry, 150 μl / well of scintillant (MICROSCINT-20 TM ; Packard), and place the flat plate on the TOPCOUNT TM The counts were taken on a gamma counter (Packard) for tens of minutes.Concentrations of each Fab that gave less than or equal to 20% of the maximal binding concentration were chosen for use in competitive binding assays.

[0213] According to another embodiment, using Kd is measured by surface plasmon resonance assay. For example, using -2000 or The assay was performed using a BIAcore-3000 (BIAcore, Inc., Piscataway, NJ) at 25°C using an immobilized antigen CM5 chip at approximately 10 response units (RU). In one aspect, a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) was activated with N-ethyl'-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Antigen was diluted to 5 μg / ml (~0.2 μM) with 10 mM sodium acetate (pH 4.8) and then injected at a flow rate of 5 μl / min to obtain approximately 10 response units (RU) of coupled protein. Following antigen injection, 1 M ethanolamine was injected to block unreacted groups. In the kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) were injected into a 0.05% polysorbate 20 (TWEEN-20 TM ) surfactant (PBST) in PBS. Using a simple one-to-one Langmuir binding model ( The association rate (kon) and the dissociation rate (koff) were calculated by simultaneously fitting the association and dissociation sensorgrams using a simple 1 : 1 binding model in the BIAevaluation software version 3.2. The equilibrium dissociation constant (Kd) was calculated as the ratio koff / kon. See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). If the association rate, as determined by the above surface plasmon resonance analysis, is greater than 106M-1s-1, the association rate can be measured using fluorescence quenching techniques in which the increase in fluorescence emission intensity of a 20 nM anti-antigen antibody (Fab form) in PBS (pH 7.2) at 25°C is measured in the presence of increasing concentrations of antigen as measured in a spectrophotometer (e.g., a stopped-flow equipped spectrometer (Aviv Instruments) or an 8000-series SLM-AMINCO TM The increase or decrease in fluorescence emission intensity of a 20 nM anti-antigen antibody (Fab form) in PBS (pH 7.2) at 25°C is measured in the presence of increasing concentrations of antigen as measured in a spectrophotometer (e.g., a stopped-flow equipped spectrometer (Aviv Instruments) or an 8000-series SLM-AMINCO

[0214] 2. Antibody Fragments

[0215] In certain embodiments, an antibody provided herein is an antibody fragment. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, and scFv fragments, and other fragments as described infra. For a review of certain antibody fragments, see Hudson, et al., Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); see also WO 93 / 16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab')2 fragments with increased in vivo half-life, see U.S. Pat. No. 5,869,046.

[0216] In some embodiments, the C-terminus of the heavy chain of the Fab fragment terminates at the amino acids "CDKTHT" (SEQ ID NO: 75), "CDKTHL" (SEQ ID NO: 76), "CDKTH" (SEQ ID NO: 77), "CDKT" (SEQ ID NO: 78), "CDK," or "CD." In some embodiments, the C-terminus of the heavy chain of the Fab fragment terminates at the sequence CDKTHX (SEQ ID NO: 79), where X is an amino acid other than T. Truncation and / or mutation at the C-terminus can be capable of reducing or eliminating AHA reactivity against the Fab without destroying thermal stability or expression. In some embodiments, the C-terminus of the heavy chain of the Fab fragment terminates at the amino acids "CDKTHTC" (SEQ ID NO: 80), "CDKTHTCPPC" (SEQ ID NO: 81), "CDKTHTCPPS" (SEQ ID NO: 82), "CDKTHTSPPC" (SEQ ID NO: 83), "CDKTHTAPPC" (SEQ ID NO: 84), "CDKTHTSGGC" (SEQ ID NO: 85), or "CYGPPC" (SEQ ID NO: 86). In some such embodiments, the free cysteine in the C-terminal amino acid can be suitable for conjugation, e.g., to a polymer such as PEG.

[0217] Diabodies are antibody fragments with two antigen-binding sites that can be bivalent or bispecific. See, e.g., EP 404097; WO 1993 / 01161; Hudson et al., Nat. Med. 9: 129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9: 129-134 (2003).

[0218] Single-domain antibodies are antibody fragments comprising all or a portion of the variable domain of the heavy chain or all or a portion of the variable domain of the light chain of an antibody. In certain embodiments, single-domain antibodies are human single-domain antibodies (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 Bl).

[0219] Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of whole antibodies as described herein, and production by recombinant host cells, e.g., E. coli or phage.

[0220] 3. Chimeric and Humanized Antibodies

[0221] In certain embodiments, an antibody provided herein is a chimeric antibody. Certain chimeric antibodies are described, e.g., in U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81 :6851-6855 (1984). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In another example, a chimeric antibody is a “class switched” antibody in which the class or subclass is altered from that of its parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0222] In certain aspects, a chimeric antibody is a humanized antibody. Typically, a non- human antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which HVRs, such as CDRs (or portions thereof) are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody optionally will comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from the antibody from which the HVR residues are derived (e.g., the antibody from which the HVR residues are derived).

[0223] Humanized antibodies and methods of making them are reviewed, e.g., in Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008), and are further described, e.g., in: Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat’l Acad. Sci. USA 86:10029-10033 (1989); US Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing CDR-grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing “surface reshaping”); Dall’Acqua et al., Methods 36:43-60 (2005) (describing “FR shuffling”); Osbourn et al., Methods 36:61-68 (2005); and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing the “guided selection” method of FR shuffling).

[0224] Human antibody framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best-fit" method (see, e.g., Sims et al. J. Immunol. 151 :2296 (1993)); framework regions derived from the consensus sequence of human antibodies of a particular subgroup or subgroup (see, e.g., Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151 :2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997); and Rosok et al., J. Biol. Chem. 271 :22611-22618 (1996)).

[0225] 4. Human Antibodies

[0226] In certain embodiments, an antibody provided herein is a human antibody. Human antibodies can be produced using various techniques well known in the art. Human antibodies are described generally in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001); and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

[0227] Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to an antigen challenge. Such animals are typically TM immunoglobulin loci, or exist extrachromosomally or integrated randomly into the animal's chromosomes. In such transgenic mice, endogenous immunoglobulin loci are typically inactivated. For review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584 (describing XENOMOUSE® technology); U.S. Patent No. 5,770,429 (describing HUMAB® technology); U.S. Patent No. 7,041,870 (describing K-M MOUSE® technology); and U.S. Patent Application Publication No. US 2007 / 0061900 (describing VELOCIMOUSE® technology). technology); and U.S. Patent Application Publication No. US 2007 / 0061900 (describing VELOCIMOUSE® technology). ​The human variable regions derived from whole antibodies produced by these animals can be further modified, for example, by combining with different human constant regions.

[0228] Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines have been described which can be used to produce human monoclonal antibodies. (See, e.g., Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).) Human antibodies generated via human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103: 3557-3562 (2006). Other methods include those described, e.g., in U.S. Patent No. 7,189,826 (describing production of monoclonal human IgM antibodies from hybridoma cell lines), and Ni, Xiandai Mianyixue, 226(4): 265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3): 927-937 (2005); and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3): 185-91 (2005).

[0229] Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from a human-derived phage display library. Such variable domain sequences can then be combined with a human constant domain of choice. Techniques for selecting human antibodies from antibody libraries are described below.

[0230] 5. Library-derived antibodies

[0231] Antibodies for use in the present application can be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, a variety of methods are known in the art for generating phage display libraries and screening these libraries for antibodies possessing the desired binding characteristics. Such methods are reviewed by, e.g., Hoogenboom et al. in Methods in Molecular Biology 178: 1-37 (O'Brien et al., eds., Human Press, Totowa, NJ, 2001) and further described, e.g., in the following publications, each of which is incorporated by reference in its entirety: McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132 (2004).

[0232] In certain phage display methods, repertoires of VHand VLgenes are separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be screened for antigen-binding phage as described in Winter et al. Ann. Rev. Immunol. 12:433-455 (1994). Phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments. Repertoires from immunized sources provide high-affinity antibodies to the immunogen without the requirement of hybridoma construction. Alternatively, one can clone all the natural components (e.g., from a human) to provide a single source of antibodies to a variety of non-self as well as self antigens without any immunization as described in Griffiths et al. EMBO J. 12:725-734 (1993). Finally, one can synthesize a naive repertoire by cloning the unrearranged V-gene segments from stem cells and using PCR primers to encode the highly variable CDR3 regions with random sequences and to complete the rearrangement in vitro as described in Hoogenboom and Winter J. Mol. Biol. 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example: U.S. Patent No. 5,750,373 and U.S. Patent Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0233] An antibody or antibody fragment isolated from a human antibody library is intended herein to be a human antibody or human antibody fragment.

[0234] 6. Multispecific Antibodies

[0235] In certain embodiments, the antibodies provided herein are multispecific antibodies, e.g., bispecific antibodies. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. In certain aspects, one of the binding specificities is for Tie2, while the other specificity is for any other antigen. In some aspects, bispecific antibodies can bind to two different epitopes of Tie2. Bispecific antibodies can be made as full length antibodies or antibody fragments.

[0236] Techniques for making multispecific antibodies include, but are not limited to, recombinant co- expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see, e.g., Milstein et al., Nature 305:537, 1983; WO 93 / 08829; and Traunecker et al., EMBO J. 10:3655, 1991), and the“plug and play” engineering (see, e.g., U.S. Patent No. 5,731,168). Multispecific antibodies can also be made by engineering electrostatic steering effects for making antibody Fc-heterodimeric molecules (WO 2009 / 089004 Al); cross-linking two or more antibodies or fragments (see, e.g., U.S. Patent No. 4,676,980; and Brennan et al., Science, 229:81 (1985)); using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5): 1547-1553 (1992)); using “diabody” technology (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (scFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)); and producing trispecific antibodies as described, e.g., in Tutt et al., J. Immunol. 147:60 (1991).

[0237] Also included among the antibodies herein are engineered antibodies having three or more antigen binding sites, including “Octopus antibodies” (see, e.g., US 2006 / 0025576 Al).

[0238] Also included among the antibodies herein are “Dual Acting FAb” or “DAF” molecules, which comprise an antigen binding site that binds to Tie2 as well as another, different antigen (see, e.g., US 2008 / 0069820).

[0239] 7. Antibody Variants

[0240] In certain aspects, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it can be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletion(s), insertion(s), and substitution(s) can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen binding.

[0241] a) Substitution, insertion, and deletion variants

[0242] In certain aspects, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include the HVRs and FRs. Conservative substitutions are listed below in the "Preferred Substitutions" section. More substantial changes, such as those that alter the binding affinity of the antibody for its antigen, are provided in the "Exemplary Substitutions" section below, and further described by reference to amino acid side chain classes below. Amino acid substitutions can be introduced into an antibody of interest and the products screened for a desired activity, for example, retained or improved antigen binding characteristic, decreased immunogenicity, or improved ADCC or CDC.

[0243] Table 1

[0244]

[0245] Amino acids can be grouped according to common side-chain properties:

[0246] (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, He;

[0247] (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;

[0248] (3) acidic: Asp, Glu;

[0249] (4) basic: His, Lys, Arg;

[0250] (5) residues that influence chain orientation: Gly, Pro;

[0251] (6) aromatic: Trp, Tyr, Phe.

[0252] Non-conservative substitutions require exchanging a member of one of these classes for a member from another.

[0253] One type of substitutional variant involves substituting one or more amino acids in the hypervariable region of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant antibody will have similar biological

[0254] Changes (e.g., substitutions) can be made in the CDRs to improve antibody affinity. Such modifications can be made in CDR "hotspots," i.e., residues encoded by codons that undergo hypermutation during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008)) and / or residues that contact antigen, and the binding affinity of the resulting variant VHand VLis tested. Affinity maturation is achieved by constructing and reselecting from secondary libraries, e.g., as described in Hoogenboom et al. in Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. This library is then screened to identify any antibody variants with the desired affinity. Another method to introduce diversity is a CDR-directed approach, in which a few CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues that specifically identify as involved in antigen binding can be identified by, e.g., alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

[0255] In certain aspects, substitutions, insertions, or deletions can occur within one or more CDRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen. For example, conservative modifications (e.g., conservative substitutions as provided herein) can be made in the CDRs that do not substantially reduce binding affinity. Such modifications can be outside of antigen contacting residues in the CDRs, for example. In certain embodiments of the variant VHand VLsequences provided above, each CDR is unaltered, or contains no more than one, two, or three amino acid substitutions.

[0256] One useful method, described by Cunningham and Wells (1989) Science, 244: 1081-1085, for identifying residues or regions of an antibody that can be targeted for mutagenesis is called "alanine scanning mutagenesis" as described there. Alternatively, or additionally, crystal structure analysis of an antigen-antibody complex can be used to identify contact points between the antibody and antigen. Such contact residues and neighboring residues can be targeted or eliminated for substitution. Variants can be screened for binding affinity to determine whether they contain the desired characteristics.

[0257] 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. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide which increases the serum half-life of the antibody.

[0258] b) Glycosylation variants

[0259] In certain embodiments, an antibody provided herein is altered to increase or decrease the extent to which it is glycosylated. Addition or deletion of glycosylation sites can be accomplished by altering the amino acid sequence to create or remove one or more glycosylation sites.

[0260] When the antibody comprises an Fc region, the carbohydrate associated with it can be altered. Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is typically linked to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharide can include various carbohydrates, such as mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the“stem” of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharide in an antibody of the application can be made in order to create antibody variants with certain improved properties.

[0261] In one embodiment, antibody variants are provided having a carbohydrate structure that lacks (directly or indirectly) fucose attached to the Fc region. For example, the content of fucose in such antibodies can be from 1% to 80%, from 1% to 65%, from 5% to 65%, or from 20% to 40%. The content of fucose is determined by calculating the average content of fucose in Asn297 glycoforms, as described in WO 2008 / 077546, for example, relative to the sum of all glycoforms attached to Asn 297 (e.g., complex, hybrid and high mannose) as measured by MALDI-TOF mass spectrometry. Asn297 refers to the asparagine residue located at about position 297 of the Fc region (EU numbering of Fc region residues); however, Asn297 can also be located about ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants can have improved ADCC function. See, e.g., US Patent Publication Nos. US 2003 / 0157108 (Presta, L.); US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Publication examples related to "defucosylated" or "fucose-deficient" antibody variants include: US 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US 2003 / 0115614; US 2002 / 0164328; US 2004 / 0093621; US 2004 / 0132140; US 2004 / 0110704; US 2004 / 0110282; US 2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO 2005 / 053742; WO 2002 / 031140; Okazaki et al., J. Mol. Biol. 336: 1239-1249 (2004); Yamane-Ohnuki et al., Biotech. Bioeng. 87: 614 (2004).Examples of cell lines capable of producing afucosylated antibodies include Lec13 CHO cells, which lack protein fucosylation (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application No. US 2003 / 0157108 Al, Presta, L; and WO 2004 / 056312 Al, Adams et al., especially in Example 11); and knockout cell lines, such as CHO cells knocked out for the a-1,6-fucosyltransferase gene FUT8 (see, e.g., Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng, 94(4):680-688 (2006); and WO 2003 / 085107).

[0262] Further provided are antibody variants with bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GIcNAc. Such antibody variants can have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described in, e.g., WO 2003 / 011878 (Jean-Mairet et al.); U.S. Pat. No. 6,602,684 (Umana et al.); and US 2005 / 0123546 (Umana et al.). Also provided are antibody variants with at least one galactose residue attached to the Fc region on the oligosaccharide. Such antibody variants can have improved CDC function. Such antibody variants are described in, e.g., WO 1997 / 30087 (Patel et al.), WO 1998 / 58964 (Raju, S.), and WO 1999 / 22764 (Raju, S.).

[0263] c) Fc region variants

[0264] In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. Fc region variants can comprise a human Fc region sequence (e.g., a human IgGl, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0265] In certain embodiments, the application contemplates an antibody variant that has some, but not all, effector functions, making it a candidate for use in applications where the half-life of the antibody in vivo is important, but certain effector functions (e.g., complement and ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be conducted to confirm the reduction / depletion of CDC and / or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the antibody is lacking in FcγRII binding (hence likely lacking in ADCC activity), but retains FcRn binding ability. NK cells, which mediate ADCC, express Fc(RIII, whereas monocytes express Fc(RI, Fc(RII, and Fc(RIII. A summary of the expression of FcRs on hematopoietic cells is provided in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro methods for assessing ADCC activity of a molecule of interest are described in U.S. Patent 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); 5,821,337 (see Bruggemann, M. et al. J. Exp. Med. 166: 1351-1361 (1987)). Alternatively, non-radioactive assays methods can be employed (see, e.g.: ACTI TM Cell Toxicity Assay (CellTechnology, Inc. Mountain View, CA); and CytoTox 24® Non-radioactive cytotoxicity assays (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 as disclosed in Clynes et al. Proc. Natl Acad. Sci. USA 95:652-656 (1998). Clq binding assays also can be carried out to confirm that the antibody is unable to bind Clq and hence lacks CDC activity. See, e.g., WO 2006 / 029879 and WO 2005 / 100402 for Clq and C3c binding ELISA. To assess complement activation, a CDC assay can be performed (see, for example: Gazzano-Santoro et al. J. Immunol. Methods 202:163 (1996); Cragg, M.S. et al. Blood 101 :1045-1052 (2003); and Cragg, M.S. and M.J. Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determination assays can also be performed using methods known in the art (see, e.g., Petkova, S.B. et al. Int'l. Immunol. 18(12): 1759-1769 (2006)).

[0266] Antibodies with reduced effector function include an antibody comprising one or more substitutions in the Fc region of the antibody at amino acids 238, 265, 269, 270, 297, 327, and 329 (US Patent No. 6,737,056). Such Fc variants include Fc variants that comprise two or more substitutions in the Fc region at amino acid positions 265, 269, 270, 297, and 327, including the so-called “DANA” Fc variant, in which residues 265 and 297 are substituted with alanine (US Patent No. 7,332,581).

[0267] Certain antibody variants with improved or diminished binding capacity to FcRs are described. (See, e.g., US Patent No. 6,737,056; WO 2004 / 056312; and Shields et al. J. Biol. Chem. 9(2):6591-6604 (2001).)

[0268] In certain embodiments, an antibody variant comprises an Fc region with one or more amino acid substitutions which improve ADCC, e.g., substitutions at positions 298, 333, and / or 334 of the Fc region (EU numbering of residues).

[0269] In some embodiments, modifications are made to the Fc region results in altered (i.e., improved or diminished) Clq binding and / or Complement Dependent Cytotoxicity (CDC), e.g., as described in US Patent No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0270] Antibodies with longer half-lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgGs to the fetus (see, e.g., Guyer et al. J. Immunol. 117:587 (1976) and Kim et al. J. Immunol. 24:249 (1994)), are described in US 2005 / 0014934A1 (Hinton et al.). Those antibodies comprise an Fc region with one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include Fc variants with substitutions 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., substitution of Fc region residue 434 (US Patent No. 7,371,826).

[0271] See also, e.g., Duncan & Winter, Nature 322:738-40 (1988); US Patent No. 5,648,260; US Patent No. 5,624,821; and WO 94 / 29351 for further examples of Fc region variants.

[0272] d) Cysteine-engineered antibody variants

[0273] In certain embodiments, it can be desirable to create cysteine engineered antibodies, e.g., "thioMAbs," in which one or more residues are substituted with cysteine residues. In particular embodiments, the substituted residues occur at accessible sites of the antibody. By substituting those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody and can be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to form an immunoconjugate, as described further herein. In certain embodiments, any one or more of the following residues can be substituted with cysteine: V205 (EU numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the Fc region of the heavy chain. Cysteine engineered antibodies can be generated according to the methods described in, e.g., US Patent No. 7,521,541.

[0274] e) Antibody derivatives

[0275] In certain embodiments, the antibodies provided herein can be further modified, e.g., to contain additional non-protein moieties known and available in the art. Moieties suitable for derivatization of the antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymer, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-l,3-dioxolane, poly-l,3,6-trioxane, ethylene / maleic acid copolymer, polyamino acids (homopolymers or random copolymers), and dextran or poly(N-2-hydroxypropyl methacrylamide) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (such as glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde can have advantages in manufacturing due to its stability in water. The polymer can have any molecular weight and can be a branched or unbranched polymer. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular property or function of the antibody to be improved, whether the antibody derivative will be used in therapy under specified conditions, etc.

[0276] In another embodiment, conjugates of antibodies and non-protein moieties that can be selectively heated by exposure to radiation are provided. In one embodiment, the non-protein moiety is a nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605, 2005). The radiation can be of any wavelength and includes, but is not limited to, wavelengths that do not damage ordinary cells but heat the non-protein moiety to a temperature close to that at which the antibody-non-protein moiety cell is killed.

[0277] B. Recombinant methods and compositions

[0278] Antibodies can be produced using recombinant methods and compositions, such as described in US 4,816,567. In one embodiment, an isolated nucleic acid encoding an anti-Tie2 antibody as described herein is provided. Such a nucleic acid encodes an amino acid sequence comprising a VL and / or an amino acid sequence comprising a VH of an antibody (e.g., a light chain and / or a heavy chain of an antibody). In a further embodiment, one or more vectors (e.g., expression vectors) comprising such a nucleic acid are provided. In a further embodiment, a host cell comprising the nucleic acid is provided. In this embodiment, the host cell comprises (e.g., has been transformed with): (1) a vector comprising the nucleic acid encodes an amino acid sequence comprising a VL of an antibody and an amino acid sequence comprising a VH of an antibody, or (2) a first vector comprising the nucleic acid encodes an amino acid sequence comprising a VL of an antibody and a second vector comprising a nucleic acid encodes an amino acid sequence comprising a VH of an antibody. In one embodiment, the host cell is a eukaryotic cell, such as a Chinese Hamster Ovary (CHO) cell or a lymphoid cell (e.g., Y0, NS0, Sp20 cell). In one aspect, a method of making an anti-Tie2 antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding an antibody as described above under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0279] In recombinant production of an anti-Tie2 antibody, a nucleic acid encoding the antibody, such as described above, is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such a nucleic acid can be readily isolated and sequenced using conventional procedures (e.g., using oligonucleotide probes that are specific to genes encoding the heavy and light chains of an antibody).

[0280] Host cells suitable for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells described herein. For example, antibodies can be produced in bacteria, in particular when glycosylation and Fc effector functions are not needed. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, describing expression of antibody fragments in E. coli.) After expression, the antibody can be isolated from the soluble fraction of the bacterial cell paste and can be further purified.

[0281] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including a strain of the fungus or yeast having a "humanized" glycosylation pathway, resulting in production of polypeptides having a partially or fully human glycosylation pattern. See: Gerngross, Nat. Biotech. 22: 1409-1414 (2004); and Li et al., Nat. Biotech. 24: 210-215 (2006).

[0282] Suitable host cells for expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which can be used in combination with insect cells, particularly for transfection of Spodoptera frugiperda cells.

[0283] Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES TM technology) for production of antibodies in transgenic plants.

[0284] Vertebrate cells can also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension can be used. Other examples of mammalian host cell lines that can be employed by the present application include: monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells as described, e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells (described, e.g., in Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR -CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines, such as Y0, NS0 and Sp2 / 0. For a review of certain mammalian host cell lines suitable for production of antibodies, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0285] C. Assays

[0286] Various assays known in the art can be employed to identify, screen or characterize the physical / chemical properties and / or biological activities of the anti-Tie2 antibodies provided herein.

[0287] 1. Binding assays and other assays

[0288] In one aspect, the antigen binding activity of the antibodies of the application is tested by known methods, e.g., ELISA, FACS or Western blot.

[0289] In another aspect, a competition assay can be used to identify antibodies that compete with an anti-Tie2 antibody, referred to herein as Tie2.1M100cF, which comprises a VH and VL for binding to Tie2, the VH comprising the sequence of SEQ ID NO: 20 and the VL comprising the sequence of SEQ ID NO: 21. In certain embodiments, the competing antibody binds to the same epitope (e.g., linear or conformational epitope) bound by the anti-Tie2 antibody comprising a VH comprising the sequence of SEQ ID NO: 20 and a VL comprising the sequence of SEQ ID NO: 21. Detailed exemplary methods for mapping epitopes to antibody binding are provided in Morris (1996) "Epitope Mapping Protocols" in Methods in Molecular Biology Vol. 66 (Humana Press, Totowa, NJ).

[0290] In one exemplary competition assay, immobilized Tie2 is incubated in a solution containing a first labeled antibody that binds to Tie2 (e.g., Tie2.1 M100cF) and a second unlabeled antibody whose ability to compete with the first antibody for binding to Tie2 is being tested. The second antibody can be present in a hybridoma supernatant. As a control, immobilized Tie2 is incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow the first antibody to bind to Tie2, excess unbound antibody is removed, and the amount of label associated with the immobilized Tie2 is measured. If the amount of label associated with the immobilized Tie2 in the test sample is significantly reduced relative to the control sample, then the second antibody is competing with the first antibody for binding to Tie2. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0291] 2. Activity Assays

[0292] In one aspect, assays are provided to identify anti-Tie2 antibodies that recognize antibodies having a desired biological activity. Biological activity can include, for example, binding to Tie2 or a fragment thereof, competing with Angl and / or Ang2 for binding to Tie2, activating phosphorylation of Tie2 protein, activating phosphorylation of Akt, and / or decreasing vascular endothelial cell permeability, in vivo, in vitro, or in a simulation of in vivo. Antibodies having such biological activity in vivo and / or in vitro are also provided. In certain embodiments, antibodies of the application are tested for such biological activity.

[0293] In some embodiments, assays are provided for determining Tie2-activating (anti-Tie2 conjugate) activity, for example, by a phospho-AKT (pAKT) assay, in which activation of AKT phosphorylation by a Tie2 conjugate is indicative of the conjugate having activating (agonist) activity. As known to the ordinarily skilled artisan and as described in Example 3 below, activation of AKT can be demonstrated by various methods, including, for example, Western blotting for phosphorylated AKT using an antibody specific for phosphorylated AKT or by a FRET assay.

[0294] In some embodiments, assays for determining the stability (e.g., thermal stability) of an anti-Tie2 antibody or antibody conjugate, fusion protein, or polymeric formulation thereof are provided. For example, the stability of an antibody or antibody conjugate, fusion protein, or polymeric formulation thereof can be determined using any method known in the art, e.g., differential scanning fluorimetry (DSF), circular dichroism spectroscopy (CD), intrinsic protein fluorescence, differential scanning calorimetry, spectroscopy, light scattering (e.g., dynamic light scattering (DLS) and static light scattering (SLS)), self-interaction chromatography (SIC).

[0295] In some embodiments, assays for determining the stability of an anti-Tie2 conjugate are provided. The stability of the assay can be determined as described herein, e.g., using capillary electrophoresis laser induced fluorescence (CE-LIF), as performed, e.g., in Example 13.

[0296] D. Immunoconjugates

[0297] The present application also provides immunoconjugates comprising an anti-Tie2 antibody as described herein conjugated (chemically linked) to one or more therapeutic agents, such as a cytotoxic agent, a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., a protein toxin derived from a bacteria, fungus, plant or animal, an enzymatically active toxin or fragments thereof) or a radioactive isotope.

[0298] E. Conjugates

[0299] The present application also provides conjugates comprising any of the anti-Tie2 antibodies or Tie2-binding fragments thereof provided herein conjugated to one or more heterologous molecules, such as a polyol.

[0300] 1. Multi-Arm Polymers

[0301] In some embodiments, the conjugates of the present disclosure can be made by derivatizing the anti-Tie2 antibodies described herein by conjugating an anti-Tie2 Fab or variant thereof to a multi-arm polymer. It will be appreciated that any multi-arm polymer that provides a conjugate described herein having a desired size or selected average molecular weight is suitable for use in constructing the antibody-polymer conjugates of the present application.

[0302] A variety of polymers are suitable for use in medicine. See, e.g., Davis et al., Biomedical Polymers: Polymeric Materials and Pharmaceuticals for Biomedical Use, pp. 441-451 (1980). In some embodiments of the present disclosure, non-proteinaceous polymers are used to form the conjugates of the present disclosure. The non-proteinaceous polymers are typically hydrophilic, synthetic polymers, i.e., polymers that are not found in nature. However, polymers that occur in nature and are manufactured by recombinant or in vitro methods can also be useful, as can polymers isolated from natural sources.

[0303] In some embodiments, the anti-Tie2 Fabs are derivatized by conjugating (e.g., covalently linking) the Fab or variant thereof to a multi-armed polyol. Thus, in some embodiments, the present disclosure is directed to conjugates comprising one or more anti-Tie2 Fabs or variants thereof disclosed herein covalently linked to one or more multi-armed polyols, preferably a six-armed polyol. The polyol employed can be any water-soluble poly(alkylene oxide) polymer, and can be linear or branched. Suitable polyols include those substituted at one or more hydroxyl positions with a chemical group, e.g., an alkyl group having between one and four carbons. Typically, the polyol is a poly(alkylene glycol), e.g., polyethylene glycol (PEG), and thus, for ease of description, the remainder of the discussion is in regard to an exemplary embodiment in which the polyol employed is PEG, and the process of conjugating the polyol to the polypeptide is referred to as "PEGylation." However, one skilled in the art will recognize that other polyols, such as polypropylene glycol and polyethylene glycol-polypropylene glycol copolymers, can be employed using techniques for conjugation similar to those described herein for PEG.

[0304] The polyol used to form the conjugates of the present disclosure is a multi-armed polyol. As used herein, "multi-armed polyol" refers to a polyol comprising a core structure, and at least two arms attached to the core structure. The multi-armed polyol can be, e.g., a dimer (two arms), a tetramer (four arms), a hexamer (six arms), an octamer (eight arms), etc. In some aspects, the multi-armed polyol is a multi-armed PEG.

[0305] The weight average molecular weight of the multi-armed PEG used in the PEGylation of the anti-Tie2 antibody or antibody variant can vary and generally can range from about 500 to about 300,000 Daltons (D). In some embodiments, the weight average molecular weight of the multi-armed PEG is from about 1,000 to about 100,000 D, from about 1,000 to about 40,000 D, from about 1,000 to about 20,000 D, from about 1,000 to about 10,000 D, from about 10,000 to about 20,000 D, from about 5,000 to about 10,000 D, or from about 1,000 to 5,000 D. In preferred embodiments, PEGylation is performed using a multi-armed PEG having a weight average molecular weight of about 6,000 D.

[0306] A variety of methods for PEGylation of proteins are known in the art. Specific methods for making PEG-conjugated proteins include those described in U.S. Patent No. 4,179,337, U.S. Patent No. 4,935,465, and U.S. Patent No. 5,849,535, all of which are incorporated herein by reference in their entireties. Generally, the protein is covalently bound to a terminal reactive group on the polymer via one or more of the amino acid residues of the protein. Herein, the polymer having the reactive groups involved is an activated or functionalized polymer (e.g., functionalized PEG). The reactive groups selectively react with free thiols or amines or other reactive groups on the antibody or antibody variant. The multi-armed PEG polymer can be coupled to the thiols or amines or other reactive groups on the antibody or antibody variant in a random or site-specific manner. However, it is understood that the type and amount of reactive groups selected, as well as the type and amount of polymer employed, will depend on the particular antibody or antibody variant employed in order to limit and preferably substantially prevent the reactive groups from reacting with too many active groups on the antibody. Since it can not be possible in some cases to sufficiently limit or prevent this, generally from about 0.05 to about 1000 moles or in some embodiments from about 0.05 to about 200 moles of functionalized polymer per mole of antibody can be employed, depending on the antibody concentration. The final amount of functionalized polymer per mole of antibody is assessed as follows: to maintain optimal activity while optimizing (if possible) the half-life of the antibody in the vitreous, retinal, and / or ocular humor.

[0307] Although the residue can be any amino acid on the antibody or antibody variant, such as the N-terminal amino acid group, in some embodiments, the reactive group is cysteine, which is linked via its free sulfhydryl group to a reactive group of a functionalized polymer, such as described in WO 99 / 03887, WO 94 / 12219, WO 94 / 22466, U.S. Pat. No. 5,206,344, U.S. Pat. No. 5,166,322, and U.S. Pat. No. 5,206,344, all of which are incorporated herein by reference in their entirety. In such embodiments, the polymer may comprise at least one terminal reactive group that is capable of reacting specifically with a sulfhydryl or sulfhydryl group on the parent antibody. Such groups include, but are not limited to, maleimide, sulfhydryl, thiol, triflate, tosylate, aziridine, epoxide, pyridyl disulfide, succinimidyl ester, -NH2, aldehyde, haloacetate, haloacetamide, and p-nitrophenyl carbonate, among others. The polymer can be coupled to the parent antibody using any scheme suitable for the chemical properties of the selected coupling system, such as the schemes and systems described in U.S. Patent No. 4,179,337, U.S. Patent No. 7,122,636, and Jevsevar, et al., Biotech J., Vol. 5, pp. 113-128 (2010). Alternatively, the reactive amino acid can be lysine, which is linked to the reactive group of the functionalized polymer via its free ε-amino group (see, for example, WO 93 / 00109, incorporated herein by reference); or glutamic acid or aspartic acid, which is linked to the polymer via an amide bond. The reactive group of the polymer can then react with, for example, α (alpha) and ε (epsilon) amines or sulfhydryl groups of proteins to form a covalent bond. It should be understood that the present disclosure is not limited to conjugates using a specific type of connection between the antibody or antibody fragment and the polymer.

[0308] Functionalized multi-arm PEGs suitable for preparing conjugates of the present disclosure can be made by a number of conventional reactions. For example, N-hydroxysuccinimidyl esters of PEG (M-NHS-PEG) can be prepared from PEG-monomethyl ether by reaction with N,N'-dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS) according to the method of Buckmann and Merr, Makromol. Chem., Vol. 182, pp. 1379-1384 (1981). In addition, PEG terminal hydroxyl groups can be converted to amino groups, for example, by reaction with thionyl bromide to form PEG-Br, followed by aminolysis using an excess of ammonia to form PEG-NH2. PEG-NH2 can then be conjugated to an antibody or antibody variant of interest using standard coupling reagents such as Woodward's Reagent K. In addition, PEG terminal CH2OH groups can be converted to aldehyde groups, for example, by oxidation using MnO2. The aldehyde groups can be conjugated to an antibody or antibody variant by reductive alkylation using reagents such as cyanoborohydride.

[0309] In some embodiments, the multi-arm PEG used to prepare conjugates of the present disclosure has the structure of Formula (I):

[0310]

[0311] wherein the PEGs are the same or different -(CH2CH2O)m- wherein each m represents the length or size of a particular arm of the polyol (PEG) and is independently an integer from about 45 to about 1000, from about 3 to about 250, or from about 50 to about 200 or from about 100 to about 150; and / is an integer > 2, preferably 2 or 3.

[0312] In some embodiments, the multi-arm PEG has the structure of Formula (I) wherein / is 2 and the multi-arm PEG is a hexamer. In another embodiment, the multi-arm PEG has the structure of Formula (I) wherein / is 3 and the multi-arm PEG is an octamer.

[0313] The multi-arm PEG having the structure of Formula (I) can be functionalized using any of the techniques disclosed above to produce a functionalized multi-arm PEG to, for example, attach a terminal reactive group suitable for reacting or conjugating with an antibody (e.g., an antibody fragment). In other embodiments, however, the multi-arm PEG can be covalently linked to an anti-Tie2 antibody by a multifunctional crosslinking agent that reacts with the PEG to be crosslinked and one or more amino acid residues of the antibody or antibody variant, as described, for example, in U.S. Patent No. 7,122,636, which is incorporated by reference herein in its entirety.

[0314] In other aspects, the multi-arm PEG used to prepare the conjugates of the present disclosure is a functionalized multi-arm PEG comprising at least one terminal reactive group. The terminal reactive group can be conjugated directly to an anti-Tie2 antibody to form the conjugates of the present disclosure. In some embodiments, the functionalized multi-arm PEG has the structure of Formula (la):

[0315]

[0316] wherein each m represents the length or size of a particular arm of the polyol (PEG) and is independently an integer from about 45 to about 1000, from about 3 to about 250, or from about 50 to about 200, or from about 20 to 30 or from about 100 to about 150; and n is an integer from about 1 to about 10; each R 1 is independently or absent or is a linking group; and each R 2 is independently or is hydrogen or is a terminal reactive group; wherein at least one R 2 is a terminal reactive group. In some embodiments, R 2 is independently selected from the group consisting of a thiol-reactive group, an amino-reactive group, and combinations thereof.

[0317] In some embodiments, the functionalized multi-arm PEG has the structure of Formula (la) wherein n is an integer from 2 to 3. In preferred embodiments, the functionalized multi-arm PEG has the structure of Formula (la) wherein n is 2 and the multi-arm PEG is a hexamer. In another embodiment, the functionalized multi-arm PEG has the structure of Formula (la) wherein n is 3 and the multi-arm PEG is an octamer. In preferred embodiments, the functionalized multi-arm PEG has the structure of Formula (la) wherein n is 2, as shown in Figure 39 .

[0318] In another embodiment, the multi-arm PEG used to prepare the conjugates of the present disclosure has the structure of Formula (II):

[0319]

[0320] wherein each m represents the length or size of a particular arm of the polyol (PEG) and is independently an integer from about 45 to about 1000, from about 3 to about 250, or from about 50 to about 200, or from about 100 to about 150; and n is an integer from about 1 to about 10.

[0321] In some embodiments, the multi -arm PEG has the structure of Formula (II), wherein n is 2, and the multi -arm PEG is a tetramer. In another embodiment, the multi -arm PEG has the structure of Formula (II), wherein n is 4, and the multi -arm PEG is a hexamer. In another embodiment, the multi -arm PEG has the structure of Formula (II), wherein n is 6, and the multi -arm PEG is an octamer.

[0322] In another embodiment, the multi -arm PEG used to make the conjugates of the present disclosure has the structure of Formula (III):

[0323]

[0324] wherein each m represents the length or size of a particular arm of the polyol (PEG) and is independently an integer from about 45 to about 1000, from about 3 to about 250, or from about 50 to about 200, or from about 100 to about 150; and n is an integer from about 1 to about 10.

[0325] In some embodiments, the multi -arm PEG has the structure of Formula (III), wherein n is 2, and the multi -arm PEG is a tetramer. In another embodiment, the multi -arm PEG has the structure of Formula (III), wherein n is 4, and the multi -arm PEG is a hexamer. In another embodiment, the multi -arm PEG has the structure of Formula (III), wherein n is 6, and the multi -arm PEG is an octamer.

[0326] In another embodiment, the multi -arm PEG used to make the conjugates of the present disclosure has the structure of Formula (IV):

[0327]

[0328] wherein each m represents the length or size of a particular arm of the polyol (PEG) and is independently an integer from about 45 to about 1000, from about 3 to about 250, or from about 50 to about 200, or from about 100 to about 150.

[0329] The multi -arm PEG having the structure of any of Formulas (I) to (IV) can be functionalized using any of the techniques described above to produce a functionalized multi -arm PEG to, for example, attach a terminal reactive group suitable for reaction or conjugation with an antibody (e.g., an antibody fragment). In other embodiments, however, the multi -arm PEG can be covalently linked to an anti-Tie2 antibody by a multifunctional crosslinking agent that reacts with the PEG to be crosslinked and one or more amino acid residues of the antibody or antibody variant, as described, for example, in U.S. Patent No. 7,122,636, which is incorporated by reference herein in its entirety.

[0330] In other aspects, the multi-arm PEG used to prepare the conjugates of the present disclosure is a functionalized multi-arm PEG comprising at least one terminal reactive group. The terminal reactive group can be conjugated directly to an anti-Tie2 antibody to form the conjugates of the present disclosure. In some embodiments, the functionalized multi-arm PEG has the structure of Formula (la):

[0331]

[0332] wherein each m represents the length or size of a particular arm of the polyol (PEG) and is independently an integer from about 45 to about 1000, from about 3 to about 250, or from about 50 to about 200 or from about 100 to about 150; and, n is an integer from about 1 to about 10; each R 1 is independently or absent or a linking group; and, each R 2 is independently or hydrogen or a terminal reactive group; wherein at least one R 2 is a terminal reactive group. In some embodiments, R 2 is independently selected from the group consisting of a thiol-reactive group, an amino-reactive group, and combinations thereof.

[0333] In some embodiments, the functionalized multi-arm PEG has the structure of Formula (la) wherein n is an integer from 1 to 3. In some embodiments, the functionalized multi-arm PEG has the structure of Formula (la) wherein n is 1, and the multi-arm PEG is a tetramer. In another embodiment, the functionalized multi-arm PEG has the structure of Formula (la) wherein n is 2, and the multi-arm PEG is a hexamer. In another embodiment, the functionalized multi-arm PEG has the structure of Formula (la) wherein n is 3, and the multi-arm PEG is an octamer. In such embodiments, the hexamer has the structure of Formula (lb):

[0334]

[0335] wherein m, R 1 and R 2 are as defined above.

[0336] The multi-arm PEG having the structure of Formula (lb) has a di-pentaerythritol (DP) core structure, and is also referred to herein as a DP hexamer.

[0337] In some embodiments, the functionalized multi-arm PEG has the structure of Formula (lb) or (lc) wherein each R 1 are the same or different when present, and when R 1 and R 2 are combined together are selected from the group consisting of as well as and combinations thereof; wherein each i is independently an integer from 0 to 10; j is an integer from 0 to 10; and R 2 As defined herein. In some embodiments, each R 1 For the connecting group.

[0338] In some embodiments, the functionalized multi-arm PEG has a structure of Formula (Ib) or (Ic), wherein when R 1 With R 2 When combined together Among them, i, j and R 2 As defined herein. In some embodiments, when R 1 With R 2 When combined together Where i is 2; j is 2 or 3, and R 2 As defined herein.

[0339] In some embodiments, the functionalized multi-arm PEG has a structure of Formula (Ib), wherein each R 2 is independently selected from maleimide, sulfhydryl, thiol, triflate, tosylate, aziridine, peroxide, pyridyl disulfide, succinimidyl ester, -NH2, aldehyde, haloacetate, haloacetamide, and p-nitrophenyl carbonate. In some embodiments, each R 2 is independently a haloacetate selected from bromoacetate, iodoacetate, chloroacetate, and combinations thereof. 2 is independently a haloacetamide selected from bromoacetamide, iodoacetamide, chloroacetamide, and combinations thereof. 2 It is maleimide.

[0340] In some embodiments, the functionalized multi-arm PEG has a structure of Formula (Ia) or (Ib), wherein each R 2 In some embodiments, the functionalized multi-arm PEG has a structure of formula (Ia) or (Ib), wherein when R 1 With R 2 When combined together wherein i and j are as defined above. In some embodiments, the functionalized multi-arm PEG has a structure of formula (Ia) or (Ib), wherein when R 1 With R 2 When combined together Where i is 2 and j is 2.

[0341] In another aspect, the functionalized multi-arm PEG used to prepare the conjugates of the present disclosure has the structure of formula (IIa):

[0342]

[0343] wherein each m represents the length or size of a particular arm of the polyol (PEG) and is independently an integer from about 45 to about 1000, from about 3 to about 250, or from about 50 to about 200, or from about 100 to about 150; and n is an integer from about 1 to about 10; each R 1 independently or absent or a linking group; and each R 2 are independently hydrogen or a terminal reactive group; wherein at least one R 2 is a terminal reactive group. In some embodiments, R 2 Independently selected from thiol-reactive groups, amino-reactive groups, and combinations thereof.

[0344] In some embodiments, the functionalized multi-arm PEG has a structure of formula (IIa), wherein n is an integer from 2 to 6. In some embodiments, the functionalized multi-arm PEG has a structure of formula (IIa), wherein n is 2, and the multi-arm PEG is a tetramer. In some embodiments, the functionalized multi-arm PEG has a structure of formula (IIa), wherein n is 3. In another embodiment, the functionalized multi-arm PEG has a structure of formula (IIa), wherein n is 4, and the multi-arm PEG is a hexamer. In another embodiment, the functionalized multi-arm PEG has a structure of formula (IIa), wherein n is 6, and the multi-arm PEG is an octamer. The octamer with the structure of formula (IIa) has a hexaglycerol (HG) core structure and is also referred to herein as an HG octamer.

[0345] In some embodiments, the functionalized multi-arm PEG has a structure of formula (IIa), wherein each R 1 When present, they are the same or different, and when R 1 With R 2 When combined, select as well as and combinations thereof; wherein each i is independently an integer from 0 to 10; j is an integer from 0 to 10; and R 2 As defined herein. In some embodiments, each R 1 For the connecting group.

[0346] In some embodiments, the functionalized multi-arm PEG has a structure of formula (IIa), wherein when R 1 With R 2 When combined together wherein i, j and R 2 as defined herein. In some embodiments, when R 1 is maleimide. 2 is maleimide. wherein i is 2; j is 2 or 3, and, R 2 as defined herein.

[0347] In some embodiments, the functionalized multi -arm PEG has the structure of Formula (IIa) wherein each R 2 is independently selected from the group consisting of maleimide, thiol, thiol, triflate, tosylate, aziridine, peroxide, pyridyl disulfide, succinimidyl ester, -NH2, aldehyde, haloacetate, haloacetamide, and p-nitrophenyl carbonate. In some embodiments, each R 2 is independently a haloacetate selected from the group consisting of bromoacetate, iodoacetate, chloroacetate, and combinations thereof. In some embodiments, each R 2 is independently a haloacetamide selected from the group consisting of bromoacetamide, iodoacetamide, chloroacetamide, and combinations thereof. In some embodiments, R 2 is maleimide.

[0348] In some embodiments, the functionalized multi -arm PEG has the structure of Formula (IIa) wherein each R 2 is maleimide. In some embodiments, the functionalized multi -arm PEG has the structure of Formula (IIa) wherein when R 1 is maleimide. 2 is maleimide. wherein i and j are as defined above. In some embodiments, the functionalized multi -arm PEG has the structure of Formula (IIa) wherein when R 1 is maleimide. 2 is maleimide. wherein i is 2 and j is 2.

[0349] In another aspect, the functionalized multi -arm PEG has the structure of Formula (IIIa):

[0350]

[0351] wherein each m represents the length or size of a particular arm of the polyol (PEG) and is independently an integer from about 45 to about 1000, or from about 3 to about 250, or from about 50 to about 200, or from about 100 to about 150; and, n is an integer from about 1 to about 10; each R 1 is independently or absent or a linking group; and, each R 2 is independently or hydrogen or a terminal reactive group; wherein at least one R2 is a terminal reactive group. In some embodiments, R 2 Independently selected from thiol-reactive groups, amino-reactive groups, and combinations thereof.

[0352] In some embodiments, the functionalized multi-arm PEG has the structure of formula (IIIa), wherein n is an integer from 2 to 6. In some embodiments, the functionalized multi-arm PEG has the structure of formula (IIIa), wherein n is 2, and the multi-arm PEG is a tetramer. In another embodiment, the functionalized multi-arm PEG has the structure of formula (IIIa), wherein n is 4, and the multi-arm PEG is a hexamer. In another embodiment, the functionalized multi-arm PEG has the structure of formula (IIIa), wherein n is 6, and the multi-arm PEG is an octamer. The octamer with the structure of formula (IIIa) has a hexaglycerol (HGEO) core structure and is also referred to herein as an HGEO octamer.

[0353] In some embodiments, the functionalized multi-arm PEG has a structure of formula (IIIa), wherein each R 1 When present, they are the same or different, and when R 1 With R 2 When combined, select as well as and combinations thereof; wherein each i is independently an integer from 0 to 10; j is an integer from 0 to 10; and R 2 As defined herein. In some embodiments, each R 1 For the connecting group.

[0354] In some embodiments, the functionalized multi-arm PEG has a structure of formula (IIIa), wherein when R 1 With R 2 When combined together Among them, i, j and R 2 As defined herein. In some embodiments, when R 1 With R 2 When combined together Where i is 2; j is 2 or 3, and R 2 As defined herein.

[0355] In some embodiments, the functionalized multi-arm PEG has a structure of formula (IIIa), wherein each R 2independently selected from maleimide, thiol, thiol, triflate, tosylate, aziridine, peroxide, pyridyl disulfide, succinimidyl ester, -NH2, aldehyde, haloacetate, haloacetamide, and p-nitrophenyl carbonate. In some embodiments, each R 2 is independently a haloacetate selected from bromoacetate, iodoacetate, chloroacetate, and combinations thereof. In some embodiments, each R 2 is independently a haloacetamide selected from bromoacetamide, iodoacetamide, chloroacetamide, and combinations thereof. In some embodiments, R 2 is maleimide.

[0356] In some embodiments, the functionalized multi -arm PEG has the structure of Formula (IIIa), wherein each R 2 is maleimide. In some embodiments, the functionalized multi -arm PEG has the structure of Formula (IIIa), wherein, when R 1 is maleimide, R 2 taken together is wherein i and j are as defined above. In some embodiments, the functionalized multi -arm PEG has the structure of Formula (IIIa), wherein, when R 1 is maleimide, R 2 taken together is wherein i is 3 and j is 2.

[0357] In another aspect, the functionalized multi -arm PEG has the structure of Formula (IVa):

[0358]

[0359] wherein each m represents the length or size of a particular arm of the polyol (PEG) and is independently an integer from about 45 to about 1000, from about 3 to about 250, or from about 50 to about 200 or from about 100 to about 150; each R 1 is independently or is absent or is a linking group; and, each R 2 is independently or is hydrogen or is a terminal reactive group; wherein at least one R 2 is a terminal reactive group. In some embodiments, R 2 is independently selected from a thiol reactive group, an amino reactive group, and combinations thereof.

[0360] The multi -arm PEG having the structure of Formula (IVa) has a butanediol core structure and is also referred to herein as a DX octamer.

[0361] In some embodiments, the functionalized multi -arm PEG has the structure of Formula (IVa), wherein each R 1 are the same or different when present and are maleimide when R1 with R 2 taken together are selected from and and combinations thereof; wherein each i is independently an integer from 0 to 10; j is an integer from 0 to 10; and R 2 as defined herein. In some embodiments, each R 1 is a linking group.

[0362] In some embodiments, the functionalized multi -arm PEG has a structure of general formula (IVa), wherein each R 1 with R 2 taken together is wherein i, j, and R 2 are as defined herein. In some embodiments, each R 1 with R 2 taken together is wherein i is 2; j is 2 or 3, and R 2 are as defined herein.

[0363] In some embodiments, each R 2 is independently selected from maleimide, thiol, thiol, triflate, tosylate, aziridine, peroxide, pyridyl disulfide, succinimidyl ester, -NH2, aldehyde, haloacetate, haloacetamide, and p-nitrophenyl carbonate. In some embodiments, each R 2 is independently a haloacetate selected from bromoacetate, iodoacetate, chloroacetate, and combinations thereof. In some embodiments, each R 2 is independently a haloacetamide selected from bromoacetamide, iodoacetamide, chloroacetamide, and combinations thereof. In some embodiments, R 2 is maleimide.

[0364] In some embodiments, the functionalized multi -arm PEG has a structure of general formula (IVa), wherein each R 2 is maleimide. In some embodiments, the functionalized multi -arm PEG has a structure of general formula (IVa), wherein each R 1 with R 2 taken together is wherein i and j are as defined above. In some embodiments, the functionalized multi -arm PEG has a structure of general formula (IVa), wherein each R 1 with R 2 taken together is wherein i is 3 and j is 2.

[0365] Other functionalized multi-arm PEGs suitable for use in the present disclosure are described in U.S. Patent Application Publication No. 2011 / 0286956 and U.S. Patent Application Publication No. 2015 / 0073155, both of which are incorporated herein by reference in their entirety.

[0366] Functionalized multi-arm PEGs suitable for use in the present disclosure are also commercially available from multiple suppliers. For example, JenKem Technology, USA sells maleimide-functionalized PEG hexamers and octamers (e.g., 6ARM(DP)-PEG-MAL and 8ARM(TP)-PEG-MAL). NOF America Corp. also sells maleimide-functionalized PEG octamers (e.g., PTE-400MA) and tetramers (e.g., HGEO-400MA; DX-400MA). HGEO-400MA; DX-400MA) and tetramers (e.g., PTE-400MA).

[0367] In particular embodiments, the active derivative of the multi-arm PEG is an active NHS ester derivative of the multi-arm PEG having the structure of Formula (IV) below:

[0368]

[0369] wherein R is dipentaerythritol.

[0370] 1. A polyol conjugate

[0371] In some embodiments, the present disclosure is directed to conjugates (e.g., Tie2 binding agents) comprising one or more of the anti-Tie2 antibodies or antibody variants disclosed herein and one or more multi-arm polyols, wherein the conjugate is prepared by covalently linking at least one anti-Tie2 Fab or Fab variant to a polyol. In some embodiments, the multi-arm polyol is PEG. In preferred embodiments, the PEG is a hexamer. In other embodiments, the PEG is an octamer. In some embodiments, the PEG has the structure of Formula (la).

[0372] The conjugates of the present disclosure can be characterized by the number of anti-Tie2 antibodies (Fabs) conjugated to each multi-arm PEG. Herein, this is referred to as "fabrylation" or "degree of fabrylation." The number of anti-Tie2 Fabs conjugated to each PEG can vary depending on a variety of factors, including: 1) the number of arms in the PEG; 2) the number of terminal reactive groups on the PEG and / or reactivity; 3) the core structure of the PEG; and / or 4) the PEGylation reaction conditions. The high polydispersity of the multi-arm PEGs used to make the conjugates can in some cases complicate analysis of the final conjugates, particularly making it more difficult and uncertain to accurately determine the number of Fabs per PEG. Accordingly, the PEGs used to form the conjugates will generally have a polydispersity in the range of about 1 to about 1.35 (determined using methods known in the art), and in various embodiments will have a polydispersity of about 1 to about 1.25, about 1 to about 1.2, about 1 to about 1.15, about 1 to about 1.1, about 1.05, or even about 1.

[0373] In some embodiments, the conjugates of the present disclosure comprise a six- arm PEG, wherein at least one anti-Tie2 Fab or variant is covalently linked to the PEG. In another embodiment, the conjugates of the present disclosure comprise a six-arm PEG, wherein at least 2, at least 3, at least 4, at least 5, or at least 6 anti-Tie2 Fabs are covalently linked to the PEG. In another embodiment, the conjugates of the present disclosure comprise an eight-arm PEG, wherein at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 anti-Tie2 Fabs are covalently linked to the PEG. In another embodiment, the conjugates of the present disclosure comprise a six-arm PEG, wherein at least 4, at least 5, or at least 6 anti-Tie2 Fabs are covalently linked to the PEG. In some embodiments, the conjugates of the present disclosure comprise a six-arm PEG, wherein 4 to 6, or 5 to 6 anti-Tie2 Fabs are covalently linked to the PEG. In another embodiment, the conjugates of the present disclosure comprise a six-arm PEG, wherein 4 to 6 anti-Tie2 Fabs are covalently linked to the PEG. In another embodiment, the conjugates of the present disclosure comprise a six-arm PEG, wherein 4 to 6, or 5 anti-Tie2 Fabs are covalently linked to the PEG.

[0374] In some embodiments, the conjugates of the present disclosure comprise a multi- arm PEG having the structure of any one of Formula (la), (Ila), (Ilia), or (IVa). In such embodiments, at least one R 2 covalently linked to an anti-Tie2 Fab or variant described herein. In some embodiments, the multi-arm PEG having the structure of any one of Formula (la), (Ila), (Ilia), or (IVa) is a hexamer, and at least 2, at least 3, at least 4, at least 5, or all 6 R2 The group is covalently attached to an anti-Tie2 Fab or variant as described herein.

[0375] In some embodiments, conjugates of the present disclosure include varieties in which the multi-armed polyol is covalently attached to one or more specific sites on the parent antibody, i.e., the polymer attachment is targeted to a particular region or one or more particular amino acid residues in the parent antibody or antibody fragment. Standard mutagenesis techniques can be used to alter the number and / or location of potential PEGylation sites in the parent antibody or antibody fragment. Thus, to the extent that amino acid substitutions introduce or replace amino acids such as cysteine and lysine, the anti-Tie2 antibodies and variants thereof of the present disclosure can contain a greater or lesser number of potential PEGylation sites than the native sequence anti-Tie2.

[0376] As described above, site-specific conjugation of polymers is most often achieved by attachment to cysteine residues in the parent antibody or antibody fragment. In such embodiments, coupling chemistry can take advantage of, for example, free thiol groups of cysteine residues in the parent antibody that are not in disulfide bonds.

[0377] In some embodiments, one or more cysteine residues naturally occurring in the parent Fab are used as attachment sites for polymer conjugation. In other embodiments, free amino groups on the Fab or variant can be thiolated using 2-imino-thiolane (Traut's reagent) and then coupled to, for example, maleimide-functionalized PEG as described in Pedley, et al., Br. J. Cancer, Vol. 70, pp. 1126-1130 (1994). In another embodiment, one or more cysteine residues are engineered into selected site(s) of the parent Fab for the purpose of providing one or more specific attachment sites for polymers.

[0378] Cysteine engineered antibodies have been previously described (U.S. Patent Publication No. 2007 / 0092940 and Junutula, J. R., et al, J. Immunol Methods, Vol. 332(l-2), pp. 41-52 (2008), which are hereby incorporated by reference in their entirety). In some embodiments, the cysteine engineered antibody can be a parent antibody. These are useful for generating antibodies with specific properties, such as increased serum half-life, increased tissue penetration, and increased tumor penetration. In some embodiments, the cysteine engineered antibody is a parent antibody that has been engineered to contain one or more cysteine residues in the variable region of the antibody. In some embodiments, the cysteine engineered antibody is a parent antibody that has been engineered to contain one or more cysteine residues in the constant region of the antibody. In some embodiments, the cysteine engineered antibody is a parent antibody that has been engineered to contain one or more cysteine residues in the variable region and the constant region of the antibody. L or C H1) antibody fragments having free cysteines. Parent antibodies engineered to contain cysteines are referred to herein as "ThioMabs," regardless of the method of manufacture, and Fab fragments made from such cysteine-engineered antibodies are referred to herein as "ThioFabs." As previously described (see, e.g., U.S. Patent Publication No. 2007 / 0092940 and Junutula, J.R., et al., J. Immunol Methods, Vol. 332(l-2), pp. 41-52 (2008)), for mutants with replaced ("engineered") cysteine (Cys) residues, the reactivity of the newly introduced engineered cysteine thiol group is assessed. The thiol reactivity value is a relative numerical term in the range of 0 to 1.0, and can be measured for any cysteine-engineered antibody. In addition to having a reactive thiol group, ThioMabs should be selected such that they retain antigen binding ability. The design, selection, and preparation of cysteine-engineered antibodies has been previously described in detail (see, e.g., WO 2011 / 069104, which is incorporated herein by reference). In some embodiments, the engineered cysteine is introduced into the constant domain of the heavy chain or light chain. Thus, the cysteine-engineered antibody retains the antigen binding ability of its wild-type, parent antibody counterpart, and is thus capable of specific binding to an antigen.

[0379] In some embodiments, the present disclosure is directed to antibody fragment-polymer conjugates, wherein the antibody fragment is a Fab, and the polymer is attached to one or more cysteine residues in the light chain or heavy chain of the Fab fragment, which would normally form an interchain disulfide bond linking the light chain to the heavy chain.

[0380] In another aspect, the present disclosure relates to antibody fragment-polymer conjugates, wherein the antibody fragment is a Fab-C, and the polymer is attached by targeting the hinge region of the Fab-C fragment. In some embodiments, one or more cysteine ​​residues naturally present in the hinge region of an antibody fragment are used to attach the polymer. In another embodiment, one or more cysteine ​​residues are engineered into the hinge region of the Fab-C fragment to provide one or more specific attachment sites for the polymer. In some embodiments, an anti-Tie2 Fab disclosed herein is modified by adding a cysteine ​​to the C-terminal end to provide one attachment site for polymer conjugation. In another embodiment, an anti-Tie2 Fab disclosed herein is modified by adding four additional residues, CPPC (SEQ ID NO: 87), to the C-terminal end to provide two attachment sites for polymer conjugation. In yet another embodiment, an anti-Tie2 Fab disclosed herein is modified by adding four additional residues, SPPC (SEQ ID NO: 88), to the C-terminal end to provide one attachment site for polymer conjugation.

[0381] The extent and site of PEGylation can also be controlled by adjusting reaction conditions such as the relative concentrations of functionalized PEG and protein and pH. Suitable conditions for a desired degree of PEGylation can be determined empirically by varying the parameters of a standard PEGylation reaction.

[0382] PEGylation of anti-Tie2 Fab and variants is performed by any conventional method. Suitable PEGylation conditions are described in detail in WO 2011 / 069104 and WO 03 / 029420, both of which are incorporated herein by reference in their entirety.

[0383] 3. Characterization of Polyol Conjugates

[0384] PEGylated proteins can be characterized by SDS-PAGE, gel filtration, NMR, peptide mapping, liquid chromatography-mass spectrometry, and in vitro biological assays. Typically, the extent of Fab is first demonstrated by SDS-PAGE. Polyacrylamide gel electrophoresis in 10% SDS is typically run in 10 mM Tris-HCl pH 8.0 with 100 mM NaCl as the elution buffer. To demonstrate which residues are PEGylated, peptide mapping can be performed using proteases such as trypsin and Lys-C protease. Thus, samples of PEGylated and non-PEGylated antibodies can be digested with a protease such as Lys-C protease, and the resulting peptides separated by techniques such as reverse-phase HPLC. The resulting peptide chromatogram can be compared to the peptide map previously determined for the anti-Tie2 polypeptide.

[0385] Each peak can then be analyzed by mass spectrometry to verify the size of the conjugate in the peak. Depending on the PEG used in the conjugation and the size of the conjugate in the peak, the amount of antibody or variant thereof conjugated to PEG can be assessed. Fragments conjugated to PEG groups often do not remain on the HPLC column after injection and are lost from the chromatogram. This loss from the chromatogram is an indication of PEGylation on the particular fragment that should contain at least one amino acid residue that can be PEGylated. The PEGylated anti-Tie Fab can be further assayed for its ability to interact with Tie2 and other biological activities using methods known in the art.

[0386] PEGylation alters the physical and chemical properties of antibody drugs and can result in improved pharmacokinetic behavior such as improved stability, reduced immunogenicity, prolonged circulation time, and increased intraocular retention time.

[0387] In some embodiments, the conjugates of the present disclosure have an increased half-life after administration into the eye of a mammal (e.g., a human) via a single intravitreal injection as compared to the corresponding unconjugated anti-Tie2 Fab. In some embodiments, the increase in half-life is at least 1.4-fold, or at least 1.8-fold or at least 2-fold of the corresponding unconjugated anti-Tie2 Fab half-life.

[0388] 3. IgM multimers as conjugates

[0389] In some embodiments, the Tie2 binding agents of the present disclosure can be made by linking more than one of the anti-Tie2 antibodies described herein to the C H1 domain of an IgM molecule, for example, using recombinant expression methods to fuse the C-terminus of the IgM C H1 domain to the N-terminus of an anti-Tie2 antibody as described herein (via a peptide bond). IgM has been demonstrated to be a viable format for antibody therapeutics (see, e.g., Hanala, 2012, MAbs, 4:555-561). The“monomeric” component of IgM is composed of two light chains (LC) each containing two Ig domains and two heavy chains (HC) containing five Ig domains and a short, truncated C-terminal tail. These four chains make up a heterodimer that forms a HC-LC homodimer. In turn, these homodimers covalently associate into a cyclic structure containing either five homodimers and a J chain (JC) (penta mer) or six homodimers (hexamer), which contain ten and twelve binding sites, respectively. Without being bound by theory, it is possible that the close association of multiple variable fragments (Fv) enables IgM to bind targets without the need for affinity maturation and thus can serve as a sentry adaptive immune receptor.

[0390] In particular embodiments, the anti-Tie2 antibody is a Fab. The IgM protein (multimer) can or can not include a J chain, such that in the presence of a J chain a pentamer can be formed (and can include up to 5 anti-Tie2 antibodies), and in the absence of a J chain a hexamer is formed (and can include up to 5 anti-Tie2 antibodies). In some embodiments, hexamers are produced by altering the ratio of IgM heavy chains or light chains (to form hexamers) or heavy chains to light chains to J chains (to form pentamers). Thus, in some embodiments, the Tie2 binding agent is a multimer that includes an IgM protein and at least 2, at least 3, at least 4, at least 5, or at least 6 anti-Tie2 antibodies described herein to form a multimer that is capable of activating Tie2. Anti-Tie2 IgM molecules are designed and shown to activate Tie2 activity (see, Example 7).

[0391] Anti-Tie2 multimer conjugates constructed using recombinant IgM formats as described herein can be useful for ocular therapeutics due to their relatively large molecular radius that potentially results in slower diffusion of the molecule out of the vitreous humor into the intraocular fluid and into the blood relative to a single Fab. As described below in Example 7, the hydrodynamic radius (R h ) was found to be approximately 12 nM, and the predicted molecular weight of the hexamer (~1050 kD) is slightly greater than that of the pentamer (~950 kD)

[0392] The systemic half-life of the recombinant IgM molecules was also explored, as it can be desirable to have rapid systemic clearance to limit the activity of the ocular therapeutic to the eye. As described in Example 7, the recombinantly expressed IgM pentamers and hexamers were cleared more rapidly than IgM isolated from human serum following intravenous injection. Further determination that the percentage of sialic acid relative to N-linked glycans was lower for these recombinant IgM molecules than for IgM isolated from serum suggests that the rate of clearance of the recombinant anti-Tie2 IgM molecules can be controlled by designing the expression system to modify the level of sialic acid in N-linked glycosylation.

[0393] It has previously been reported that IgM effectively recruits Clq and induces target cell killing via complement-dependent cytotoxicity (CDC). This activity can be undesirable for an ocular therapeutic. Accordingly, as described in Example 7, an IgM variant P434G (EU numbering) was designed and shown to have removed all detectable complement activity.

[0394] 3. Hexameric peptide multimers as conjugates

[0395] The present disclosure also encompasses conjugates comprising a plurality of antigen binding agents (e.g., antibodies or antigen binding fragments thereof), each of which is linked to a peptide that naturally forms a multimer, e.g., an NDK peptide. In some embodiments, a Tie2 binding agent of the present disclosure can be made by linking more than one anti-Tie2 Fab described herein to a peptide multimer. The peptide multimer comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 peptides that, when expressed in a recombinant expression system, naturally fold to form a single multi-armed structure. Conventional molecular engineering techniques and materials are used to express the peptides, which will form multimers as fusions, with the antigen binding protein (e.g., antibody or fragment thereof) expressed at the N- or C-terminus of the peptide that forms the multimer. In particular embodiments, the peptides in the multimer are identical, and the expression vector is constructed to link the C- or N-terminus of the peptide to the anti-Tie2 antibody or fragment thereof (e.g., via a peptide bond), respectively, as taught by the present disclosure.

[0396] In particular embodiments, the peptide of the multimeric peptide is a portion of a eukaryotic nucleoside diphosphate kinase (NDK) enzyme having a homohexameric quaternary structure. There are several NDK enzymes that can be used to design multimers, including NDK1 (e.g., SEQ ID NO: 69), NDK2 (e.g., SEQ ID NO: 70), NDK3 (e.g., SEQ ID NO: 71), NDK4 (e.g., SEQ ID NO: 72), NDK5 (e.g., SEQ ID NO: 73). In preferred embodiments, the NDK is an NDK3 peptide derived from, e.g., UniProt accession number P22887. The sequence of Tie2.1.M100cF with a Tie2.1.M100cF linked at its C-terminus to an NDK3 N-terminus is provided in SEQ ID NO: 74. In this preferred embodiment, the Fab light chain comprises SEQ ID NO: 21. (See, e.g., Example 8 below.)

[0397] E. Methods and compositions for diagnosis and detection

[0398] In certain embodiments, any of the anti-Tie2 antibodies provided herein can be used to detect the presence of Tie2 in a biological sample. The term "detecting" as used herein encompasses quantitative or qualitative detection. In certain embodiments, the biological sample comprises cells or tissue, e.g., retinal tissue (photoreceptors and underlying retinal pigment epithelial cells (RPE) and choroidal capillaries).

[0399] In one embodiment, an anti-Tie2 antibody for use in a diagnostic or detection method is provided. In another aspect, a method of detecting the presence or absence of Tie2 in a biological sample is provided. In certain embodiments, the method comprises contacting a biological sample with an anti-Tie2 antibody described herein under conditions that allow binding of the anti-Tie2 antibody to Tie2, and detecting whether a complex is formed between the anti-Tie2 antibody and Tie2. Such a method can be an in vitro or in vivo method. In one embodiment, an anti-Tie2 antibody is used to select a subject suitable for treatment with an anti-Tie2 antibody, e.g., Tie2 is a biomarker for selecting a patient.

[0400] In certain aspects, labeled anti-Tie2 antibodies are provided. Labels include, but are not limited to, labels or moieties that are directly detectable (e.g., fluorescent, chromogenic, electron-dense, chemiluminescent, and radioactive labels), as well as moieties that are indirectly detectable (e.g., via an enzymatic reaction or molecular interaction), such as enzymes or ligands. Exemplary labels include, but are not limited to: radioisotopes 32 P、 14 C、 125 I、 3 H and 131 I; fluorophores such as rare earth chelates or fluoresceins and their derivatives; rhodamines and their derivatives; dansyl; umbelliferone; luciferases such as firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456); luciferin; 2,3-dihydrophthalazinediones; horseradish peroxidase (HRP); alkaline phosphatase; beta-galactosidase; glucoamylase; lysozyme; saccharide oxidizing enzymes, such as glucose oxidase, galactose oxidase, and glucose 6-phosphate dehydrogenase; heterocyclic oxidizing enzymes, such as uricase and xanthine oxidase, used in combination with an oxidizing dye precursor, such as HRP, lactoperoxidase, or microperoxidase; biotin / avidin; a spin label; a phage label; stable free radicals; and the like.

[0401] F. Pharmaceutical Formulations

[0402] Pharmaceutical formulations of anti-Tie2 antibodies or Tie2 conjugates as described herein are prepared by mixing a magnetic anti-Tie2 antibody or Tie2 conjugate having the desired purity with one or more optional pharmaceutical carriers (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980)) in the form of a lyophilized formulation or an aqueous solution. Pharmaceutical carriers are generally non-toxic to recipients at the dosages and concentrations employed and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl alcohol, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol, and m-cresol); low molecular weight (less than about 10 residues) polypeptides; Proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrin; chelating agents (such as EDTA); sugars, such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions, such as sodium; metal complexes (such as zinc protein complexes); and / or non-ionic surfactants, such as polyethylene glycol (PEG). Exemplary pharmaceutical carriers in the present application further include interstitial drug dispersants, such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), such as human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 ( Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use (including rHuPH20) are described in U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinase.

[0403] Exemplary lyophilized antibody formulations are described in US Patent No. 6,267,958. Aqueous antibody formulations include those described in US Patent No. 6,171,586 and WO 2006 / 044908, the latter formulations including a histidine-acetate buffer.

[0404] The formulations described herein can also comprise more than one active ingredient suitable for the particular indication being treated, preferably those with complementary activities. For example, it can be desirable to further provide a second biomolecule selected from the group consisting of: IL-6; IL-6R; IL-13; IL-13R; PDGF; Angiopoietin; Ang2; Tie2; S1P; Integrins αvβ3, αvβ5 and α5β1; Betacellulin; Apelin / APJ; Erythropoietin; Complement Factor D; TNFα; HtrA1; VEGF receptor; ST-2 receptor; and proteins genetically associated with AMD risk, such as complement pathway components C2, Factor B, Factor H, CFHR3, C3b, C5, C5a and C3a; HtrA1; ARMS2; TIMP3; HLA; Interleukin-8 (IL-8); CX3CR1; TLR3; TLR4; CETP; LIPC; COL10A1; and TNFRSF10A. Alternatively or additionally, the second biomolecule is an antibody or fragment thereof that specifically binds to a sub selected from the group consisting of: IL-6; IL-6R; IL-13; IL-13R; PDGF; Angiopoietin; Ang2; Tie2; S1P; Integrins αvβ3, αvβ5 and α5β1; Betacellulin; Apelin / APJ; Erythropoietin; Complement Factor D; TNFα; HtrA1; VEGF receptor; ST-2 receptor; and proteins genetically associated with AMD risk, such as complement pathway components C2, Factor B, Factor H, CFHR3, C3b, C5, C5a and C3a; HtrA1; ARMS2; TIMP3; HLA; Interleukin-8 (IL-8); CX3CR1; TLR3; TLR4; CETP; LIPC; COL10A1; and TNFRSF10A. In certain embodiments, the additional compound is an antibody, or antigen-binding fragment thereof, that binds to VEGF and / or Ang2 and / or IL-1 β. Such active ingredients suitably are present in combination in amounts that are effective for the purpose intended.

[0405] The active ingredients can be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano- particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0406] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

[0407] Formulations to be used for in vivo administration are typically sterile. Sterility can be readily achieved, e.g., by filtration through a sterile filtration membrane.

[0408] The conjugates described herein for the prevention or treatment of an ocular disease or disorder are typically administered by ocular, intraocular, and / or intravitreal injection, and / or retrobulbar injection, and / or subtenon injection, and / or suprachoroidal injection, and / or topical administration as eye drops and / or ointment. Such compositions of the disclosure can be delivered by a variety of methods, e.g., intravitreal delivery as a device and / or depot that allows slow release of the compound into the vitreous, including those described in reference texts such as Intraocular Drug Delivery, Jaffe, Jaffe, Ashton, and Pearson, editors, Taylor & Francis (March 2006). In one example, the device can be a micro-pump and / or a matrix and / or a passive diffusion system and / or encapsulated units that release the compound over an extended period of time (Intraocular Drug Delivery, Jaffe, Jaffe, Ashton, and Pearson, editors, Taylor & Francis (March 2006). Other methods of administration can also be used, including, but not limited to, topical, parenteral, subcutaneous, intraperitoneal, intrapulmonary, intranasal, and intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.

[0409] Formulations for ocular, intraocular, or intravitreal administration can be prepared by methods known in the art and using ingredients known in the art. The primary requirement for effective treatment is proper penetration through the eye. Different ocular anterior segment diseases for which drugs can be delivered topically require more site-specific approaches. Eye drops and ointments penetrate very little into the posterior segment of the eye, and the blood- eye barrier impedes penetration of systemically administered drugs into ocular tissues. Thus, the method of choice for drug delivery to treat retinal diseases such as DME and AMD is typically direct intravitreal injection. Intravitreal injections are typically repeated at intervals that depend on the patient’s condition and the properties and half-life of the delivered drug. For intraocular (e.g., intravitreal) penetration, smaller size molecules are generally preferred.

[0410] In some embodiments, the antibodies and conjugates described herein can be formulated for delivery using an implantable port delivery system (PDS). As previously described, a PDS is a refillable device in which release into the vitreous is controlled by a porous metal membrane containing titanium frit. In some embodiments, because of the small volume of the reservoir, effective delivery using a PDS requires a high concentration of protein. Accordingly, in some embodiments, the antibodies and conjugates described herein are formulated at a high concentration. In some embodiments, the antibodies and conjugates described herein are formulated at a concentration of at least 150 mg / ml, at least 160 mg / ml, at least 170 mg / ml, at least 180 mg / ml, at least 190 mg / ml, at least 200 mg / ml, or at least 210 mg / ml, or at least 220 mg / ml, or at least 230 mg / ml, or at least 240 mg / ml, or at least 250 mg / ml, or at least 260 mg / ml, or at least 270 mg / ml, or at least 280 mg / ml, or at least 290 mg / ml, or at least 300 mg / ml. In some embodiments, the antibodies and conjugates described herein are formulated at a concentration between 150 mg / ml and 350 mg / ml, between 150 mg / ml and 300 mg / ml, between 170 mg / ml and 300 mg / ml, between 200 mg / ml and 300 mg / ml, or between 170 mg / ml and 220 mg / ml.

[0411] G. Methods of Treatment and Compositions

[0412] Any of the anti-Tie2 antibodies or conjugates provided herein can be used in a method of treatment. An "individual," "patient," or "subject" according to any of the embodiments herein can be a human.

[0413] The anti-Tie2 conjugates of the present disclosure can be used to treat a mammal. In some embodiments, for example, the anti-Tie2 conjugates are administered to a non-human mammal for the purpose of obtaining preclinical data. Exemplary non-human mammals to be treated include non-human primates, dogs, cats, pigs, rodents, and other mammals on which preclinical studies are performed. Such mammals can be animal models established in connection with the disease to be treated using the antibody, or can be used to study the toxicity of the antibody of interest. In each of these embodiments, dose escalation studies can be performed on the mammal.

[0414] The anti-Tie2 conjugates can be administered by any suitable means, including parenterally, subcutaneously, intraperitoneally, intravitreally, intrapulmonarily, and intranasally, and, if needed for local immunosuppressive treatment, intralesionally. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, intravitreal, and subcutaneous administration. In addition, the conjugates are suitable for administration by pulsatile infusion, especially for decreasing doses of the antibody or antibody variant or fragment thereof (e.g., antigen-binding fragment) thereof. In some embodiments, administration can be by injection, e.g., intravenous or subcutaneous injection, depending in part on whether short-term or long-term administration.

[0415] For the prevention or treatment of disease, the appropriate dosage of an anti-Tie2 antibody or conjugate will depend on the type of disease to be treated, the severity and course of the disease, and the stage of the disease, the patient's clinical history and response to the antibody, and the judgment of the treating physician.

[0416] Depending on the type and severity of the disease, about 1-25 mg / eye (0.015 mg / kg - 0.36 mg / kg per eye) of the antibody can be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations or by continuous infusion. For repeated administrations of doses 2 or more times a day, the

[0417] In one aspect, an anti-Tie2 antibody or conjugate is provided for use as a medicament. In a further aspect, an anti-Tie2 antibody or conjugate is provided for use in the treatment of an ocular disease or condition. In certain embodiments, an anti-Tie2 antibody or conjugate is provided for use in a method of treatment. In certain embodiments, the present application provides an anti-Tie2 antibody for use in a method of treating an individual having an ocular disease or condition, the method comprising administering to the individual an effective amount of an anti-Tie2 antibody or conjugate. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent (e.g., described below). In a further embodiment, the present application provides an anti-Tie2 antibody or conjugate for use in increasing vascular endothelial cell membrane integrity and / or decreasing vascular leakage. In certain embodiments, the present application provides an anti-Tie2 antibody or conjugate for use in a method of increasing vascular endothelial cell membrane integrity and / or decreasing vascular leakage in an individual, the method comprising administering to the individual an effective anti-Tie2 antibody or conjugate to increase vascular endothelial cell membrane integrity and / or decrease vascular leakage. The "individual" according to any of the above embodiments is preferably a human.

[0418] As used herein, the term "ocular disease" includes any ocular disease associated with pathological angiogenesis and / or atrophy (also referred to herein interchangeably as "ocular disease"). An ocular disease can be characterized by altered or unregulated expansion of new blood vessels and / or invasion into structures of the eye, such as the retina or cornea. An ocular disease can be characterized by atrophy of retinal tissue (photoreceptors and underlying retinal pigment epithelial cells (RPE) and choriocapillaris).

[0419] Diabetic macular edema (DME) is caused by a complication of diabetes known as diabetic retinopathy (DR). This eye condition can occur in people who are diagnosed with either type 1 or type 2 diabetes. DME is defined as a thickening of the macular retina of 2 disc diameters or less, and can be localized or diffuse. DME is associated with alterations in retinal microvessels with disruption of the blood-retinal barrier, causing leakage of plasma components into the surrounding retina, leading to retinal edema.

[0420] Non-limiting ocular diseases include, for example, diabetic macular edema (DME) (e.g., focal non-center DME and diffuse DME involving the fovea), diabetic retinopathy (DR) (e.g., proliferative DR (PDR), non-proliferative DR (NPDR), and high-altitude DR), non-edemic retinopathy, other ischemia-related retinopathy, AMD (e.g., wet AMD, dry AMD, intermediate AMD, advanced AMD, and geographic atrophy (GA)), macular degeneration, macular edema, retinopathy, ROP, retinal vein occlusion (RVO) (e.g., central (CRVO) and branched (BRVO) forms), CNV (e.g., myopic CNV), corneal neovascularization, diseases associated with corneal neovascularization, retinal neovascularization, diseases associated with retinal / choroidal neovascularization, central serous retinopathy (CSR), pathologic myopia, von Hippel-Lindau disease, ocular histoplasmosis, FEVR, Coats' disease, Norrie's disease, retinal abnormalities associated with osteoporosis-pseudoglioma syndrome (OPPG), subconjunctival hemorrhage, ocular rubeosis, ocular neovascular disease, neovascular glaucoma, retinitis pigmentosa (RP), hypertensive retinopathy, retinal angiomatous proliferation, macular telangiectasia, iridal neovascularization, intraocular neovascularization, retinal degeneration, cystoid macular edema (CME), vasculitis, papilloedema, retinitis (including but not limited to CMV retinitis), ocular melanoma, retinoblastoma, conjunctivitis (e.g., infectious conjunctivitis and non-infectious (e.g., allergic) conjunctivitis), Leber congenital amaurosis (also known as Leber's congenital amaurosis or LCA), uveitis (including infectious and non-infectious uveitis), choroiditis (e.g., multifocal choroiditis), ocular histoplasmosis, blepharitis, dry eye, traumatic eye injury, von Hippel-Lindau disease and other ophthalmic diseases in which the disease or disease is associated with ocular neovascularization, vascular leakage, and / or retinal edema or retinal atrophy. Additional exemplary ocular diseases include retinoschisis (abnormal splitting of the retinal neurosensory layer), diseases associated with rubeosis (neovascularization of the angle of the eyelid), and diseases caused by abnormal proliferation of fibrovascular or fibrous tissue (including all forms of proliferative vitreoretinopathy).

[0421] Exemplary diseases associated with corneal neovascularization include, but are not limited to, epidemic keratoconjunctivitis, vitamin A deficiency, contact lens overwear, atopic keratitis, superior limbic keratitis, terygium keratitis sicca, sjogrens, acne, phylectenulosis, syphilis, mycobacterial infections, lipid degeneration, chemical burns, bacterial ulcers, fungal ulcers, herpes simplex infections, herpes zoster infections, protozoan infections, Kaposi's sarcoma, mooren ulcers, terrien's marginal degeneration, marginal keratolysis, rheumatoid arthritis, systemic lupus erythematosis, polyarteritis, trauma, Wegener's granulomatosis, scleritis, Steven's Johnson disease, pemphigoid, radial keratotomy, and corneal graph rejection.

[0422] Exemplary diseases associated with choroidal neovascularization and defects in the retinal vascul system, including increased vascular leakage, aneurysms, and capillary droplets, include, but are not limited to, diabetic retinopathy, macular degeneration, sickle cell anemia, sarcoid, syphilis, pseudoxanthoma elasticum, Paget's disease, vein occlusion, artery occlusion, carotid obstructive disease, chronic uveitis / vitritis, mycobacterial infections, Lyme's disease, systemic lupus erythematosus, retinopathy of prematurity, retinal edema (including macular edema), Eales' disease, Behcet's disease, infections causing retinitis or choroiditis, presumed ocular histoplasmosis, Best's disease (vitelliform macular degeneration), myopia, optic pits, pars planitis, retinal detachments (e.g., chronic retinal

[0423] Exemplary diseases associated with retinal tissue (photoreceptors and underlying RPE) include, but are not limited to, atrophic or non-exudative AMD (e.g., geographic atrophy or advanced dry AMD), macular atrophy (e.g., atrophy associated with neovascularization and / or geographic atrophy), diabetic retinopathy, Stargardt's disease, Skorsby's chorioretinal atrophy, retinoschisis, and retinitis pigmentosa.

[0424] For example, in certain embodiments, any of the aforementioned methods further comprises administering one or more additional compounds. In certain embodiments, the Tie2 binder or conjugate, or polymeric formulation thereof, is administered concurrently with the additional compound. In certain embodiments, the Tie2 binder or conjugate, or polymeric formulation thereof, is administered before or after the additional compound. In certain embodiments, the additional compound binds to a second biomolecule selected from the group consisting of IL-1β; IL-6; IL-6R; IL-13; IL-13R; PDGF; angiopoietin; Ang2; Tie2; S1P; integrins αvβ3, αvβ5, and α5β1; betacellulin; apelin / APJ; erythropoietin; complement factor D; TNFα; HtrA1; VEGF receptor; ST-2 receptor; and proteins genetically associated with AMD risk, such as complement pathway components C2, factor B, factor H, CFHR3, C3b, C5, C5a, and C3a; HtrA1; ARMS2; TIMP3; HLA; interleukin-8 (IL-8); CX3CR1; TLR3; TLR4; CETP; LIPC; COL10A1; and TNFRSF10A. In certain embodiments, the additional compound is an antibody or antigen-binding fragment thereof. In certain embodiments according to (or as applied to) any of the above embodiments, the eye disease is an intraocular neovascular disease selected from the group consisting of: proliferative retinopathy, choroidal neovascularization (CNV), age-related macular degeneration (AMD), diabetic and other ischemic retinopathy, diabetic macular edema, pathological myopia, Hippel-Lindau disease, ocular histoplasmosis, retinal vein occlusion (RVO) (including CRVO and BRVO), corneal neovascularization, retinal neovascularization and retinopathy of prematurity (ROP). For example, in some cases, the additional compound is a bispecific antibody (e.g., an anti-VEGF / anti-Ang2 bispecific antibody, such as RG-7716 or any of the bispecific anti-VEGF / anti-Ang2 bispecific antibodies disclosed in WO 2010 / 069532 or WO 2016 / 073157, or variants thereof. In another example, in some cases, the additional compound is an anti-IL-6 antibody, such as EBI-031 (eleven biotherapeutic agents, see, e.g., WO 2016 / 073890), siltuximab ), olokizumab, clazakizumab, sirukumab, elsilimomab, OPR-003, MEDI5117, PF-04236921, or a variant thereof. In still further examples, in some cases the additional compound is an anti-IL-6R antibody, e.g., tocilizumab (Actemra®) (see, e.g., WO 1992 / 019579), sarilumab, ALX-0061, SA237, or a variant thereof.

[0425] In some cases, the Tie2 binding agent or conjugate of the present disclosure and / or a polymeric formulation thereof can be administered in combination with at least one additional therapeutic agent for the treatment of an ocular disease, e.g., an ocular disease described herein (e.g., DME, DR, AMD (e.g., wet AMD), RVO, or GA). Exemplary additional therapeutic agents for use in combination therapy for the treatment of an ocular disease include, without limitation, anti-angiogenic agents, e.g., VEGF antagonists, including, e.g., anti-VEGF antibodies (e.g., anti-VEGF Fabs (ranibizumab), soluble receptor fusion proteins (e.g., recombinant soluble receptor fusion proteins (aflibercept, also known as VEGF Trap Eye; Regeneron / Aventis), aptamers (e.g., anti-VEGF pegylated aptamers (pegaptanib sodium; NeXstar Pharmaceuticals / OSI Pharmaceuticals)), and VEGFR tyrosine kinase inhibitors (e.g., 4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(1-methylpiperidin-4-ylmethoxy)quinazoline (ZD6474), 4-(4-fluoro-2-methylindol-5-yloxy)-6-methoxy-7-(3-pyrrolidin-1-ylpropoxy)quinazoline (AZD2171), vatalanib (PTK787), semaxaminib (SU5416; SUGEN), and (sunitinib); tryptophanyl-tRNA synthetase (TrpRS); squalamine; (anecortave for depot suspensions; Alcon, Inc.); Combretastatin A4 prodrug (CA4P); (Levonorgestrel-ru486); triamcinolone; intravitreal crystalline triamcinolone; matrix metalloproteinase inhibitors (e.g., Prinomastat (AG3340; Pfizer)); fluocinolone (including fluocinolone intraocular implant; Bausch & Lomb / Controlled Delivery System); linomide; inhibitors of integrin beta 3 function; angiostatin; and combinations thereof. Such and other therapeutic agents that can be administered in combination with the Tie2 binding agents or conjugates of the present application are described in U.S. Patent Application No. US2014 / 0017244, which is incorporated by reference herein in its entirety.

[0426] Further examples of additional therapeutic agents that can be administered in combination with the Tie2 binding agents or conjugates and / or polymeric formulations thereof to treat ocular diseases (e.g., DME, DR, AMD, RVO, or GA) include, but are not limited to, (verteporfin; a light-activated drug that is typically used in conjunction with photodynamic therapy using a non-thermal laser), PKC412, Endovion (NS 3728; NeuroSearch A / S), neurotrophic factors (e.g., glial cell-derived neurotrophic factor (GDNF) and ciliary neurotrophic factor (CNTF)), diltiazem, dorzolamide, 9-cis retinal, ophthalmic drugs (e.g., phospholine iodide, echothiophate, or carbonic anhydrase inhibitors), veovastat (AE-941; AEterna Laboratories, Inc.), Sirna-027 (AGF-745; Sima Therapeutics, Inc.), neurotrophins (including, by way of example only, NT-4 / 5, Genentech), Cand5 (Acuity Pharmaceuticals), INS-37217 (Inspire Pharmaceuticals), integrin antagonists (including those from Jerini AG and Abbott Laboratories), EG-3306 (Ark Therapeutics Ltd.), BDM-E (BioDiem Ltd.), thalidomide (e.g., as used by EntreMed, Inc.), cardiotrophin-1 (Genentech), 2-methoxyestradiol (Allergan / Oculex), DL-8234 (Toray Industries), NTC-200 (Neurotech), tetrathiomolybdate (University of Michigan), LYN-002 (Lynkeus Biotech), microalgae compounds (Aquasearch / Albany, Mera Pharmaceuticals), D-9120 (Celltech Group plc), ATX-S10 (Hamamatsu Photonics), TGF-β2 (Genzyme / Celtrix), tyrosine kinase inhibitors (e.g., those from Allergan, SUGEN, or Pfizer), NX-278-L (NeXstar Pharmaceuticals / Gilead Sciences), Opt-24 (OPTIS France SA), retinal cell ganglion neuroprotectants (Cogent Neurosciences), N-nitropyrazole derivatives (Texas A&M University System), KP-102 (Krenitsky Pharmaceuticals), cyclosporin A, therapeutic agents used in photodynamic therapy (e.g., Receptor-targeted PDT, Bristol-Myers Squibb, Co.; porfimer sodium for injection with PDT; verteporfin, QLT Inc.; rostaporfin for use with PDT, Miravent Medical Technologies; talaporfin sodium for use with PDT, Nippon Petroleum; and motexafin lutetium, Pharmacyclics, Inc.), antisense oligonucleotides (including, for example, products tested by Novagali Pharma SA and ISIS-13650, Ionis Pharmaceuticals) and combinations thereof.

[0427] The Tie2 binding agent or conjugate and / or polymeric formulation thereof can be administered in conjunction with a therapeutic or surgical procedure for the treatment of an ocular disease (e.g., DME, DR, AMD, RVO, or GA), including, for example, laser photocoagulation (e.g., panretinal photocoagulation (PRP)), endo-photocoagulation, macular hole surgery, macular translocation surgery, implantable mini-telescope, PHI-movement angiography (also known as micro laser therapy and feeder vessel treatment), photonic beam therapy, microstimulation therapy, retinal detachment and vitreous surgery, scleral buckle, submacular surgery, transpupillary thermotherapy, photosystem I therapy, use of RNA interference (RNAi), extracorporeal rheology procedure (also known as membrane filtration and rheology therapy), microchip implantation, stem cell therapy, gene replacement therapy, ribozyme gene therapy (including gene therapy for hypoxia response element, Oxford Biomedica; Lentipak, Genetix; and PDEF gene therapy, GenVec), photoreceptor / retinal cell transplantation (including transplantable retinal epithelial cells, Diacrin, Inc.; retinal cell grafts, e.g., Astellas Pharma US, Inc., ReNeuron, CHA Biotech), acupuncture, and combinations thereof.

[0428] In some cases, the Tie2 binding agents or conjugates of the application and / or polymeric formulations thereof can be administered in combination with an anti-angiogenic agent for the treatment of ocular diseases (e.g., DME, DR, AMD, RVO, or GA). Any suitable anti-angiogenic agent can be used in combination with the Tie2 binding agents or conjugates of the application, including but not limited to, those listed in Carmeliet et al. Nature 407:249-257, 2000. In some embodiments, the anti-angiogenic agent is a VEGF antagonist, including but not limited to, an anti-VEGF antibody (e.g., anti-VEGF Fab (Ranibizumab), RTH-258 (formerly ESBA-1008, an anti-VEGF single-chain antibody fragment; Novartis), or a bispecific anti-VEGF antibody (e.g., an anti-VEGF / anti-Ang2 bispecific antibody such as RG-7716; Roche), a soluble recombinant receptor fusion protein (e.g., VEGF Trap; aflibercept), a VEGF variant, a soluble VEGFR fragment, an aptamer capable of blocking VEGF (e.g., pegaptanib), or a VEGFR, a neutralizing anti-VEGFR antibody, a small molecule inhibitor of VEGFR tyrosine kinase, an anti-VEGF (antibody fragment; Novartis), or a bispecific anti-VEGF antibody (e.g., an anti-VEGF / anti-Ang2 bispecific antibody such as RG-7716; Roche), a soluble recombinant receptor fusion protein (e.g., VEGF Trap; aflibercept), a VEGF variant, a soluble VEGFR fragment, an aptamer capable of blocking VEGF (e.g., pegaptanib), or a VEGFR, a neutralizing anti-VEGFR antibody, a small molecule inhibitor of VEGFR tyrosine kinase, an anti-VEGF (e.g., abicipar pegol, Molecular Partners AG / Allergan), a small interfering RNA that inhibits the expression of VEGF or VEGFR, a VEGFR tyrosine kinase inhibitor (e.g., 4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(1-methylpiperidin-4-ylmethoxy)quinazoline (ZD6474), 4-(4-fluoro-2-methylindol-5-yloxy)-6-methoxy-7-(3-pyrrolidin-1-ylpropoxy)quinazoline (AZD2171), vatalanib (PTK787), semaxanib (SU5416; SUGEN), and In some cases, the anti-Tie2 antibody or fragment thereof can be used in combination with an antibody or fragment thereof or other therapeutic agent targeting a second biological molecule, including but not limited to IL-1 beta; IL-6; IL-6R; PDGF (e.g., PDGF-BB); angiopoietin; angiopoietin 2; Tie2; S1P; integrin alpha v beta 3, alpha v beta 5, and alpha 5 beta 1; betacellulin; apelin / APJ; erythropoietin; complement factor D; TNF alpha; HtrAl; VEGF receptors (e.g., VEGFR1, VEGFR2, VEGFR3, mbVEGFR, or sVEGFR); ST-2 receptor; and proteins genetically linked to the risk of age-related macular degeneration (AMD), such as complement pathway components C2, Factor B, Factor H, CFHR3, C3b, C5, C5a, and C3a; HtrAl; ARMS2; TIMP3; HLA; IL-8; CX3CR1; TLR3; TLR4; CETP; LIPC; COL10A1; and TNFRSF10A. For example, in some cases, the additional compound is a bispecific antibody (e.g., an anti-VEGF / anti-Ang2 bispecific antibody, such as RG-7716 or any of the anti-VEGF / anti-Ang2 bispecific antibodies or variants thereof disclosed in WO 2010 / 069532 or WO 2016 / 073157).

[0429] Other suitable anti-angiogenic agents that can be administered in combination with an anti-Tie2 conjugate and / or polymeric formulation thereof for the treatment of an ocular disease (e.g., DME, DR, AMD, RVO, or GA) include corticosteroids, angiostatic steroids, anecortave acetate, vasculostatin, endostatin, tyrosine kinase inhibitors, matrix metalloproteinase (MMP) inhibitors, insulin-like growth factor binding protein 3 (IGFBP3), stromal-derived factor (SDF-1) antagonists (e.g., anti-SDF-1 antibodies), pigment epithelium-derived factor (PEDF), insulin-like growth factor 1, integrin antagonists, hypoxia-inducible factor (HIF)-1 alpha antagonists, protein kinase CK2 antagonists, agents that inhibit stem cells (e.g., endothelial progenitor cells) at sites of neovascularization (e.g., anti-vascular endothelial cadherin (CD-144) antibodies and / or anti-SDF-1 antibodies), and combinations thereof.

[0430] In further examples, in some cases, a Tie2-binding conjugate and / or polymeric formulation thereof can be administered in combination with an agent having anti-neovascularization activity for the treatment of an ocular disease (e.g., DME, DR, AMD, RVO, or GA), such as an anti-inflammatory agent, a mammalian target of rapamycin (mTOR) inhibitor (e.g., rapamycin, (everolimus) and (temsirolimus), cyclosporine, a tumor necrosis factor (TNF) antagonist (e.g., an anti-TNFa antibody or antigen-binding fragment thereof (e.g., infliximab, adalimumab, certolizumab pegol, and golimumab) or a soluble receptor fusion protein (e.g., etanercept)), an anti-complement agent, a non-steroidal anti-inflammatory agent (NSAID), or a combination thereof.

[0431] In still further examples, in some cases, the Tie2-binding conjugate and / or polymeric formulation thereof can be administered in combination with a neuroprotective drug that is effective in reducing the progression of dry AMD to wet AMD, such as a class of drugs known as “neurosteroids,” which include drugs such as dehydroepiandrosterone (DHEA) (brand name: PRASTERA TM and ), dehydroepiandrosterone sulfate, and pregnenolone sulfate.

[0432] Any suitable AMD therapeutic agent can be administered as an additional therapeutic agent in combination with the Tie2-binding conjugate and / or polymeric formulation thereof of the present application to treat an ocular disease (e.g., DME, DR, AMD, RVO, or GA), including, but not limited to, a VEGF antagonist, such as an anti-VEGF antibody (e.g., (ranibizumab), RTH-258 (formerly ESBA-1008, an anti-VEGF single-chain antibody fragment; Novartis), or a bispecific anti-VEGF antibody (e.g., an anti-VEGF / anti-angiopoietin 2 bispecific antibody such as RG-7716; Roche), a soluble VEGF receptor fusion protein (e.g., (aflibercept), an anti-VEGF (e.g., abicipar pegol; Molecular Partners AG / Allergan), or an anti-VEGF aptamer (e.g., (pegaptanib sodium); a platelet-derived growth factor (PDGF) antagonist, such as an anti-PDGFR antibody (e.g., REGN2176-3), an anti-PDGF-BB pegylated aptamer (e.g., Ophthotech / Novartis), soluble PDGFR receptor fusion proteins or dual PDGF / VEGF antagonists (e.g., small molecule inhibitors (e.g., DE-120 (Santen) or X-82 (TyrogeneX)) or bispecific anti-PDGF / anti-VEGF antibodies); in combination with photodynamic therapy (verteporfin); antioxidants; complement system antagonists, e.g., complement factor C5 antagonists (e.g., small molecule inhibitors (e.g., ARC-1905; Ophthotech) or anti-C5 antibodies (e.g., LFG-316; Novartis), properdin antagonists (e.g., anti-properdin antibodies, e.g., CLG-561; Alcon) or complement factor D antagonists (e.g., anti-complement factor D antibodies, e.g., lampalizumab; Roche); C3 binding peptides (e.g., APL-2, Appellis); visual cycle modulators (e.g., emixustat hydrochloride); squalamine (e.g., OHR-102; Ohr Pharmaceutical); vitamin and mineral supplements (e.g., those described in Age-Related Eye Disease Study 1 (AREDS1; zinc and / or antioxidants) and Study 2 (AREDS2; zinc, antioxidants, lutein, zeaxanthin and / or omega-3 fatty acids)); cell-based therapies, e.g., NT-501 (Renexus); PH-05206388 (Pfizer), huCNS-SC cell transplants (StemCells), CNTO-2476 (umbilical cord stem cell line; Janssen), OpRegen (suspension of RPE cells; Cell Cure Neurosciences) or MA09-hRPE cell transplants (Ocata Therapeutics); tissue factor antagonists (e.g., hI-conl; Iconic Therapeutics); alpha-adrenergic receptor agonists (e.g., brimonidine tartrate; Allergan); peptide vaccines (e.g., S-646240; Shionogi); beta-amyloid antagonists (e.g., anti-beta-amyloid monoclonal antibodies, e.g., GSK-933776); S1P antagonists (e.g., anti-S1P antibodies, e.g., iSONEP TM; a ROBO4 antagonist (e.g., an anti-ROBO4 antibody, e.g., DS-7080a; Daiichi Sankyo); a lentiviral vector expressing endostatin and angiostatin (e.g., RetinoStat); and combinations thereof. In some cases, an AMD therapeutic (including any of the foregoing AMD therapeutics) can be co-formulated. For example, the anti-PDGFR antibody REGN2176-3 can be co-formulated with aflibercept In some cases, such co-formulation can be administered in combination with a Tie2 binding agent or conjugate of the present application. In some cases, the ocular disease is DME and / or DR. In some cases, the ocular disease is AMD (e.g., wet AMD).

[0433] A Tie2 binding conjugate and / or a polymeric formulation thereof can be administered in combination with (ranibizumab) for the treatment of an ocular disease (e.g., DME, DR, AMD, RVO, or GA). In some cases, the ocular disease is DME and / or DR. In some cases, the ocular disease is AMD (e.g., wet AMD). In some cases, the ocular disease is GA.

[0434] A Tie2 binding conjugate and / or a polymeric formulation thereof can be administered in combination with (afibercept) for the treatment of an ocular disease (e.g., DME, DR, AMD, RVO, or GA). In some cases, the ocular disease is DME and / or DR. In some cases, the ocular disease is AMD (e.g., wet AMD). In some cases, the ocular disease is GA.

[0435] A Tie2 binding conjugate and / or a polymeric formulation thereof can be administered in combination with (pegaptanib sodium) for the treatment of an ocular disease (e.g., DME, DR, AMD, RVO, or GA). In some cases, the ocular disease is DME and / or DR. In some cases, the ocular disease is AMD (e.g., wet AMD). In some cases, the ocular disease is GA.

[0436] A Tie2 binding conjugate and / or a polymeric formulation thereof can be administered in combination with (verteporfin) and photodynamic therapy for the treatment of an ocular disease (e.g., DME, DR, AMD, RVO, or GA). In some cases, the ocular disease is DME and / or DR. In some cases, the ocular disease is AMD (e.g., wet AMD). In some cases, the ocular disease is GA.

[0437] The Tie2-binding conjugate and / or polymeric formulation thereof can be administered in combination with a PDGF antagonist for the treatment of an ocular disease (e.g., DME, DR, AMD, RVO, or GA). Exemplary PDGF antagonists that can be administered in combination with the Tie2-binding conjugate of the application include anti-PDGF antibodies, anti-PDGFR antibodies, small molecule inhibitors (e.g., squalamine), anti-PDGF-B pegylated aptamers such as (E10030; Ophthotech / Novartis) or dual PDGF / VEGF antagonists (e.g., small molecule inhibitors (e.g., DE-120 (Santen) or X-82 (TyrogeneX)) or bispecific anti-PDGF / anti-VEGF antibodies). For example, can be administered as an adjunct therapy to the Tie2-binding agent or conjugate of the application. OHR-102 can be administered in combination with a VEGF antagonist such as or In some embodiments, the Tie2-binding agent or conjugate of the application can be administered in combination with OHR-102, and / or In some cases, the ocular disease is DME and / or DR. In some cases, the ocular disease is AMD (e.g., wet AMD). In some cases, the ocular disease is GA.

[0438] The Tie2-binding conjugate and / or polymeric formulation thereof can be administered in combination with RTH-258 for the treatment of an ocular disease (e.g., DME, DR, AMD, RVO, or GA). For example, RTH-258 can be administered by intravitreal injection or ocular infusion. In some cases, the ocular disease is DME and / or DR. In some cases, the ocular disease is AMD (e.g., wet AMD). In some cases, the ocular disease is GA.

[0439] The Tie2-binding conjugate and / or polymeric formulation thereof can be administered in combination with abicipar pegol for the treatment of an ocular disease (e.g., DME, DR, AMD, RVO, or GA). In some cases, the ocular disease is DME and / or DR. In some cases, the ocular disease is AMD (e.g., wet AMD). In some cases, the ocular disease is GA.

[0440] The Tie2-binding conjugate and / or polymeric formulation thereof can be administered in combination with abicipar pegol for the treatment of an ocular disease (e.g., DME, DR, AMD, RVO, or GA). In some cases, the ocular disease is DME and / or DR. In some cases, the ocular disease is AMD (e.g., wet AMD). In some cases, the ocular disease is GA.

[0441] Any suitable DME and / or DR therapeutic agent can be administered in combination with the Tie2-binding conjugates and / or polymeric formulations thereof for the treatment of an ocular disease (e.g., DME, DR, DME, RVO, or GA), including but not limited to, VEGF antagonists (e.g., or ), corticosteroids (e.g., sebum-based malfunction implants (e.g., (dexamethasone intravitreal implant) or (fluocinolone acetonide intravitreal implant)) or a corticosteroid formulated for administration by intravitreal injection (e.g., fluocinolone acetonide intravitreal implant)) or a combination thereof. In some instances, the ocular disease is DME and / or DR.

[0442] Tie2 binding conjugates and / or polymeric formulations thereof can be used with (ranibizumab) for the treatment of DME and / or DR (eg, NPDR or PDR).

[0443] Tie2 binding conjugates and / or polymeric formulations thereof can be used with (aflibercept) for the treatment of DME and / or DR (e.g., NPDR or PDR).

[0444] Tie2 binding conjugates and / or polymeric formulations thereof can be used with (dexamethasone intravitreal implant) for the treatment of DME and / or DR.

[0445] Tie2 binding conjugates and / or polymeric formulations thereof can be used with (dexamethasone intravitreal implant) for the treatment of DME and / or DR.

[0446] In some cases, a TAO / PRN treatment regimen or a TAE treatment regimen can be used to administer an AMD therapeutic agent (e.g., ranibizumab or aflibercept) in combination with a Tie2-binding conjugate and / or a polymeric formulation thereof. In some cases, the eye disease is DME and / or DR. In some cases, the eye disease is AMD (e.g., wet AMD). In some cases, the eye disease is GA.

[0447] Such combination therapies contemplated above encompass combined administration (where two or more therapeutic agents are included in the same or separate pharmaceutical compositions), and separate administration, in which case the administration of a Tie2-binding conjugate of the present application can occur prior to, simultaneously with, and / or following the administration of the additional therapeutic agent or agents. In one embodiment, the administration of a Tie2-binding conjugate or polymeric formulation and the administration of the additional therapeutic agent occur within about 1, 2, 3, 4, or 5 months, or within about 1, 3, or 4 weeks, or within about 1, 2, 3, 4, 5, or 6 days of each other.

[0448] It is further contemplated that the Tie2-binding conjugates and / or polymeric formulations thereof are used to treat glaucoma. Glaucoma is a group of eye diseases characterized by progressive damage to the eye due, at least in part, to elevated intraocular pressure (IOP) (Merck Manual of Diagnosis and Therapy (1999)). Additionally, glaucoma is characterized by retinal ganglion cell (RGC) death, axonal loss, and increased prevalence of optic nerve head cupping (Alward, "Medical Management of Glaucoma," N Eng J Med, 1998; 339: 1298-1307). Glaucoma can be diagnosed before vision loss occurs by visual field testing and by ophthalmoscopic examination of the optic nerve for "cupping." The mean IOP in normal adults is 15 to 16 mm Hg; the normal range is 10 to 21 mm Hg. One form of glaucoma management is based on lowering IOP using typically applied pharmaceuticals ("Glaucoma", Lancet, 1999; 354: 1803-1810).

[0449] There are currently five major classes of drugs used to reduce IOP: beta-adrenergic antagonists, adrenergic agonists, parasympathomimetic agents, prostamide analogs, and carbonic anhydrase inhibitors. Although most drugs are applied externally to the eye, they can cause serious systemic side effects and negatively impact the patient's quality of life. If additional reduction of IOP is indicated or if the drugs fail to successfully reduce IOP, the next step is generally laser trabeculoplasty. If IOP is still not adequately controlled, incisional glaucoma surgery is indicated. Reduction of IOP, while significantly reducing the extent of neuronal loss, does not ensure a halt in disease progression, as RGCs can continue to be lost. Recent studies on the correlation between IOP regulation and visual field loss following medical or surgical intervention have shown that if IOP is lower, the continued neuronal loss reflected in visual field testing can be reduced. Glaucomatous optic neuropathy can result from specific pathophysiological changes and subsequent death of RGCs and their axons. The RGC death process is thought to be a two-stage process, with an initial insult causing damage, followed by a slow secondary degeneration that can be attributed to a hostile environment around the degenerating cell.

[0450] Another aspect of the application provides use of an anti-Tie2 conjugate of the application in the manufacture or preparation of a medicament. In one embodiment, the medicament is for treating an ocular disease (e.g., DME, DR, AMD, RVO, or GA). In a preferred embodiment, the medicament is for treating DME and / or DR. In a further embodiment, the medicament is for use in a method of treating an ocular disease (e.g., DME, DR, AMD, RVO, or GA), the method comprising administering to an individual having the ocular disease an effective amount of the medicament. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent (e.g., described below). In a further embodiment, the medicament is for use in a method of reducing vascular permeability, particularly in an eye of an individual, the method comprising administering to the individual an effective amount of the medicament to reduce vascular permeability, particularly in the eye. The "individual" according to any of the above embodiments can be a human.

[0451] In a further aspect, the application provides a method for treating an ocular disease (e.g., DME, DR, AMD, RVO, or GA). In one embodiment, the method comprises administering to an individual having such an ocular disease (e.g., DME, DR, AMD, RVO, or GA) an effective amount of a Tie2-binding conjugate of the application. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent (as described below). The "individual" according to any of the above embodiments can be a human.

[0452] In a further aspect, the present application provides methods for reducing vascular permeability in an individual, particularly in the eye. In one embodiment, the method comprises administering to the individual an effective amount of a Tie2 binding conjugate of the present application to reduce vascular permeability, particularly in the eye. In one embodiment, the "individual" is a human.

[0453] In a further aspect, the present application provides a pharmaceutical formulation comprising any Tie2 binding conjugate of the present application provided herein, e.g., for use in any of the therapeutic methods above. In one embodiment, the pharmaceutical formulation comprises any Tie2 binding conjugate of the present application provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical formulation comprises any Tie2 binding conjugate of the present application provided herein and at least one additional therapeutic agent, e.g., as described above.

[0454] Such combination therapy as mentioned above encompasses combined administration (where two or more therapeutic agents are administered within the same or separate pharmaceutical compositions), and separate administration, in which case administration of the antibody of the present application can occur either prior to, simultaneously with, and / or following, administration of the additional therapeutic agent or agents. In one embodiment, administration of the anti-Tie2 conjugate and administration of the additional therapeutic agent occur within about 1 month of each other, or within about 1, 2, or 3 weeks of each other, or within about 1, 2, 3, 4, 5, or 6 days of each other.

[0455] The Tie2 conjugates of the present application (and any other therapeutic agents) can be administered by any suitable means, including parenterally, intrapulmonary, and intranasally, and, if local treatment is required, intralesionally. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Administration can be by any suitable route, for example, by injection, for example, intravenous or subcutaneous injection, depending, in part, on whether short or long term administration is desired. Various dosing schedules are contemplated herein, including, but not limited to, single or multiple administrations at various time points, bolus injection administration, and pulsed infusion.

[0456] The Tie2 binding conjugates of the application will be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the part of the body to be treated, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The antibody need not, but can optionally be, formulated with one or more agents currently used in preventing or treating the disorder in question. The effective amount of such other agents depends on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and according to the same schedules as the antibodies described herein or about from 1 to 99% of the dosages as the agents described herein.

[0457] For the prevention or treatment of disease, the appropriate dosage of a conjugate of the application (administered alone or in combination with one or more other agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, and the stage / extent of the disease, the patient's clinical history and response to the antibody, and the judgment of the treating physician. The conjugate is suitably administered to the patient at one or more doses. Depending on the type and severity of the disease, about 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg - 10 mg / kg) of the conjugate can be an initial candidate dosage, e.g., for a patient weighing 70 kg, for example, 0.1 mg to 1 g (e.g., 0.3 mg, 2 mg, 5 mg, 10 mg, 20 mg, 40 mg, 100 mg, 150 mg, 200 mg, 250 mg, 500 mg, or 1 g) of the conjugate. Typically, the physician will determine the dosage that is appropriate for the patient, depending on the factors mentioned above. A typical daily dose might range from about 1 μg / kg to 100 mg / kg or more. For repeated administrations

[0458] It should be understood that any formulation and data method described above can be used with or instead of the Tie2 binding conjugates of the application or the immunoconjugates of the application other than the Tie2 binding conjugates of the application.

[0459] H. Manufactured Products

[0460] Another aspect of the application provides an article of manufacture comprising a Tie2 binding conjugate of the application for use in treating, preventing and / or diagnosing the above-mentioned diseases. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers can be formed from a variety of materials such as glass or plastic. The container can hold a composition, which is by itself or combined with another composition for treating, preventing and / or diagnosing diseases, and can have a sterile access port (for example the container can be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is a Tie2 binding agent or conjugate of the application. The label or package insert indicates that the composition is used for treating the disease of choice. Moreover, the article of manufacture can further comprise (a) a first container wherein is contained a composition comprising a Tie2 binding agent or conjugate of the application; and (b) a second container wherein is contained a composition comprising another cytotoxic or other therapeutic agent. The article of manufacture of this embodiment of the application can further include a package insert indicating that the compositions can be used to treat the particular disease. Alternatively, or additionally, the article of manufacture can further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It can further include other materials from which a

[0461] It is understood that any of the above articles of manufacture can include an immunoconjugate of the application in place of or in addition to a Tie2 binding conjugate.

[0462] I. Specific embodiments of the application

[0463] The following specific embodiments of the application are set forth.

[0464] 1. An isolated antibody or fragment thereof that specifically binds to Tie2, wherein the antibody comprises:

[0465] a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence NTDIS (SEQ ID NO:3), (b) CDR-H2 comprising the amino acid sequence RISPSDGNTYYADSVKG (SEQ ID NO:4), and (c) CDR-H3 comprising the amino acid sequence RTRWASXiAX2DY (SEQ ID NO:5), wherein Xi is M, L, K, F, Y, R, N, Q, H, or W and / or X2 is F, Y, L, Q, I, K, or H; and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence RASQDVSTAVA (SEQ ID NO:8), (e) CDR-L2 comprising the amino acid sequence SASFLYS (SEQ ID NO:9), and (f) CDR-L3 comprising the amino acid sequence QQSYTTPPT (SEQ ID NO: 10).

[0466] 2. The antibody of any of the preceding embodiments, wherein the CDR-H3 comprises the amino acid sequence RTRWASWAMDY (SEQ ID NO:6).

[0467] 3. The antibody of embodiment 1, wherein the CDR-H3 comprises the amino acid sequence RTRWASWAFDY (SEQ ID NO:7).

[0468] 4. The antibody of any of the preceding embodiments, which is a monoclonal antibody.

[0469] 5. The antibody of any of the preceding embodiments, which is a humanized or chimeric antibody.

[0470] 6. The antibody of any of the preceding embodiments, which is an antibody fragment that binds to Tie2.

[0471] 7. The antibody of any of the preceding embodiments, which is a Fab fragment.

[0472] 10. The antibody of any of embodiments 1-7, comprising a VL domain comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:21 and a VH domain comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:20.

[0473] 11. The antibody of any one of embodiments 1 to 7, comprising: a VL domain comprising an amino acid sequence having at least 96% sequence identity to the amino acid sequence of SEQ ID NO: 21; and a VH domain comprising an amino acid sequence having at least 96% sequence identity to the amino acid sequence of SEQ ID NO: 20.

[0474] 12. The antibody of any one of embodiments 1 to 7, comprising: a VL domain comprising an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO: 21; and a VH domain comprising an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO: 20.

[0475] 13. The antibody of any one of embodiments 1 to 7, comprising: a VL domain comprising an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 21; and a VH domain comprising an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 20.

[0476] 14. The antibody of any one of embodiments 1 to 7, comprising: a VL domain comprising an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 21; and a VH domain comprising an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 20.

[0477] 15. The antibody of any one of the preceding embodiments, wherein the antibody comprises: a VL sequence of SEQ ID NO: 21 and a VH sequence of SEQ ID NO: 19; a VL sequence of SEQ ID NO: 21 and a VH sequence of SEQ ID NO: 22; or a VL sequence of SEQ ID NO: 21 and a VH sequence of SEQ ID NO: 20.

[0478] 16. The antibody of any one of the preceding embodiments, wherein the antibody comprises an engineered cysteine.

[0479] 17. The antibody of embodiment 16, wherein

[0480] the engineered cysteine is selected from T120C, G166C, G178C, T187C, and T209C in the HC; or the engineered cysteine is selected from Q124C, R142C, Q155C, L201C, T206C, K107C, K126C, and K149C in the LC; wherein the residue number is according to EU numbering.

[0481] 18. The antibody of embodiment 16 or 17, wherein the engineered cysteine is selected from T209C in the HC and T206C in the LC.

[0482] 19. The antibody of any one of embodiments 16-18, wherein the engineered cysteine is T206C in the LC.

[0483] 20. The antibody of any one of embodiments 16-18, wherein the engineered cysteine is T209C in the HC.

[0484] 21. The antibody of any of the preceding embodiments, comprising: a LC comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 25; and a HC comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 55.

[0485] 22. The antibody of any of the preceding embodiments, comprising: a LC comprising an amino acid sequence having at least 96% sequence identity to the amino acid sequence of SEQ ID NO: 25; and a HC comprising an amino acid sequence having at least 96% sequence identity to the amino acid sequence of SEQ ID NO: 55.

[0486] 23. The antibody of any of the preceding embodiments, comprising: a LC comprising an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO: 25; and a HC comprising an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO: 55.

[0487] 24. The antibody of any of the preceding embodiments, comprising: a LC comprising an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 25; and a HC comprising an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 55.

[0488] 25. The antibody of any of the preceding embodiments, comprising: a LC comprising an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 25; and a HC comprising an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 55.

[0489] 26. The antibody of any of the preceding embodiments, wherein the antibody comprises: a LC comprising the sequence of SEQ ID NO: 25 and a HC comprising the sequence of SEQ ID NO: 55.

[0490] 27. The antibody of any one of embodiments 1 to 25, wherein the antibody comprises: a LC comprising the sequence of SEQ ID NO: 25 and a HC comprising a sequence selected from SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, and SEQ ID NO: 92.

[0491] 28. The antibody of any one of embodiments 1 to 20, comprising: a LC comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 56; and a HC comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 23.

[0492] 29. The antibody of any one of embodiments 1 to 20, comprising: a LC comprising an amino acid sequence having at least 96% sequence identity to the amino acid sequence of SEQ ID NO: 56; and a HC comprising an amino acid sequence having at least 96% sequence identity to the amino acid sequence of SEQ ID NO: 23.

[0493] 30. The antibody of any one of embodiments 1 to 20, comprising: a LC comprising an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO: 56; and a HC comprising an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO: 23.

[0494] 31. The antibody of any one of embodiments 1 to 20, comprising: a LC comprising an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 56; and a HC comprising an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 23.

[0495] 32. The antibody of any one of embodiments 1 to 20, comprising: a LC comprising an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 56; and a HC comprising an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 23.

[0496] 33. The antibody of any one of embodiments 1 to 20, wherein the antibody comprises: a LC comprising the sequence of SEQ ID NO: 56 and a HC comprising the sequence of SEQ ID NO: 90, SEQ ID NO: 91, or SEQ ID NO: 23.

[0497] 34. An antibody that specifically binds to Tie-2, wherein the antibody comprises a HC comprising the sequence of SEQ ID NO: 55 and a LC comprising the sequence of SEQ ID NO: 25.

[0498] 35. An isolated nucleic acid encoding an antibody of any of the preceding embodiments.

[0499] 36. An isolated host cell comprising the nucleic acid of embodiment 38.

[0500] 37. A method of making an antibody that binds to Tie-2, comprising culturing the host cell of embodiment 39 under conditions suitable for expression of the antibody.

[0501] 38. A conjugate that binds to Tie2, wherein the conjugate comprises at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight antibodies of any of embodiments 1-34, wherein each of the antibodies is linked to a multimerization moiety.

[0502] 39. The conjugate of embodiment 38, wherein the conjugate activates phosphorylation of Tie2 in an in vitro or in vivo cell assay.

[0503] 40. The conjugate of embodiment 38 or 39, wherein the conjugate causes a decrease in Tie2 protein levels of less than 25%, less than 50%, or less than 75% in an in vitro assay; or wherein the conjugate does not cause a decrease in Tie-2 protein levels of more than 25%, more than 50%, or more than 75% in an in vitro assay.

[0504] 41. The conjugate of embodiments 38-40, wherein the conjugate decreases vascular permeability as determined by an in vitro barrier function assay.

[0505] 42. The conjugate of any of embodiments 38-42, wherein the multimerization moiety comprises a polyol, a polypeptide, and / or a peptide.

[0506] 43. The conjugate of embodiment 42, wherein the polyol is a multi-armed polyol selected from a dimer, a tetramer, a hexamer, and an octamer.

[0507] 44. The conjugate of embodiment 43, wherein the multi-armed polyol is a hexamer.

[0508] 45. The conjugate of embodiment 43 or 44, wherein the multi-armed polyol is an octamer.

[0509] 46. The conjugate of any of embodiments 42-45, wherein the polyol is a polyethylene glycol (PEG).

[0510] 47. The conjugate of any one of embodiments 42-45, wherein the polyol is covalently linked to the at least two antibodies via a free thiol group of a cysteine amino acid.

[0511] 48. The conjugate of embodiment 47, wherein the cysteine amino acid is an engineered cysteine.

[0512] 49. The conjugate of embodiment 48, wherein the engineered cysteine is located within a HC and / or LC constant region of the antibody.

[0513] 50. The conjugate of embodiments 48 or 49, wherein the engineered cysteine is selected from T120C, G166C, G178C, T187C, and T209C in the HC; or the engineered cysteine is selected from the group consisting of Q124C, R142C, Q155C, L201C, T206C, K107C, K126C, and K149C in the LC; wherein the residue numbers are according to EU numbering.

[0514] 51. The conjugate of any one of embodiments 48-50, wherein the engineered cysteine is T206C in the LC, wherein the residue numbers are according to EU numbering.

[0515] 52. The conjugate of any one of embodiments 48-50, wherein the engineered cysteine is T209C in the HC, wherein the residue numbers are according to EU numbering.

[0516] 53. The conjugate of any one of embodiments 42-45, wherein the polyol is covalently linked to the at least one antibody via a free amino group of a lysine amino acid.

[0517] 54. The conjugate of embodiment 53, wherein the lysine amino acid is located within a HC or LC constant region of the antibody, and / or the lysine amino acid is located at the C-terminus of a heavy chain or light chain of the antibody.

[0518] 55. The conjugate of any one of embodiments 46-54, wherein the PEG has a weight average molecular weight of about 500 daltons (Da) to about 300,000 Da or about 500 Da to about 20,000 Da.

[0519] 56. The conjugate of any one of embodiments 46-55, wherein the PEG has a weight average molecular weight of about 6000 Da.

[0520] 57. The conjugate of any one of embodiments 46-56, wherein the PEG comprises a dipentaerythritol hexamer or octamer core.

[0521] 58. The conjugate of any one of embodiments 46 to 57, wherein the PEG has the structure of Formula (la):

[0522]

[0523] wherein n is an integer from 1 to 10; each m is independently an integer from 3 to 250; each R 1 is independently absent or a linking group; and each R 2 is independently hydrogen or a terminal reactive group;

[0524] wherein at least one R 2 is a terminal reactive group and is covalently linked to the antibody of any one of claims 1 to 14.

[0525] 59. The conjugate of any one of embodiments 46 to 57, wherein the PEG has the structure of Formula (lb):

[0526]

[0527] wherein each m is independently an integer from 3 to 250; each R 1 is independently absent or a linking group; and each R 2 is independently hydrogen or a terminal reactive group;

[0528] wherein at least one R 2 is a terminal reactive group and is covalently linked to the antibody of any one of claims 1 to 14.

[0529] 60. The conjugate of embodiment 59, wherein each m is independently an integer from 15 to 35, preferably from about 20 to 30.

[0530] 61. The conjugate of any one of embodiments 43 to 60, wherein the conjugate is prepared by covalently linking at least one antibody of any one of embodiments 1 to 34 to a multi-armed polyol.

[0531] 62. A conjugate comprising an antibody that specifically binds to Tie-2, wherein the antibody comprises a VH sequence of SEQ ID NO: 20 and a VL sequence of SEQ ID NO: 21, wherein the antibody is covalently linked to a polyethylene glycol having the structure of Formula (lb):

[0532]

[0533] wherein each m is independently an integer from 3 to 250; each R 1 is independently absent or a linking group; and each R 2 is independently hydrogen or a terminal reactive group;

[0534] wherein at least one R 2 is a terminal reactive group and is covalently attached to the antibody.

[0535] 63. The conjugate of embodiment 62, wherein m is an integer from 10 to 200.

[0536] 64. The conjugate of embodiment 62 or 63, wherein m is an integer from 20 to 30.

[0537] 65. The conjugate of any one of embodiments 58 to 64, wherein R 1 is absent, or wherein R 1 is selected from the group consisting of: and combinations thereof; wherein each i is independently an integer from 0 to 10; j is an integer from 0 to 10; and R 2 is a terminal reactive group selected from the group consisting of a thiol-reactive group, an amine-reactive group, and combinations thereof.

[0538] 66. The conjugate of any one of embodiments 58 to 65, each R 2 is independently selected from the group consisting of a maleimide, a mercaptan, a thiol, a triflate, a tosylate, an aziridine, a peroxide, a pyridyl disulfide, a succinimidyl ester, -NH2, an aldehyde, a haloacetate, a haloacetamide, and a p-nitrophenyl carbonate.

[0539] 67. The conjugate of any one of embodiments 58 to 66, wherein R 2 is a maleimide.

[0540] 68. The conjugate of any one of embodiments 43 to 67, wherein the conjugate is prepared by covalently attaching at least one antibody according to any one of claims 1 to 34 to a multi-armed polyol.

[0541] 69. A pharmaceutical composition comprising: a conjugate according to any one of embodiments 38 to 68; and a pharmaceutically acceptable carrier.

[0542] 70. The pharmaceutical composition according to embodiment 69, wherein the concentration of the conjugate is from about 50 mg / ml to about 300 mg / ml.

[0543] 71. The pharmaceutical composition according to embodiment 69 or 70, further comprising an additional therapeutic agent.

[0544] 72. The pharmaceutical composition according to embodiment 71, wherein the additional therapeutic agent is selected from the group consisting of a VEGF antagonist, an Ang2 antagonist, an HtrAl antagonist, and an IL33 antagonist, a complement component antagonist, and a second Tie2 agonist.

[0545] 73. The pharmaceutical composition according to embodiment 72, wherein the VEGF antagonist is selected from the group consisting of a VEGF trap and an anti-VEGF antibody.

[0546] 74. A long-acting delivery device for ocular delivery comprising a pharmaceutical composition according to embodiments 79-73 and means for delivering the composition intravitreally to a patient, wherein the composition remains effective in situ for an extended period of time.

[0547] 75. A method of treating a Tie2 pathway-mediated disease in a subject in need thereof comprising administering to the subject an effective amount of an antibody of any one of embodiments 1-34, a conjugate of any one of embodiments 38-68, or a pharmaceutical composition of any one of embodiments 69-73.

[0548] 76. The method of embodiment 75, wherein the Tie2 pathway-mediated disease is a vascular permeability disease.

[0549] 77. The method of embodiment 75 or 76, wherein the Tie2 pathway-mediated disease is an ocular condition.

[0550] 78. The method of embodiment 77, wherein the ocular condition is selected from diabetic macular edema (DME), diabetic retinopathy, age-related macular degeneration (AMD) (including dry and wet (non-exudative and exudative) forms), choroidal neovascularization (CNV), uveitis, ischemia-related retinopathy, pathologic myopia, von Hippel-Lindau disease, ocular histoplasmosis, central retinal vein occlusion (CRVO), corneal neovascularization, glaucoma, and retinal neovascularization.

[0551] 79. The method of any one of embodiments 75-78, wherein the ocular condition is DME.

[0552] 80. The method of any one of embodiments 75-79, wherein the method comprises administering the antibody, conjugate, or pharmaceutical preparation using an implantable port delivery system.

[0553] 81. The method of any one of embodiments 75-79, wherein the method comprises administering the antibody, conjugate, or pharmaceutical preparation by intravenous administration.

[0554] 82. The method of embodiment 81, wherein the intravenous administration is through a narrow gauge needle.

[0555] 83. The method of embodiment 82, wherein the narrow gauge needle is about 30, 29, 28, 27, 26, 25, 24, 23, or 22 gauge.

[0556] 84. The method of any one of embodiments 75-83, further comprising administering an additional therapeutic agent to the individual.

[0557] Example III

[0558] The following are examples of the methods and compositions of the present application. It will be understood that various modifications can be made to the embodiments described herein, in light of the foregoing description.

[0559] Example 1 - Generation of Anti-Tie 2 Antibodies from a Natural Phage Library

[0560] Antibodies that bind to the extracellular domain (ECD) of Tie2 were initially selected from a phage display synthetic antibody library constructed on a single human framework by introducing synthetic diversity at solvent exposed positions within the heavy chain CDRs as described below. Phage was screened against the ECD and various subdomains of the ECD.

[0561] Phage for library construction

[0562] Phagemids pV0350-2b and pV0350-4 were designed to display Fab templates monovalently or bivalently, respectively, on the surface of M13 phage particles. The Fab templates were based on the h4D5 antibody, which is a humanized antibody that recognizes a cancer-related antigen known as Her-2 (erbB2). The h4D5 sequence was obtained by polymerase chain reaction using the humAb4D5 version 8 ("humAb4D5-8") sequence (Carter et al., (1992) PNAS 89: 4285-4289). The h4D5 nucleic acid sequence encodes modified CDR regions from a mouse monoclonal antibody that has specificity for Her-2 in the human consensus sequence Fab framework. Specifically, the sequence contains a kappa light chain (LC region) upstream of the VH and CH1 domains (HC region). Methods of making anti-Her-2 antibodies and the identity of the variable domain sequences are provided in U.S. Patent Nos. 5,821,337 and 6,054,297.

[0563] The vector pV0350-2b was constructed by modifying the phagemid (pHGHam-gIII) previously described for phage display of human growth hormone (hGH) under the control of the phoA promoter. The open reading frame in phGHam-gIII encoding the stII secretion signal sequence and hGH fused to the C-terminal domain of M13 minor coat protein P3 (cP3) was replaced with a DNA fragment containing two open reading frames. The first open reading frame encoded the h4D5 light chain (version 8) and the second open reading frame encoded the variable domain (VH) and the first constant domain (CHI) of the h4D5 heavy chain fused to cP3; each protein was directed for secretion by the N-terminal stII signal sequence. The amber stop codon located between the heavy chain fragment and cP3 was deleted as this modification has been shown to increase the level of Fab displayed on phage. An epitope tag was added to the C-terminus of the h4D5 light chain (gD tag). The vector for bivalent display (pV0350-4) was identical to pV0350-2b but a DNA fragment encoding the GCN4 leucine zipper was inserted directly after the CHI domain of the heavy chain cP3 as described. The light chain gene was further modified in both phagemids at three positions to encode amino acids most commonly found in the Kabat database of natural antibody sequences; specifically, Arg66 was changed to Gly and Asn30 and His91 were changed to Ser. These changes were found to increase the expression and display of Fab on phage. Site-directed mutagenesis was performed using the method of Kunkel et al. (Kunkel, J. D., et al. (1987) Methods Enzymol 154:367-82).

[0564] As described, phage display libraries were generated using oligonucleotide site-directed mutagenesis and "stop templates" of pV0350-2b or pV0350-4 (Lee, C. V., et al. (2004) J. Immunol. Methods 284: 119-132; Lee, C. V., et al. (2004) JMB 340: 1073-1093). A stop codon (TAA) was embedded in all three heavy chain CDRs. This was repaired by a mixture of degenerate oligonucleotides that anneal to the sequence encoding CDR-H1, CDR-H2, and CDR-H3 during the mutagenesis reaction and replace the codons at the positions selected for randomization with custom degenerate codons. The mutagenesis reaction was electroporated into E. coli SS320 cells and the culture was grown overnight at 30°C in 2YT broth supplemented with h KO7 helper phage, 50 g / ml carbenicillin, and 50 g / ml kanamycin. As described, phage was harvested from the culture medium by precipitation using PEG / NaCl (Sidhu, S. S. et al. (2000), Methods Enzymol. 328: 333-363). Each electroporation reaction used ~1011E. coli cells and ~10 ug of DNA and resulted in 1 x 109-5 x 109transformants.

[0565] A special library was made using custom degenerate oligonucleotides to mimic the natural diversity of CDR-H1 hexyl CDR-H2 (Table 1 in Lee, C. V, et al. (2004), JMB above): Library 3 with Fab.zip template (Lib-3). See Lib-3 described in Lee, C. V, et al. (2004) above. Two to four oligonucleotides for CDR-H1 and CDR-H2 were combined to increase coverage of natural diversity. Lib-3 used oligonucleotides H1a and H1b (ratio 2:1) and H2a-c (ratio 1:2:0.1) for CDR-H1 and CDR-H2, respectively (see Table 1 in Lee, C. V. et al. (2004), JMB above for description of oligonucleotides).

[0566] For positions 95-100 in CDR-H3, Lib-3 consists of a set of libraries with expanded CDR-H3 lengths containing NNS codons (or NNK codons) or modified versions of NNS codons (XYZ codons) that contain unequal nucleotide ratios at each position of the codon triplet. NNS codons encompass 32 codons and encode all 20 amino acids. X contains 38% G, 19% A, 26% T, and 17% C; Yc contains 31% G, 34% A, 17% T, and 18% C; and Z contains 24% G and 76% C. The CDR-H3 design for Lib-3 is described in Table 5 of Lee, C. V. et al. (2004) supra. Independent mutagenesis reactions were performed for each CDR-H3 length and electroporation was performed, but electroporation was performed together for lengths of 7 and 8 residues.

[0567] Phage display levels of full Fabs in each library were examined by measuring binding of 48 randomly picked clones to anti-gD antibody. For Lib-3, similar display levels were observed for different CDR-H3 lengths, but libraries incorporating the longest CDR-H3 (15-19 residues) had a reduced percentage of Fab-displaying clones (15-30%). This can reflect reduced mutagenesis efficiency when using super-long synthetic oligonucleotides.

[0568] Phage sorting

[0569] Lib-3 described above was sorted against a variety of Tie2 extracellular domain (ECD) proteins. Tie2 ECD consists of, from the distal membrane to the proximal membrane, 3 IgG domains (Ig1 and Ig2), 3 EGF domains (EGF1-3), a third IgG domain (Ig3), and 3 fibronectin type III domains (FN3) (see, e.g., Figure 1 ) Constructs were generated to encode the full extracellular domain (ECD), the membrane proximal FN3 domains, or the ECD without the FN3 domains (referred to as ECD5). Thus, the ligand binding domain Ig2 is encoded by both the ECD construct and the ECD5 construct, but not by the FN3 construct. The encoded proteins were fused at the C-terminus to the Fc region of hIgG1 (for human and cynomolgus proteins) or mIgG2a (for murine and rat proteins) or to a C-terminal Flag tag (for all species). Figure 1 Proteins used for panning are shown. Full ECD constructs for human, murine, and rat Tie1 receptors were also generated using both C-terminal Fc fusions or Flag tags.

[0570] Expression and purification of Tie2 ECD protein

[0571] Flag-tagged Tie2 ECD was expressed and purified from Chinese hamster ovary (CHO) cell conditioned media. After 11-14 days, the conditioned media was harvested and concentrated approximately ten-fold. The concentrate was loaded onto an anti-flag-tag column and washed with binding buffer containing 25 mM TRIS, 150 mM NaCl, 1 mM EDTA, 0.1% Triton X-114, and 0.1% Triton X-110, pH 7.5. The flag-tagged protein was then eluted with 50 mM Na-citrate, pH 3.0, 150 mM NaCl, then neutralized to pH 5.0 using 1 M arginine, 400 mM succinate, pH 9.0. The eluted protein was loaded onto a size exclusion column (Superdex 200 or Superdex 75) in phosphate buffered saline and fractions were collected; monomeric peak fractions were pooled, concentrated, and 0.2 um filtered.

[0572] For panning, 96-well Nunc Maxisorp plates were coated with target Tie2 antigen (5 ug / ml) in PBS at 100 ul / well overnight at 4°C. The plates were blocked with 65 ul 1% blocking protein for 30 minutes (min) and re-blocked with 40 ul 1% Tween 20 for 30 min (blocking protein: Round 1: Bovine Serum Albumin (BSA); Round 2: Casein; Round 3: Bovine Serum Albumin (BSA); Round 4: Casein). Afterwards, the phage library was diluted to ~3-5 OD / ml (1 OD = 1.13 x 10 13Typically, phage input is: 3-5 OD / ml for round 1, 3 OD / ml for round 2, ~0.5-1 OD / ml for round 3 and ~0.1-0.5 OD / ml for round 4. Diluted phage is incubated for 30 min at room temperature. The wells are washed at least 5 times consecutively with PBS and 0.05% Tween 20. Blocked phage library is added to 8 target antigen coated wells and 2 uncoated wells at 100 ul / well for 2 hours (hr) at room temperature. The plates are washed at least 10 times consecutively with PBS and 0.05% Tween 20. Starting with the 3rd round of panning, 1 uM omalizumab is added to the phage library as an irrelevant Fc containing protein, Tie2.ECD5 or antibody anti-Tie2.20 (ligand blocking) for 1 hour before the mixture is applied to Tie2 coated wells for 2 hr. Phage is diluted with 100 mM HC1 at 100 ul / well for 20 min at room temperature. Diluted phage from coated wells and background phage from uncoated wells are collected into separate tubes. The diluted collections are neutralized by adding 1 / 10 volume of 1 M Tris pH 11.0 to both tubes. BSA is added to the phage from coated wells tube at a final concentration of 0.1%. To determine phage titer, 90 ul of log phase XL-1 (OD 600nm ~ 0.1-0.3) is infected with 10 ul of diluted phage or background phage for 30 min at 37°C. Thereafter, the infected cells are serially diluted in 10-fold increments with 90 ul of 2YT. Ten ul aliquots of infected cells are plated on carbenicillin plates.

[0573] To propagate phage between panning rounds, ~4 ml of log phase XL-1 (OD 600nm ~ 0.1-0.3) is infected with approximately 400 ul of diluted phage for 30-45 min at 37°C. Helper phage KO7 and carbenicillin are added to the infected cells at 1 x 10 10Final concentrations of 50 pfu / ml KO7 and 50 ug / ml carbenicillin were added to the infection, one hour after the addition time. Cultures were grown in a final volume of 20-25 ml of 2YT media with carbenicillin 50 ug / ml and kanamycin 50 ug / ml, at 37°C for 4 hr and at 30°C overnight (or at least 18 hr). The next day, the phage were purified by spinning down the cells at 8000 rpm for 10 min. The supernatant was collected. 20% PEG / 2.5 M NaCl was added at a volume of 1 / 5 of the supernatant volume, mixed, and left on ice for 5 min. The phage were pelleted into a pellet at 12000 rpm for 15 min. The pellet was spun again at 5000 rpm for 5 min. The pellet was resuspended in 1 ml PBS and spun down at 12000 rpm for 15 min to clarify debris, and PEG / NaCl was added for precipitation. The phage pellet was resuspended in PBS. The OD of the resuspended phage pellet was read at 268 nm.

[0574] Screening ELISA assay

[0575] Clones from the fourth round were screened for Tie2 binding and specificity by ELISA. Screening ELISAs were performed by coating wells of 96-well microtiter plates overnight at 4°C with Tie2 protein or irrelevant protein at 65 ul / well (1 ug / ml in coating buffer). In 96-tube plates, clones from the fourth round were grown overnight at 37°C in 400 ul 2YT media with 50 ug / ml carbenicillin and helper phage KO7. These plates were spun down at 3000 rpm for 10 min. Thirty ul of culture supernatant was added to Tie2-coated plates with 60 ul of ELISA buffer (PBS with 0.5% BSA and 0.05% Tween20) and incubated at room temperature for 1 hr. Plates were washed with PBS-0.05% Tween20 and 100 ul / well of horseradish peroxidase (HRP)-conjugated anti-M13 antibody (1 / 5000 dilution in PBS plus 0.5% BSA and 0.05% Tween20) at room temperature for 30 min (Sidhu et al., supra). The wells were washed with PBS-0.05% Tween20 and then 100 ul / well of a 1:1 ratio of 3,3',5,5'-tetramethylbenzidine (TMB) peroxidase substrate and peroxidase solution B (H202) (Kirkegaard-Perry Laboratories (Gaithersburg, MD)) was added to each well and incubated at room temperature for 5 min. The reaction was stopped by adding 100 ul 1 M phosphoric acid (H3PO4) to each well and the OD of the wells was determined at 450 nm using a standard ELISA plate reader.

[0576] Further analysis was performed on clones that had Tie2 binding in combination with the above background and possessed Tie2 binding species specificity and low binding to irrelevant Fc-containing proteins (omalizumab). The following Tables 2-5 summarize the binding data. None of Tables 2-5 show binding to omalizumab.

[0577] Table 2

[0578]

[0579]

[0580] Table 3

[0581]

[0582] Table 4

[0583]

[0584]

[0585] Table 5

[0586]

[0587] Anti-Tie2 IgG expression and purification

[0588] The positive binders identified above with the desired species specificity and low non- related protein binding were sequenced and the variable domains of the anti-Tie2 heavy chains were cloned into a previously involved vector for transient human IgGl expression in mammalian cells. (Lee et al., 2004a).

[0589] Transient transfections of 293 cells were performed using the heavy chains encoded by the constructs generated above and the 4D5 light chain (SEQ ID NO: 25) to express the resulting anti-Tie2 human IgG. The IgG was purified from the transfection supernatant using protein A affinity chromatography and screened by ELISA for Tie2 binding, Tie1 binding and epitope profile. Eight antibodies were not further analyzed because the hlgG proteins were either not expressed or poorly expressed.

[0590] Example 2 - Characterization of anti-Tie 2 antibodies derived from phage library Tie2 binding

[0591] To confirm Tie2 binding by the anti-Tie2 antibodies, an ELISA format was used in which the Tie2 ecto-construct configured as an IgGl as described above was immobilized at 2 ug / ml in 65 ul PBS on Maxisorp immunoplates overnight at 4°C. Serial dilutions of the anti-Tie2 IgG were applied to the plates with immobilized Tie2 which had been previously blocked with a solution of 1% BSA in PBS and incubated for 20 min at room temperature. The plates were washed and detected with an anti-huFC conjugated HRP secondary antibody using the method described above. Figure 2A and Figure 2B Various clones were shown to bind to human Tie2 ECD.

[0592] Tie1 binding was also assessed using a binding ELISA using immobilized Tie1 protein. The results provided in Figure 3A and Figure 3B showed the lack of Tie1 binding by several of the anti-Tie2 antibodies, demonstrating that these anti-Tie2 antibodies specifically bind to Tie2.

[0593] Ligand blocking

[0594] To assess the Angl and Ang2 ligand blocking activity of anti-Tie2 antibodies, a competitive ELISA format was used. In this assay, hAng2 (GenBank Accession No. NP_001137) or hAngl (GenBank Accession No. NP_000450) was immobilized on Maxisorp immunoplates (2 ug / ml) and biotinylated huTie2 ECD.Fc was equilibrated in solution with serial dilutions of anti-Tie2 antibodies, then unbound biotin-Tie2.ECD.Fc was captured with immobilized hAngl or hAng2 and detected using streptavidin-conjugated HRP. These results show that both Angl and Ang2 are blocked by at least anti-Tie2 antibodies Tie2.1, Tie2.12, and Tie2.20 (see, Figure 4A and Figure 4B ).

[0595] Example 3 - Functional analysis of anti-Tie2 antibodies

[0596] The anti-Tie2 antibodies identified above as being able to specifically bind Tie2 were further analyzed to identify those that are able to function as Tie2 agonists. Both human umbilical vein endothelial cells (HUVECs) and rat aortic endothelial cells (RAECs), which are known to express Tie2, were used to assess the functional activity of anti-Tie2 IgGs with demonstrated Tie2 binding (see, Figure 5A and Figure 5B ).

[0597] Stimulation of phospho-AKT (pAKT)

[0598] Experiments were performed to assess the function of anti-Tie2 antibodies with respect to Tie2 activation. Tie2 conjugates according to the present disclosure that are Tie2 agonists are expected to bind to and activate Tie2, leading to downstream AKT enzyme activation. Activation of AKT can be evidenced by AKT protein phosphorylation to produce pAKT, as described below. Phosphorylation of AKT was determined by Western blot analysis using an antibody specific for phosphorylated AKT or by a FRET assay, as described below.

[0599] Cells and Antibodies: HUVECs were purchased from Lonza (Catalog No. CC-2517; Lonza, Ltd., Basel, Switzerland). RAECs were purchased from VEC Technologies and cultured in growth medium (Catalog No. MCDB-13110; VEC Technologies, Inc., Rensselaer, NY). Anti-Tie2 antibodies were prepared at Genentech, Inc. (South San Francisco, CA). Polyclonal goat anti-hIgG was used as a cross-linker (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA).

[0600] Preparation of HUVEC: HUVEC (human vascular endothelial cells) were trypsinized and plated at 0.4 × 10 5 Cells / well were seeded in 100 μl of culture medium in a sterile 96-well plate ( The plate was incubated overnight in a 37°C, 5% CO2 incubator. The medium was removed, and 100 μL of prewarmed serum-starved medium (Basal Medium EndoGRO™; catalog number SCME-BM, MilliporeSigma) was added to each well of the plate. The plate was incubated in a 37°C, 5% CO2 incubator for 3 hours, followed by incubation with anti-Tie2 antibody.

[0601] Preparation of RAECs: RAECs (rat aortic endothelial cells) were seeded at a density of 12,000 cells / well in 96-well cell culture plates and cultured overnight in 100 μl of EGM2 MV medium (Lonza, Ltd.) at 37°C and 5% CO. After overnight culture, the cells were starved for 3 hours in EBM2 basal medium (Lonza, Ltd.) containing 0.1% BSA and then incubated with anti-Tie2 antibody.

[0602] To test the effect of cross-linking anti-Tie2 antibodies, 20 μg / ml of cross-linker (polyclonal goat anti-hIgG1) in assay buffer (basal medium + 0.2% BSA) was mixed with an equal volume of 60 μg / ml anti-Tie2 bivalent antibody and incubated for 1 hour at RT. After incubation, the mixture of hIgG1 and anti-Tie2-IgG antibodies was serially diluted 3-fold.

[0603] To test other Tie2 antibody agents, these molecules were diluted in assay buffer to the starting stock concentration (typically 30 - 1000 μg / ml) followed by serial dilutions (typically 2 to 10 fold). After removal of serum-starved media, the dilutions (50 μΐ) were added to each well and the plate was incubated at 37°C, 5% C02for 15 minutes. The solution was removed and 50 μΐ of lysis buffer containing blocking buffer from the Phospho-AKT1 / 2 / 3 Ser473 Cell Kit (Cat. No. 64AKSPEH; Cisbio, Codolet, France) was added to the cells. The plate was incubated at room temperature for about 30 min to 45 min with gentle shaking, frozen at -80°C until use, or used directly in the FRET assay.

[0604] Western Blot Assay: HUVEC cells were plated at 1 x 10 6 cells / well in Endogro media and incubated at 37°C for 16-18 hours. The media was changed to 0.1% BSA Endogro basal media for a duration of 4-5 hours followed by stimulation. Cells were incubated at 37°C for 30 min with the relevant Tie-2 agonist, washed three times with cold phosphate buffered saline, pH 7.4. Cells were placed on ice and incubated with 100 ul / well of RIPA buffer (Sigma, Cat. No. 20-188) containing Roche Complete Protease and Phosphatase Inhibitors (Thermo Scientific, Cat. No. 1861281) for 5 min. The lysate was harvested from the well using a cell scraper and centrifuged at 17,800 x g for 10 min. The supernatant was evaluated by SDS-PAGE (8% NuPAGE Bis-Tris (Invitrogen, NW800085)) and then transferred to nitrocellulose membranes. The membranes were blocked at room temperature with 5% BSA in TBS-T for 1 hr and probed with rabbit anti-pAKT (Cell Signaling Technologies, Cat. No. 9271S) in blocking buffer. After 4 washes with TBS-T, the membranes were probed with HRP anti-rabbit Ig (1 : 10,000) (GE Healthcare, NA934V) at RT for 1 hr. The membranes were washed 3 times with TBS-T and incubated with ECL reagent (Thermo Scientific, 32132) at room temperature for 5 min before the blots were exposed to film.

[0605] FRET assay: Cell lysates (15 μΐ) were thawed on ice and then mixed with 5 μΐ of 1 :40 dilution of each of the phospho-AKT Ser473 kit's phospho-AKT d2 antibody and phospho-AKT Cryptate antibody in a 384-well microplate (catalog # 784080; Greiner Bio-One North America, Inc., Monroe, NC) and the plate was incubated at room temperature for 4 hr or at 4°C overnight and read on a CLARIOstar (BMG LABTECH, software version: 5.01 R2) at 620 nm and 665 nm. For each well, data was calculated as the ratio of acceptor to donor emission signals multiplied by 104.

[0606] In an attempt to identify anti-Tie2 antibodies capable of activating Tie2 activity, the Tie-2 binding antibodies identified in detail above were formalized as human IgGl (hIgGl) antibodies and tested for their ability to stimulate AKT phosphorylation (to generate pAKT), which is downstream of Tie2 activation. The following recombinant anti-Tie2 antibodies (hIgGl) were tested for their ability to stimulate AKT phosphorylation in RAECs: Tie2.1, Tie2.4, Tie2.5, Tie2.16, and Tie2.20, for a 10 minute time period. Cell lysates were subjected to Western Blot (WB) analysis for pAKT. Figure 6A It was shown that incubation of the anti-Tie2 antibodies Tie2.1 and Tie2.20 increased AKT phosphorylation in this cell-based in vitro assay, and that Tie2.1 had a stronger Tie2 agonist effect than Tie2.20.

[0607] Subsequently, experiments were performed to assess the effect of cross-linking on the agonist activity of the anti-Tie2 antibodies. Anti-hIgGl cross-linking antibodies were incubated with Tie2.1 or Tie2.20 in the pAKT assay. As shown in Figure 6B Cross-linking of Tie2.1 further increased its ability to activate Tie2, as determined by increased AKT phosphorylation, as shown in

[0608] In an attempt to identify additional agonistic anti-Tie2 antibodies, a HTRF-based assay (Cisbio) was also used. RAECs were treated with recombinant anti-Tie2 antibodies (hIgGl) for 10 minutes. Cell lysates were subjected to HTRF analysis (cisbio) for pAKT. As shown in Figure 7A As shown in

[0609] It is believed that the level of Tie2 activation when bound by Tie2 IgG (full length) antibody can be due in part to non-specific aggregation of IgG molecules within the reaction mixture. As shown in Example 2, Tie2.1 antibody formulations with a higher percentage (e.g., 3.5%) of aggregates exhibited greater agonistic activity compared to Tie2.1 antibody formulations with a lower percentage (e.g., 0.15%) of aggregates. Figure 7B It is further shown that cross-linking anti-hlgGl enhances the agonistic activity of Tie2.1. Without being bound by theory, it is believed that activation of Tie2 by anti-Tie2 agonist antibodies can be facilitated by cross-linking of the Tie2-bound antibodies. Figure 7B

[0610] Example 4 - Classification of Anti-Tie2 Antibodies Derived from a Native Phage Library

[0611] Classification of anti-Tie2 antibodies was performed using both phage ELISA and Octet measurements to further characterize the binding of anti-Tie2 antibodies to the Tie2 receptor protein and to identify those antibodies with a common epitope.

[0612] For phage ELISA, huTie2 ECD.Fc protein was immobilized at 2 ug / ml in 65 ul PBS on Maxisorp immunoplates overnight at 4°C. Serial dilutions of anti-Tie2 antibodies Tie2.1, Tie2.20, Tie2.34 or anti-Tie2 antibody 13H10 (anti-Tie2 receptor agonist antibody described in U.S. Patent No. 6,365,154) formatted as IgG were applied to the plates with immobilized huTie2 ECD.Fc that had been previously blocked with 1% BSA in PBS and incubated for 1 hour at room temperature. Anti-Tie2 phage were added at 0.1 OD268nm / mL for 15 min at room temperature. The plates were then washed and detected using anti-M13 conjugated HRP secondary antibody using the method described above. The signal increased with serial dilution of the antibody, indicating that the test phage competed with the serially diluted antibody, suggesting that they bound to the same site or epitope.

[0613] ​Epitope binding as determined by the above ELISA was confirmed by Octet Epitope Binning Assay using an Octet RED384 (Pall Forte Bio Corporation, Menlo Park, CA) and standard sandwich format binning assay. Specifically, biotinylated hTie2.ECD.Fc protein (10 ug / mL) was used to coat streptavidin biosensors (Pall Forte Bio Corporation) and then exposed to benchmark anti-Tie2 hlgGl (50 ug / mL Ab1, Ab20, 13H10) followed by exposure to a second (test) anti-Tie2 hlgGl. Data was processed using Forte Bio’s data analysis software. Additional binding by the second (test) antibody indicated unoccupied epitopes (non-competitors), while lack of binding indicated epitope blockade (competitors). A graphical representation of the experiment with data obtained from Ab20 (Tie2.20) is provided in Figure 8A and Figure 8B Data for other anti-Tie2 antibodies is not shown.

[0614] Results from the ELISA and Octet binning assay analysis are summarized and shown in Figure 9 which shows which anti-Tie2 antibodies compete with each other and likely bind the same or similar epitopes. Specifically, the binding assays described above demonstrated that phage-derived antibodies Tie2.1 (Ab1), Tie2.12 (Ab12), Tie2.24 (Ab24), and Tie2.33 (Ab33) all bind to the Tie2 IgG2 domain, block binding of Ang1 and Ang2 to Tie2, and are Tie2 agonists (e.g., enhance AKT and / or Tie2 phosphorylation, and / or enhance vascular endothelial cell membrane integrity).

[0615] Overall, the results indicate that there are at least 3 classes of epitopes bound by the anti-Tie2 antibodies described herein. These at least 3 classes of epitopes are shown in Figure 9 .

[0616] Affinity determination of anti-Tie2 IgG antibodies

[0617] The monovalent affinities of selected anti-Tie2 hlgGs were determined using a Biacore T200 instrument (GE Life Sciences). Anti-human Fc was covalently immobilized on a Series S CM5 Biacore sensor chip to enable non-covalent capture of anti-Tie2 antibodies, and binding of human or rat Tie2 ECD.flag was monitored in real-time at 25°C using the multi-cycle kinetic experiment format. Between cycles, the surface was regenerated using 3M MgCl2. The Biacore evaluation software (GE Life Sciences) was used to fit a 1 :1 binding model to the data, and monovalent affinities were determined by kinetic analysis. Results are summarized in Table 6.

[0618] Table 6

[0619]

[0620]

[0621] Example 5 - In vivo generation of anti-Tie2 antibodies

[0622] In addition to phage libraries, the generation of anti-Tie2 antibodies useful for therapeutic applications is also performed by animal immunization.

[0623] Rabbit immunization. New Zealand white rabbits were immunized with the ECD of human and cynomolgus Tie2 (SEQ ID NO: 1, residues 23-442) and individual B cells were isolated using a modified protocol related to the published literature, see, e.g., Seeber et al., PLoS ONE 9(2), 2014. B cell culture supernatants were analyzed by ELISA for binding to human, rat and cynomolgus Tie2 as well as an irrelevant control protein and by FACS for binding to HUVEC cells. Tie2-specific B cells were lysed and immediately stored frozen at -80°C until molecular cloning. The variable regions (VH and VL) of various monoclonal antibodies from rabbit B cells were cloned into expression vectors from extracted mRNA as described previously, see, e.g., Seeber et al., PLoS ONE 9(2), 2014. Individual recombinant rabbit antibodies were expressed in Expi293 cells and then purified using protein A, and the purified anti-Tie2 antibodies were subjected to functional activity assays and kinetic screening.

[0624] Immunization of rats.Rats were similarly immunized and hybridomas generated using the modified fusion partner (see, e.g., Price et al., J Immunol Methods 31;343(1):28-41 (2009)). Various conditions were optimized to enable sorting of individual IgG+huTie2 fusion partners into individual wells, followed by additional culturing post-sorting. Resulting hybridoma supernatants were analyzed for binding to human, murine, and cynomolgus Tie2, as well as an unrelated control protein by ELISA, and to HUVEC cells by FACS, and positive samples were purified using Protein A for subsequent functional and kinetic characterization.

[0625] Acute functional screening of rat and rabbit clones using an anti-IgG crosslinker.

[0626] The antibodies generated from rat and rabbit immunizations described above were characterized for pAKT induction downstream of Tie2 agonism.

[0627] Cells and Antibodies: Human umbilical vein endothelial (HUVEC) cells were purchased from Lonza (Cat# CC-2517; Lot# 0000321046) and rat aortic endothelial cells (RAEC) were purchased from VEC Technologies and cultured in growth medium (VECTechnologies, INC, Cat# MCDB-13110). Anti-Tie2 antibodies were produced at Genentech. Species-specific antibodies used as cross-linking reagents were purchased from Jackson ImmunoResearch Laboratories Inc.

[0628] Preparation of HUVEC: HUVEC were trypsinized and plated at 0.4 x 10 5 Cells / well were seeded in sterile 96-well plates (Costar, Cat# 3997) in 100 μΐ of medium and the plates were incubated overnight at 37°C, 5% CO2incubator. The medium was removed and 100 μΐ of pre-warmed Basal Medium EndoGRO TM ; Cat# SCME-BM) was added to each well of the plate. The plate was incubated for 3 hours at 37°C, 5% CO2incubator, then incubated with Tie-2 agonist.

[0629] Preparation of RAEC: RAEC were seeded at a density of 12,000 cells / well in 96-well cell culture plates and cultured in 100 μΐ of EGM2 MV medium at 37°C and 5% CO2overnight. Following overnight incubation, the cells were starved for 3 hours in EBM2 basal medium with 0.1% BSA, then incubated with Tie-2 agonist.

[0630] To test the cross-linked anti-Tie2 antibody, 20 pg / ml cross-linker in assay buffer (basal media from GNE media preparation facility + 0.2% BSA) was mixed with an equal volume of 60 pg / ml anti-Tie2 bivalent antibody and incubated at RT for 1 hr. After incubation, the antibody was serially diluted 3-fold.

[0631] To test other Tie2 agonists, these molecules were diluted in assay buffer to the starting stock concentration (typically 30 - 1000 pg / ml) and then serially diluted (typically 2 to 10 fold). After removal of serum-starved media, these dilutions (50 mΐ) were added to each well and the plate was incubated at 37°C, 5% C02 for 15 min. The solution was removed and 50 ul of lysis buffer containing blocking buffer from the pAKT Ser473 kit (Cisbio, ref 64AKSPEH) was added to the cells. The plate was incubated at room temperature for about 30 to 45 min with gentle shaking, stored at -80°C until use, or used directly in the FRET assay.

[0632] FRET assay: Cell lysates (15 mΐ) were thawed on ice and then mixed with 5 mΐ of 1 :40 dilution of each phospho-AKT d2 antibody and phospho-AKT cryptate antibody from the pAKT Ser473 kit in a 384-well microplate (Greiner Bio-One North America, Inc., Cat# 784080) and the plate was incubated at room temperature for 4 hr or overnight at 4°C in the fridge, and read on a CLARIOstar (BMG LABTECH, software version: 5.01 R2) at 620 nm and 665 nm. Data was calculated for each well as the ratio of acceptor to donor emission signals multiplied by 10 4 .

[0633] Recombinant antibody production.

[0634] DNA encoding the heavy chain variable domain and the light chain variable domain were generated by gene synthesis and inserted into mammalian vectors for IgGl antibody heavy chain and light chain expression, respectively. The variable domain sequences were edited to remove apparently unpaired cysteine residues as well as NX[S / T]N-glycosylation motifs. Recombinant antibodies were produced by transient transfection of Expi293 cells using mammalian expression vectors encoding the antibody heavy and light chains. The heavy and light chains were encoded on separate vectors and transfection was performed using a 1 :2 ratio of heavy chain expression vector to light chain expression vector. Antibodies were purified from cell culture supernatant by affinity chromatography. In some cases, antibodies were subjected to an additional purification step based on SEC.

[0635] Antibodies binding to human and cynomolgus macaque Tie2 were screened using a Biacore T200 instrument (GE Life Sciences). Briefly, human antibody capture chips were generated using an S-series CM5 chip, a human antibody capture kit, and an amine coupling kit (GE Life Sciences). Antibodies diluted to 5 μg / ml were captured using a flow rate of 10 μl / min and a contact time of 20 seconds. Binding of 300 nM and 1500 nM recombinant human and cynomolgus macaque Tie2 extracellular domains to the captured antibodies was analyzed at 37°C using a single-cycle kinetic method with a flow rate of 50 μl / min, a contact time of 60 seconds, and a dissociation time of 120 seconds. Between cycles, the chip was regenerated using a 30 μl / min injection of 3 M MgCl2 for 30 seconds. Data were evaluated using Biacore T200 evaluation software (GE Life Sciences). Kinetic constants were obtained using a 1:1 binding model with the parameter RI set to zero. Selected antibodies (Tables 7A-7B; ch indicates rabbit Ab; TEK indicates rat Ab) were subjected to secondary activity screening in the Fab-NDK format (see Example 8 below).

[0636] Table 7A

[0637]

[0638]

[0639] Table 7B

[0640]

[0641]

[0642] HTP epitope classification.

[0643] Antibodies generated by animal immunization as well as the selected phage-derived antibody format described in Example 1 were formalized into a hlgGl backbone and sorted using a CFM2 / MX96 SPR system (Wasatch Microfluidics, now Carterra) equipped with DAv6.19.3, IBIS SUIT, SprintX & Carterra Epitope Tool software. Antibodies were immobilized on SPR sensor prisms CMD 200M (Xantec Bioanalytics) by amine coupling using 10 mM sodium acetate pH 4.5 immobilization buffer. Immobilization was performed using the CFM2 instrument, then the sensor prisms were transferred to the IBIS MX96 instrument for SPR-based competition analysis. Using HBS-EP running buffer (10 mM HEPES, 150 mM NaCl, 0.05% Tween20, pH 7.4, 1 mM EDTA), the immobilized antibodies were first exposed to 1 mM recombinant human Tie2 extracellular domain, then to antibodies in 20 ug / ml solution. Results are shown in Figures 11A to 11B and indicate several different Tie2 sandwich features. Figures 11A to 11B Results in Example 5 are generally interpreted as demonstrating the presence of multiple (e.g., at least 8) different Tie2 epitopes in the analyzed antibody panel. Interestingly, the data show that Tie2.1, Tie2.1M100cF (described in Example 11 below), Tie2.12 and Tie2.24 are all capable of acting as Tie2 agonists, and are grouped together.

[0644] Example 6 - Generation of PEG-conjugated anti-Tie2 Fab multimers

[0645] As shown in Example 3 above, activation of Tie2 by anti-Tie2 antibodies is facilitated by cross-linking to the anti-Tie2 antibodies. In light of this, multimeric Tie2 binding compositions were designed and generated to determine multimeric configurations that would have optimized therapeutic efficacy.

[0646] One multimerization approach employed was the use of a multi-armed polyethylene glycol (PEG) molecule as a core, with each arm linked or conjugated to a single anti-Tie2 Fab molecule. Various multimer configurations were tested using Tie2.1 Fab, which was shown to bind specifically to Tie2 with high affinity and to act as a Tie2 agonist (activating AKT phosphorylation when interacting with Tie2). Specifically, Tie2.1 Fab was modified such that the C-terminus of the heavy chain was modified to include the amino acid residues SPPC (SEQ ID NO: 89), providing a linker and cysteine to which a PEG moiety could be conjugated. This "Tie2.1-SPPC" Fab was conjugated to a PEG-maleimide backbone with variable number and length of arms (multi-armed PEG-maleimide obtained from JenKem Technology USA, Plano, TX).

[0647] Fab purification and deblocking

[0648] All chromatography resins were sourced from GE Healthcare.

[0649] Fabs were expressed in E. coli. E. coli pellets were resuspended in 25 mM Tris HCI, 150 mM NaCI, 5 mM EDTA (EQ) pH 7.5 at 1.5 L / Kg and microfluidized twice at 1000 bar. PEI was added slowly to a final concentration of 0.4% and then stirred overnight at 4°C. The suspension was then centrifuged at 15,000 g for 60 min and the supernatant 0.22 um filtered. The E. coli filtrate was then loaded onto a 1.2 L Gammabind Plus (GBP) column, equilibrated in EQ at 30 ml / min, then washed with 2 CV of EQ at 30 ml / min, then 4 CV of EQ containing 0.1% TX114 and 0.1% TX100 at 5 ml / min. This was followed by a 2 CV wash of EQ at 30 ml / min, then a 2 CV wash of 25 mM succinate pH 6.0 at 30 ml / min, and elution with 0.15 M acetic acid at 30 ml / min. As the eluate flowed off the column, it was neutralized to pH 5.0 with 1 M Tris pH 9.0.

[0650] Alternatively, the E. coli filtrate was loaded onto a 1.7 L Capto L column, equilibrated in EQ at 35 ml / min, then washed with 2 CV of EQ at 50 ml / min, 5 CV of EQ containing 0.1% (v / v) Triton X114 and 0.1% (v / v) Triton X100 at 4 ml / min, then 50 ml / min 2 CV EQ, then 25 mM succinate pH 6.0 at 50 ml / min, and eluted with 0.15 M acetic acid at 50 ml / min. As the eluate flowed off the column, it was neutralised to pH 5.0 with 1 M Tris pH 9.0.

[0651] The neutralised GBP or Capto L eluate was diluted one to three times with 20 mM sodium acetate pH 5.0 (A) and loaded onto a cation exchange resin (SPHP). The column was washed with 2 CV (A) 5 CV (A) containing 0.1% Triton X114 and 0.1% Triton X100, followed by 2 CV (A), then gradient eluted using a 10 CV 0-20% gradient of (A) containing 1 M NaCl (B) 0-20%, collecting fractions. Fractions containing the Fab peak were pooled.

[0652] The purified Fab pool was adjusted to pH 8.0 using 1 M Tris pH 8.5 and EDTA was added to a final concentration of 2 mM. To reduce the Fab, a 50-fold excess of DTT was added to the solution which was then incubated overnight at 22°C and checked for the removal of adducts by mass spectrometry.

[0653] After adjusting the pH to 5.2 using 10% acetic acid, the reduced Fab was bound to the SPHP, washed with 10 CV of 25 mM sodium acetate pH 5.0, and eluted with 50 mM Tris, 150 mM NaCl pH 8.0. The eluted Fab pool was brought to pH 8.0 using 1 M Tris pH 8.5, 2 mM EDTA was added, and re-oxidation was performed using a 15-fold molar excess of DHAA. The Fab was checked for re-oxidation by mass spectrometry 1.5 hours after; if necessary, a further 10-fold molar excess of DHAA was added, the sample was incubated for 1 hour and checked again. The re-oxidised Fab was then adjusted to pH 5 using 10% acetic acid and purified on a SPHP column as described above, but eluted using a 20 CV 10-60% gradient (B = 25 mM sodium acetate, 300 mM sodium chloride, pH 5.0), the main peak was 10 kD concentrated and 0.2 um filtered. Other Fabs can be purified and de-blocked in essentially the same way.

[0654] Hexamer conjugation and purification

[0655] Tie2.1-SPPC was conjugated to 6KDa PEG hexamer from JenKem at a concentration of around 10 mg / mL in 25mM NaOAC, 150mM NaCl, 2mM EDTA at pH 5.0. After equilibration to room temperature, 6KDa PEG hexamer from JenKem was suspended in 25mM sodium acetate at pH 5.0 at a concentration of 3mM. The pH was kept below pH 6 to avoid maleimide ring opening. Once the PEG was solubilized, it was added to the blocked Fab of Tie2.1-SPPC at a molar ratio of 9: 1 (Fab:PEG). The mixture was then left overnight at room temperature with gentle shaking. After conjugation, Tie2.1 Fab PEG hexamer was purified using size exclusion chromatography (SEC) on a Superdex-200 column in 20mM His-acetate, 150mM NaCl at pH 5.5, which removed excess Fab and aggregates from the conjugation mixture. A NuPage 4-12% Bis tris gel was run without reducing agent to determine which conjugate fractions needed further purification to enrich for hexamers. Conjugate fractions were pooled and further purified using cation exchange (CEX) with SP Sepharose High Performance strong cation exchange resin from GE to enrich for 6 Fab / PEG. CEX was run in 25mM sodium acetate at pH 5.0 and a gradient of 20% to 33.4% in buffer B (buffer B is 25mM NaOAC, 300mM NaCl at pH 5.0) was eluted at 44.5 CV, then a gradient of 33.4% to 50% B was eluted for 10 min and finally eluted with 100% B. Mini-pools of a few different fractions were run on a gel as described above and the data was used to decide which fractions to pool. Mini-pool 3 was pooled and formulated at 40 mg / mL in PBS at pH 7.2.

[0656] The final sample was then run on analytical SEC using a TSKgel G3000SW xl column with 0.2M KPO4, 0.25M KCl, 15% isopropanol at pH 6.2 as buffer to determine the percentage of aggregates present. It was also run on a gel as described earlier.

[0657] FabIgG purification

[0658] CHO conditioned media was purified over a MabSelect Sure affinity column (GE Healthcare) followed by size exclusion chromatography (SEC) S200 column. Alternatively, affinity eluate was diluted and loaded onto a cation exchange column (SPHP), then washed and eluted with a salt gradient. Purified monomeric peaks were then concentrated and dialyzed into formulation buffer and 0.2um sterile filtered.

[0659] Comparison of FabIgG and hexameric pAKT activities

[0660] Tie2.1.38 is a bi-epitope Fab-IgG that activates Tie2 (HC is provided herein as SEQ ID NO 54, LC is provided herein as SEQ ID NO 53). Experiments were performed to assess the ability of Tie2.1 in PEG-hexamer form (6kDa PEG attached to the C-terminus of HC via SPPC) and Tie2.1.38 (cross-linking provided by IgG format). Higher potency activation was observed with Tie2.1-PEG-hexamer Figure 12A ) than with Tie2.1.38.

[0661] Agonist activity of anti-Tie2 Fab polymerized with multi-arm PEG

[0662] PEG-multimers were incubated with RAECs at 10μg / ml and changes in pAKT levels assessed as described (Example 3). There was a tendency for more arms and / or shorter arms to have higher activity, with the 6-arm, 6kDa core and 8-arm, 20kDa core showing near maximal activity Figure 12B ).

[0663] Example 7 - Multimerization of anti-Tie2 Fabs using IgM format

[0664] Another approach to multimerize anti-Tie2 Fabs was developed using the IgM format. DNA encoding the VH from Tie2.1 was fused to IgM CH1. Without being bound by theory, it is believed that the close association of multiple variable fragments (Fv) enables IgM to bind targets without the need for affinity maturation (Boes, 2000, Mol Immunol, 37:1141-1149).

[0665] Optimization of IgM expression and purification.

[0666] Human IgM (huIgM) was expressed in 30 ml of Expi293 cell cultures as recommended by the manufacturer (e.g., see “Expi293 Expression System, User Guide”, print code MAN0007814 from Thermo Fisher Scientific). Various ratios of IgM heavy chain to light chain (hexamer) or heavy chain to light chain to J chain (pentamer) were transfected into the cells. The supernatant was affinity purified using Capto L resin (GE healthcare) according to the manufacturer’s recommendations (e.g., see Lombana et al., 2019, mAbs, 11:1122-1138). Total protein yield was assessed by A280 and purity by analytical SEC using a Waters Xbridge BEH SEC 450 Angstrom column. Figure 13A The effect of various ratios of huIgM heavy chain to light chain to J chain (if included) on total protein yield from the affinity column was shown.

[0667] Hexamer optimization was continued by transfecting cells with plastids containing LC and HC at a ratio of 1:1 or 2:1 with plastid DNA. Each ratio resulted in similar total protein yields (2.5-3 mg from 30 ml expression), however, the quality of the product appeared to be improved at the 1:1 ratio and the amount of LC dimer present was reduced. Expression of putative pentamers was performed by including J chain in the transfection. At the optimal ratio of 4:4:1 LC:HC:JC, both expression and product quality were maximized 13A to 13B It is notable that at the optimal conditions, the high levels of LC and HC DNA relative to JC approximated the 10:10:1 chain ratio in the final IgM pentamer.

[0668] Since the expression of pentameric and hexameric IgM had an optimized chain ratio, the expression and purification of human and murine hexameric and pentameric IgM was scaled up. Approximately 20 mg / L of purified hexameric or pentameric IgM was isolated from CHO cells via a two-step purification. Although the transient yield of IgG expressed in this process was 3- to 4-fold lower than typical, the relatively easy purification together with the low material requirements for ocular drugs should facilitate the feasibility of manufacturing. The chain composition of human IgM was confirmed by SDS-PAGE and LC-MS of the reductant deglycosylated material showed the presence of LC, HC, and JC if present. Size exclusion chromatography showed monodisperse peaks corresponding to pentameric and hexameric murine IgM and a monodisperse peak corresponding to hexameric human IgM. Pentameric human IgM eluted from SEC in two close related peaks, both containing J chain and having a high SEC expression molecular weight. The hydrodynamic radius (R h) was approximately 12 nM, and the predicted molecular weight of the hexamer (~1050 kDa) was slightly higher than those of the pentamer (~950 kDa) (Table 9 below).

[0669] Table 9

[0670]

[0671]

[0672] The ratio of the gyration radius to the hydrodynamic radius (Rg / Rh) provides some insight into the shape of the molecule, with spherical molecules averaging ~0.775, while more elongated molecules tend to be higher. The polydispersity of the monodisperse IgM was in the range of 0.85-0.91, consistent with the expected structure. To further assess the polymerization signature of IgM, negative stain TEM and reference-free 2D classification were used to describe the architecture of IgM particles, and the results appeared to match the expected geometry of the particles. No significant differences were observed between the two peaks corresponding to putative human pentamers. The human IgM hexamer peak was selected for further exploration. A total of 1500 particles were obtained from 110 TEM micrographs collected manually. After three rounds of reference-free 2D classification using the Relion suite (Scheres, 2015, J Struct Biol, 189:114-122), it was found that the majority of the particles organized into hexameric structures. A small number of particles (<2%) were refined as pentamers. Consistent with previous structural exploration of IgM pentamers (Czajkowsky and Shao, 2009, Proc Natl, Acad Sci USA, 106:14960-14965), the CH3 and CH4 domains appeared to form a stalk-like structure orthogonal to the imaging plane. Furthermore, while the pairs of Fabs attached to the same Fc appeared to be co-planar, there were some landmarks that indicated the Fabs on adjacent Fcs were offset. The partial offset can allow the hexamer to accommodate all the chains in a spatially crowded environment. Based on these data, human hexameric IgM was selected as the preferred therapeutic scaffold, as both the improved production purity and the absence of JCs simplify manufacturing and avoid the need to consider polymeric Ig receptor-mediated binding in vivo.

[0673] Characterization of IgM for intraocular administration

[0674] Subsequently, the suitability of IgM for ocular applications was explored using New Zealand white rabbits as a model system. PK parameters in rabbits have previously been correlated with results in cynomolgus monkeys and humans. To minimize the risk of anti-drug antibodies interfering with the pharmacokinetic assessment of IgM, whole rabbit IgM hexamers (rabbit IgM Fc with a targeting intracellular location rabbit Fv) were generated. To avoid frequent repeated injections, the amount of protein injected, and thus the concentration of protein injected, was maximized. In this regard, protein viscosity is of considerable importance. The viscosity of IgM hexamers was assessed and compared to other multivalent compounds that have been explored previously (Shatz et al., 2019, PLoS ONE, 14: e0218613-e0218613), specifically, 8-arm 40 kDa branched PEG and four-arm 20 kDa branched PEG terminated with 8 and 4 Fabs, respectively. Figure 13C ) The viscometer readings for all three substances showed the expected log-linear response. At viscosities that allow for easy intravitreal administration without patient pain or excessive syringe back pressure (~10-50 cP), approximately twice the mass of IgM can be administered compared to other formats.

[0675] SEC-MALS analysis of rabIgM found a hydrodynamic radius of 12.9 + / - 1.1 nM, significantly exceeding the 2.5 nM radius observed previously for Fabs. To assess the functional consequences of this increase in molecular size, rabbit Fabs and IgM were labeled with Alexafluor 488 and 0.5 mg or 1.2 mg, respectively, were administered intravitreally to New Zealand white rabbits. In vivo quantitative fluorescence measurements were taken over 28 days, indicating sustained exposure of each of the two therapeutic agents. Figure 13D ) In this experiment, the Fab showed a half-life of 3.5 days, comparable to the typical half-life of ~3.4 days observed for other Fabs in the rabbit vitreous (Crowe...

Claims

1. An isolated antibody or antigen-binding fragment thereof that specifically binds to Tie2, wherein the antibody or fragment thereof comprises: a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of NTDIS (SEQ ID NO: 3), (b) CDR-H2 comprising the amino acid sequence of RISPSDGNTYYADSVKG (SEQ ID NO: 4), and (c) CDR-H3 comprising the amino acid sequence of RTRWASX1AX2DY (SEQ ID NO: 5), wherein X1 is M, L, K, F, Y, R, N, Q, H or W and / or X2 is F, Y, L, Q, I, K or H; and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of RASQDVSTAVA (SEQ ID NO: 8), (e) CDR-L2 comprising the amino acid sequence of SASFLYS (SEQ ID NO: 9), and (f) CDR-L3 comprising the amino acid sequence of QQSYTTPPT (SEQ ID NO: 10).

2. A conjugate that binds to Tie2, wherein the conjugate comprises at least two, at least three, at least four, at least five, at least six, at least seven or at least eight antibodies or antigen-binding fragments thereof according to any one of claims 1 to 18, wherein each of the antibodies or antigen-binding fragments thereof is linked to a multimerization moiety.

3. A Tie2-binding conjugate comprising: A Fab comprising an HC having SEQ ID NO: 55 and an LC having SEQ ID NO: 25; conjugated to a polyol having the structure of Formula 1(b): wherein each m is independently an integer from 20 to 30, wherein R 1 and R 2 Taken together, they have structure where R 2 is diamine maleate; and wherein the polyol is linked to the Fab HC at residue C209 (EU numbering).

4. A pharmaceutical composition comprising: the conjugate according to claim 2 or 3; and a pharmaceutically acceptable carrier.

5. The pharmaceutical composition of claim 4, further comprising an additional therapeutic agent, wherein the additional therapeutic agent is selected from the group consisting of a VEGF antagonist, an Ang2 antagonist, an HtrA1 antagonist, and an IL33 antagonist, a complement component antagonist, and a second Tie2 agonist.

6. A long-acting delivery device for transocular delivery, comprising a pharmaceutical composition according to claim 4 or 5 and means for delivering the composition intravitreally to a patient, wherein the composition remains effective in situ for an extended period of time.

7. A method for treating a Tie2 pathway-mediated disease in a subject in need thereof, comprising administering to the subject an effective amount of the antibody or antigen-binding fragment thereof according to claim 1, the conjugate according to claim 2 or 3, or the pharmaceutical composition according to claim 4. The method according to claim 7 , wherein the disease mediated by the Tie2 pathway is a vascular permeability disease.

9. The method according to claim 7 or 8, wherein the disease mediated by the Tie2 pathway is an ocular disorder.

10. Use of the antibody according to claim 1 or the conjugate according to claim 2 or 3 in the preparation of a medicament for treating a Tie2 pathway-mediated disease and / or an ocular disease in a subject in need thereof.

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