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 widely studied Tie2 ligands are angiopoietin 1 (Ang1) and angiopoietin 2 (Ang2). Ang1 is a strong Tie2 agonist and has been shown to inhibit ocular neovascularization and the 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 increased expression of Ang2 is associated with a variety of ocular diseases, including DME, wet AMD, DR, metastasis, sepsis, and inflammation. In addition, Ang2 competitively binds to Tie2 and inhibits Ang1 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 Tie1 and Tie2, are type 1 transmembrane protein receptor tyrosine kinases (RTKs) (Ramsauer, M. & D'Amore, PA J Clin. Invest. (2002); 110: 1615–1617). Tie represents a tyrosine kinase receptor with immunoglobulin and EGF homology domains. Tie2 is located in all vascular endothelial cells and in the endocardium of mouse embryos (Korhonen et al., Blood (1992); 80: 2548–2555). The extracellular domain or extracellular domain (ECD) of Tie2 includes three immunoglobulin (Ig) domains (Ig1, Ig2, and Ig3), three epidermal growth factor (EGF) domains, and a fibronectin type III domain (FNIII). The Ig-EGF region of Tie2 mediates angiogenin recognition and binding (Fiedler, U. et al., J. Biol. Chem. (2003); 278: 1721-1727; Barton, WA, 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 to 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) 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), (b) RTRWASWAMDY (SEQ ID NO: 6) or (c) RTRWASWAFDY (SEQ ID NO: 7); and the VL domain comprises: a CDR-H3 comprising the amino acid sequence of RASQDVSTAVA (SEQ ID NO: 8). In some embodiments, the CDR-H3 comprises the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 7. In certain embodiments, the CDR-H3 comprises the amino acid sequence of 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 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), (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, CDR-H3 comprises SEQ ID NO: 6 or SEQ ID NO: 7. In specific embodiments, CDR-H3 comprises SEQ ID NO: 7. In other embodiments, the VH domain is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:20, and the VL domain is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:21.

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

[0017] In some embodiments, the anti-Tie2 antibody comprises the VH domain sequence of SEQ ID NO: 20. In other embodiments, the anti-Tie2 antibody comprises the VL domain sequence of SEQ ID NO: 21. In other embodiments, the anti-Tie2 antibody comprises the VH domain sequence of SEQ ID NO: 20 and the 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, an antibody or antigen-binding fragment thereof that specifically binds Tie2 is provided, 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; and 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, an antibody or antigen-binding fragment thereof that specifically binds Tie2 is provided, 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; and 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 Tie2 is provided, which comprises two VH domains and two VL domains, the VH domain comprising: in the 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 the 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 located 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 IgG1 or 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 a preferred embodiment, the anti-Tie2 antibody or fragment thereof is Fab.

[0030] In some embodiments, the anti-Tie2 antibody is an antibody or Fab that competes with an anti-Tie2 antibody comprising the VH sequence of SEQ ID NO: 22 and the 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 monkey Tie2. In other embodiments, the cynomolgus monkey 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, methods are provided for preparing antibodies or antigen-binding fragments thereof that bind to Tie2. In some embodiments, the methods comprise culturing a host cell containing a nucleic acid encoding the anti-Tie2 antibody under conditions suitable for expression of the anti-Tie2 antibody. In other embodiments, the methods further comprise recovering the anti-Tie2 antibody from the host cell.

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

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

[0040] In some embodiments, the conjugate comprises an anti-Tie2 antibody that binds to Tie2, and a multi-arm portion, wherein the multi-arm portion is linked to at least two anti-Tie2 Fabs. In some embodiments, the multi-arm portion is linked to at least two, three, four, five, six, seven, eight, nine, or ten anti-Tie2 Fabs. In other embodiments, the multi-arm portion is linked to two, three, four, five, six, seven, eight, nine, or ten anti-Tie2 Fabs. In other embodiments, the multi-arm portion is linked to six or eight anti-Tie2 Fabs. In other embodiments, the multi-arm portion is linked to eight anti-Tie2 Fabs. In preferred embodiments, the multi-arm portion is linked to six anti-Tie2 Fabs.

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

[0042] In some embodiments, the Tie2-binding conjugate activates AKT phosphorylation. In other embodiments, activation of AKT phosphorylation is demonstrated by an increase in phosphorylated AKT protein in an in vitro assay. In other embodiments, the Tie2-binding agent activates Tie2 phosphorylation. In other embodiments, 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 polyethylene glycol (PEG). In some embodiments, the PEG is a functionalized multi-arm PEG.

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

[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 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.

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

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

[0058] In some embodiments, the PEG has the structure of formula (Ia), wherein n is 2, and the multi-arm PEG is a hexamer. In such embodiments, the hexamer has the structure of formula (Ib):

[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; and 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 2is a terminal reactive group and is covalently linked to the 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 from about 22.

[0061] In some embodiments, R 1 With R 2 Together with structure Among them, R 2 is diamine maleate;

[0062] In some embodiments, the PEG has the structure of formula (Ia), wherein n is 3, and the multi-arm PEG is an octamer. In such embodiments, the octamer has the structure of formula (Ic):

[0063]

[0064] wherein each m is independently an integer from 3 to 250; 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 and is covalently linked to the 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-arm polyol. In other embodiments, the multi-arm polyol of the conjugate is covalently linked to the at least two anti-Tie2 antibody fragments or Fabs via a free sulfhydryl 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 its HC and / or LC. 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 numbering of the engineered cysteine ​​is according to EU numbering.

[0067] In some embodiments, the conjugate comprises a multi-arm polyol covalently linked to the at least two anti-Tie2 antibody fragments or Fabs via free amino groups of lysine amino acids. In other embodiments, the lysine amino acids are located in the constant region of the anti-Tie2 antibody fragments or Fabs. In other embodiments, the lysine amino acids are located at the C-terminus of the heavy or light chain of the anti-Tie2 antibody fragments or Fabs. In alternative embodiments, the Tie2 binder is not covalently linked to the at least two anti-Tie2 Fabs via free amino groups of lysine amino acids.

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

[0069] In some embodiments, the conjugate is stable over extended 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 is provided comprising an anti-Tie2 antibody or antigen-binding fragment thereof and a multi-arm portion, wherein the multi-arm portion comprises an IgM molecule. In other embodiments, the IgM molecule comprises a J chain, and the multi-arm portion comprises five anti-Tie2 Fabs, wherein approximately each of the five anti-Tie2 Fabs is linked to the IgM molecule. In other embodiments, the IgM molecule does not comprise a J chain, and the multi-arm portion comprises six anti-Tie2 Fabs, wherein each of the six 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 a preferred embodiment, the IgM variant comprises a P436G substitution based on EU numbering.

[0072] In one aspect, a conjugate is provided comprising an anti-Tie2 antibody or antigen-binding fragment thereof and a multi-arm portion, wherein the multi-arm portion comprises at least 2, 4, 6, 8 or 10 peptides, wherein approximately each of these peptides is covalently linked to an anti-Tie2 Fab. In some embodiments, the anti-Tie2 Fab sequence ends at residues 221, 222, 223, 224 or 225 (EU numbering). In other embodiments, each of these peptides is a nucleoside diphosphate kinase (NDK) peptide. In other embodiments, each of these NDK peptides comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% identity 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 or light chain. In other embodiments, a linker is present between the Fab heavy 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 portion comprises 6 or 8 peptides. In other embodiments, the multi-arm portion comprises 6 peptides. In a preferred embodiment, the multi-arm portion comprises 6 NDK peptides.

[0075] In one aspect, a conjugate is provided comprising an antibody or antigen-binding fragment thereof and a multi-arm portion, wherein the multi-arm portion comprises an IgM molecule. In other embodiments, the IgM molecule comprises a J chain, and the multi-arm portion comprises 5 antibody fragments, Fabs, or antigen-binding fragments thereof, wherein approximately each of the 5 antibody fragments, Fabs, or antigen-binding fragments thereof is connected to the IgM molecule. In other embodiments, the IgM molecule does not comprise a J chain, and the multi-arm portion comprises 6 antibody fragments, Fabs, or antigen-binding fragments thereof, wherein each of the 6 antibody fragments, Fabs, or antigen-binding fragments thereof is connected 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 a preferred embodiment, the IgM variant comprises a P436G substitution based on EU numbering.

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

[0078] In some embodiments, each of these NDK peptides is connected at its N-terminus to the C-terminus of the heavy or light chain of the antibody, antibody fragment, Fab or its antigen-binding fragment. In other embodiments, a linker is present between the Fab heavy 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 portion comprises 6 or 8 peptides. In other embodiments, the multi-arm portion comprises 6 peptides. In a preferred embodiment, the multi-arm portion comprises 6 NDK peptides.

[0080] In one aspect, a pharmaceutical composition is provided, 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 binder 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 binder 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 aforementioned 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 aforementioned pharmaceutical compositions can be used to reduce or inhibit pathological vascular permeability in a subject suffering from 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, a method for treating an individual in need of treatment is provided, comprising administering to the patient an anti-Tie2 antibody and / or Tie2 binder as described herein. In some embodiments, the method comprises 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 invention features a method of inhibiting vascular permeability in a patient suffering from a disease associated with improper vascular permeability, the method comprising administering to the subject an effective amount of any of the aforementioned antibodies or conjugates, thereby inhibiting vascular permeability in the subject.

[0089] In another aspect, the invention features a method of treating a disease associated with improper 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, pathological myopia, Hippel-Lindau disease, ocular histoplasmosis, central retinal vein occlusion (CRVO), corneal neovascularization, glaucoma, retinopathy without edema, and retinal neovascularization.

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

[0094] Figure 1 A schematic diagram of the Tie2 domain used in panning the VH library is provided.

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

[0096] Figures 3A to 3B 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 ) and human Tie1 binding assay results.

[0097] Figures 4A to 4B Evaluation of the effect of anti-Tie2 antibodies Tie2.1, Tie2.12, and Tie2.20 on the interaction between Tie2 and Ang1 is shown. Figure 4A ) and the interaction between Tie2 and Ang2 ( Figure 4B )’s measurement results.

[0098] Figures 5A to 5B Figure 2 shows the evaluation of the binding of anti-Tie2 antibodies Tie2.1, Tie2.10, Tie2.11 and Tie2.12 to HUVEC ( Figure 5A ) and RAEC ( Figure 5B )’s measurement results.

[0099] Figures 6A to 6B The results show that the anti-Tie2 antibodies Tie2.1, Tie2.4, Tie2.5 and Tie2.20 activated AKT phosphorylation in RAECs ( Figure 6A ) and to evaluate the effect of anti-IgG cross-linking of anti-Tie2 antibodies on AKT phosphorylation ( Figure 6BThe level of phosphorylated AKT was determined by Western blot analysis.

[0100] 7A to 7B Results of assays evaluating activation of AKT phosphorylation by 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 are shown ( Figure 7A ). Figure 7B The effect of anti-Tie2 antibody aggregation or anti-IgG cross-linking on the activity of the 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 A method for classifying anti-Tie2 antibodies using ELISA is shown ( Figure 8A ) and results ( Figure 8B ).

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

[0103] Figure 10A and Figure 10B The heavy chain variable regions (VH) of the 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) are shown ( Figure 10A ) and the light chain variable regions (VL) of anti-Tie2 antibodies Tie2.1 (SEQ ID NO: 21), Tie2.1.M100cF (SEQ ID NO: 21), Tie2.12 (SEQ ID NO: 21), Tie2.24 (SEQ ID NO: 21), Tie2.33 (SEQ ID NO: 21) and Tie2.38 (SEQ ID NO: 21) ( Figure 10B ) amino acid sequence alignment.

[0104] Figures 11A to 11B Shown are the results of sorting assays using various anti-Tie2.1 antibodies generated via phage display or animal immunization. Figure 11A A list of covalently immobilized antibodies and several of these antibodies in solution is provided, while Figure 11B The remaining listings in this series of antibodies in solution are provided.

[0105] FIG. 12A to FIG. 12B Shown are the results of assays evaluating the agonist activity of multimeric anti-Tie2 antibodies. Figure 12A The AKT phosphorylation activities of Tie2.1 as a PEG hexamer and as a bi-epitope Fab IgG (1.38) were compared. Figure 12B Multimeric forms of anti-Tie2 antibodies were compared.

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

[0107] Figure 13C Shown are rheological measurements of IgM compared to an ocular-targeted multivalent PEG form of factor D (fD).

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

[0109] Figure 13E Shown is the systemic pharmacokinetic analysis of IgM comparing non-binding recombinant hIgM pentamer and recombinant hIgM hexamer to IgM isolated from human serum following intravenous injection into female SCID mice. Figure 13E Serum IgM levels are shown.

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

[0111] Figures 13G to 13H Shown are the results of an assay characterizing anti-Tie2 antibodies using characteristic IgM multimeric forms. Figure 13G Shown are the results of complement assays evaluating mutations in the IgM constant domain. Figure 13H Agonist activity of anti-Tie antibodies in the form of IgM hexamer is shown.

[0112] FIG. 14A to FIG. 14B Shown is the design and analysis of hexameric forms of anti-Tie2 antibodies linked via peptide moieties. Figure 14A Schematic diagram designed for the multimer. Figure 14B Results of an AKT phosphorylation assay are shown, comparing hexameric forms of anti-Tie2 antibodies linked via NDK peptide, IgM, and multi-arm PEG.

[0113] FIG. 15A to FIG. 15B Results of AKT phosphorylation assays evaluating the agonist activity of various anti-Tie2 antibodies in hexameric form are shown. Results of AKT phosphorylation assays evaluating the agonist activity of various anti-Tie2 antibodies in hexameric form are shown. Figure 15A ) and Tie2.1, Tie2.1.M100cF, Tie2.12, Tie2.24( Figure 15B ) results.

[0114] 16A to 16C Shown are the results of assays evaluating the effects of anti-Tie2 antibodies on cellular levels of Tie2 protein in an in vitro assay. Figure 16A 、 Figure 16B and Figure 16C Tie2 levels were compared when HUVEC were exposed to anti-Tie2 antibodies generated via phage display and animal immunization, respectively. Figure 16A 、 Figure 16B and Figure 16C All assays in were analyzed by Western blotting.

[0115] Figure 17 Shown are the results of in vivo assays evaluating the effects of anti-Tie2 antibodies on the cellular level of Tie2 protein levels.

[0116] 18A to 18B The assay method of in vitro endothelial cell assay to study the effect of anti-Tie2.1 antibodies on endothelial cell barrier permeability is shown ( Figure 18A ) and results ( Figure 18B ).

[0117] Figures 19A to 19B The method of in vivo vascular permeability assay for studying the effect of anti-Tie2.1 antibody on VEGF-induced vascular leakage is shown ( Figure 19A ) and results ( Figure 19B ).

[0118] FIG. 20A to FIG. 20B The method of in vivo vascular permeability assay for studying the effect of anti-Tie2.1 antibody or anti-VEGF antibody on VEGF-induced vascular leakage is shown ( Figure 20A ) and results ( Figure 20B ).

[0119] Figures 21A to 21B Shown are the in vivo effects of a bi-epitope anti-Tie2 agonist (anti-Tie2Fab-IgF 1.38) on the protein level of Tie2 as determined by Western blot analysis ( Figure 21A ), and in vivo vascular permeability assay results to investigate the effect of anti-Tie2 Fab-IgG 1.38 on VEGF-induced vascular leakage ( Figure 21B ).

[0120] Figure 22 Shown are the effects of anti-Tie2 agonists on the cellular organization of VE-cadherin (upper panel) and F-actin (lower panel) in cultured HUVECs.

[0121] Figure 23 Shown are the results of an AKT phosphorylation assay evaluating the agonist activity of the IG1 form of the Tie2.1 anti-Tie2 antibody variant.

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

[0123] Figure 25 Shown are the pharmacokinetics of an anti-Tie2 Fab hexamer conjugate following ocular injection into cynomolgus monkeys.

[0124] Figures 26 to 27 Electropherogram analysis of anti-Tie2.1 PEG conjugates is shown, where the monomer peak is counted ( Figure 26 ) or not included ( Figure 27 ) in relative quantification.

[0125] Figure 28 Shown is an electropherogram analysis of anti-Tie2.1 PEG conjugates in samples taken from the vitreous humor of cynomolgus monkeys.

[0126] Figure 29 The relative amounts of anti-Tie2 PEG conjugate hexamers and pentamers are shown.

[0127] FIG. 30A to FIG. 30B Provided are samples taken from the eyes of cynomolgus monkeys over a period of 21 days ( Figure 30B ) in M100c oxidation ( Figure 30A ) data.

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

[0129] Figures 32 to 33 The effect of deconjugation on pAKT activity was shown ( Figure 32 ), the standardized values ​​are shown in Figure 33 middle.

[0130] Figure 34 The effect of the position of the PEG conjugation site on deconjugation is shown.

[0131] Figure 35A The effect of the position of the PEG conjugation site on activity is shown.

[0132] Figure 35B The effect of PEG conjugation site location on activity is shown.

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

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

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

[0136] Figure 39 The structure of the 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 alterations 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 of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity," as used herein, refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed in terms of a dissociation constant (Kd). Affinity can be measured by conventional 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 binds to Tie2 with sufficient affinity so that the antibody can be used as a therapeutic and / or diagnostic agent targeting Tie2. In one embodiment, the extent to which the anti-Tie2 antagonist antibody binds to unrelated, non-Tie2 proteins is less than about 10% of the binding of the antibody to Tie2, as measured by, for example, radioimmunoassay (RIA). In certain embodiments, the dissociation constant (K) of the antibody binding to Tie2 is D ) is ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM or ≤0.001 nM (e.g., 10 -8 M or lower, such as 10 -8 M to 10 -13 M, for example 10 -9 to 10 -13 M). In certain embodiments, the anti-Tie2 antagonist antibody binds to an epitope of Tie2 that is conserved among Tie2 of different species.

[0144] The term "antibody" herein is used in the broadest sense and encompasses 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] An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds to 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 antibody molecules (e.g., scFv), and multispecific antibodies formed from antigen fragments.

[0146] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure or an antibody 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 comprises 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 contain a CH2 domain or a CH3 domain. As used herein, Fab may comprise one or more amino acids in the hinge region. Therefore, as used herein, the term "Fab" encompasses Fab' antibodies. Fab may comprise additional non-natural amino acids, such as a C-terminal cysteine, in which case it may be referred to as Fab-C. As described below, the term Fab-C also encompasses Fab comprising natural amino acids in the hinge region (including natural cysteine ​​at the C-terminus). In some embodiments, Fab comprises engineered cysteine ​​(i.e., Fab may be THIOMAB). In some embodiments, engineered cysteine ​​is a cysteine ​​amino acid residue in the Fab HC and / or LC polypeptide sequence that is substituted with a non-cysteine ​​amino acid residue.

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

[0149] "Fab-SH" refers to a Fab with a free sulfhydryl group. In some embodiments, the free sulfhydryl group is located at the last 10 amino acids of the C-terminus of the Fab. Fab-C antibodies are also commonly referred to as Fab-SH antibodies.

[0150] An antibody that "binds to the same epitope as a reference antibody" refers to an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competition assay, and conversely, the reference antibody blocks the binding of the antibody to its antigen by 50% or more in a competition assay. An exemplary competition 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 domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of those can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0153] The term "conjugate" as used herein, according to its broadest definition, means joined or linked together. When molecules act or operate as if they were joined together, the molecules are "conjugated". A "conjugate" is an antibody (e.g., Fab) conjugated to one or more heterologous molecules, which include but are not limited to polyols. In specific embodiments, a "conjugate" refers to an antibody (e.g., an antibody fragment, as described in detail herein) covalently bound to a multi-arm portion. In specific embodiments, the multi-arm portion is a peptide in the form of a polyol, an IgM molecule, or a polymer (e.g., a hexamer).

[0154] Unless otherwise indicated, the term "Tie2" as used herein 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 obtained in cell manipulation. The term also encompasses naturally occurring Tie2 variants, such as splice variants or allelic variants. The amino acid sequence of an exemplary human Tie2 protein has the NCBI reference number NP_000450 (SEQ ID NO: 1).

[0155] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cell function and / or causes cell death or destruction. 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 radioisotopes of Lu); 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 nucleases; antibiotics; toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof, as well as various anti-tumor or anti-cancer agents disclosed below.

[0156] "Effector function" refers to those biological activities attributed to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation 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 the C-terminal region of an immunoglobulin heavy chain comprising at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the 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) in the Fc region may or may not be present. Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region or constant region is carried out according to the EU numbering system (also referred to as the EU index), as described by Kabat et al. (Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service, National Institutes of Health, Bethesda, MD, 1991) (see also above).

[0159] "Framework" or "FR" refers to the variable domain residues excluding the hypervariable region (HVR) residues. The FR of a variable domain is typically composed of four FR domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences typically appear in the following order in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

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

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

[0162] "Human consensus framework" is a framework that represents the most common amino acid residues in a series of human immunoglobulin VL or VH framework sequences. Typically, 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 described by Kabat et al. in Sequences of Proteins of Immunological Interest (5th Edition, NIH Publication 91-3242, Bethesda MD (1991), Volumes 1-3). In one embodiment, for VL, the subgroup is subgroup κI as described by Kabat et al. in the aforementioned literature. In one embodiment, for VH, the subgroup is subgroup III as described by Kabat et al. in the aforementioned literature.

[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 include substantially all of at least one (and typically two) variable domains, wherein all or substantially all HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all FRs correspond to those of a human antibody. A humanized antibody optionally may 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 chain 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 natural antibodies 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., WH Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, VH or VL domains can be used to separate antibodies that bind to a specific antigen from antibodies that bind to 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] As used herein, the term "hypervariable region", "HVR" or "HV" refers to a region of an antibody variable domain whose sequence has high variability (also referred to herein as "complementarity determining region" or "CDR") and / or forms a structurally defined loop. In general, an antibody comprises six HVRs; three in VH (H1, H2, H3) and three in VL (L1, L2, L3). In natural antibodies, H3 and L3 show the most diversity among the six HVRs, and H3 in particular is believed to play a unique role in conferring excellent specificity on antibodies. See, for example, Xu et al., Immunity 13:37-45 (2000); Johnson and Wu, in Methods in Molecular Biology 248:1-25 (Lo, ed., Human Press, Totowa, NJ, 2003). In fact, in the absence of light chains, natural camelid antibodies composed only of heavy chains have function and stability. See, eg, Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).

[0166] Many 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 Edition. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia refers to the positions of structural loops instead (Chothia and Lesk J. Mol. Biol. 196: 901-917 (1987)). AbMHVRs represent intermediates between Kabat HVRs and Chothia structural loops and are adopted by Oxford Molecular's AbM antibody modeling software. The "contact" HVRs are based on analysis of available complex crystal structures. The residues of each of these HVRs are as follows.

[0167] RingKabat AbM Chothia Contact

[0168] L1 L24-L34 L24-L34 L26-L32 L30-L36

[0169] L2 L50-L56 L50-L56 L50-L52 L46-L55

[0170] L3 L89-L97 L89-L97 L91-L96 L89-L96

[0171] H1 H31-H35B H26-H35B H26-H32 H30-H35B (Kabat numbering)

[0172] H1 H31-H35 H26-H35 H26-H32 H30-H35 (Chothia numbering)

[0173] H2 H50-H65 H50-H58 H53-H55 H47-H58

[0174] H3 H95-H102 H95-H102 H96-H101 H93-H101

[0175] HVRs may comprise an "extended HVR" as follows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in VL, and 26-35 (H1), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) in VH. For each of these definitions, the variable domain residues are numbered according to Kabat et al. (supra).

[0176] The terms "variable domain residue numbering as in Kabat" or "amino acid position numbering as in Kabat" and variations thereof refer to the numbering system used for the heavy chain variable domain or light chain variable domain of the antibody compilation in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of or insertion into a FR or HVR of the variable domain. For example, the heavy chain variable domain may include a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and inserted residues after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c, etc. according to Kabat). The Kabat numbering of residues for a given antibody can be determined by aligning the region of sequence homology of the antibody to the "standard" Kabat numbering sequence.

[0177] When referring to residues in the variable domain, the Kabat numbering system is generally used (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., above). When referring to residues in the immunoglobulin heavy chain constant region, the "EU numbering system" or "EU index" is generally used (e.g., Kabat et al., EU index reported above). The "EU index as in Kabat" refers to the residue numbering of human IgG1 EU antibodies. Unless otherwise indicated herein, reference to the residue numbering in the antibody variable domain refers to the residue numbering of the Kabat numbering system. Unless otherwise indicated herein, reference to the residue numbering in the antibody constant domain refers to the residue numbering of the EU numbering system (e.g., see U.S. Patent Application Publication No. 2008 / 0181888, for EU numbering diagrams).

[0178] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules, including but not limited to a cytotoxic agent.

[0179] A "subject" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, 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 subject or individual is a human.

[0180] An "isolated" antibody is one that has been separated from components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0181] 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 ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0182] "Isolated nucleic acid encoding an antibody, such as an anti-Tie2 antibody" refers to one or more nucleic acid molecules encoding the heavy and light chains of an antibody (or fragments thereof), including such nucleic acid molecules in a single vector or separate vectors, and such nucleic acid molecules are present at one or more locations in a host cell.

[0183] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies contained in the population are identical and / or bind to the same epitope, but do not include (for example) possible variant antibodies containing naturally occurring mutations or produced during the production of the monoclonal antibody preparation, which variants are usually present in small amounts. In contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. Therefore, the modifier "monoclonal" indicates that the characteristic of the antibody is obtained from a substantially homogeneous antibody population and should not be interpreted as requiring the production of antibodies by any particular method. For example, the monoclonal antibodies intended to be used according to the present invention can be manufactured by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals comprising all or part of the human immunoglobulin loci. These methods and other exemplary methods for preparing monoclonal antibodies are described herein.

[0184] A "naked antibody" is an antibody that is not conjugated to a heterologous moiety (eg, a cytotoxic moiety) or a radiolabel. Naked antibodies can be present in pharmaceutical formulations.

[0185] "Natural antibodies" refer to naturally occurring immunoglobulin molecules with different structures. For example, natural IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two identical light chains and two identical heavy chains bonded by disulfide bonds. From the N-terminus to the 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 the N-terminus to the 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. Based on the amino acid sequence of its constant domain, the light chain of an antibody can be classified into one of two types, called kappa (κ) and lambda (λ).

[0186] 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.

[0187] "Percent (%) amino acid sequence identity" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing differences (if necessary) to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in various ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. One skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required to achieve maximum alignment over the full length of the compared sequences. However, for the purposes of this article, the sequence comparison computer program ALIGN-2 is used to generate % amino acid sequence identity values. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and the source code has been deposited with user documentation in the U.S. Copyright Office, Washington, D.C. 20559, and is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc. in South San Francisco, California, and can also be compiled from 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 are not varied.

[0188] In cases where ALIGN-2 is employed for amino acid sequence comparisons, 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 has or comprises a certain % of amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows:

[0189] 100 times the fraction X / Y

[0190] where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in the alignment of A and B, and Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A, as the comparison of A to B is not identical to the comparison of B to A. All % amino acid sequence identity values used herein were obtained using the ALIGN-2 computer program noted in the preceding paragraph, unless otherwise specifically indicated.

[0191] The term "pharmaceutical formulation" refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.

[0192] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation other than an active ingredient which is not toxic to a subject to which the formulation would be administered. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizing agents, or preservatives.

[0193] The term "polyol" refers broadly to a polyhydroxylic 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), and preferably is 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, as well as those which can be obtained, for example, from publicly available sources.

[0194] As used herein, "treatment" (and grammatical variations thereof, such as "treat" or "treating") refers to a clinical intervention that attempts to alter the natural course of a disease in a treated subject and can be performed prophylactically or during clinical pathology. Desired therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, alleviating any direct or indirect pathological consequences of a disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating the disease state, alleviating or improving prognosis. In some embodiments, the antibodies of the invention are used to delay the development of a disease or slow the progression of a disease.

[0195] As used herein, the term "vector" refers to a nucleic acid molecule capable of delivering another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. These vectors are referred to herein as "expression vectors."

[0196] 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 constructed with a refill port communicating with the reservoir and a release-control element that determines the rate at which the drug is released into the eye. See, e.g., US20100174272, 8,277,830, 8,399,006, 8,795,712, and 8,808,727.

[0197] "Small-gauge needle" refers to a needle for injection of fluid compositions that is about 30, 29, 28, 27, 26, 25, 24, 23, or 22 gauge or higher, such as a 30 gauge needle. In some embodiments, the small-gauge needle has a standard-sized needle wall. In another embodiment, the small-gauge needle has a thin needle wall, which may be preferred for viscous solutions.

[0198] II. Compositions and Methods

[0199] Provided herein are novel Tie2 agonists that activate Tie2 function, as demonstrated by, for example, Tie2 phosphorylation. In the absence or insufficiency of the endogenous agonist Ang1, direct agonism more effectively activates Tie2 signaling, driving improved vision, and thus may offer therapeutic advantages. Because Tie2 activators may both block Ang2 binding (blocking Ang2 antagonist function) and directly bind to Tie2 to activate Tie2 activity, for example, by increasing Tie2 and / or Akt phosphorylation, they may offer advantages over Ang2 inhibitors. Tie2 activation has been shown to require Tie2 aggregation upon ligand binding, and accordingly, the Tie2 agonists provided herein are multimeric, preferably hexameric or octameric. The multimeric Tie2 agonists described herein may have the additional and unexpected advantage of not significantly reducing cellular Tie2 levels in vitro or in vivo. Furthermore, the multimeric Tie2 agonists described herein may be formulated for intravitreal injection and possess a molecular size that confers advantageous pharmacokinetics and, therefore, pharmacodynamics. Data are provided showing that multimeric Tie2 agonists reduce endothelial cell membrane permeability and enhance cell-cell junctions (see, eg, Example 10).

[0200] In one aspect, the present invention 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, such as, but not limited to, anti-Tie2 Fab. It may be preferred that the binding agent comprises more than four (e.g., 5, 6, 7, 8, 9, or 10) anti-Tie2 antibodies, such as more than four anti-Tie2 Fabs, such that binding of the Tie2 binding agent to Tie2 localized on the cell surface is correlated with Tie2 activation. Tie2 aggregation may also occur upon binding of the binding agent to Tie2.

[0201] In some embodiments, it is advantageous to have a Tie2 binder that, when binding to Tie2 on the surface of the protein, does not cause a significant decrease in the level of Tie2 protein on the cell surface. In some embodiments, the Tie2 binder that activates Tie2 comprises an anti-Tie2 antibody (Fab) having an affinity for Tie2 in the range of about 0.1 μM to 10 μM.

[0202] Activation of the Tie2 protein can be measured in vitro by measuring increased phosphorylation of the Tie2 protein (e.g., by Western blotting) or increased phosphorylation of the related Akt protein (AKT serine / threonine kinase 1, e.g., GenBank accession number NP_001014431) using materials and methods known to those of ordinary skill. In certain embodiments, antibodies that bind to Tie2 and compositions comprising more than one antibody that binds to Tie2 are provided. The antibodies of the present invention and compositions comprising multiple antibodies can be used, for example, to diagnose or treat vascular permeability diseases associated with Tie2 function, particularly ocular diseases.

[0203] A. Exemplary Anti-Tie2 Binders

[0204] In one aspect, the present invention provides a Tie2 binder comprising an isolated antibody that binds to Tie2. In some embodiments, the Tie2 binder comprises a multi-arm portion, wherein each of these arms is conjugated or linked to an anti-Tie2 antibody or fragment thereof. In a preferred embodiment, the anti-Tie2 antibody or fragment thereof is an anti-Tie2 Fab. Examples of multi-arm portions include, but are not limited to, multi-arm polyols (e.g., polyethylene glycol (PEG)), IgM, and multimers such as hexameric peptides. In certain embodiments, a Tie2 binder is provided that comprises more than one anti-Tie2 antibody or fragment thereof, wherein the anti-Tie2 antibody (when not part of the Tie2 binder, but optionally formatted 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. D and / or K exceeding 1 μM D. It should be understood that affinity is measured using antibodies rather than Tie2 binders comprising more than one anti-Tie2 antibody. In some embodiments, affinity is a monovalent affinity. In addition, Tie2 binders and Tie2 antibodies are provided that activate Tie2 activity (e.g., act as Tie2 agonists). For example, activation of Tie2 is measured by measuring an increase in Tie2 phosphorylation and / or an increase in AKT phosphorylation in an in vitro assay. In some embodiments, the Tie2 binder does not downregulate (reduce) 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 binder reduces 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 binder reduces vascular permeability. The reduction in vascular endothelial cell permeability can be measured in vivo or in vitro. In some embodiments, the Tie2 binder can increase the translocation of Tie2 to cell-cell junctions and / or promote the structural organization of actin and / or cadherin. In preferred embodiments, the Tie2 binder comprises a hexameric PEG molecule, wherein each of the six arms binds to an anti-Tie2 Fab. Alternatively, the PEG molecule comprises eight arms. In some embodiments, the Tie2 binder comprises more than one anti-Tie2 antibody or fragment thereof described herein. The present invention 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 present at least partially within amino acid residues 23 to 120 of SEQ ID NO: 1).

[0205] In one aspect, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds 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 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), 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 specific embodiment, the CDR-H3 comprises SEQ ID NO: 7.

[0206] In one aspect, the present invention provides an antibody comprising at least one, at least two, or all three VH domain CDR sequences selected from the group consisting of: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 3; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 4; and (c) a CDR-H3 comprising the amino acid sequence of 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. 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.

[0207] In another aspect, the present invention 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.

[0208] In another aspect, an antibody of the invention comprises: (a) a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from the group consisting of: (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 3; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 4; and (iii) a CDR-H3 comprising the amino acid sequence of 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), SEQ ID NO: 6, and SEQ ID NO: 7; and (b) a VL domain comprising at least one, at least two, or all three VLCDR sequences selected from the group consisting of: (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8; (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9; and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10.

[0209] In another aspect, the present invention provides an antibody comprising a VH domain and a VL domain, wherein the VH domain comprises: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 3; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 4; (c) an HVR-H3 comprising the amino acid sequence of 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), SEQ ID NO: 6 or SEQ ID NO: 7; and the VL domain comprises: (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, and (f) a CDR-L3 comprising an 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.

[0210] In any of the above embodiments, the anti-Tie2 antibody can be humanized. In one embodiment, the anti-Tie2 antibody comprises the CDRs described in any of the above embodiments and further comprises a human acceptor framework, such as a human immunoglobulin framework or a human consensus framework. In another embodiment, the anti-Tie2 antibody comprises the CDRs 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.

[0211] 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 comprises substitutions (e.g., conservative substitutions), insertions or deletions relative to the reference sequence, but an anti-Tie2 antibody comprising the sequence retains the ability to bind to Tie2. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and / or deleted in SEQ ID NO: 20. In certain embodiments, the substitutions, insertions or deletions occur in regions outside of the CDRs (i.e., in the FRs). Optionally, the anti-Tie2 antibody comprises the VH sequence of SEQ ID NO: 20, including post-translational modifications of that sequence. In a specific embodiment, the VH comprises one, two, or three 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; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 7.

[0212] In another aspect, an anti-Tie2 antibody is provided, wherein the antibody comprises a light chain variable domain (VL) 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. In certain embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity comprises substitutions (e.g., conservative substitutions), insertions or deletions relative to the reference sequence, but the anti-Tie2 antibody comprising the sequence retains the ability to bind to Tie2. In certain embodiments, in SEQ ID NO: 21, a total of 1 to 10 amino acids are substituted, inserted and / or deleted. In certain embodiments, the substitutions, insertions or deletions occur in regions outside the HVR (i.e., in the FR). Optionally, the anti-Tie2 antibody comprises the VL sequence of SEQ ID NO: 21, including post-translational modifications of that sequence. In a specific embodiment, the VL comprises one, two, or three HVRs selected from the group consisting of: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 8; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 9; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 10.

[0213] In another aspect, an anti-Tie2 antibody is provided, wherein the antibody comprises the VH of any one of the embodiments provided above and the 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 these sequences.

[0214] In yet another aspect, the present invention provides an antibody that binds to the same epitope as the anti-Tie2 antibodies provided herein. For example, in certain embodiments, an antibody that binds to the same epitope as the anti-Tie2 antibodies is provided, comprising a VH sequence of SEQ ID NO: 20 and a VL sequence of SEQ ID NO: 21. In certain embodiments, an antibody that binds to an epitope within a Tie2 fragment, such as the Ig1 domain of Tie2, is provided, wherein the Ig1 domain comprises amino acids 23-120 of SEQ ID NO: 1.

[0215] In another aspect of the present invention, 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 an Fv, Fab, Fab', scFv, a diabody, or a F(ab')2 fragment. In another embodiment, the antibody is a full-length antibody, such as a complete IgG1 antibody or other antibody class or isotype as defined herein.

[0216] In yet another aspect, the anti-Tie2 antibody according to any of the above embodiments may incorporate any of the features, alone or in combination, as described in Sections 1-7 below:

[0217] 1. Antibody affinity

[0218] In certain embodiments, the dissociation constant (Kd) of the antibodies provided herein is ≤10 μM, ≤100 μM, ≤10 μM, ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM or ≤0.001 nM and / or ≥0.01 μM, 0.1 μM or 1 μM (e.g., 10 -5 M or lower, 10 -6 M or more, 10 -8 M or less, for example, 1 μM to 10 μM, for example, 0.1 μM to 10 μM, for example, 10 -6 M to 10 -9 M, for example, 10 -8 M to 10 -13 M, for example, 10 -9 M to 10 -13 M).

[0219] In one embodiment, Kd is measured by radiolabeled antigen binding assay (RIA). In one aspect, RIA is performed using a Fab form of the antibody of interest and its antigen. For example, by titrating the antibody against a minimal concentration of ( 125 The solution binding affinity of Fab for antigen was measured by equilibrating Fab with 1) labeled antigen and then capturing the bound antigen with an anti-Fab antibody coated plate (see, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). To determine the conditions of the assay, 5 μg / ml of capture anti-Fab antibody (Cappel Labs) was used to capture the antigen in 50 mM sodium carbonate (pH 9.6). 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.

[0220] According to another embodiment, using Kd is measured using surface plasmon resonance assays. For example, -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 dissociation rate (koff) are calculated by simultaneously fitting the binding and dissociation sensor profiles using the ELISA software version 3.2. The equilibrium dissociation constant (Kd) is calculated as the ratio of koff / kon. See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). If the association rate determined by the surface plasmon resonance analysis described above exceeds 106 M-1 s-1, the association rate can be measured using a fluorescence quenching technique, which is measured on a spectrometer (e.g., a flow-stop equipped spectrophotometer with a stirred cuvette (Aviv Instruments) or an 8000-series SLM-AMINCO TM The increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) of 20 nM anti-antigen antibody (Fab form) in PBS (pH 7.2) was measured at 25°C in the presence of increasing concentrations of antigen as measured in a spectrophotometer (ThermoSpectronic).

[0221] 2. Antibody fragments

[0222] In certain embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv and scFv fragments, as well as other fragments described below. For a review of certain antibody fragments, see Hudson et al., Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, for example, Pluckthün, 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. Patents Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab')2 fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Patent No. 5,869,046.

[0223] In some embodiments, the C-terminus of the heavy chain of the Fab fragment ends at the amino acid "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 ends at the sequence CDKTHX (SEQ ID NO: 79), wherein X is an amino acid other than T. Truncations and / or mutations at the C-terminus may be able to reduce or eliminate AHA reactivity against the Fab without compromising thermal stability or expression. In some embodiments, the C-terminus of the heavy chain of the Fab fragment ends at 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 ​​at the C-terminal amino acid may be suitable for conjugation, for example, to a polymer such as PEG.

[0224] Diabodies are antibody fragments with two antigen-binding sites (which 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).

[0225] Single-domain antibodies are antibody fragments that comprise all or part of the heavy chain variable domain of an antibody or all or part of the light chain variable domain 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).

[0226] Antibody fragments can be made by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies as described herein and production in recombinant host cells (eg, E. coli or phage).

[0227] 3. Chimeric and humanized antibodies

[0228] In certain embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, 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 has been changed compared to its parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0229] In some aspects, chimeric antibodies are humanized antibodies. Generally, non-human antibodies are humanized antibodies to reduce immunogenicity to people while retaining the specificity and affinity of the parent non-human antibody. Generally, humanized antibodies include one or more variable domains, wherein HVR such as CDR (or part thereof) is derived from non-human antibodies, and FR (or part thereof) is derived from human antibody sequences. Humanized antibodies are optionally at least partially comprised of human constant regions. In certain embodiments, some FR residues in humanized antibodies are replaced with corresponding residues from non-human antibodies (e.g., antibodies derived from HVR residues), for example, to restore or improve antibody specificity or affinity.

[0230] Humanized antibodies and methods for their preparation are reviewed, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described, for example, in: Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (specifically describing SDR transplantation); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "resurfacing"); 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).

[0231] 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 of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89: 4285 (1992); and Presta et al. J. Immunol., 151: 2623 (1993)). 93)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro & 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)).

[0232] 4. Human Antibodies

[0233] In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be produced using various techniques known in the art. Human antibodies are generally described in: van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001); and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

[0234] Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce complete human antibodies or complete antibodies with human variable regions in response to an antigenic attack. Such animals typically contain all or part of a human immunoglobulin locus that replaces the endogenous immunoglobulin locus, or is present outside the chromosome or is randomly integrated into the chromosome of the animal. In such transgenic mice, the endogenous immunoglobulin locus is typically inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23: 1117-1125 (2005). See also, for example, U.S. Patent Nos. 6,075,181 and 6,150,584 (describing XENOMOUSE TM technology); U.S. Patent No. 5,770,429 (describing technology); U.S. Patent No. 7,041,870 (describing KM technology); and U.S. Patent Application Publication No. US2007 / 0061900 (describing Technology). Human variable regions derived from intact antibodies produced by these animals can be further modified, for example by combining them with different human constant regions.

[0235] Human antibodies can also be prepared by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described. (See, for example, 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 produced by 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 in, for example, U.S. Patent No. 7,189,826 (describing the production of monoclonal human IgM antibodies by 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).

[0236] Human antibodies can also be produced by separating the Fv clone variable domain sequence selected from the human phage display library. This variable domain sequence can then be combined with the required human constant domain. The technology of selecting human antibodies from the antibody library is described below.

[0237] 5. Antibodies from the library

[0238] Antibodies for use in the present invention can be isolated by screening combinatorial libraries for antibodies having one or more desired activities. For example, various methods known in the art are used to generate phage display libraries and screen these libraries for antibodies having the desired binding properties. These methods are reviewed in, for example, Hoogenboom et al., in Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), and are further described in, for example, 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 et al., 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).

[0239] In certain phage display methods, the VH and VL gene repertoires are cloned separately by polymerase chain reaction (PCR) and randomly recombined in the phage library, and then the antigen-binding phage can be screened according to the methods described in the following literature: Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Phage usually display antibody fragments as single-chain Fv (scFv) fragments or Fab fragments. The library from the immune source can provide high-affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, all natural components (e.g., from humans) can be cloned without any immunization to provide a single source of antibodies to various non-self and self antigens, as described by Griffiths et al. in EMBO J. 12: 725-734 (1993). Finally, natural libraries can also be synthesized by cloning unrearranged V gene segments in stem cells and using PCR primers containing random sequences to encode highly variable CDR3 regions and rearrange them in vitro, as described by Hoogenboom and Winter in J. Mol. Biol., 227:381-388 (1992). Patent disclosures 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.

[0240] Antibodies or antibody fragments isolated from human antibody repertoires are referred to herein as human antibodies or human antibody fragments.

[0241] 6. Multispecific Antibodies

[0242] 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 specificities are for any other antigen. In some aspects, a bispecific antibody can bind to two different epitopes of Tie2. A bispecific antibody can bind to two different epitopes of Tie2. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0243] Techniques for preparing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see: Milstein et al., Nature 305:537, 1983; WO 93 / 08829; and Traunecker et al., EMBO J. 10:3655, 1991) and "protrusion-into-hole" engineering (see, e.g., U.S. Patent No. 5,731,168). Multispecific antibodies can also be prepared by the following methods: engineering electrostatic steering effects for preparing antibody Fc-heterodimer molecules (WO 93 / 08829). 2009 / 089004A1); 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 generate bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); using "diabody" technology to prepare double-chain antibody fragments ( See, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)); and preparing trispecific antibodies as described, e.g., in Tutt et al., J. Immunol. 147:60 (1991).

[0244] Also included herein are engineered antibodies with three or more antigen binding sites, including "Octopus antibodies" (see, eg, US 2006 / 0025576 A1).

[0245] The antibodies or fragments herein also include "dual-acting FAbs" or "DAFs," which comprise an antigen binding site that binds to Tie2 as well as another, different antigen (see, eg, US 2008 / 0069820).

[0246] 7. Antibody variants

[0247] In some aspects, it is contemplated that the amino acid sequence variants of the antibodies provided herein may be modified. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Suitable modifications may be introduced into the nucleotide sequence encoding the antibody, or the amino acid sequence variants of the antibody may be prepared by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of the residues in the amino acid sequence of the antibody. Any combination of deletion, insertion, and substitution may be implemented to obtain the final construct, provided that the final construct has desired characteristics, for example, antigen-binding characteristics.

[0248] a) Substitution, insertion, and deletion variants

[0249] 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.

[0250] Table 1

[0251]

[0252] Amino acids can be grouped according to common side-chain properties:

[0253] (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, He;

[0254] (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;

[0255] (3) acidic: Asp, Glu;

[0256] (4) basic: His, Lys, Arg;

[0257] (5) residues that influence chain orientation: Gly, Pro;

[0258] (6) aromatic: Trp, Tyr, Phe.

[0259] Non-conservative substitutions require exchanging a member of one of these classes for a member from another class.

[0260] 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

[0261] Changes (e.g., substitutions) can be made in 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.

[0262] 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 CDRs that do not substantially reduce binding affinity. Such modifications can be outside of antigen contacting residues in 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.

[0263] 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” (see also, e.g., F. W. Smith, in Protein

[0264] Amino acid sequence insertions include amino and / or carboxyl terminal fusions ranging in length from one residue to polypeptides comprising one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertion variants of the antibody molecule include enzymes fused to the N-terminus or C-terminus of the antibody (e.g., for ADEPT) or polypeptides that increase the serum half-life of the antibody.

[0265] b) Glycosylation variants

[0266] In certain embodiments, the antibodies provided herein are altered to increase or decrease the extent to which the antibodies are glycosylated. Adding or deleting glycosylation sites in an antibody can be conveniently achieved by altering the amino acid sequence to create or remove one or more glycosylation sites.

[0267] When antibody comprises Fc district, can change the carbohydrate connected thereto.Natural antibody that mammalian cell produces usually comprises branched biantennary oligosaccharide, and it is usually connected to the Asn297 of Fc district CH2 domain by N key.See, for example, Wright et al. TIBTECH 15:26-32 (1997).Oligosaccharide can comprise various carbohydrates, for example mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid and the fucose that is connected to GlcNAc in the " stem " of biantennary oligosaccharide structure.In certain embodiments, can modify the oligosaccharide in the antibody of the present invention, to produce the antibody variant with some improved characteristic.

[0268] In one embodiment, antibody variants are provided that lack carbohydrate structures that are fucose bound (directly or indirectly) to the Fc region. For example, the fucose content in such antibodies may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The content of fucose relative to the sum of all glycostructures (e.g., complex, hybrid, and high mannose) bound to Asn 297 as measured by MALDI-TOF mass spectrometry is determined by calculating the average fucose content in the Asn297 sugar chain, as described, for example, in WO 2008 / 077546. Asn297 refers to an asparagine residue located near position 297 of the Fc region (EU numbering of Fc region residues); however, Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300 due to minor sequence variations in the antibody. Such fucosylated variants may have improved ADCC function. See, eg, US Patent Publication Nos. US 2003 / 0157108 (Presta, L.); US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to "defucosylated" or "fucose-deficient" antibody variants include: US2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US2003 / 0115614; US2002 / 0164328; US2004 / 0093621; US2004 / 0132140; US2004 / 0110704; US2004 / 0110282; US2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO2005 / 053742; WO2002 / 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 defucosylated antibodies include Lec13 CHO cells that lack protein fucosylation (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application No. US2003 / 0157108A1, Presta, L; and WO 2004 / 056312 A1, Adams et al., particularly in Example 11); and knockout cell lines, such as CHO cells in which the α-1,6-fucosyltransferase gene FUT8 is knocked out (see, e.g., Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng, 94(4):680-688 (2006); and WO2003 / 085107).

[0269] Further provided are antibody variants having bisected oligosaccharides, for example, wherein the biantennary oligosaccharides attached to the Fc region of the antibody are bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described in, for example, WO 2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and US2005 / 0123546 (Umana et al.). Also provided are antibody variants having at least one galactose residue attached to the Fc region on the oligosaccharide. Such antibody variants may have improved CDC function. Such antibody variants are described in, for example, WO 1997 / 30087 (Patel et al.), WO 1998 / 58964 (Raju, S.), and WO 1999 / 22764 (Raju, S.).

[0270] c) Fc region variants

[0271] 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. The Fc region variant can comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0272] In certain embodiments, the present invention contemplates an antibody variant that has some, but not all, effector functions, making it a desirable candidate antibody for applications where in vivo half-life of the antibody is important but certain effector functions (e.g., complement and ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduction / depletion of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the antibody lacks FcγR binding (and therefore may lack ADCC activity) but retains FcRn binding ability. NK cells, the primary cells that mediate ADCC, express only Fc(RIII), whereas monocytes express Fc(RI, Fc(RII), and Fc(RIII). FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of the paper by Ravetch and Kinet (Annu. Rev. Immunol. 9:457-492 (1991)). Non-limiting examples of in vitro assays for evaluating ADCC activity of target molecules are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986). 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays can be used (see, for example, ACTI for flow cytometry). TM Nonradioactive cytotoxicity assay (CellTechnology, Inc. Mountain View, CA); and CytoTox Non-radioactive cytotoxicity assay (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of the target molecule can be assessed in vivo in an animal model such as that disclosed in Clynes et al., Proc. Natl Acad. Sci. USA 95:652-656 (1998). A C1q binding assay can also be performed to confirm that the antibody is unable to bind to C1q and therefore lacks CDC activity. See, for example, C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996); Cragg, MS et al., Blood 101: 1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103: 2738-2743 (2004)). FcRn binding and in vivo clearance / half-life assays can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12): 1759-1769 (2006)).

[0273] Antibodies with reduced effector function include antibodies in which one or more Fc region residues 238, 265, 269, 270, 297, 327, and 329 are substituted (U.S. Patent No. 6,737,056). Such Fc variants include Fc variants with two or more substitutions 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 (U.S. Patent No. 7,332,581).

[0274] Certain antibody variants with improved or reduced FcR binding are described. (See, e.g., U.S. Patent No. 6,737,056; WO 2004 / 056312 and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001).)

[0275] In certain embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, such as substitutions at positions 298, 333, and / or 334 (EU numbering of residues) of the Fc region.

[0276] In some embodiments, modifications are made in the Fc region resulting in modified (ie, improved or reduced) C1q binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Patent No. 6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol. 164:4178-4184 (2000).

[0277] Antibodies with longer half-lives and improved binding to the neonatal Fc receptor (FcRn) (which is responsible for the transfer of maternal IgG to the fetus, see Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) are described in US 2005 / 0014934 A1 (Hinton et al.). Those antibodies comprise an Fc region having one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more of the 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 ( U.S. Pat. No. 7,371,826 ).

[0278] See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351 for other examples of Fc region variants.

[0279] d) Cysteine-engineered antibody variants

[0280] In certain embodiments, it may be desirable to create cysteine ​​engineered antibodies, such as "thioMAbs," in which one or more residues of an antibody are replaced by cysteine ​​residues. In a specific embodiment, the substituted residues appear at accessible sites of the antibody. By replacing those residues with cysteine, reactive sulfhydryl groups are thereby positioned at accessible sites of the antibody and can be used to conjugate the antibody to other moieties (e.g., drug moieties or linker-drug moieties) to form immunoconjugates, as further described herein. In certain embodiments, any one or more of the following residues may be substituted by cysteine: V205 (EU numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region. Cysteine ​​engineered antibodies can be produced according to the methods described, for example, in U.S. Patent No. 7,521,541.

[0281] e) Antibody derivatives

[0282] 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.

[0283] 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.

[0284] B. Recombinant methods and compositions

[0285] 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).

[0286] 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).

[0287] 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.

[0288] In addition to prokaryotes, eukaryotic microorganisms (such as filamentous fungi or yeast) are also suitable cloning or expression hosts for antibody encoding vectors, including fungi and yeast strains whose glycosylation pathways have been "humanized", resulting in the production of polypeptides with partially or fully human glycosylation patterns. See: Gerngross, Nat. Biotech. 22: 1409-1414 (2004); and Li et al., Nat. Biotech. 24: 210-215 (2006).

[0289] Suitable host cells for expressing glycosylated antibodies also come from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Many baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfecting Spodoptera frugiperda cells.

[0290] Plant cell cultures can also be used as hosts. See, for example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing plant antigens produced in transgenic plants). TM technology).

[0291] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for growth in suspension can be used. Other examples of useful mammalian host cell lines include: monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (e.g., 293 or 293 cells described in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse testicular Sertoli cells (e.g., TM4 cells described 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 hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); TRI cells (e.g., Mather et al., Annals of NY Acad. Sci. 383: 44-68 (1982)); MRC 5 cells; and FS4 cells. Other mammalian host cell lines that can be used 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).

[0292] C. Assays

[0293] 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.

[0294] 1. Binding assays and other assays

[0295] 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.

[0296] 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).

[0297] In an exemplary competition assay, immobilized Tie2 is incubated in a solution containing a first labeled antibody that binds to Tie2 (e.g., Tie2.1M100cF) and a second unlabeled antibody (being tested for its ability to compete with the first antibody for binding to Tie2). The second antibody can be present in the 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 binding of the first antibody 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 immobilized Tie2 in the test sample is significantly reduced relative to the control sample, it indicates that 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).

[0298] 2. Activity Assay

[0299] In one aspect, assays are provided for identifying anti-Tie2 antibodies that possess a desired biological activity. Biological activities may include, for example, binding to Tie2 or its fragments, competing with Ang1 and / or Ang2 for Tie2 binding, activating phosphorylation of Tie2 proteins, activating phosphorylation of Akt, and / or reducing vascular endothelial cell permeability in vivo, in vitro, or pseudo-in vivo. Antibodies that possess such biological activities in vivo and / or in vitro are also provided. In certain embodiments, the antibodies of the invention are tested for such biological activities.

[0300] In some embodiments, assays for determining Tie2 activation (anti-Tie2 conjugate) activity are provided, such as by a phospho-AKT (pAKT) assay, wherein activation of AKT phosphorylation by a Tie2 conjugate indicates that the conjugate has activation (agonist) activity. As known to those of ordinary skill and as described in Example 3 below, activation of AKT can be demonstrated by various methods, including, for example, Western blot detection of phosphorylated AKT using an antibody specific for phosphorylated AKT or by a FRET assay.

[0301] In some embodiments, an assay for determining the stability (e.g., thermal stability) of an anti-Tie2 antibody or antibody conjugate, fusion protein, or polymeric formulation thereof is 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, such as differential scanning fluorimetry (DSF), circular dichroism (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).

[0302] In some embodiments, an assay for determining the stability of an anti-Tie2 conjugate is provided. The stability of the assay can be determined as described herein, for example, using capillary electrophoresis laser induced fluorescence (CE-LIF), as performed, for example, as described in Example 13.

[0303] D. Immunoconjugates

[0304] The present invention also provides immunoconjugates comprising an anti-Tie2 antibody as described herein, which is conjugated (chemically bonded) 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, an enzymatically active toxin, or a fragment thereof derived from bacteria, fungi, plants, or animals), or a radioactive isotope.

[0305] E. Conjugates

[0306] The present invention also provides conjugates comprising any anti-Tie2 antibody or Tie2-conjugated fragment thereof provided herein conjugated to one or more heterologous molecules, such as a polyol.

[0307] 1. Multi-arm polymers

[0308] In some embodiments, the conjugates of the present disclosure can be made by derivatizing an anti-Tie2 antibody described herein by conjugating an anti-Tie2 Fab or variant thereof to a multi-arm polymer. It should be understood that any multi-arm polymer that provides a conjugate of a desired size or selected average molecular weight as described herein is suitable for use in constructing the antibody-polymer conjugates of the present invention.

[0309] A variety of polymers are suitable for use in medicine. See, for example, 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 polymer is typically a hydrophilic synthetic polymer, i.e., a polymer not found in nature. However, polymers that exist in nature and are produced by recombinant or in vitro methods may also be useful, as may polymers isolated from natural sources.

[0310] In some embodiments, the anti-Tie2 Fab is derivatized by conjugating (e.g., covalently linking) the Fab or variant thereof to a multi-arm polyol. Thus, in some embodiments, the present disclosure is directed to conjugates comprising one or more anti-Tie2 Fab or variants thereof disclosed herein covalently linked to one or more multi-arm polyols (preferably a six-arm polyol). The polyol employed can be any water-soluble poly(alkylene oxide) polymer and can have a linear or branched chain. Suitable polyols include those substituted at one or more hydroxyl positions with a chemical group, such as an alkyl group having between one and four carbon atoms. Typically, the polyol is a poly(alkylene glycol), such as polyethylene glycol (PEG), and thus, for ease of description, the remainder of the discussion is with respect 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.

[0311] The polyol used to form the conjugate of the present disclosure is a multi-arm polyol. As used herein, "multi-arm polyol" refers to a polyol comprising a core structure, and at least two arms are attached to the core structure. The multi-arm polyol can be, for example, a dimer (two arms), a tetramer (four arms), a hexamer (six arms), an octamer (eight arms), etc. In some aspects, the multi-arm polyol is a multi-arm PEG.

[0312] The weight average molecular weight of the multi-arm PEG used in the PEGylation of the anti-Tie2 antibody or antibody variant can vary and generally ranges from about 500 to about 300,000 Daltons (D). In some embodiments, the multi-arm PEG has a weight average molecular weight of about 1,000 to about 100,000 D, about 1,000 to about 40,000 D, about 1,000 to about 20,000 D, about 1,000 to about 10,000 D, about 10,000 to about 20,000 D, about 5,000 to about 10,000 D, or about 1,000 to 5,000 D. In a preferred embodiment, a multi-arm PEG with a weight average molecular weight of about 6,000 D is used for PEGylation.

[0313] A variety of methods for protein PEGylation are known in the art. Specific methods for manufacturing proteins conjugated to PEG include methods 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 entirety. Typically, the protein is covalently bound to the terminal reactive groups on the polymer via one or more of the amino acid residues of the protein. Herein, polymers with these reactive groups are directed to activated or functionalized polymers (e.g., functionalized PEG). The reactive group selectively reacts with the free sulfhydryl or amino or other reactive groups on the antibody or antibody variant. The multi-arm PEG polymer can be coupled to the sulfhydryl or amino or other reactive groups on the antibody or antibody variant in a random or site-specific manner. However, it should be understood that in order to obtain optimal results, the type and amount of the reactive group selected and the type and amount of the polymer used will depend on the specific antibody or antibody variant used, to limit and preferably substantially prevent the reactive group from reacting with too many reactive groups on the antibody. Since it may not be possible to fully limit or prevent this in some cases, about 0.05 to about 1000 moles, or in some embodiments, about 0.05 to about 200 moles of functionalized polymer per mole of antibody may generally be used, depending on the antibody concentration. The final amount of functionalized polymer per mole of antibody is a measure of maintaining optimal activity while optimizing (if possible) the half-life of the antibody in the vitreous humor, retina, and / or amyotrophic fluid.

[0314] 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.

[0315] 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.

[0316] In some embodiments, the multi-arm PEG used to prepare conjugates of the present disclosure has the structure of Formula (I):

[0317]

[0318] 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 independently is 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.

[0319] 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.

[0320] 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.

[0321] 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 directly conjugated to an anti-Tie2 antibody to form the conjugates of the present disclosure. In some embodiments, the functionalized multi-arm PEG has a structure of formula (Ia):

[0322]

[0323] 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 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.

[0324] In some embodiments, the functionalized multi-arm PEG has the structure of formula (Ia), wherein n is an integer from 2 to 3. In a preferred embodiment, the functionalized multi-arm PEG has the structure of formula (Ia), 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 (Ia), wherein n is 3, and the multi-arm PEG is an octamer. In a preferred embodiment, the functionalized multi-arm PEG has the structure of formula (Ia), wherein n is 2, such as Figure 39 As shown in .

[0325] In another embodiment, the multi-arm PEG used to prepare the conjugates of the present disclosure has the structure of formula (II):

[0326]

[0327] 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.

[0328] In some embodiments, the multi-arm PEG has a structure of formula (II), wherein n is 2, and the multi-arm PEG is a tetramer. In another embodiment, the multi-arm PEG has a structure of formula (II), wherein n is 4, and the multi-arm PEG is a hexamer. In another embodiment, the multi-arm PEG has a structure of formula (II), wherein n is 6, and the multi-arm PEG is an octamer.

[0329] In another embodiment, the multi-arm PEG used to prepare the conjugates of the present disclosure has the structure of formula (III):

[0330]

[0331] 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.

[0332] In some embodiments, the multi-arm PEG has a structure of formula (III), wherein n is 2, and the multi-arm PEG is a tetramer. In another embodiment, the multi-arm PEG has a structure of formula (III), wherein n is 4, and the multi-arm PEG is a hexamer. In another embodiment, the multi-arm PEG has a structure of formula (III), wherein n is 6, and the multi-arm PEG is an octamer.

[0333] In another embodiment, the multi-arm PEG used to prepare the conjugates of the present disclosure has the structure of formula (IV):

[0334]

[0335] 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.

[0336] A multi-arm PEG having a structure of any one of formulas (I) to (IV) can be functionalized using any of the techniques described above to produce a functionalized multi-arm PEG, for example, to attach terminal reactive groups suitable for reaction or conjugation with an antibody (e.g., an antibody fragment). However, in other embodiments, the multi-arm PEG can be covalently linked to an anti-Tie2 antibody via a multifunctional cross-linker that reacts with the PEG to be cross-linked 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 herein by reference in its entirety.

[0337] 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):

[0338]

[0339] 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.

[0340] 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):

[0341]

[0342] wherein m, R 1 and R 2 are as defined above.

[0343] 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.

[0344] 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

[0345] 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 (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.

[0347] 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.

[0348] 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.

[0349] In another aspect, the functionalized multi-arm PEG used to prepare the conjugates of the present disclosure has the structure of formula (IIa):

[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, 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.

[0352] 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.

[0353] 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 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 (IIa), 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 (IIa), 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.

[0356] In some embodiments, the functionalized multi-arm PEG has a structure of Formula (IIa), wherein each R 2 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 and j are as defined above. In some embodiments, the functionalized multi-arm PEG has a structure of formula (IIa), wherein when R 1 With R 2 When combined together Where i is 2 and j is 2.

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

[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, 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 independently or absent or a linking group; and each R 2 are independently hydrogen or a terminal reactive group; wherein at least one R 2is a terminal reactive group. In some embodiments, R 2 Independently selected from thiol-reactive groups, amino-reactive groups, and combinations thereof.

[0360] 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.

[0361] 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

[0362]

[0363] 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.

[0364] 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.

[0365] In some embodiments, the functionalized multi-arm PEG has a structure of formula (IIIa), wherein each R 2is 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.

[0366] In some embodiments, the functionalized multi-arm PEG has the structure of formula (IIIa), wherein each R 2 In some embodiments, the functionalized multi-arm PEG has a structure of formula (IIIa), 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 (IIIa), wherein when R 1 With R 2 When combined together Here, i is 3 and j is 2.

[0367] In another aspect, the functionalized multi-arm PEG has the structure of Formula (IVa):

[0368]

[0369] 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 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.

[0370] 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.

[0371] In some embodiments, the functionalized multi-arm PEG has a structure of formula (IVa), wherein each R 1 When present, they are the same or different, and when R1 With R 2 When combined, select

[0372] 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.

[0373] In some embodiments, the functionalized multi-arm PEG has a structure of formula (IVa), 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.

[0374] In some embodiments, 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.

[0375] In some embodiments, the functionalized multi-arm PEG has a structure of Formula (IVa), wherein each R 2 In some embodiments, the functionalized multi-arm PEG has a structure of formula (IVa), 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 (IVa), wherein when R 1 With R 2 When combined together Here, i is 3 and j is 2.

[0376] 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 herein incorporated by reference in their entireties.

[0377] Functionalized multi-arm PEGs suitable for use in the present disclosure are also commercially available from a variety of 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., HGEO-400MA; DX-400MA) and tetramers (e.g., PTE-400MA).

[0378] In a specific embodiment, the active derivative of the multi-arm PEG is an active NHS ester derivative of the multi-arm PEG, having the following general formula (IV):

[0379]

[0380] Wherein, R is dipentaerythritol.

[0381] 1. Polyol conjugates

[0382] In some embodiments, the present disclosure is directed to conjugates (e.g., Tie2 binders) comprising one or more 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 a structure of Formula (Ia).

[0383] The conjugates of the present disclosure can be characterized by the number of anti-Tie2 antibodies (Fabs) conjugated to each multi-arm PEG. This is referred to herein as "fabization" or "degree of fabization." 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 and / or reactivity of terminal reactive groups on the PEG; 3) the core structure of the PEG; and / or 4) the PEGylation reaction conditions. The high polydispersity of the multi-arm PEGs used to prepare the conjugates may, in some cases, complicate analysis of the final conjugates, particularly making accurate determination of the number of Fabs per PEG more difficult and uncertain. Accordingly, the PEGs used to form the conjugates will typically 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.

[0384] 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 conjugate of the present disclosure comprises a six-arm PEG, wherein 4 to 6 or 5 anti-Tie2 Fabs are covalently linked to the PEG.

[0385] In some embodiments, the conjugates of the present disclosure comprise a multi-arm PEG having a structure of any one of Formula (Ia), (IIa), (IIIa), 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 a structure of any one of Formula (Ia), (IIa), (IIIa), 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.

[0386] 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.

[0387] 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.

[0388] 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.

[0389] 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 can be a Fab fragment. These are useful for generating antibodies with specific properties, such as increased serum half-life, increased tissue penetration, and increased tumor penetration. L or C H1) with free cysteine. Parent antibodies engineered to contain cysteine ​​are referred to herein as "ThioMabs," and Fab fragments produced from such cysteine-engineered antibodies are referred to herein as "ThioFabs," regardless of the manufacturing method. As previously described (see, e.g., U.S. Patent Publication No. 2007 / 0092940 and Junutula, JR, et al., J. Immunol Methods, Vol. 332 (1-2), pp. 41-52 (2008)), the reactivity of the newly introduced engineered cysteine ​​thiol groups is assessed for mutants with replaced ("engineered") cysteine ​​(Cys) residues. The thiol reactivity value is a relative numerical term ranging from 0 to 1.0 and can be measured for any cysteine-engineered antibody. In addition to having reactive thiol groups, ThioMabs should be selected so that they retain antigen binding ability. The design, selection, and preparation of cysteine ​​engineered antibodies have been previously described in detail (see, e.g., WO 2011 / 069104, which is incorporated herein by reference). In some embodiments, engineered cysteines are introduced into the constant domains of the heavy or light chains. Thus, the cysteine ​​engineered antibodies retain the antigen-binding ability of their wild-type, parental antibody counterparts and are therefore capable of specific binding to the antigen.

[0390] In some embodiments, the disclosure relates 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 or heavy chain of the Fab fragment, which cysteine ​​residues would normally form an interchain disulfide bond linking the light chain to the heavy chain.

[0391] 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.

[0392] 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.

[0393] 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.

[0394] 3. Characterization of Polyol Conjugates

[0395] 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.

[0396] Each peak can then be analyzed by mass spectrometry to verify the size of the conjugate in that peak. Based on the PEG used in the conjugation and the size of the conjugate in that peak, the amount of antibody or variant conjugated to PEG can be estimated. Fragments conjugated to PEG groups often are not retained on the HPLC column after injection and disappear from the chromatogram. This disappearance from the chromatogram is a marker of PEGylation at a specific fragment that should contain at least one PEGylatable amino acid residue. The ability of the PEGylated anti-Tie Fab to interact with Tie2 and other biological activities can be further assayed using methods known in the art.

[0397] 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.

[0398] In some embodiments, the conjugates of the present disclosure have an increased half-life after administration to the eye of a mammal (e.g., a human) via a single intravitreal injection compared to a 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 greater than the half-life of the corresponding unconjugated anti-Tie2 Fab.

[0399] 3. IgM multimers as conjugates

[0400] In some embodiments, the Tie2 binders of the present disclosure may be prepared by combining more than one anti-Tie2 antibody described herein with the C H1 domains are linked, for example, using recombinant expression methods to link (via peptide bonds) IgM C H1 The C-terminus of the Ig domain is fused to the N-terminus of an anti-Tie2 antibody as described herein. IgM has been shown to be a viable form of antibody therapeutic (see, e.g., Hanala, 2012, MAbs, 4:555-561). The "monomer" component of IgM consists 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 form a heterodimer to form HC-LC homodimers. These homodimers are then covalently associated into a ring structure containing five homodimers and a J chain (JC) (pentamer) or six homodimers (hexamer), with pentamers and hexamers containing 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 to targets without affinity maturation and thus to serve as a sentinel adaptive immune receptor.

[0401] 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).

[0402] 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)

[0403] 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.

[0404] 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.

[0405] 3. Hexameric peptide multimers as conjugates

[0406] The present disclosure also encompasses conjugates comprising multiple antigen binding agents (e.g., antibodies or antigen binding fragments thereof), each antigen binding agent linked to a peptide that naturally forms a multimer, e.g., an NDK peptide. In some embodiments, the Tie2 binders 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 naturally fold to form a single multi-arm structure when expressed in a recombinant expression system. Conventional molecular engineering techniques and materials are used to express the peptides, which will form a multimer as a fusion, wherein the antigen binding protein (e.g., an antibody or fragment thereof) is expressed at the N-terminus or C-terminus of the peptides that form the multimer. In specific embodiments, the multiple peptides in the multimer are identical, and the expression vector is constructed to link the C-terminus or N-terminus of the peptide to the anti-Tie2 antibody or fragment thereof (e.g., via a peptide bond), respectively, as taught in the present disclosure.

[0407] In certain embodiments, the peptide of the multimeric peptide is a portion of a eukaryotic nucleoside diphosphate kinase (NDK) enzyme having a homologous hexameric quaternary structure. Several NDK enzymes exist that can be used to design multimeric NDKs, 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), and NDK5 (e.g., SEQ ID NO: 73). In a preferred embodiment, the NDK is derived from, for example, the NDK3 peptide of UniProt accession number P22887. SEQ ID NO: 74 provides the sequence of Tie2.1.M100cF linked at its C-terminus to the N-terminus of NDK3. In this preferred embodiment, the Fab light chain comprises SEQ ID NO: 21. (See, for example, Example 8 below.)

[0408] E. Methods and compositions for diagnosis and detection

[0409] In certain embodiments, any of the anti-Tie2 antibodies provided herein can be used to detect the presence of Tie2 in a biological sample. As used herein, the term "detection" encompasses quantitative or qualitative detection. In certain embodiments, the biological sample comprises cells or tissues, such as retinal tissue (photoreceptors and underlying retinal pigment epithelium (RPE) and choroidal capillaries).

[0410] In one embodiment, an anti-Tie2 antibody for use in a diagnostic or detection method is provided. In another aspect, a method for detecting the presence of Tie2 in a biological sample is provided. In certain embodiments, the method comprises contacting the biological sample with an anti-Tie2 antibody described herein under conditions permissive for binding of the anti-Tie2 antibody to Tie2, and detecting whether a complex is formed between the anti-Tie2 antibody and Tie2. This method can be in vitro or in vivo. In one embodiment, the anti-Tie2 antibody is used to select subjects suitable for treatment with the anti-Tie2 antibody, for example, Tie2 is used as a biomarker for patient selection.

[0411] In certain aspects, labeled anti-Tie2 antibodies are provided. Labels include, but are not limited to, directly detectable labels or moieties (e.g., fluorescent, chromogenic, electron-dense, chemiluminescent, and radioactive labels), as well as indirect detectable moieties (e.g., via enzymatic reactions or molecular interactions), 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 fluorescein and its derivatives; rhodamine and its derivatives; dansyl; umbelliferone; luciferases such as firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456); luciferin; 2,3-dihydrophthalazinone; horseradish peroxidase (HRP); alkaline phosphatase; β-galactosidase; glucoamylase; lysozyme; carbohydrate oxidases such as glucose oxidase, galactose oxidase, and glucose 6-phosphate dehydrogenase; heterocyclic oxidases such as urate oxidase and xanthine oxidase, used in conjunction with enzymes that oxidize dye precursors (e.g., HRP, lactoperoxidase, or microperoxidase) using hydrogen peroxide; biotin / avidin; spin labels; phage labels; stable free radicals, etc.

[0412] F. Pharmaceutical Preparations

[0413] 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.

[0414] 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.

[0415] The formulations described herein may also contain more than one active ingredient as appropriate for the particular indication being treated, preferably having complementary active ingredients that do not adversely affect each other. For example, it may 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-molecule 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 that binds to VEGF and / or Ang2 and / or IL-1β, or an antigen-binding fragment thereof.Such active ingredients are suitably present in combination in amounts that are effective for the intended purpose.

[0416] The active ingredient can be entrapped in microcapsules (e.g., hydroxymethylcellulose microcapsules or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively) prepared, for example, by coacervation techniques or by interfacial polymerization, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions. This technology is disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0417] 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.

[0418] Formulations to be used for in vivo administration are generally sterile. Sterility is readily achieved, e.g., by filtration through a sterile filtration membrane.

[0419] The conjugates described herein for preventing or treating an ocular disease or disorder are generally 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.

[0420] 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 generally direct intravitreal injection. Intravitreal injections are generally 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.

[0421] In certain embodiments, antibodies and conjugates as herein described can be formulated for use with an implantable port delivery system (PDS) for delivery. As previously mentioned, PDS is a refillable device in which release into the vitreous body is controlled by a porous metal membrane comprising titanium glass. In certain embodiments, due to the small volume of the reservoir, PDS is used to effectively deliver the required protein concentration. Therefore, in certain embodiments, antibodies and conjugates as herein described are formulated at high concentrations. In certain embodiments, antibodies and conjugates as herein described can be 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 can be 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.

[0422] G. Methods of Treatment and Compositions

[0423] Any anti-Tie2 antibody or conjugate provided herein can be used in a method of treatment. An "individual," "patient," or "subject" according to any embodiment herein can be a human.

[0424] The anti-Tie2 conjugates disclosed herein can be used to treat mammals. In some embodiments, for example, the anti-Tie2 conjugates are administered to non-human mammals to obtain 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 conducted. Such mammals can be established animal models for the disease to be treated with the antibody, or can be used to study the toxicity of the antibody of interest. In each of these embodiments, a dose escalation study can be performed on the mammal.

[0425] The anti-Tie2 conjugate can be administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intravitreal, intrapulmonary and intranasal administration, and, if local immunosuppressive therapy is required, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, intravitreal and subcutaneous administration. In addition, the conjugate is suitable for administration by pulse infusion, especially for decreasing doses of the antibody or its antibody variant or fragment thereof (e.g., antigen binding fragment). In some embodiments, administration can be performed by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-term or long-term.

[0426] For the prevention or treatment of disease, the appropriate dosage of the anti-Tie2 antibody or conjugate will depend on the type of disease to be treated, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the judgment of the attending physician.

[0427] 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 may be an initial candidate dose for administration to the patient, for example, by one or more separate administrations or by continuous infusion. For repeated administrations over several days or longer, depending on the condition, the treatment is continued until the desired suppression of disease symptoms occurs. However, other dosage regimens may be used. The progress of this treatment is readily monitored by conventional techniques and assays. Exemplary methods of administration are disclosed in WO 94 / 04188.

[0428] In one aspect, an anti-Tie2 antibody or conjugate for use as a medicament is provided. In a further aspect, an anti-Tie2 antibody or conjugate for use in treating an ocular disease or disorder is provided. In certain embodiments, an anti-Tie2 antibody or conjugate for use in a method of treating a patient suffering from an ocular disease or disorder is provided. In certain embodiments, an anti-Tie2 antibody or conjugate is provided. In certain embodiments, the present invention provides an anti-Tie2 antibody for use in a method of treating a patient suffering from an ocular disease or disorder, the method comprising administering to the patient an effective amount of the anti-Tie2 antibody or conjugate. In one such embodiment, the method further comprises administering to the patient an effective amount of at least one additional therapeutic agent (e.g., as described below). In a further embodiment, the present invention provides an anti-Tie2 antibody or conjugate for increasing vascular endothelial cell membrane integrity and / or reducing vascular leakage. In certain embodiments, the present invention provides an anti-Tie2 antibody or conjugate for use in a method of increasing vascular endothelial cell membrane integrity and / or reducing vascular leakage in a patient, the method comprising administering to the patient an effective anti-Tie2 antibody or conjugate to increase vascular endothelial cell membrane integrity and / or reduce vascular leakage. The "patient" according to any of the above embodiments is preferably a human.

[0429] As used herein, the term "ocular disease" includes any ocular disease associated with pathological angiogenesis and / or atrophy (also interchangeably referred to herein as "ocular disease"). An ocular disease can be characterized by altered or unregulated proliferation and / or invasion of new blood vessels into structures of ocular tissues, such as the retina or cornea. An ocular disease can be characterized by atrophy of retinal tissues (photoreceptors and underlying retinal pigment epithelium (RPE) and choriocapillaris).

[0430] Diabetic macular edema (DME) is caused by a complication of diabetes known as diabetic retinopathy (DR). This eye condition can occur in people diagnosed with either type 1 or type 2 diabetes. DME is defined as retinal thickening within the fovea of ​​the eye that is less than 2 disc diameters and can be focal or diffuse. DME is associated with changes in the retinal microvasculature accompanied by a breakdown of the blood-retinal barrier, resulting in leakage of plasma components into the peripheral retina, leading to retinal edema.

[0431] Non-limiting eye diseases include, for example, diabetic macular edema (DME) (e.g., localized non-central DME and diffuse DME involving the fovea), diabetic retinopathy (DR) (e.g., proliferative DR (PDR), non-proliferative DR (NPDR) and high altitude DR), retinopathy without edema, 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 branching (BRVO) forms), CNV (e.g., myopic CNV), corneal neovascularization, diseases associated with corneal neovascularization, retinal neovascularization, diseases associated with retinal / choroidal neovascularization, central serous chorioretinopathy (CSR), pathological myopia, Hippel-Lincoln disease, ocular histoplasmosis, FEVR, Coats' disease, Norrie's disease, retinal abnormalities associated with osteoporosis-pseudoglioma syndrome (OPPG), subconjunctival hemorrhage, redness of the skin, ocular neovascular disease, neovascular glaucoma, retinitis pigmentosa (RP), hypertensive retinopathy, retinal angiomatous proliferation, macular telangiectasia, iris neovascularization, intraocular neovascularization, retinal degeneration, cystoid macular edema (CME), vasculitis, optic disc edema, retinitis (including but not limited to CMV retinitis), ocular melanoma, retinoblastoma, conjunctivitis (e.g., infectious conjunctivitis and non-infectious (e.g., allergic) conjunctivitis), Leber's 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, Diseases of the eye include retinal edema or retinal atrophy, and diseases of the eye associated with ocular neovascularization, vascular leakage, and / or retinal swelling or atrophy. Additional exemplary eye diseases include retinoschisis (abnormal division of the neurosensory layer of the retina), diseases associated with redness of the skin (neovascularization of the eyelid corners), and diseases caused by abnormal proliferation of fibrovascular or fibrous tissue (including all forms of proliferative vitreoretinopathy).

[0432] Exemplary diseases associated with corneal neovascularization include, but are not limited to, epidemic keratoconjunctivitis, vitamin A deficiency, excessive contact lens wear, atopic keratitis, superior limbal keratitis, terygium keratitis sicca, Sjögren's syndrome, acne, phylectenulosis, syphilis, mycobacterial infection, lipid degeneration, chemical burns, bacterial ulcers, fungal ulcers, herpes simplex infection, herpes zoster virus infection, protozoal infection, Kaposi's sarcoma, molluscum corneal ulcer, Trion's marginal corneal degeneration, marginal keratolysis, rheumatoid arthritis, systemic lupus erythematosus, polyarteritis, trauma, Wegener's sarcoidosis, scleritis, Steven's Johnson disease, pemphigoid radial keratotomy, and corneal graph rejection.

[0433] Exemplary diseases associated with choroidal neovascularization and defects in the retinal vasculature, including increased vascular leakage, aneurysms, and capillary dripping, include, but are not limited to, diabetic retinopathy, macular degeneration, sickle cell anemia, sarcoidosis, syphilis, pseudoxanthoma elasticum, Paget's disease, venous occlusion, arterial occlusion, carotid occlusive disease, chronic uveitis / vitritis, mycobacterial infection, Lyme disease, systemic lupus erythematosus, retinopathy of prematurity, retinal edema (including macular edema), Eal's disease, Behçet's disease, infection due to retinitis or choroiditis, presumed ocular histoplasmosis, Best's disease (vitelliform macular degeneration), myopia, optic pits, pars planitis, retinal detachment (e.g., chronic retinal detachment), hyperviscosity syndrome, toxoplasmosis, trauma, and post-laser complications.

[0434] 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 and / or geographic atrophy associated with neovascularization), diabetic retinopathy, Stargardt's disease, Skorsby's fundus atrophy, retinoschisis, and retinitis pigmentosa.

[0435] 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 variants thereof. In yet further examples, in some cases, the additional compound is an anti-IL-6R antibody, e.g., tocilizumab ) (see, e.g., WO 1992 / 019579), sarilumab, ALX-0061, SA237, or variants thereof.

[0436] In some cases, the Tie2 binders or conjugates and / or polymeric formulations thereof disclosed herein may be administered in combination with at least one additional therapeutic agent for the treatment of an ocular disease, such as an ocular disease described herein (e.g., DME, DR, AMD (e.g., wet AMD), RVO, or GA). Exemplary additional therapeutic agents used in combination therapies for the treatment of ocular diseases include, without limitation, anti-angiogenic agents such as VEGF antagonists, including, for example, anti-VEGF antibodies (e.g., anti-VEGF Fab (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 suspension; Alcon, Inc.); Combretastatin A4 prodrug (CA4P); (mifepristone-ru486); triamcinolone acetonide; intravitreal crystalline triamcinolone acetonide; matrix metalloproteinase inhibitors (e.g., Prinomastat (AG3340; Pfizer)); fluocinolone acetonide (including fluocinolone intraocular implant; Bausch & Lomb / controlled delivery system); linomide; inhibitors of integrin β3 function; angiostatin, and combinations thereof. These and other therapeutic agents that can be administered in combination with the Tie2 binders or conjugates of the present invention are described in U.S. Patent Application No. US2014 / 0017244, which is incorporated herein by reference in its entirety.

[0437] Further examples of additional therapeutic agents that can be administered in combination with the Tie2 binder or conjugate and / or polymeric formulation thereof to treat ocular diseases (e.g., DME, DR, AMD, RVO or GA) include, but are not limited to, (verteporfin; a light-activated drug often used in conjunction with photodynamic therapy using non-thermal lasers), 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.

[0438] 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.

[0439] In some cases, the Tie2 binders or conjugates of the present invention and / or their polymeric formulations may 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 may be used in combination with the Tie2 binders or conjugates of the present invention, including but not limited to those described 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., an anti-VEGF Fab). (ranibizumab), RTH-258 (formerly known as ESBA-1008, an anti-VEGF single-chain antibody fragment; Novartis), or bispecific anti-VEGF antibodies (e.g., anti-VEGF / anti-angiopoietin 2 bispecific antibodies such as RG-7716; Roche), soluble recombinant receptor fusion proteins (e.g., (aflibercept), VEGF variants, soluble VEGFR fragments, aptamers capable of blocking VEGF (e.g., pegaptanib) or VEGFR, neutralizing anti-VEGFR antibodies, small molecule inhibitors of VEGFR tyrosine kinases, anti-VEGF (e.g., abicipar pegol, Molecular Partners AG / Allergan), small interfering RNA that inhibits the expression of VEGF or VEGFR, 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), varatranil (PTK787), semaminid (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).

[0440] Other suitable anti-angiogenic agents that can be administered in combination with the anti-Tie2 conjugate and / or polymeric formulation thereof for the treatment of ocular diseases (e.g., DME, DR, AMD, RVO, or GA) include corticosteroids, angiostatic steroids, alitretinoin, 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.

[0441] In further examples, in some cases, the Tie2-binding conjugate and / or polymeric formulation thereof can be administered in combination with an agent having anti-neovascularization activity for the treatment of ocular diseases (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, tumor necrosis factor (TNF) antagonists (e.g., anti-TNFα antibodies or antigen-binding fragments thereof (e.g., infliximab, adalimumab, certolizumab pegol, and golimumab) or soluble receptor fusion proteins (e.g., etanercept)), anti-complement agents, nonsteroidal anti-inflammatory agents (NSAIDs), or combinations thereof.

[0442] In yet further examples, in some cases, the Tie2-binding conjugates and / or polymeric formulations 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) (trade name: PRASTERA TM and ), dehydroepiandrosterone sulfate and pregnenolone sulfate.

[0443] Any suitable AMD therapeutic agent can be administered as an additional therapeutic agent in combination with the Tie2-binding conjugates of the present invention and / or polymeric formulations thereof to treat an ocular disease (e.g., DME, DR, AMD, RVO, or GA), including but not limited to, VEGF antagonists, e.g., anti-VEGF antibodies (e.g., (ranibizumab), RTH-258 (formerly known as ESBA-1008, an anti-VEGF single-chain antibody fragment; Novartis) or bispecific anti-VEGF antibodies (e.g., anti-VEGF / anti-angiopoietin 2 bispecific antibodies such as RG-7716; Roche)), soluble VEGF receptor fusion proteins (e.g., (Aflibercept), anti-VEGF (e.g., abicipar pegol; Molecular Partners AG / Allergan) or anti-VEGF aptamers (e.g., (pegaptanib sodium)); platelet-derived growth factor (PDGF) antagonists, e.g., anti-PDGFR antibodies (e.g., REGN2176-3), anti-PDGF-BBpegated aptamers (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); combined with photodynamic therapy (verteporfin); antioxidants; complement system antagonists, e.g., complement factor C5 antagonists (e.g., small molecule inhibitors (e.g., ARC-1905; Opthotech) 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 blocking peptides (e.g., APL-2, Appellis); visual cycle modifiers (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 (AREDS 1); zinc and / or antioxidants) and Study 2 (e.g., squalamine); 2 (AREDS2; zinc, antioxidants, lutein, zeaxanthin, and / or omega-3 fatty acids); cell-based therapies, for example, NT-501 (Renexus); PH-05206388 (Pfizer), huCNS-SC cell transplantation (StemCells), CNTO-2476 (umbilical cord stem cell line; Janssen), OpRegen (suspension of RPE cells; Cell Cure Neurosciences), or MA09-hRPE cell transplantation (Ocata Therapeutics); tissue factor antagonists (e.g., hI-con1; Iconic Therapeutics); alpha-adrenergic receptor agonists (e.g., brimonidine tartrate; tartrate); Allergan); peptide vaccines (e.g., S-646240; Shionogi); β-amyloid antagonists (e.g., anti-β-amyloid monoclonal antibodies, e.g., GSK-933776); S1P antagonists (e.g., anti-S1P antibodies, e.g., iSONEP TM; Lpath Inc); ROB04 antagonists (e.g., anti-ROBO4 antibodies, e.g., DS-7080a; Daiichi Sankyo); lentiviral vectors expressing endostatin and angiostatin (e.g., RetinoStat), and combinations thereof. In some cases, AMD therapeutics (including any of the aforementioned AMD therapeutics) can be co-formulated. For example, the anti-PDGFR antibody REGN2176-3 can be co-administered with aflibercept ( ) are co-formulated. In some cases, this co-formulation can be administered in conjunction with a Tie2 binder or conjugate of the present invention. In some cases, the eye disease is DME and / or DR. In some cases, the eye disease is AMD (e.g., wet AMD).

[0444] Tie2 binding conjugates and / or polymeric formulations thereof can be used with 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.

[0445] Tie2 binding conjugates and / or polymeric formulations thereof can be used with 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.

[0446] Tie2 binding conjugates and / or polymeric formulations thereof can be used with (pegaptanib sodium) is administered in combination with dapoxetine for the treatment of an eye disease (e.g., DME, DR, AMD, RVO, or GA). 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] Tie2 binding conjugates and / or polymeric formulations thereof can be used with (verteporfin) and photodynamic therapy in combination with for the treatment of eye diseases (e.g., DME, DR, AMD, RVO or GA). 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.

[0448] A 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 a 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, OHR-102 can be administered as an adjunct therapy to a 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, a 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.

[0449] A 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.

[0450] A 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.

[0451] A 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.

[0452] 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.

[0453] 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).

[0454] 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).

[0455] Tie2 binding conjugates and / or polymeric formulations thereof can be used with (dexamethasone intravitreal implant) for the treatment of DME and / or DR.

[0456] Tie2 binding conjugates and / or polymeric formulations thereof can be used with (dexamethasone intravitreal implant) for the treatment of DME and / or DR.

[0457] 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.

[0458] Such combination therapies described above encompass combined administration (where two or more therapeutic agents are contained in the same or separate pharmaceutical compositions), as well as separate administration, in which case administration of the Tie2 conjugate of the present invention may occur before, simultaneously with, and / or after administration of the additional therapeutic agent(s). In one embodiment, administration of the Tie2 binding conjugate or polymeric formulation and 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.

[0459] It is further contemplated that the Tie2-binding conjugates and / or polymeric formulations thereof are useful for treating 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 death of retinal ganglion cells (RGCs), loss of axons, and increased appearance of the optic nerve head (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 to detect "cupping." The mean IOP for 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).

[0460] There are currently five main categories of medications used to lower IOP: beta-adrenergic antagonists, adrenergic agonists, parasympathomimetics, prostaglandin analogs, and carbonic anhydrase inhibitors. Although most medications are applied externally to the eye, they can cause serious systemic side effects and negatively impact the patient's quality of life. If additional IOP reduction is indicated or if the medication fails to successfully reduce IOP, the next step is generally laser trabeculoplasty. If IOP is still not adequately controlled, incisional glaucoma surgery is indicated. Although a reduction in IOP significantly reduces the extent of neuronal loss, it does not guarantee cessation of disease progression because RGCs may continue to be lost. Recent studies on the association between IOP regulation and visual field loss after medical or surgical intervention have shown that if IOP is lower, the persistent neuronal loss reflected in visual field testing can be reduced. Glaucomatous optic neuropathy may be due to specific pathophysiological changes in RGCs and their axons and subsequent death. The RGC death process is considered a two-stage process, with an initiation of damage caused by a primary insult, followed by a slow secondary degeneration that is attributed to the harsh environment surrounding the degenerating cells.

[0461] Another aspect of the present invention provides the use of an anti-Tie2 conjugate of the present invention in the manufacture or preparation of a medicament. In one embodiment, the medicament is used to treat an eye disease (e.g., DME, DR, AMD, RVO or GA). In a preferred embodiment, the medicament is used to treat DME and / or DR. In a further embodiment, the medicament is used in a method for treating an eye disease (e.g., DME, DR, AMD, RVO or GA), the method comprising administering an effective amount of the medicament to an individual suffering from the eye disease. In one such embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent (e.g., as described below) to the individual. In a further embodiment, the medicament is used to reduce vascular permeability, especially in the eye. In a further embodiment, the medicament is used in a method for reducing vascular permeability in an individual, especially in the eye, the method comprising administering an effective amount of the medicament to the individual to reduce vascular permeability, especially in the eye. The "individual" according to any of the above embodiments may be a human.

[0462] In a further aspect, the present invention provides a method for treating an ocular disease (e.g., DME, DR, AMD, RVO or GA). In one embodiment, the method comprises administering an effective amount of a Tie2-binding conjugate of the present invention to an individual suffering from such an ocular disease (e.g., DME, DR, AMD, RVO or GA). In one such embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent (as described below) to the individual. The "individual" according to any of the above embodiments can be a human.

[0463] In a further aspect, the present invention provides a method for reducing vascular permeability in a subject, particularly in the eye. In one embodiment, the method comprises administering to the subject an effective amount of a Tie2-binding conjugate of the invention to reduce vascular permeability, particularly in the eye. In one embodiment, the "subject" is a human.

[0464] In a further aspect, the present invention provides a pharmaceutical formulation comprising any of the Tie2-binding conjugates of the invention provided herein, e.g., for use in any of the above treatment methods. In one embodiment, the pharmaceutical formulation comprises any of the Tie2-binding conjugates of the invention provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical formulation comprises any of the Tie2-binding conjugates of the invention provided herein and at least one additional therapeutic agent, e.g., as described above.

[0465] The combination therapy mentioned above encompasses combined administration (wherein two or more therapeutic agents are contained in the same or separate pharmaceutical compositions), as well as separate administration, in which case administration of the antibody of the invention may occur before, simultaneously with, and / or after administration of the additional therapeutic agent(s). In one embodiment, administration of the anti-Tie2 conjugate and administration of the additional therapeutic agent occur within about 1 month, or within about 1, 2, or 3 weeks, or within about 1, 2, 3, 4, 5, or 6 days of each other.

[0466] The Tie2 conjugates of the present invention (and any other therapeutic agents) can be administered by any suitable means, including parenteral, intrapulmonary and intranasal administration, and if local treatment is required, intralesional administration can be used. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration can be carried out by any suitable route, for example by injection, such as intravenous or subcutaneous injection, depending in part on whether it is a short-term administration or a long-term administration. Various dosing regimens are contemplated herein, including but not limited to single or multiple administrations at various time points, rapid injection administration and pulse infusion.

[0467] The Tie2-binding conjugates of the present invention will be formulated, dosed, and administered in a manner consistent with good medical practice. Factors considered in this context include the specific disorder to be treated, the specific mammal to be treated, the clinical condition of the individual patient, the cause of the disorder, the site of drug delivery, the method of administration, the schedule of administration, and other factors known to medical practitioners. The antibody is not required to be, but can optionally be, formulated with one or more agents currently used to prevent or treat the disease in question. The effective amount of these other therapeutic agents depends on the amount of antibody present in the formulation, the type of disorder or treatment, and the other factors discussed above. These are generally used in the same dosages and by the same routes of administration as described herein, or about 1% to 99% of the dosages described herein, or in any dosage and by any route determined empirically / clinically to be appropriate.

[0468] For the prevention or treatment of a disease, the appropriate dose of the conjugate of the invention (used alone or in combination with one or more other therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, the administration of the conjugate for the purpose of prevention or treatment, previous treatment, the patient's clinical history and response to the antibody, and the judgment of the attending physician. The conjugate is suitably administered to the patient in one or a series of treatments. Depending on the type and severity of the disease, a conjugate of about 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg–10 mg / kg) may be an initial candidate dose for administration to the patient, for example, by one or more separate administrations or by continuous infusion. Depending on the above factors, a typical daily dose may be in the range of about 1 μg / kg to 100 mg / kg or more. For repeated administration over several days or longer, depending on the condition, treatment will generally continue until the desired suppression of disease symptoms occurs. An exemplary dose of the conjugate will be in the range of from 0.05 mg / kg to about 10 mg / kg. Thus, the patient may be administered one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, or 10 mg / kg (or any combination thereof). Such doses may be administered intermittently, for example weekly or every three weeks (e.g., so that the patient receives from about 2 to about 20, or for example, about 6, doses of the conjugate). An initial higher loading dose may be administered, followed by one or more lower doses. The progress of this treatment is readily monitored by conventional techniques and assays.

[0469] It will be understood that any of the formulations and data methods described above may be performed using an immunoconjugate of the invention instead of or in addition to the Tie2-binding conjugate of the invention.

[0470] H. Finished products

[0471] Another aspect of the present invention provides an article of manufacture comprising a composition for treating, preventing, and / or diagnosing the aforementioned 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, and the like. The container can be formed from a variety of materials, such as glass or plastic. The container can hold a composition, which can be used alone or in combination with another composition effective for treating, preventing, and / or diagnosing the disease, and may have a sterile access port (e.g., the container can be an intravenous solution bag or cannula with a stopper pierceable by a hypodermic needle). At least one active agent in the composition is a Tie2 binder or conjugate of the present invention. The label or package insert indicates that the composition is used to treat a selected disease. In addition, the article of manufacture may include (a) a first container containing a composition, wherein the composition contains a Tie2 binder or conjugate of the present invention; and (b) a second container containing a composition, wherein the composition contains another cytotoxic or other therapeutic agent. The article of manufacture in this embodiment of the present invention may further include a package insert indicating that the composition can be used to treat a specific disease. Alternatively or additionally, the article of manufacture may 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. From a commercial and user perspective, it may further comprise other materials, including other buffers, diluents, filters, needles, and syringes.

[0472] It will be understood that any of the above-described articles of manufacture may include an immunoconjugate of the invention instead of or in addition to a Tie2-binding conjugate.

[0473] Ⅰ Specific embodiments of the present invention

[0474] Specific embodiments of the present invention are described below.

[0475] 1. An isolated antibody or fragment thereof that specifically binds to Tie2, wherein the antibody comprises:

[0476] 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).

[0477] 2. The antibody of the preceding embodiment, wherein the CDR-H3 comprises the amino acid sequence RTRWASWAMDY (SEQ ID NO: 6).

[0478] 3. The antibody of embodiment 1, wherein the CDR-H3 comprises the amino acid sequence RTRWASWAFDY (SEQ ID NO: 7).

[0479] 4. The antibody according to any one of the preceding embodiments, which is a monoclonal antibody.

[0480] 5. The antibody of any preceding embodiment, which is a humanized antibody or a chimeric antibody.

[0481] 6. The antibody of any preceding embodiment, which is an antibody fragment that binds to Tie2.

[0482] 7. The antibody of any preceding embodiment, which is a Fab fragment.

[0483] 10. The antibody of any one of embodiments 1 to 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.

[0484] 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.

[0485] 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.

[0486] 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 with the amino acid sequence of SEQ ID NO: 21; and a VH domain comprising an amino acid sequence having at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 20.

[0487] 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 with the amino acid sequence of SEQ ID NO: 21; and a VH domain comprising an amino acid sequence having at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 20.

[0488] 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.

[0489] 16. The antibody of any preceding embodiment, wherein the antibody comprises an engineered cysteine.

[0490] 17. The antibody of embodiment 16, wherein

[0491] The engineered cysteine ​​is selected from T120C, G166C, G178C, T187C and T209C in HC; or the engineered cysteine ​​is selected from Q124C, R142C, Q155C, L201C, T206C, K107C, K126C and K149C in LC; wherein the residue number of the engineered cysteine ​​is according to EU numbering.

[0492] 18. The antibody of embodiment 16 or 17, wherein the engineered cysteine ​​is selected from T209C in HC and T206C in LC.

[0493] 19. The antibody of any one of embodiments 16 to 18, wherein the engineered cysteine ​​is T206C in LC.

[0494] 20. The antibody of any one of embodiments 16 to 18, wherein the engineered cysteine ​​is T209C in HC.

[0495] 21. The antibody of any one of the preceding embodiments, comprising: an LC comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 25; and an HC comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 55.

[0496] 22. The antibody of any one of the preceding embodiments, comprising: an LC comprising an amino acid sequence having at least 96% sequence identity to the amino acid sequence of SEQ ID NO: 25; and an HC comprising an amino acid sequence having at least 96% sequence identity to the amino acid sequence of SEQ ID NO: 55.

[0497] 23. The antibody of any one of the preceding embodiments, comprising: an LC comprising an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO: 25; and an HC comprising an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO: 55.

[0498] 24. The antibody of any one of the preceding embodiments, comprising: an LC comprising an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 25; and an HC comprising an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 55.

[0499] 25. The antibody of any one of the preceding embodiments, comprising: an LC comprising an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 25; and an HC comprising an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 55.

[0500] 26. The antibody of any one of the preceding embodiments, wherein the antibody comprises: an LC comprising the sequence of SEQ ID NO: 25 and an HC comprising the sequence of SEQ ID NO: 55.

[0501] 27. The antibody of any one of embodiments 1 to 25, wherein the antibody comprises: an LC comprising the sequence of SEQ ID NO: 25 and an HC comprising a sequence selected from the group consisting of SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91 and SEQ ID NO: 92.

[0502] 28. The antibody of any one of embodiments 1 to 20, comprising: an LC comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 56; and an HC comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 23.

[0503] 29. The antibody of any one of embodiments 1 to 20, comprising: an LC comprising an amino acid sequence having at least 96% sequence identity with the amino acid sequence of SEQ ID NO: 56; and an HC comprising an amino acid sequence having at least 96% sequence identity with the amino acid sequence of SEQ ID NO: 23.

[0504] 30. The antibody of any one of embodiments 1 to 20, comprising: an LC comprising an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO: 56; and an HC comprising an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO: 23.

[0505] 31. The antibody of any one of embodiments 1 to 20, comprising: an LC comprising an amino acid sequence having at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 56; and an HC comprising an amino acid sequence having at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 23.

[0506] 32. The antibody of any one of embodiments 1 to 20, comprising: an LC comprising an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 56; and an HC comprising an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 23.

[0507] 33. The antibody of any one of embodiments 1 to 20, wherein the antibody comprises: an LC comprising the sequence of SEQ ID NO: 56 and an HC comprising the sequence of SEQ ID NO: 90, SEQ ID NO: 91 or SEQ ID NO: 23.

[0508] 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.

[0509] 35. An isolated nucleic acid encoding the antibody of any one of the preceding embodiments.

[0510] 36. An isolated host cell comprising the nucleic acid of embodiment 38.

[0511] 37. A method for producing an antibody that binds to Tie-2, comprising culturing the host cell of Example 39 under conditions suitable for expression of the antibody.

[0512] 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 one of embodiments 1 to 34, wherein each of the antibodies is linked to a multimerization moiety.

[0513] 39. The conjugate of embodiment 38, wherein the conjugate activates the phosphorylation of Tie2 in an in vitro or in vivo cell assay.

[0514] 40. The conjugate of embodiment 38 or 39, wherein the conjugate causes a decrease in Tie-2 protein levels by 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 by more than 25%, more than 50%, or more than 75% in an in vitro assay.

[0515] 41. The conjugate of embodiments 38 to 40, wherein the conjugate reduces vascular permeability as measured by an in vitro barrier function assay.

[0516] 42. The conjugate of any one of embodiments 38 to 42, wherein the multimerization moiety comprises a polyol, a polypeptide and / or a peptide.

[0517] 43. The conjugate of embodiment 42, wherein the polyol is a multi-arm polyol selected from a dimer, a tetramer, a hexamer, and an octamer.

[0518] 44. The conjugate of embodiment 43, wherein the multi-arm polyol is a hexamer.

[0519] 45. The conjugate of embodiment 43 or 44, wherein the multi-arm polyol is an octamer.

[0520] 46. ​​The conjugate of any one of embodiments 42 to 45, wherein the polyol is polyethylene glycol (PEG).

[0521] 47. The conjugate of any one of embodiments 42 to 45, wherein the polyol is covalently linked to at least two antibodies via free thiol groups of cysteine ​​amino acids.

[0522] 48. The conjugate of embodiment 47, wherein the cysteine ​​amino acid is an engineered cysteine.

[0523] 49. The conjugate of embodiment 48, wherein the engineered cysteine ​​is located within the HC and / or LC constant region of the antibody.

[0524] 50. The conjugate of embodiment 48 or 49, wherein the engineered cysteine ​​is selected from T120C, G166C, G178C, T187C, and T209C in HC; or the engineered cysteine ​​is selected from the group consisting of Q124C, R142C, Q155C, L201C, T206C, K107C, K126C, and K149C in LC; wherein the residue numbering is according to EU numbering.

[0525] 51. The conjugate of any one of embodiments 48 to 50, wherein the engineered cysteine ​​is T206C in LC, wherein the residue numbering is according to EU numbering.

[0526] 52. The conjugate of any one of embodiments 48 to 50, wherein the engineered cysteine ​​is T209C in HC, wherein the residue numbering is according to EU numbering.

[0527] 53. The conjugate of any one of embodiments 42 to 45, wherein the polyol is covalently linked to at least one antibody via a free amino group of a lysine amino acid.

[0528] 54. The conjugate of embodiment 53, wherein the lysine amino acid is located within the HC or LC constant region of the antibody, and / or the lysine amino acid is located at the C-terminus of the heavy chain or light chain of the antibody.

[0529] 55. The conjugate of any one of embodiments 46 to 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.

[0530] 56. The conjugate of any one of embodiments 46 to 55, wherein the PEG has a weight average molecular weight of about 6000 Da.

[0531] 57. The conjugate of any one of embodiments 46 to 56, wherein the PEG comprises a dipentaerythritol hexamer or octamer core.

[0532] 58. The conjugate of any one of embodiments 46 to 57, wherein the PEG has the structure of formula (Ia):

[0533]

[0534] wherein n is an integer from 1 to 10; each m is independently an integer from 3 to 250; each R 1 independently absent or a linking group; and each R 2 are independently hydrogen or a terminal reactive group;

[0535] Among them, at least one R 2 is a terminal reactive group and is covalently linked to the antibody according to any one of claims 1 to 14.

[0536] 59. The conjugate of any one of embodiments 46 to 57, wherein the PEG has the structure of formula (Ib):

[0537]

[0538] wherein each m is independently an integer from 3 to 250; each R 1 independently absent or a linking group; and each R 2 are independently hydrogen or a terminal reactive group;

[0539] Among them, at least one R 2 is a terminal reactive group and is covalently linked to the antibody according to any one of claims 1 to 14.

[0540] 60. The conjugate of embodiment 59, wherein each m is independently an integer from 15 to 35, preferably from about 20 to 30.

[0541] 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-arm polyol.

[0542] 62. A conjugate comprising an antibody that specifically binds to Tie-2, wherein the antibody comprises the VH sequence of SEQ ID NO: 20 and the VL sequence of SEQ ID NO: 21, wherein the antibody is covalently linked to polyethylene glycol having the structure of general formula (Ib):

[0543]

[0544] wherein each m is independently an integer from 3 to 250; each R 1 independently absent or a linking group; and each R 2 are independently hydrogen or a terminal reactive group;

[0545] Among them, at least one R 2 It is a terminal reactive group and is covalently linked to the antibody.

[0546] 63. The conjugate of embodiment 62, wherein m is an integer from 10 to 200.

[0547] 64. The conjugate of embodiment 62 or 63, wherein m is an integer from 20 to 30.

[0548] 65. The conjugate of any one of embodiments 58 to 64, wherein R 1 is not present, or, where R 1 Select 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.

[0549] 66. The conjugate of any one of embodiments 58 to 65, wherein each R 2 Independently selected from the group consisting of maleimide, sulfhydryl, thiol, triflate, tosylate, aziridine, peroxide, pyridyl disulfide, succinimidyl ester, -NH2, aldehyde, haloacetate, haloacetamide, and p-nitrophenyl carbonate.

[0550] 67. The conjugate of any one of embodiments 58 to 66, wherein R 2 It is maleimide.

[0551] 68. The conjugate of any one of embodiments 43 to 67, wherein the conjugate is prepared by covalently linking at least one antibody according to any one of claims 1 to 34 to a multi-arm polyol.

[0552] 69. A pharmaceutical composition comprising: the conjugate according to any one of embodiments 38 to 68; and a pharmaceutically acceptable carrier.

[0553] 70. The pharmaceutical composition of embodiment 69, wherein the concentration of the conjugate is about 50 mg / ml to about 300 mg / ml.

[0554] 71. The pharmaceutical composition of embodiment 69 or 70, further comprising an additional therapeutic agent.

[0555] 72. The pharmaceutical composition of embodiment 71, 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.

[0556] 73. The pharmaceutical composition of embodiment 72, wherein the VEGF antagonist is selected from the group consisting of a VEGF capture agent and an anti-VEGF antibody.

[0557] 74. A long-acting delivery device for transocular delivery comprising a pharmaceutical composition according to embodiments 79 to 73 and means for delivering the composition intravitreally to a patient, wherein the composition remains effective in situ for an extended period of time.

[0558] 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 the antibody of any one of embodiments 1 to 34, the conjugate of any one of embodiments 38 to 68, or the pharmaceutical composition of any one of embodiments 69 to 73.

[0559] 76. The method of embodiment 75, wherein the disease mediated by the Tie2 pathway is a vascular permeability disease.

[0560] 77. The method of embodiment 75 or 76, wherein the Tie2 pathway-mediated disease is an ocular disorder.

[0561] 78. The method of embodiment 77, wherein the ocular disorder 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, pathological myopia, von Hippel-Lindau disease, ocular histoplasmosis, central retinal vein occlusion (CRVO), corneal neovascularization, glaucoma, and retinal neovascularization.

[0562] 79. The method of any one of embodiments 75 to 78, wherein the eye disorder is DME.

[0563] 80. The method of any one of embodiments 75 to 79, wherein the method comprises administering the antibody, conjugate, or pharmaceutical formulation using an implantable port delivery system.

[0564] 81. The method of any one of embodiments 75 to 79, wherein the method comprises administering the antibody, conjugate, or pharmaceutical formulation by intravenous administration.

[0565] 82. The method of embodiment 81, wherein the intravenous administration is performed through a narrow-gauge needle.

[0566] 83. The method of embodiment 82, wherein the narrow-gauge needle is about 30, 29, 28, 27, 26, 25, 24, 23, or 22 gauge.

[0567] 84. The method of any one of embodiments 75 to 83, further comprising administering an additional therapeutic agent to the subject.

[0568] III. Example

[0569] The following are examples of methods and compositions of the present invention. It will be understood that various other embodiments may be practiced, given the general description given above.

[0570] Example 1 - Generation of anti-Tie 2 antibodies from a natural phage library

[0571] Antibodies that bind to the extracellular domain (ECD) of Tie2 were initially selected from a phage-displayed 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 were screened for the ECD and various subdomains of the ECD.

[0572] Phage for library construction

[0573] Phage plasmids pV0350-2b and pV0350-4 are designed to display Fab templates monovalently or bivalently, respectively, on the surface of M13 phage particles. The Fab templates are based on the h4D5 antibody, a humanized antibody that recognizes a cancer-associated antigen called 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 with specificity for Her-2 in a 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 for making anti-Her-2 antibodies and the identity of variable domain sequences are provided in US Pat. Nos. 5,821,337 and 6,054,297.

[0574] 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).

[0575] 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.

[0576] 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 (Lib-3) with Fab.zip template. 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).

[0577] For positions 95–100 in CDR-H3, Lib-3 consists of a set of libraries containing NNS codons (or NNK codons) or modified versions of NNS codons (XYZ codons) with expanded CDR-H3 lengths, containing unequal nucleotide ratios at each position of the codon triplet. NNS codons cover 32 codons and encode all 20 amino acids. X contains 38% G, 19% A, 26% T, and 17% C; Y c 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, CV, et al. (2004), supra. Separate mutagenesis reactions and electroporation were performed for each CDR-H3 length, but electroporation was performed together for lengths of 7 and 8 residues.

[0578] The level of phage display of the complete Fab in each library was examined by measuring the binding of 48 randomly picked clones to anti-gD antibodies. For Lib-3, similar display levels were observed for different CDR-H3 lengths, but libraries incorporating the longest CDR-H3s (15–19 residues) had a reduced percentage of Fab-displaying clones (15–30%). This likely reflects the reduced mutagenesis efficiency when using ultra-long synthetic oligonucleotides.

[0579] Phage sorting

[0580] The Lib-3 proteins were sorted for the Tie2 extracellular domain (ECD) proteins. The Tie2 ECD consists of, from membrane distal to membrane proximal, three IgG domains (Ig1 and Ig2), three EGF domains (EGF1-3), a third IgG domain (Ig3), and three fibronectin type III domains (FN3) (see, e.g., Figure 1 Constructs were generated to encode the full extracellular domain (ECD), the membrane-proximal FN3 domain, or an ECD without the FN3 domain (referred to as ECD5). Thus, the ligand-binding domain, Ig2, was encoded by both the ECD and ECD5 constructs, but not by the FN3 construct. The encoded proteins were C-terminally fused to the Fc region of hIgG1 (for human and cynomolgus monkey 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 were also generated for human, murine and rat Tie1 receptors using either two C-terminal Fc fusions or a Flag tag.

[0581] Expression and purification of Tie2 ECD protein

[0582] Flag-tagged Tie2ECD was expressed and purified from Chinese hamster ovary (CHO) cell conditioned medium. After 11-14 days, the conditioned medium was harvested and concentrated approximately tenfold. The concentrate was loaded onto an anti-flag-tag column and washed with binding buffer containing 25mM TRIS, 150mM NaCl, 1mM EDTA, 0.1% Triton X-114, and 0.1% Triton X-110, pH 7.5. The flag-tagged protein was then eluted with 50mM N-citrate and 150mM NaCl, pH 3.0, and then neutralized to pH 5.0 using 1M arginine and 400mM 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; monomer peak fractions were combined, concentrated, and filtered through 0.2 μm.

[0583] For panning, 96-well Nunc Maxisorp plates were coated with target Tie2 antigen (5 μg / ml) in PBS at 100 μl / well overnight at 4° C. The plates were blocked for 30 minutes (min) with 65 μl of 1% blocking protein and blocked for another 30 min with 40 μl of 1% Tween 20 (blocking protein: round 1: bovine serum albumin (BSA); round 2: casein; round 3: bovine serum albumin (BSA); round 4: casein). Afterwards, the phage pool was diluted to ~3-5 OD / ml (1 OD = 1.13 x 10 13phage / ml). Typically, the phage input was: 3-5 OD / ml in round 1, 3 OD / ml in round 2, ~0.5-1 OD / ml in round 3, and ~0.1-0.5 OD / ml in round 4. The diluted phage were incubated at room temperature for 30 minutes. The wells were washed at least five times with PBS and 0.05% Tween 20. The blocked phage pool was added to eight target antigen-coated wells and two uncoated wells at 100 μl / well for two hours at room temperature. The plates were washed at least ten times with PBS and 0.05% Tween 20. Starting in round 3 of panning, 1 μM omalizumab was added to the phage pool as an unrelated Fc-containing protein, Tie2.ECD5, or an antibody against Tie2.20 (ligand blocking) for one hour. The mixture was then applied to the Tie2-coated wells and allowed to bind for two hours. Phage were diluted with 100 μl / well of 100 mM HCl at room temperature for 20 minutes. Diluted phage (from coated wells) and background phage (from uncoated wells) were collected into separate tubes. The diluted pools were neutralized by adding 1 / 10 volume of 1M Tris pH 11.0 to both tubes. BSA was added to the tube containing diluted phage at a final concentration of 0.1%. To determine phage titer, 90 μl of log-phase XL-1 (OD 600 nm ~0.1-0.3) was infected with 10 μl of diluted phage or background phage at 37°C for 30 minutes. The infected cells were then serially diluted in 10-fold increments using 90 μl of 2YT. A 10-μl aliquot of the infected cells was plated onto carbenicillin plates.

[0584] To propagate phage between panning rounds, approximately 400 ul of diluted phage was used to infect ~4 ml of log-phase XL-1 (OD 600 nm ~0.1-0.3) at 37°C for 30-45 min. Helper phage KO7 and carbenicillin were added at 1 x 10 10A final concentration of 10 pfu / ml KO7 and 50 ug / ml carbenicillin was added to the infection for one hour. The culture was grown in 2YT medium with 50 ug / ml carbenicillin and 50 ug / ml kanamycin in a final volume of 20-25 ml, grown at 37°C for 4 hours and grown overnight at 30°C (or at least 18 hours). The next day, the library phage was purified by spinning the cells at 8000 rpm for 10 minutes. The supernatant was collected. 20% PEG / 2.5M NaCl was added to 1 / 5 of the supernatant volume, mixed, and allowed to stand on ice for 5 minutes. The phage was centrifuged at 12000 rpm for 15 minutes. The pellet was spun again at 5000 rpm for 5 minutes. The pellet was resuspended in 1 ml PBS and spun at 12000 rpm for 15 minutes to clarify the 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.

[0585] Screening ELISA assay

[0586] Clones from round 4 were screened for Tie2 binding and specificity by ELISA. Screening ELISAs were performed by coating the wells of a 96-well microtiter plate with 65 μl / well (1 μg / ml in coating buffer) of Tie2 protein or an irrelevant protein overnight at 4°C. Clones from round 4 were grown overnight in 400 μl of 2YT medium supplemented with 50 μg / ml carbenicillin and helper phage KO7 in a 96-well plate at 37°C. The plates were spun down at 3000 rpm for 10 minutes. 30 μl of culture supernatant was added to the Tie2-coated plate along with 60 μl of ELISA buffer (PBS with 0.5% BSA and 0.05% Tween 20) and incubated at room temperature for 1 hour. The plates were washed with PBS-0.05% Tween 20 and 100 ul / well of horseradish peroxidase (HRP)-conjugated anti-M13 antibody (1 / 5000 diluted in PBS plus 0.5% BSA and 0.05% Tween 20) at room temperature for 30 min (Sidhu et al., supra). The wells were washed with PBS-0.05% Tween 20, and then 100 ul / well of 3,3',5,5'-tetramethylbenzidine (TMB) peroxidase substrate and peroxidase solution B (H2O2) (Kirkegaard-Perry Laboratories (Gaithersburg, MD)) at a 1:1 ratio were added to each well and incubated for 5 min at room temperature. The reaction was stopped by adding 100 ul of 1 M phosphoric acid (H3PO4) to each well, and the OD of the wells was measured at 450 nm using a standard ELISA plate reader.

[0587] A clone (omalizumab) with Tie2 binding above background, species specificity for Tie2 binding, and low binding to unrelated Fc-containing proteins was further analyzed. The binding data are summarized in Tables 2 to 5 below. None of the clones in Tables 2 to 5 showed binding to omalizumab.

[0588] Table 2

[0589]

[0590]

[0591] Table 3

[0592]

[0593] Table 4

[0594]

[0595]

[0596] Table 5

[0597]

[0598] Anti-Tie2 IgG expression and purification

[0599] 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).

[0600] 293 transient transfections 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 confirmation, Tie1 binding and epitope profile. Eight antibodies were not further analyzed because the hlgG proteins were either not expressed or poorly expressed.

[0601] Example 2 - Characterization of anti-Tie 2 antibodies derived from phage library Tie2 binding

[0602] 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.

[0603] 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.

[0604] Ligand blocking

[0605] To evaluate the Ang1 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 hAng1 (GenBank Accession No. NP_000450) was immobilized on Maxisorp immunoplates (2 ug / ml) and biotinylated huTie2ECD.Fc was equilibrated with a solution of serial dilutions of anti-Tie2 antibodies. Unbound biotin-Tie2.ECD.Fc was then captured with immobilized hAng1 or hAng2 and detected using streptavidin-conjugated HRP. These results show that both Ang1 and Ang2 were blocked by at least the anti-Tie2 antibodies Tie2.1, Tie2.12, and Tie2.20 (see, Figure 4A and Figure 4B ).

[0606] Example 3 - Functional analysis of anti-Tie2 antibodies

[0607] The anti-Tie2 antibodies identified above as being able to specifically bind to Tie2 were further analyzed to identify those that could function as Tie2 agonists. The functional activity of anti-Tie2 IgG with demonstrated Tie2 binding was assessed using human umbilical vein endothelial cells (HUVEC) and rat aortic endothelial cells (RAEC), both of which are known to express Tie2 (see, Figure 5A and Figure 5B ).

[0608] Stimulation of phospho-AKT (pAKT)

[0609] Experiments were performed to evaluate the function of anti-Tie2 antibodies with respect to Tie2 activation. Tie2 conjugates according to the present disclosure, which are Tie2 agonists, are expected to bind to and activate Tie2, leading to downstream activation of the AKT enzyme. AKT activation can be demonstrated by phosphorylation of the AKT protein to produce pAKT, as described below. AKT phosphorylation is determined by Western blot analysis using an antibody specific for phosphorylated AKT or by FRET assays, as described below.

[0610] 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).

[0611] 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.

[0612] 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.

[0613] 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.

[0614] To test other Tie2 antibodies, these molecules were diluted in assay buffer to a starting plateau concentration (typically 30-1000 μg / ml) and then serially diluted (typically 2- to 10-fold). After removing the serum starvation medium, these dilutions (50 μl) were added to each well and the plate was incubated at 37°C, 5% CO2 for 15 minutes. The solution was removed and 50 μl 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 approximately 30 to 45 minutes with gentle shaking and frozen at -80°C until use or used directly in the FRET assay.

[0615] Western blot assay: HUVEC cells were plated at 1 x 10 6 Cells / well were plated in Endogro medium and incubated at 37°C for 16-18 hours. The medium was changed to 0.1% BSA Endogro basal medium for 4-5 hours and then stimulated. The cells were incubated at 37°C with the relevant Tie-2 agonist for 30 minutes and washed three times with cold phosphate-buffered saline (pH 7.4). The cells were placed on ice and incubated for 5 minutes with 100ul / well RIPA buffer (Sigma, catalog number 20-188) containing Roche complete protease and phosphatase inhibitors (ThermoScientific, catalog number 1861281). Lysates were harvested from the wells using a cell scraper and centrifuged at 17,800xg for 10 minutes. The supernatant was evaluated by SDS-PAGE (8% NuPAGE Bis-Tris (Invitrogen, NW800085)) and then transferred to a nitrocellulose membrane. The membrane was blocked with 5% BSA in TBS-T for 1 hour at room temperature and probed with rabbit anti-pAKT (Cell Signaling Technologies, catalog number 9271S) in blocking buffer. After washing four times with TBS-T, the membrane was probed with HRP anti-rabbit Ig (1:10,000) (GE Healthcare, NA934V) for 1 hour at room temperature. The membrane was washed three times with TBS-T and incubated with ECL reagent (Thermo Scientific, 32132) for 5 minutes at room temperature before the blot was exposed to the membrane.

[0616] FRET assay: Cell lysate (15 μl) was thawed on ice and then mixed with 5 μl of a 1:40 dilution of each phospho-AKT d2 antibody and phospho-AKT cryptate antibody from the phospho-AKT Ser473 kit in a 384-well microplate (Cat. No. 784080; Greiner Bio-One North America, Inc., Monroe, NC). The plates were incubated at room temperature for 4 hours or overnight at 4°C and read at 620 nm and 665 nm on a CLARIOstar (BMG LABTECH, software version: 5.01R2). For each well, data were calculated as the ratio of the acceptor to donor emission signal multiplied by 104.

[0617] In an attempt to identify anti-Tie2 antibodies capable of activating Tie2 activity, the Tie-2-binding antibodies identified in detail above were formatted as human IgG1 (hIgG1) antibodies and tested for their ability to stimulate AKT phosphorylation (to generate pAKT), which is downstream of Tie2 activation. RAECs were tested using the following recombinant anti-Tie2 antibodies (hIgG1): Tie2.1, Tie2.4, Tie2.5, Tie2.16, and Tie.2.20 for 10 minutes. Cell lysates were subjected to Western blot analysis for pAKT. Figure 6A We show that incubation of anti-Tie2 antibodies Tie2.1 and Tie2.20 increased AKT phosphorylation in this cell-based in vitro assay, and that Tie2.1 has a stronger Tie2 agonist effect than Tie2.20.

[0618] Next, experiments were performed to evaluate the effect of cross-linking on the agonist activity of anti-Tie2 antibodies. In the pAKT assay, anti-hIgG1 cross-linking antibodies were incubated with Tie2.1 or Tie2.20. Figure 6B As shown in , cross-linking of Tie2.1 further increased its ability to activate Tie2, as measured by increased AKT phosphorylation.

[0619] In an attempt to identify additional agonistic anti-Tie2 antibodies, an HTRF-based assay (Cisbio) was also used. RAEC were treated with recombinant anti-Tie2 antibody (hIgG1) for 10 minutes. Cell lysates were subjected to HTRF analysis of pAKT (cisbio). Figure 7A As shown in , antibodies found to significantly increase pAKT levels (used as Tie2 agonists) include at least Tie2.24, Tie2.31, Tie2.32, Tie2.33, Tie2.38 and Tie2.1, with Tie2.1 having potent activity.

[0620] It is thought that the level of Tie2 activation when bound by Tie2 IgG (full length) antibodies may be due in part to nonspecific aggregation of IgG molecules within the reaction mixture. Figure 7B As shown in , Tie2.1 antibody preparations with a higher percentage (eg, 3.5%) of aggregates exhibited stronger agonist activity compared to Tie2.1 antibody preparations with a lower percentage (eg, 0.15%) of aggregates. Figure 7B It was further shown that cross-linking anti-hlgG1 enhanced the agonist activity of Tie2.1. Without being bound by theory, it is believed that activation of Tie2 by anti-Tie2 agonist antibodies may be facilitated by cross-linking of Tie2 binding antibodies.

[0621] Example 4 - Classification of anti-Tie2 antibodies derived from a natural phage library

[0622] Sorting 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 identify those antibodies with a shared epitope.

[0623] For phage ELISA, huTie2ECD.Fc protein was immobilized overnight at 4°C on Maxisorp immunoplates at 2ug / ml in 65ul PBS. Serial dilutions of anti-Tie2 antibodies Tie2.1, Tie2.20, Tie2.34, or anti-Tie2 antibody 13H10 (an anti-Tie2 receptor agonist antibody described in U.S. Patent No. 6,365,154), formatted as IgG, were applied to the plate with immobilized huTie2ECD.Fc, which had been previously blocked with 1% BSA in PBS and incubated for 1 hour at room temperature. Anti-Tie2 phage was added at an OD268nm / mL of 0.1 for 15 minutes at room temperature. The plate was then washed and detected with an anti-M13-conjugated HRP secondary antibody using the method described above. The signal increased as the serial dilution of the antibody decreased, indicating that the test phage competed with the serial dilution of the antibody, indicating that they bound to the same site or epitope.

[0624] Epitope binding as determined by the above ELISA was confirmed by the Octet epitope sorting assay using OctetRED384 (Pall Forte Bio Corporation, Menlo Park, CA) and a standard sandwich format sorting assay. Specifically, streptavidin biosensors (Pall Forte Bio Corporation) were coated with biotinylated hTie2.ECD.Fc protein (10 ug / mL) and then exposed to benchmark anti-Tie2 hIgG1 (50 ug / mL Ab1, Ab20, 13H10) followed by exposure to secondary (test) anti-Tie2 hIgG1. Data were processed using Forte Bio's data analysis software. Additional binding by the secondary (test) antibody indicates an unoccupied epitope (non-competitor), while no binding indicates epitope blocking (competitor). 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 are not shown.

[0625] The results of ELISA and Octet typing assay analyses are summarized and shown in Figure 9 The present invention shows which anti-Tie2 antibodies compete with each other and may bind to the same or similar epitopes. Specifically, the binding assays described above demonstrated that the phage-derived antibodies Tie2.1 (Abl), Tie2.12 (Abl2), Tie2.24 (Ab24), and Tie2.33 (Ab33) all bind to the Tie2 IgG2 domain, block the binding of Ang1 and Ang2 to Tie2, and are Tie2 agonists (e.g., enhance the phosphorylation of AKT and / or Tie2, and / or enhance the integrity of vascular endothelial cell membranes).

[0626] Overall, the results indicate that there are at least three epitope classes that are bound by the anti-Tie2 antibodies described herein. These at least three epitope classes are shown in Figure 9 middle.

[0627] Affinity determination of anti-Tie2 IgG antibodies

[0628] The monovalent affinities of selected anti-Tie2 hIgGs 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 a multi-cycle kinetic assay format. Between cycles, the surface was regenerated using 3 M MgCl2. Monovalent affinities were determined by kinetic analysis using Biacore evaluation software (GE Life Sciences) to fit a 1:1 binding model to the data. The results are summarized in Table 6.

[0629] Table 6

[0630]

[0631]

[0632] Example 5 - In vivo generation of anti-Tie2 antibodies

[0633] In addition to phage libraries, generation of anti-Tie2 antibodies useful for therapeutic applications is also performed by animal immunization.

[0634] Rabbit immunization. New Zealand white rabbits were immunized with the ECD of human and cynomolgus monkey Tie2 (SEQ ID NO: 1, residues 23-442), and single B cells were isolated using a modified protocol from the literature, e.g., Seeber et al., PLoS ONE 9(2), 2014. B cell culture supernatants were analyzed for binding to human, rat, and cynomolgus monkey Tie2 and irrelevant control proteins by ELISA, and for binding to HUVEC cells by FACS. Tie2-specific B cells were lysed and immediately 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 the extracted mRNA as previously described, e.g., Seeber et al., PLoS ONE 9(2), 2014. Individual recombinant rabbit antibodies were expressed in Expi293 cells and then purified with protein A. The purified anti-Tie2 antibodies were then subjected to functional activity assays and kinetic screening.

[0635] Immunization of rats.Rats were immunized in a similar manner and hybridomas were generated using modified fusion partners (e.g., see Price et al., J Immunol Methods 31;343(1):28-41 (2009)). Various conditions were optimized to enable sorting of individual IgG+huTie2 fusions into individual wells, followed by additional culture after sorting. The resulting hybridoma supernatants were analyzed by ELISA for binding to human, mouse, and cynomolgus monkey Tie2, as well as irrelevant control proteins, and by FACS analysis for binding to HUVEC cells. Positive samples were purified using protein A for subsequent functional and kinetic characterization.

[0636] Acute functional screening of rat and rabbit clones using an anti-IgG crosslinker.

[0637] The antibodies generated from rat and rabbit immunizations described above were characterized with respect to pAKT induction downstream of Tie2 agonism.

[0638] Cells and Antibodies: Human umbilical vein endothelial (HUVEC) cells were purchased from Lonza (Catalog No. CC-2517; Lot No. 0000321046), while rat aortic endothelial cells (RAEC) were purchased from VEC Technologies and cultured in growth medium (VEC Technologies, Inc., Catalog No. MCDB-13110). Anti-Tie2 antibodies were prepared at Genentech. Species-specific antibodies used as cross-linking agents were purchased from Jackson ImmunoResearch Laboratories Inc.

[0639] Preparation of HUVECs: HUVECs were trypsinized and plated at 0.4 × 10 5 Cells / well were seeded in a sterile 96-well plate (Costar, catalog number 3997) in 100 μl of culture medium and the plate was incubated overnight at 37°C in a 5% CO2 incubator. The culture medium was removed and 100 μl of pre-warmed serum starvation medium (Basal Medium EndoGRO TM SCME-BM) was added to each well of the plate. The plate was incubated in a 37° C., 5% CO 2 incubator for 3 hours and then incubated with Tie-2 agonist.

[0640] Preparation of RAECs: RAECs 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 at 37°C and 5% CO. After overnight culture, the cells were starved for 3 hours in EBM2 basal medium with 0.1% BSA and then incubated with Tie-2 agonists.

[0641] To test cross-linked anti-Tie2 antibodies, 20 μg / ml cross-linker in assay buffer (basal medium from the GNE medium manufacturer + 0.2% BSA) was mixed with an equal volume of 60 μg / ml anti-Tie2 bivalent antibody and incubated for 1 hr at RT. Following incubation, the antibodies were serially diluted 3-fold.

[0642] To test other Tie2 agonists, these molecules were diluted in assay buffer to a starting plateau concentration (typically 30-1000 μg / ml), followed by serial dilutions (typically 2- to 10-fold). After removing the serum-starved medium, 50 μl of these dilutions were added to each well, and the plate was incubated at 37°C, 5% CO₂ for 15 minutes. The solution was removed, and 50 μl of lysis buffer containing blocking buffer from the pAKT Ser473 kit (Cisbio, reference number 64AKSPEH) was added to the cells. The plate was incubated at room temperature with gentle shaking for approximately 30-45 minutes and stored at -80°C until use, or used directly in the FRET assay.

[0643] FRET assay: Cell lysate (15 μl) was thawed on ice and then mixed with 5 μl of a 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., catalog number 784080). The plate was incubated at room temperature for 4 hours or in a refrigerator at 4°C overnight and read at 620 nm and 665 nm on a CLARIOstar (BMG LABTECH, software version: 5.01R2). For each well, data was calculated as the ratio of the acceptor to donor emission signal multiplied by 10 4 .

[0644] Recombinant antibody production.

[0645] DNA encoding the heavy and light chain variable domains was generated by gene synthesis and inserted into mammalian vectors for expression of the IgG1 heavy and light chains, respectively. The variable domain sequences were edited to remove significant unpaired cysteine ​​residues and the NX[S / T] N-glycosylation motif. Recombinant antibodies were produced by transiently transfecting Expi293 cells with mammalian expression vectors encoding the heavy and light chains of the antibody. The heavy and light chains were encoded on separate vectors and transfected using a 1:2 ratio of heavy chain expression vector to light chain expression vector. Antibodies were purified from cell culture supernatants by affinity chromatography. In some cases, the antibodies underwent an additional purification step based on SEC.

[0646] 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).

[0647] Table 7A

[0648]

[0649]

[0650] Table 7B

[0651]

[0652]

[0653] HTP epitope classification.

[0654] 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.

[0655] Example 6 - Generation of PEG-conjugated anti-Tie2 Fab multimers

[0656] 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.

[0657] One multimeric approach employed was to use a multi-arm polyethylene glycol (PEG) molecule as the core, with each arm attached or conjugated to a single anti-Tie2 Fab molecule. Various multimeric configurations were tested using the Tie2.1 Fab, which has been shown to specifically bind to Tie2 with high affinity and act as a Tie2 agonist (activating AKT phosphorylation upon interaction with Tie2). Specifically, the Tie2.1 Fab was modified such that the C-terminus of the heavy chain was modified to include the amino acid residue SPPC (SEQ ID NO: 89), providing a linker and cysteine ​​to which the PEG moiety could be conjugated. This "Tie2.1-SPPC" Fab was conjugated to a PEG-maleimide backbone with a variable number and length of arms (multi-arm PEG-maleimide was obtained from JenKem Technology USA, Plano, TX).

[0658] Fab purification and deblocking

[0659] All chromatography resins were sourced from GE Healthcare.

[0660] Fab was expressed in E. coli. The E. coli pellet was again suspended in 25 mM TrisHCl, 150 mM NaCl, 5 mM EDTA (EQ) at pH 7.5 at 1.5 L / Kg and microfluidized twice at 1000 bar. PEI was slowly added 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 was filtered through 0.22 μm. 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, and washed with 4 CV of EQ containing 0.1% TX114 and 0.1% TX100 at 5 ml / min. This was followed by a 2CV wash of EQ at 30 ml / min, followed by a 2CV 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.

[0661] 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, washed with 5 CV of EQ containing 0.1% (v / v) Triton X114 and 0.1% (v / v) Triton X100 at 4 ml / min, then washed with 50 ml / min of 2 CV of EQ, then washed with 25 mM succinate at pH 6.0 at 50 ml / min, and eluted with 0.15 M acetic acid at 50 ml / min. As the eluate flowed out of the column, it was neutralized to pH 5.0 with 1 M Tris at pH 9.0.

[0662] The neutralized GBP or Capto L eluate was diluted one to three times with 20 mM sodium acetate (A) at pH 5.0 and loaded onto a cation exchange resin (SPHP). The column was washed with 2 CV (A) and 5 CV (A) containing 0.1% Triton X114 and 0.1% Triton X100, followed by 2 CV (A), and then gradient eluted using a 10 CV 0-20% gradient of (A) containing 1 M NaCl (B), and fractions were collected. Fractions containing the Fab peak were pooled.

[0663] 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 pull down the Fab, a 50-fold excess of DTT was added to the solution, followed by overnight incubation at 22°C and removal of the adduct checked by mass spectrometry.

[0664] After adjusting the pH to 5.2 with 10% acetic acid, the reduced Fab was bound to 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 with 1 M Tris, pH 8.5, 2 mM EDTA was added, and reoxidized with a 15-fold molar excess of DHAA. 1.5 hours after reoxidation of the Fab was checked by mass spectrometry; if necessary, an additional 10-fold molar excess of DHAA was added, the sample incubated for 1 hour, and checked again. The reoxidized Fab was then adjusted to pH 5 with 10% acetic acid and purified on an 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 concentrated to 10 kD and filtered through 0.2 μm. Other Fabs can be purified and blocked in essentially the same manner.

[0665] Hexamer conjugation and purification

[0666] Tie2.1-SPPC was conjugated to a 6KDa PEG hexamer from JenKem at a concentration of approximately 10 mg / mL in 25 mM NaOAC, 150 mM NaCl, and 2 mM EDTA at pH 5.0. After equilibration to room temperature, the 6KDa PEG hexamer from JenKem was suspended in 25 mM sodium acetate at pH 5.0 at a concentration of 3 mM. The pH was maintained below pH 6 to avoid maleimide ring opening. Once PEG was soluble, it was added to the Tie2.1-SPPC-blocked Fab at a molar ratio of 9:1 (Fab:PEG). The mixture was then left at room temperature overnight with gentle shaking. After conjugation, the Tie2.1 FabPEG hexamer was purified using size exclusion chromatography (SEC) on a Superdex-200 column with 20 mM His-acetate, 150 mM NaCl, pH 5.5. This purification step removed excess Fab and aggregates from the conjugation mixture. A NuPage 4-12% Bis tris gel, unreduced with a reducing agent, was run to determine which conjugate fractions required further purification to enrich for the hexamer. The conjugate fractions were pooled and further purified by cation exchange (CEX) using SP Sepharose high-performance strong cation exchange resin from GE to enrich for the six Fab / PEG species. CEX was run in 25 mM sodium acetate, pH 5.0, and eluted with a gradient from 20% to 33.4% in buffer B (buffer B was 25 mM NaOAC, 300 mM NaCl, pH 5.0) over 44.5 CV, followed by a gradient from 33.4% to 50% B over 10 min, and finally eluted with 100% B. Several different mini-pools of different fractions were run on the gel as described above, and this data was used to decide which fractions to pool. Mini-pool 3 was combined and then prepared at 40 mg / mL in PBS, pH 7.2.

[0667] The final sample was then run on analytical SEC using a TSKgel G3000SW xl column with 0.2 M KPO4, 0.25 M KCl pH 6.2, 15% isopropanol as buffer to determine the percentage of aggregates present. This was also run on the gel as described earlier.

[0668] FabIgG purification

[0669] 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.

[0670] Comparison of FabIgG and hexameric pAKT activities

[0671] 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.

[0672] Agonist activity of anti-Tie2Fab via multi-arm PEG polymerization

[0673] 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 ).

[0674] Example 7 - Multimerization of anti-Tie2 Fabs using IgM format

[0675] 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).

[0676] Optimization of IgM expression and purification.

[0677] As recommended by the manufacturer, human IgM (huIgM) was expressed in 30 ml of Expi293 cell culture (e.g., see "Expi293 Expression System, User Guide", print number MAN0007814, from ThermoFisherScientific). 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 supernatants harvested were 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). The total yield of the protein was assessed by A280, and the purity was assessed by analytical SEC using a Waters Xbridge BEH SEC 450 angstrom column. Figure 13A Shown is the effect of various ratios of huIgM heavy chain to light chain to J chain (if included) on the total yield of protein isolated from the affinity column.

[0678] Hexamer optimization was continued by transfecting cells with plasmids containing LC and HC at a ratio of 1:1 or 2:1 to plasmid DNA. Each ratio resulted in similar total protein yields (2.5-3 mg from 30 ml expression), however, product quality appeared improved at the 1:1 ratio and the amount of LC dimer present was reduced. Expression of the putative pentamer was achieved by including the 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 Notably, under optimal conditions, the high levels of LC and HC DNA relative to JC approximate a 10:10:1 chain ratio in the final IgM pentamer.

[0679] Since the expression of pentameric and hexameric IgM has an optimized chain ratio, the expression and purification of human and mouse hexameric and pentameric IgM have been 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 under this process is 3 to 4 times lower than typical, the relatively easy purification together with the low material requirements for ocular drugs should promote manufacturing feasibility. The chain composition of human IgM was confirmed by SDS-PAGE, and LC-MS of the reducing agent deglycosylated material showed the presence of LC, HC and JC (if any). Size exclusion chromatography showed monodisperse peaks corresponding to pentameric and hexameric mouse IgM and a monodisperse peak corresponding to hexameric human IgM. The pentameric human IgM eluted from SEC was in two closely related peaks, both of which contained J chains and had high SEC expressed molecular weights. The hydrodynamic radius (R h) is approximately 12 nM, and the predicted molecular weight of the hexamer (~1050 kDa) slightly exceeds that of the pentamer (~950 kDa) (Table 9 below).

[0680] Table 9

[0681]

[0682]

[0683] The ratio of the radius of gyration to the hydrodynamic radius (Rg / Rh) provides some insight into molecular shape, averaging ~0.775 for spherical molecules and tending to be higher for more elongated molecules. The dispersity of monodisperse IgM ranged from 0.85 to 0.91, consistent with the expected structure. To further assess the polymeric identity of IgM, negative stain TEM and reference-free 2D classification were used to characterize 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 investigation. 110 TEM micrographs were manually collected, yielding a total of 1500 particles. After three rounds of reference-free 2D classification using the Relion suite (Scheres, 2015, J Struct Biol, 189:114-122), the majority of particles were found to be organized into hexameric structures. A small number of particles (<2%) were refined into pentamers. Consistent with previous structural studies of IgM pentamers (Czajkowsky and Shao, 2009, Proc Natl, Acad Sci USA, 106: 14960-14965), the CH3 and CH4 domains appear to form a stem-like domain orthogonal to the imaging plane. Furthermore, although multiple pairs of Fabs attached to the same Fc appear to be coplanar, there are some signs of Fab offset on adjacent Fcs. Some of this offset may allow the hexamer to accommodate all chains in a sterically crowded environment. Based on these data, human hexameric IgM was selected as a preferred therapeutic scaffold because the improved production purity and the absence of JC both simplify manufacturing and avoid the need to consider polymeric Ig receptor-mediated in vivo binding.

[0684] Characterization of IgM for intraocular administration

[0685] Subsequently, the suitability of IgM for ocular applications was explored using New Zealand white rabbits as a model system. PK para...

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. The antibody or antigen-binding fragment thereof according to claim 1, wherein the CDR-H3 comprises the amino acid sequence RTRWASWAMDY (SEQ ID NO: 6).

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the CDR-H3 comprises the amino acid sequence RTRWASWAFDY (SEQ ID NO: 7).

4. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, which is a monoclonal antibody, a humanized antibody, an antibody fragment and / or a chimeric antibody.

5. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, which is a Fab fragment that binds Tie2.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, comprising: a VL domain comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 21; and a VH domain comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:

20.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, comprising: a VL domain comprising an amino acid sequence having at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 21; and a VH domain comprising an amino acid sequence having at least 99% sequence identity with the amino acid sequence of SEQ ID NO:

20.

8. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antibody comprises the VL sequence of SEQ ID NO: 21 and the VH sequence of SEQ ID NO:

20.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein the antibody comprises an engineered cysteine, wherein The engineered cysteine ​​is selected from T120C, G166C, G178C, T187C and T209C in HC; or The engineered cysteine ​​is selected from Q124C, R142C, Q155C, L201C, T206C, K107C, K126C and K149C in LC; The residue numbers of the engineered cysteine ​​residues are according to EU numbering.

10. The antibody or antigen-binding fragment thereof according to claim 9, wherein the engineered cysteine ​​is selected from T209C in the HC and T206C in the LC.

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