Tie2 binder and method of use

Abstract

To provide a Tie-2 antibody and a fragment and a conjugate thereof, and a method for using them.SOLUTION: Provided is an isolated antibody that specifically binds to Tie2, or an antigen-binding fragment thereof, which includes: a heavy chain variable domain (VH) which includes CDR-H1 including an amino acid sequence NTDIS, CDR-H2 including an amino acid sequence RISPSDGNTYYADSVKG, and CDR-H3 including an amino acid sequence RTRWASX1AX2DY, where X1 denotes M, L, K, F, Y, R, N, Q, H or W, and / or X2 denotes F, Y, L, Q, I, K or H; and a light chain variable domain (VL) which includes (d) CDR-L1 including an amino acid sequence RASQDVSTAVA, (e) CDR-L2 including amino acid sequence SASFLYS, and (f) CDR-L3 including an amino acid sequence QQSYTTPPT.SELECTED DRAWING: None

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 993930, filed March 24, 2020, and U.S. Provisional Patent Application No. 63 / 046318, filed June 30, 2020, which are incorporated herein by reference in their entireties.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on March 17, 2021, is named P35891-WO_SeqList.txt and is 107,354 bytes in size. [Technical Field]

[0003] FIELD OF THE INVENTION The subject matter of the present disclosure relates to Tie2 binding agents and conjugates, including anti-Tie2 antibodies, and methods of using same. [Background technology]

[0004] background Tie2 is a promising therapeutic target for the treatment of various ocular disorders (see, e.g., Campochiaro and Peters, 2016, Curr Diab Rep, 16:126; Whitehead et al., 2019, J Diabetes Res, 2019:5140-521; Hussain et al., 2019, Expert Opin Investig Drug, 28:861-869). Tie2 is a receptor tyrosine kinase 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 disorders, including, but not limited to, diabetic macular edema (DME), diabetic retinopathy (DR), and age-related macular degeneration (AMD).

[0005] The most widely studied ligands for Tie2 are angiopoietin 1 (Ang1) and angiopoietin 2 (Ang2). Ang1 is a potent Tie2 agonist and has been shown to inhibit ocular neovascularization and blood-retinal barrier breakdown (see, e.g., Nambu et al., 2004, Gene Therapy, 11:865-873). Ang2 is a context-dependent antagonist of Tie2, and its expression is increased in association with several ocular disorders, including DME, wet AMD, DR, metastasis, sepsis, and inflammation. Specifically, Ang2 competitively binds to Tie2 and inhibits Ang1 signaling, leading to endothelial and vascular destabilization, blood-retinal barrier breakdown, and inflammation (Klaassen et al., 2013, Prog Retin Eye Res, 34:19-48; Saharinen et al., 2017, Nat Rev Drug Discov, 16:635-661).

[0006] Tie receptors, including Tie1 and Tie2, are type 1 transmembrane protein receptor tyrosine kinases (RTKs) (Ramsauer, M. & D'Amore, PAJ Clin. Invest. (2002); 110:1615-1617). Tie represents a tyrosine kinase receptor with immunoglobulin and EGF homology domains. Tie2 is located in the endothelial cells of all forming blood vessels and the endocardium of mouse embryos (Korhonen et al., Blood (1992); 80:2548-2555). The ectodomain or extracellular domain ("ECD") of Tie2 contains three immunoglobulin (Ig) domains (Ig1, Ig2, and Ig3), three epidermal growth factor (EGF) domains, and a fibronectin type III domain (FNIII). The Ig-EGF domain of Tie2 mediates the recognition and binding of angiopoietin (Fiedler, U. et al., J. Biol. Chem. (2003); 278:1721-1727; Barton, WA, et al., Structure (2005); 13:825-832).

[0007] Two ligands for the Tie2 receptor have been identified: angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2). Ang-1, a Tie2 agonist, binds to and induces tyrosine phosphorylation of Tie2, whose expression in vivo is located in close proximity to developing blood vessels (Davis et al., Cell (1996); 87:1161-1169). Mice lacking Ang-1 exhibit angiogenesis defects similar to those seen in mice lacking Tie2, supporting the notion that Ang-1 is the primary physiological ligand for Tie2 and that Tie2 has important in vivo angiogenic effects (Suri et al., Cell (1996); 87:1171-1180). Ang-1 is anti-inflammatory, promotes vascular integrity, and reduces vascular permeability. Ang2 has been identified as a naturally occurring antagonist of Tie2. Transgenic overexpression of Ang2 disrupts angiogenesis in mouse embryos (Maisonpierre et al., Science 277:55-60, 1997). Ang2 is pro-inflammatory, disrupts EC quiescence, and can increase vascular permeability. Taken together, these studies support the Ang1 / Ang2 / Tie2 system as a critical player in angiogenesis. Given the important role of Tie2 in angiogenesis, agents that recognize Tie2 and methods for using such agents are desirable. Furthermore, compositions that function as Tie2 agonists and can reduce vascular permeability or enhance vascular integrity have great potential as therapeutic agents, particularly for the treatment of ocular disorders. Summary of the Invention

[0008] overview The present invention provides anti-Tie2 antibodies, compositions (eg, conjugates) comprising anti-Tie2 antibodies or fragments thereof, and methods of use thereof.

[0009] The presently disclosed subject matter provides isolated antibodies or antigen-binding fragments thereof that specifically bind to Tie2, compositions comprising at least one or more anti-Tie2 antibodies or antigen-binding fragments thereof, and methods of use thereof. In an exemplary embodiment, the anti-Tie2 antibody is a Fab.

[0010] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds to Tie2 is provided, wherein the anti-Tie2 antibody comprises a CDR-H1 comprising the amino acid sequence NTDIS (SEQ ID NO: 3), a CDR-H2 comprising the amino acid sequence RISPSDGNTYYADSVKG (SEQ ID NO: 4), and a CDR-H3 comprising the amino acid sequence (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 a light chain (LC) variable domain (VL domain) comprising a CDR-L1 comprising the amino acid sequence RASQDVSTAVA (SEQ ID NO: 8), a CDR-L2 comprising the amino acid sequence SASFLYS (SEQ ID NO: 9), and a CDR-L3 comprising the amino acid sequence QQSYTTPPT (SEQ ID NO: 10). In some embodiments, the CDR-H3 comprises SEQ ID NO: 6 or SEQ ID NO: 7. In certain embodiments, the CDR-H3 comprises SEQ ID NO: 7.

[0011] 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.

[0012] In some embodiments, an anti-Tie2 antibody comprises a VH domain comprising 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 a 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 other embodiments, the CDR-H3 comprises SEQ ID NO: 6 or SEQ ID NO: 7. In a specific embodiment, the CDR-H3 comprises SEQ ID NO: 7. In still 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.

[0013] 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.

[0014] 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.

[0015] In some embodiments, the anti-Tie2 antibody or fragment thereof comprises the heavy chain (HC) domain sequence of SEQ ID NO: 55 and / or the light chain (LC) domain sequence of SEQ ID NO: 25. In other embodiments, the anti-Tie2 antibody or fragment thereof comprises the heavy chain (HC) domain sequence of SEQ ID NO: 23 and / or the light chain (LC) domain sequence of SEQ ID NO: 56.

[0016] In some aspects, an antibody or antigen-binding fragment thereof that specifically binds to Tie2 is provided, comprising a VH domain comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO:28, CDR-H2 comprising the amino acid sequence of SEQ ID NO:29, and CDR-H3 comprising the amino acid sequence of SEQ ID NO:30, and a VL domain comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO:8, CDR-L2 comprising the amino acid sequence of SEQ ID NO:9, and CDR-L3 comprising the amino acid sequence of SEQ ID NO:10. In some embodiments, the VH domain comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:31, and the VL domain comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:21. In yet 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.

[0017] In some aspects, an antibody or antigen-binding fragment thereof that specifically binds to Tie2 is provided, comprising a VH domain comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 33, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 34, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 35, and a VL domain comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10. In some embodiments, the VH domain comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 36, and the VL domain comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 21. In yet 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.

[0018] In some aspects, an antibody or antigen-binding fragment thereof that specifically binds to Tie2 is provided, the antibody or antigen-binding fragment thereof comprising a VL domain comprising CDR-L1 comprising SEQ ID NO:8, CDR-L2 comprising SEQ ID NO:9, and CDR-L3 comprising SEQ ID NO:10, and a VH domain comprising (a) CDR-H1 comprising SEQ ID NO:38, CDR-H2 comprising SEQ ID NO:39, and CDR-H3 comprising SEQ ID NO:40; (b) CDR-H1 comprising SEQ ID NO:43, CDR-H2 comprising SEQ ID NO:44, and CDR-H3 comprising SEQ ID NO:45; or (c) CDR-H1 comprising SEQ ID NO:48, CDR-H2 comprising SEQ ID NO:49, and CDR-H3 comprising SEQ ID NO:50. In other embodiments, the VL domain comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 21, and the VH domain consists of (a), (b), or (c) and comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to (d) SEQ ID NO: 41, (e) SEQ ID NO: 46, or (f) SEQ ID NO: 51, respectively.

[0019] In some embodiments, the antibody or antigen-binding fragment thereof comprises an LC of SEQ ID NO:21 and an HC comprising SEQ ID NO:42, SEQ ID NO:46, or SEQ ID NO:52.

[0020] In some aspects, an antibody that specifically binds to Tie2, or an antigen-binding fragment thereof, is provided, comprising two VH domains comprising, from N-terminus to C-terminus, CDR-H1 comprising SEQ ID NO:6, CDR-H2 comprising SEQ ID NO:8, CDR-H3 comprising SEQ ID NO:9, CDR-H1 comprising SEQ ID NO:48, CDR-H2 comprising SEQ ID NO:49, and CDR-H3 comprising SEQ ID NO:50, and two VL domains comprising, from N-terminus to C-terminus, CDR-L1 comprising the amino acid sequence of SEQ ID NO:8, CDR-L2 comprising the amino acid sequence of SEQ ID NO:9, and 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.

[0021] In some embodiments, the anti-Tie2 antibody or antigen-binding fragment thereof comprises an engineered cysteine, wherein the engineered cysteine ​​is in the HC constant domain and / or 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, where residue numbers are according to EU numbering. In other embodiments, the anti-Tie2 antibody or antigen-binding fragment thereof is a Fab in which the HC of the Fab terminates with the amino acid sequence CDKTHTSPPC (SEQ ID NO:83). In some embodiments, the Fab terminates with 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.

[0022] In some embodiments, the anti-Tie2 antibody is a monoclonal antibody.

[0023] In other embodiments, the anti-Tie2 antibody is a humanized or human antibody.

[0024] In some embodiments, the anti-Tie2 antibody is a full-length IgG1 or full-length IgM antibody.

[0025] In some embodiments, the anti-Tie2 antibody or fragment thereof binds to Tie2, where Tie2 is a protein having at least 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1.

[0026] In a preferred embodiment, the anti-Tie2 antibody or fragment thereof is a Fab.

[0027] 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.

[0028] 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 yet other embodiments, the anti-Tie2 antibody or fragment thereof does not bind to the Ig3 domain of Tie2. In yet other embodiments, the anti-Tie2 antibody or fragment thereof does not bind to the FNIII domain of Tie2.

[0029] 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.

[0030] 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 yet other embodiments, the multispecific antibody binds to Tie2 and Ang2.

[0031] 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, and the multispecific antibody is capable of activating Tie2. In other embodiments, the multispecific antibody binds to Tie2 and factor D, and the multispecific antibody is capable of activating Tie2. In yet other embodiments, the multispecific antibody binds to Tie2 and Ang2, and the multispecific antibody is capable of activating Tie2.

[0032] In one aspect, an isolated nucleic acid encoding an anti-Tie2 antibody or antigen-binding fragment thereof is provided.

[0033] In one aspect, a host cell is provided that comprises an isolated nucleic acid encoding an anti-Tie2 antibody or antigen-binding fragment thereof.

[0034] In one aspect, methods for producing an antibody or antigen-binding fragment thereof that binds to Tie2 are provided. In some embodiments, the method comprises culturing a host cell containing nucleic acid encoding the anti-Tie2 antibody under conditions suitable for expression of the anti-Tie2 antibody. In other embodiments, the method further comprises recovering the anti-Tie2 antibody from the host cell.

[0035] In one aspect, an anti-Tie2 antibody produced by the above method is provided.

[0036] In one aspect, there is provided a conjugate comprising at least two antibodies that specifically bind to Tie2 or an antigen-binding fragment thereof and a multi-arm moiety according to embodiments disclosed herein, hi a preferred embodiment, the at least two anti-Tie2 antibodies are each Fabs.

[0037] In some embodiments, the conjugate comprises an anti-Tie2 Fab that binds to Tie2 and a multi-arm portion linked to at least two anti-Tie2 Fabs. In some embodiments, the multi-arm portion is linked to at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 anti-Tie2 Fabs. In other embodiments, the multi-arm portion is linked to 2, 3, 4, 5, 6, 7, 8, 9, or 10 anti-Tie2 Fabs. In yet other embodiments, the multi-arm portion is linked to 6 or 8 anti-Tie2 Fabs. In yet other embodiments, the multi-arm portion is linked to 8 anti-Tie2 Fabs. In a preferred embodiment, the multi-arm portion is linked to 6 anti-Tie2 Fabs.

[0038] In one aspect, the conjugate binds to Tie2 and activates Tie2 activity.

[0039] In some embodiments, the conjugate that binds to Tie2 activates AKT phosphorylation. In other embodiments, the 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 yet other embodiments, the activation of Tie2 phosphorylation is measured in vitro.

[0040] In some embodiments, exposure of Tie2-expressing cells to the conjugate does not reduce Tie2 protein levels in the cells. In other embodiments, exposure does not reduce Tie2 protein levels in the cells by more than 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, or 75%. In other embodiments, exposure reduces Tie2 protein levels in the cells by less than 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, or 75%. In still other embodiments, exposure reduces Tie2 protein levels in the cells by more than about 25% and less than about 75%, more than about 50% and less than about 75%, or more than about 60% and less than about 80%. In some embodiments, Tie2 protein levels are measured by Western blot before and after in vitro incubation of Tie2-expressing cells with a Tie2-binding agent. In other embodiments, the incubation is at 37° C. for 10 to 36 hours or 12 to 24 hours.

[0041] In some embodiments, the anti-Tie2 antibody or antigen-binding fragment thereof has an equilibrium dissociation constant (Kd) in the range of 0.1 uM to 10 uM, 0.01 uM to 10 uM, or 0.1 to 100 uM.

[0042] In some embodiments, a conjugate comprising a multi-arm moiety and an anti-Tie2 antibody or antigen-binding fragment thereof increases translocation of Tie2 to cell-cell junctions.

[0043] In some embodiments, the conjugate reduces vascular permeability.

[0044] In some embodiments, the conjugate promotes vascular stability and / or increases vascular integrity.

[0045] In some embodiments, the conjugate does not inhibit or reduce the binding of Ang1 to Tie2.

[0046] In some embodiments, the conjugate inhibits or reduces the binding of Ang2 to Tie2.

[0047] In some embodiments, conjugate activity is measured using an in vitro assay.

[0048] In some embodiments, the multi-arm polyol is selected from 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.

[0049] In some embodiments, the polyol is a poly(alkylene oxide) polymer. In some embodiments, the polyol is a poly(alkylene glycol). In still other embodiments, the polyol is polyethylene glycol (PEG). In some embodiments, the PEG is a functionalized multi-arm PEG. In some embodiments, PEG has the general formula (Ia): JPEG2025138630000001.jpg42170 (wherein each m represents the length or size of a particular arm of the polyol (PEG) and is independently an integer of about 45 to about 1000, about 20 to about 1000, about 10 to about 1000, about 3 to about 250, about 3 to about 200, about 3 to about 100, about 10 to about 50, about 10 to about 30, about 20 to about 30, about 50 to about 200, or about 100 to about 150; n is an integer of about 1 to about 10; and each R 1 is independently either absent or a linking group, and each R 2 are independently either hydrogen or a terminal reactive group, and at least one R 2 is a terminal reactive group. 2 is independently selected from thiol-reactive groups, amino-reactive groups, and combinations thereof.

[0050] In some embodiments, PEG has a structure of general formula (Ia), wherein n is an integer from 1 to 3.

[0051] In some embodiments, the PEG has the structure of general formula (Ia), n is 1, and the multi-arm PEG is a tetramer. In some embodiments, the PEG has a structure of general formula (Ia), n is 2, and the multi-arm PEG is a hexamer. In such embodiments, the octamer has the structure of general formula (Ib): JPEG2025138630000002.jpg46170 (wherein each m is independently an integer of about 45 to about 1000, about 20 to about 1000, about 10 to about 1000, about 3 to about 250, about 3 to about 200, about 3 to about 100, about 10 to about 50, about 10 to about 30, about 20 to about 30, about 50 to about 200, or about 100 to about 150; and 1 is independently either absent or a linking group, and each R 2 are independently either hydrogen or a terminal reactive group, where at least one R 2 is a terminal reactive group, covalently attached to an anti-Tie2 antibody fragment or Fab, as described above. In some embodiments, each m is independently an integer between about 15 and 35 or between about 20 and 30. In other embodiments, each m is independently an integer between about 22.

[0052] In some embodiments, R 1 and R 2 Together, the structure JPEG2025138630000003.jpg15170, where R 2 is maleamide.

[0053] In some embodiments, the multi-arm PEG has a structure of general formula (Ia), n is 3, and the multi-arm PEG is an octamer. In such embodiments, the octamer has the general formula (Ic): JPEG2025138630000004.jpg46170 (wherein each m is independently an integer of 3 to 250, and each R 1 is independently either absent or a linking group, and each R 2 are independently either hydrogen or a terminal reactive group, and at least one R 2 is a terminal reactive group and is covalently attached to the anti-Tie2 antibody. In some embodiments, each m is independently an integer between 15 and 35. In other embodiments, each m is independently an integer of about 22.

[0054] In some embodiments, at least two anti-Tie2 antibody fragments or Fabs described herein are covalently linked to a multi-armed polyol. In other embodiments, the multi-armed polyol of the conjugate is covalently linked to at least two anti-Tie2 antibody fragments or Fabs via the free sulfhydryl groups of cysteine ​​amino acids. In other embodiments, the cysteine ​​amino acid is an engineered cysteine. In yet other embodiments, the cysteine ​​amino acid is in the anti-Tie2 constant domain. In other embodiments, the cysteine ​​amino acid is 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 at the N-terminus or C-terminus of the HC or LC.

[0055] In some embodiments, the conjugate comprises an anti-Tie2 antibody or antigen-binding fragment thereof comprising 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 numbers of the engineered cysteines are according to EU numbering.

[0056] In some embodiments, the conjugate comprises a multi-arm polyol covalently attached to 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 in the constant region of the anti-Tie2 antibody fragments or Fabs. In yet other embodiments, the lysine amino acids are at the C-terminus of the heavy or light chain of the anti-Tie2 antibody fragments or Fabs. In alternative embodiments, the Tie2-binding agent is not covalently attached to at least two anti-Tie2 Fabs via free amino groups of lysine.

[0057] In some embodiments, the conjugate deconjugates less than 20%, less than 15%, or less than 10% per month under physiological conditions in vitro, hi other embodiments, the conjugate deconjugates less than 20%, less than 15%, or less than 10% per month under physiological conditions in vivo.

[0058] In some embodiments, the conjugate is stable over long periods of time, losing less than 20%, less than 15%, or less than 10% of its TIe2 binding capacity per month under physiological conditions.

[0059] In one aspect, a conjugate is provided that includes 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 yet 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.

[0060] In some embodiments, the conjugate comprises an IgM molecule, wherein the IgM variant has an amino acid substitution that reduces or eliminates complement-dependent cytotoxicity (CDC) activity. In a preferred embodiment, the IgM variant comprises the substitution P436G, according to EU numbering.

[0061] In one aspect, a conjugate is provided that includes 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, about each of the peptides being covalently linked to an anti-Tie2 Fab. In some embodiments, the anti-Tie2 Fab sequence terminates at residue 221, 222, 223, 224, or 225 (EU numbering). In other embodiments, each peptide is a nucleoside diphosphate kinase (NDK) peptide. In still other embodiments, each of the NDK peptides comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 71.

[0062] In some embodiments, each NDK peptide is linked at its N-terminus to the C-terminus of an 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 still other embodiments, the linker is an amino acid linker. In still other embodiments, the amino acid linker comprises 2, 3, 4, 5, 6, 7, 8, 2-20, 5-10, or 4-10 amino acids. In other embodiments, the linker comprises glycine.

[0063] 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.

[0064] In one aspect, a conjugate is provided that includes 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 includes a J chain, and the multi-arm portion includes five antibody fragments, Fab fragments, or antigen-binding fragments thereof, wherein approximately each of the five antibody fragments, Fab fragments, or antigen-binding fragments thereof is linked to the IgM molecule. In yet other embodiments, the IgM molecule does not include a J chain, and the multi-arm portion includes six antibody fragments, Fab fragments, or antigen-binding fragments thereof, wherein each of the six antibody fragments, Fab fragments, or antigen-binding fragments thereof is linked to the IgM molecule.

[0065] In some embodiments, the conjugate comprises an IgM molecule, wherein the IgM variant has an amino acid substitution that reduces or eliminates complement-dependent cytotoxicity (CDC) activity. In a preferred embodiment, the IgM variant comprises the substitution P436G, according to EU numbering.

[0066] In one aspect, a conjugate is provided that includes an antibody, antibody fragment, Fab, or antigen-binding fragment thereof, and a multi-arm moiety, wherein the multi-arm moiety comprises at least 2, 4, 6, 8, or 10 peptides, approximately each of which is covalently attached to an anti-Tie2 antibody, antibody fragment, Fab, or antigen-binding fragment thereof. In some embodiments, the sequence of the antibody, antibody fragment, Fab, or antigen-binding fragment thereof terminates at residue 221, 222, 223, 224, or 225 (EU numbering). In other embodiments, each peptide is a nucleoside diphosphate kinase (NDK) peptide. In still other embodiments, each of the NDK peptides comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 71.

[0067] In some embodiments, each NDK peptide is linked at its N-terminus to the C-terminus of the heavy or light chain of an antibody, antibody fragment, Fab, or antigen-binding fragment thereof. In other embodiments, a linker is present between the Fab heavy or light chain and the peptide. In still other embodiments, the linker is an amino acid linker. In still other embodiments, the amino acid linker comprises 2, 3, 4, 5, 6, 7, 8, 2-20, 5-10, or 4-10 amino acids. In other embodiments, the linker comprises glycine.

[0068] 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.

[0069] 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 binding agent comprising multiple anti-Tie2 Fabs linked to a multi-arm moiety described herein and a pharmaceutically acceptable carrier.

[0070] In some embodiments, the pharmaceutical composition comprising an anti-Tie2 antibody or Tie2 binding agent further comprises an additional therapeutic agent. In other embodiments, the additional therapeutic agent is selected from the group consisting of an anti-VEGF antibody, an anti-Tie2 antibody, and an anti-Ang2 antibody. In some embodiments, the additional therapeutic agent is selected from an Ang2 antagonist, a VEGF antagonist, a VEGF trap, an anti-VEGF antibody, an anti-Ang2 antibody, and a complement component antagonist.

[0071] In some embodiments, any of the aforementioned pharmaceutical compositions can be used as a medicament.

[0072] In some embodiments, any of the aforementioned pharmaceutical compositions can be used in the manufacture of a medicament for treating an ocular disorder in a subject.

[0073] In some embodiments, any of the aforementioned pharmaceutical compositions can be used to reduce or inhibit pathological vascular permeability in a subject with an ocular disorder.

[0074] In other aspects, any of the aforementioned pharmaceutical compositions can be used to treat an ocular disorder in a subject.

[0075] In one aspect, provided are methods of treating an individual in need thereof, comprising administering to the patient an anti-Tie2 antibody and / or Tie2 binding agent described herein. In some embodiments, the method comprises administering to the individual a pharmaceutical composition described herein comprising an anti-Tie2 conjugate described herein.

[0076] In some embodiments, the individual has been diagnosed with a vascular disorder, hi other embodiments, the individual has been diagnosed with a vascular disorder of the eye.

[0077] In another aspect, the invention features a method of inhibiting vascular permeability in a subject suffering from a disorder associated with undesirable vascular permeability, the method comprising administering to the subject an effective amount of any one of the foregoing antibodies or conjugates, thereby inhibiting vascular permeability in the subject.

[0078] In another aspect, the invention features a method of treating a disorder associated with undesirable vascular permeability, comprising administering to a subject in need of such treatment an effective amount of any one of the foregoing antibodies or conjugates.

[0079] In some embodiments, the individual has been diagnosed with a disorder associated with the Tie2 pathway.

[0080] In some embodiments, the individual has been diagnosed with an ocular disorder 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, von Hippel-Lindau disease, ocular histoplasmosis, central retinal vein occlusion (CRVO), corneal neovascularization, glaucoma, retinopathy in the absence of edema, and retinal neovascularization.

[0081] In some embodiments, the individual has undergone treatment with an anti-VEGF antibody. In other embodiments, the individual does not experience the therapeutic benefit of the anti-VEGF antibody, experiences a decrease in the therapeutic benefit of the anti-VEGF antibody, and / or has stopped experiencing the therapeutic benefit of the anti-VEGF antibody.

[0082] In some embodiments, the method further comprises administering to the individual a second therapeutic agent, hi other embodiments, the second therapeutic agent is selected from the group consisting of an anti-VEGF antibody, an anti-Ang2 antibody, an anti-VEGF / Ang2 bispecific antibody, a VEGF antagonist, and an Ang2 antagonist. [Brief explanation of the drawings]

[0083] [Figure 1] FIG. 1 provides a schematic diagram of the Tie2 domains used for panning the VH library. [Figure 2A] Figures 2A-2B show the results of assays assessing binding by anti-Tie2 antibodies Tie2.1, Tie2.10, Tie2.11, and Tie2.12 to the mouse Tie2 ECD5 domain (Figure 2A) and human Tie2 ECD5 domain (Figure 2B). The ECD5 domain contains the Ig1, Ig2, EGF, and Ig3 domains of Tie2. [Figure 2B]Figures 2A-2B show the results of assays assessing binding by anti-Tie2 antibodies Tie2.1, Tie2.10, Tie2.11, and Tie2.12 to the mouse Tie2 ECD5 domain (Figure 2A) and human Tie2 ECD5 domain (Figure 2B). The ECD5 domain contains the Ig1, Ig2, EGF, and Ig3 domains of Tie2. [Figure 3A] Figures 3A-3B show the results of assays to assess binding by 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) to human Tie1. [Figure 3B] Figures 3A-3B show the results of assays to assess binding by 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) to human Tie1. [Figure 4A] Figures 4A-4B show the results of assays to assess the blocking of the interaction between Tie2 and Ang1 (Figure 4A) and the interaction between Tie2 and Ang2 (Figure 4B) by the anti-Tie2 antibodies Tie2.1, Tie2.12, and Tie2.20. [Figure 4B] Figures 4A-4B show the results of assays to assess the blocking of the interaction between Tie2 and Ang1 (Figure 4A) and the interaction between Tie2 and Ang2 (Figure 4B) by the anti-Tie2 antibodies Tie2.1, Tie2.12, and Tie2.20. [Figure 5A] 5A-5B show the results of assays to assess binding by anti-Tie2 antibodies Tie2.1, Tie2.10, Tie2.11, and Tie2.12 to HUVEC (FIG. 5A) and RAEC (FIG. 5B). [Figure 5B]5A-5B show the results of assays to assess binding by anti-Tie2 antibodies Tie2.1, Tie2.10, Tie2.11, and Tie2.12 to HUVEC (FIG. 5A) and RAEC (FIG. 5B). [Figure 6A] Figure 6A-6B show the results of assays to assess activation of AKT phosphorylation by anti-Tie2 antibodies Tie2.1, Tie2.4, Tie2.5, and Tie2.20 in RAECs (Figure 6A) and to assess the effect of anti-IgG cross-linking of anti-Tie2 antibodies on AKT phosphorylation (Figure 6B). The levels of phosphorylated AKT were determined by Western blot analysis. [Figure 6B] Figure 6A-6B show the results of assays to assess activation of AKT phosphorylation by anti-Tie2 antibodies Tie2.1, Tie2.4, Tie2.5, and Tie2.20 in RAECs (Figure 6A) and to assess the effect of anti-IgG cross-linking of anti-Tie2 antibodies on AKT phosphorylation (Figure 6B). The levels of phosphorylated AKT were determined by Western blot analysis. [Figure 7A] Figures 7A-7B show the results of an assay to assess 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, Tie2.38, and Tie2.1 (Figure 7A). Figure 7B shows the effect of anti-Tie2 antibody aggregation or anti-IgG cross-linking on the activity of anti-Tie2 antibody Tie2.1 to induce AKT phosphorylation. The level of phosphorylated AKT was determined by FRET analysis. [Figure 7B]Figures 7A-7B show the results of an assay to assess 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, Tie2.38, and Tie2.1 (Figure 7A). Figure 7B shows the effect of anti-Tie2 antibody aggregation or anti-IgG cross-linking on the activity of anti-Tie2 antibody Tie2.1 to induce AKT phosphorylation. The level of phosphorylated AKT was determined by FRET analysis. [Figure 8A] 8A-8B show results from a binning study of anti-Tie2 antibodies using the method (FIG. 8A) and ELISA (FIG. 8B). [Figure 8B] 8A-8B show results from a binning study of anti-Tie2 antibodies using the method (FIG. 8A) and ELISA (FIG. 8B). [Figure 9] FIG. 9 is a schematic diagram showing the epitope groups on Tie2 to which the anti-Tie2 antibodies of the present disclosure bind. [Figure 10A] Figures 10A and 10B show sequence alignments of the amino acid sequences of the heavy chain variable regions (VH) of the anti-Tie2 antibodies Tie2.1 (sequence number 22), Tie2.1.M100cF (sequence number 20), Tie2.12 (sequence number 31), Tie2.24 (sequence number 36), Tie2.33 (sequence number 41), and Tie2.38 (sequence number 51) (Figure 10A), and the light chain variable regions (VL) of the anti-Tie2 antibodies Tie2.1 (sequence number 21), Tie2.1.M100cF (sequence number 21), Tie2.12 (sequence number 21), Tie2.24 (sequence number 21), Tie2.33 (sequence number 21), and Tie2.38 (sequence number 21) (Figure 10B). [Figure 10B]Figures 10A and 10B show sequence alignments of the amino acid sequences of the heavy chain variable regions (VH) of the anti-Tie2 antibodies Tie2.1 (sequence number 22), Tie2.1.M100cF (sequence number 20), Tie2.12 (sequence number 31), Tie2.24 (sequence number 36), Tie2.33 (sequence number 41), and Tie2.38 (sequence number 51) (Figure 10A), and the light chain variable regions (VL) of the anti-Tie2 antibodies Tie2.1 (sequence number 21), Tie2.1.M100cF (sequence number 21), Tie2.12 (sequence number 21), Tie2.24 (sequence number 21), Tie2.33 (sequence number 21), and Tie2.38 (sequence number 21) (Figure 10B). [Figure 11A] Figures 11A-11B show the results of a binning assay using various anti-Tie2.1 antibodies generated by phage display or animal immunization. Figure 11A provides a list of covalently immobilized antibodies and some antibodies in solution, while Figure 11B provides a list of the remaining antibodies in solution. [Figure 11B] Figures 11A-11B show the results of a binning assay using various anti-Tie2.1 antibodies generated by phage display or animal immunization. Figure 11A provides a list of covalently immobilized antibodies and some antibodies in solution, while Figure 11B provides a list of the remaining antibodies in solution. [Figure 12A] Figures 12A-12B show the results of assays evaluating the agonist activity of multimeric anti-Tie2 antibodies. Figure 12A compares AKT phosphorylation activation by Tie2.1 as a PEG hexamer and as a bi-epitope FabIgG(1.38). Figure 12B compares the multimeric formats of anti-Tie2 antibodies. [Figure 12B] Figures 12A-12B show the results of assays evaluating the agonist activity of multimeric anti-Tie2 antibodies. Figure 12A compares AKT phosphorylation activation by Tie2.1 as a PEG hexamer and as a bi-epitope FabIgG(1.38). Figure 12B compares the multimeric formats of anti-Tie2 antibodies. [Figure 13A]Figures 13A-B show the effect of various ratios of huIgM heavy chain to light chain, with or without J chain, on the total yield of protein isolated from the affinity column as measured by total protein A280 (Figure 13A) and SEC profile (Figure 13B). [Figure 13B] Figures 13A-B show the effect of various ratios of huIgM heavy chain to light chain, with or without J chain, on the total yield of protein isolated from the affinity column as measured by total protein A280 (Figure 13A) and SEC profile (Figure 13B). [Figure 13C] FIG. 13C shows rheological measurements of IgM compared to ocular multivalent PEG formats targeting factor D (fD). [Figure 13D] FIG. 13D shows intravitreal pharmacokinetic analysis of unconjugated IgM and unconjugated Fab. [Figure 13E] Figure 13E shows the whole body pharmacokinetic analysis of IgM, comparing non-binding recombinant hIgM pentamer and recombinant hIgM hexamer with IgM isolated from human serum injected intravenously into female SCID mice. Figure 13E shows serum IgM levels. [Figure 13F] FIG. 13F shows LC-MS analysis of the global N-linked glycan profile from serum samples. [Figure 13G] Figures 13G-13H show the results of assays characterizing anti-Tie2 antibodies in IgM multimer formats. Figure 13G shows the results of a complementation assay to evaluate mutations in the IgM constant domain. Figure 13H shows the agonist activity of anti-Tie antibodies in IgM hexamer format. [Figure 13H] Figures 13G-13H show the results of assays characterizing anti-Tie2 antibodies in IgM multimer formats. Figure 13G shows the results of a complementation assay to evaluate mutations in the IgM constant domain. Figure 13H shows the agonist activity of anti-Tie antibodies in IgM hexamer format. [Figure 14A]Figures 14A-14B show the design and analysis of anti-Tie2 antibodies in a hexameric format via a peptide moiety. Figure 14A is a schematic of the multimeric design. Figure 14B shows the results of an AKT phosphorylation assay comparing anti-Tie2 antibodies in a hexameric format via an NDK peptide, IgM, and a multi-arm PEG. [Figure 14B] Figures 14A-14B show the design and analysis of anti-Tie2 antibodies in a hexameric format via a peptide moiety. Figure 14A is a schematic of the multimeric design. Figure 14B shows the results of an AKT phosphorylation assay comparing anti-Tie2 antibodies in a hexameric format via an NDK peptide, IgM, and a multi-arm PEG. [Figure 15A] Figures 15A-15B show the results of an AKT phosphorylation assay to assess the agonist activity of various anti-Tie2 antibodies in a hexameric format. Results are shown for Tie2.1, Tie2.38, and Tie2.33 (Figure 15A) and Tie2.1, Tie2.1.M100cF, Tie2.12, and Tie2.24 (Figure 15B). [Figure 15B] Figures 15A-15B show the results of an AKT phosphorylation assay to assess the agonist activity of various anti-Tie2 antibodies in a hexameric format. Results are shown for Tie2.1, Tie2.38, and Tie2.33 (Figure 15A) and Tie2.1, Tie2.1.M100cF, Tie2.12, and Tie2.24 (Figure 15B). [Figure 16A] Figures 16A-16C show the results of assays to evaluate the effect of anti-Tie2 antibodies on cellular levels of Tie2 protein in an in vitro assay. Figures 16A, 16B, and 16C compare Tie2 levels upon exposure of HUVECs to anti-Tie2 antibodies generated via phage display and animal immunization, respectively. All assays in Figures 16A, 16B, and 16C were analyzed by Western blot. [Figure 16B]Figures 16A-16C show the results of assays to evaluate the effect of anti-Tie2 antibodies on cellular levels of Tie2 protein in an in vitro assay. Figures 16A, 16B, and 16C compare Tie2 levels upon exposure of HUVECs to anti-Tie2 antibodies generated via phage display and animal immunization, respectively. All assays in Figures 16A, 16B, and 16C were analyzed by Western blot. [Figure 16C] Figures 16A-16C show the results of assays to evaluate the effect of anti-Tie2 antibodies on cellular levels of Tie2 protein in an in vitro assay. Figures 16A, 16B, and 16C compare Tie2 levels upon exposure of HUVECs to anti-Tie2 antibodies generated via phage display and animal immunization, respectively. All assays in Figures 16A, 16B, and 16C were analyzed by Western blot. [Figure 17] FIG. 17 shows the results of an in vivo assay to assess the effect of anti-Tie2 antibodies on cellular levels of Tie2 protein levels. [Figure 18A] 18A and 18B show the assay method (FIG. 18A) and results (FIG. 18B) of an in vitro endothelial cell assay to study the effect of anti-Tie2.1 antibodies on endothelial cell barrier permeability. [Figure 18B] 18A and 18B show the assay method (FIG. 18A) and results (FIG. 18B) of an in vitro endothelial cell assay to study the effect of anti-Tie2.1 antibodies on endothelial cell barrier permeability. [Figure 19A] 19A-19B show the assay method (FIG. 19A) and results (FIG. 19B) of an in vivo vascular permeability assay to study the effect of anti-Tie2.1 antibodies on VEGF-induced vascular leakage. [Figure 19B] 19A-19B show the assay method (FIG. 19A) and results (FIG. 19B) of an in vivo vascular permeability assay to study the effect of anti-Tie2.1 antibodies on VEGF-induced vascular leakage. [Figure 20A]20A-20B show the assay method (FIG. 20A) and results (FIG. 20B) of an in vivo vascular permeability assay to study the effect of anti-Tie2.1 or anti-VEGF antibodies on VEGF-induced vascular leakage. [Figure 20B] 20A-20B show the assay method (FIG. 20A) and results (FIG. 20B) of an in vivo vascular permeability assay to study the effect of anti-Tie2.1 or anti-VEGF antibodies on VEGF-induced vascular leakage. [Figure 21A] Figures 21A-B show the in vivo effect of a bi-epitope anti-Tie2 agonist (anti-Tie2 Fab-IgG1.38) on Tie2 protein levels determined by Western blot analysis (Figure 21A), and the results of an in vivo vascular permeability assay to study the effect of anti-Tie2 Fab-IgG1.38 on VEGF-induced vascular leakage (Figure 21B). [Figure 21B] Figures 21A-B show the in vivo effect of a bi-epitope anti-Tie2 agonist (anti-Tie2 Fab-IgG1.38) on Tie2 protein levels determined by Western blot analysis (Figure 21A), and the results of an in vivo vascular permeability assay to study the effect of anti-Tie2 Fab-IgG1.38 on VEGF-induced vascular leakage (Figure 21B). [Figure 22] FIG. 22 shows the effect of anti-Tie2 agonists on the cellular organization of VE-cadherin (upper panel) and F-actin (lower panel) in cultured HUVECs. [Figure 23] FIG. 23 shows the results of an AKT phosphorylation assay to assess the agonist activity of Tie2.1 anti-Tie2 antibody variants in the IG1 format. [Figure 24] FIG. 24 shows the results of an AKT phosphorylation assay to assess the agonist activity of variants of Tie2.1 anti-Tie2 conjugates in hexamer format compared to non-PEG conjugated Tie2.1 Fab (no hexamer). [Figure 25]FIG. 25 shows the pharmacokinetics of anti-Tie2 Fab hexamer conjugates after ocular injection in cynomolgus monkeys. [Figure 26] Figures 26-27 show electropherogram analyses of anti-Tie2.1 PEG conjugates with (Figure 26) and without (Figure 27) the monomer peak being considered for relative quantification. [Figure 27] Figures 26-27 show electropherogram analyses of anti-Tie2.1 PEG conjugates with (Figure 26) and without (Figure 27) the monomer peak being considered for relative quantification. [Figure 28] FIG. 28 shows an electropherogram analysis of anti-Tie2.1 PEG conjugates in samples taken from cynomolgus monkey vitreous humor. [Figure 29] FIG. 29 shows the relative amounts of anti-Tie2 PEG-conjugated hexamers and pentamers. [Figure 30A] 30A-B provide data showing the oxidation of M100c (FIG. 30A) in samples taken from cynomolgus monkey eyes over a 21 day period (FIG. 30B). [Figure 30B] 30A-B provide data showing the oxidation of M100c (FIG. 30A) in samples taken from cynomolgus monkey eyes over a 21 day period (FIG. 30B). [Figure 31] FIG. 31 shows the effect of oxidation on pAKT activity. [Figure 32] 32-33 show the effect of deconjugation on pAKT activity (FIG. 32), with normalized values ​​graphed in FIG. [Figure 33] 32-33 show the effect of deconjugation on pAKT activity (FIG. 32), with normalized values ​​graphed in FIG. [Figure 34] Figure 34 shows the effect of the location of the PEG conjugation site on deconjugation. [Figure 35A] Figure 35A shows the effect of the location of the PEG conjugation site on activity. [Figure 35B] Figure 35B shows the effect of the location of the PEG conjugation site on activity. [Figure 36]FIG. 36 shows the pharmacokinetics of different Tie2.1M100cF PEG conjugates. [Figure 37] Figure 37 compares the in vivo stability of different Tie2.1M100cF PEG conjugates. [Figure 38A] Figures 38A-B compare the activity of different Tie2.1M100cF PEG conjugates: Figure 38A shows pAKT activity, and Figure 38B shows total conjugate levels by ELISA. [Figure 38B] Figures 38A-B compare the activity of different Tie2.1M100cF PEG conjugates: Figure 38A shows pAKT activity, and Figure 38B shows total conjugate levels by ELISA. [Figure 39] FIG. 39 shows the structure of a PEG-conjugated hexameric core molecule conjugated to an anti-Tie2 antibody according to the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0084] Detailed Description of Embodiments of the Invention I. Definition Unless otherwise defined herein, the term "comprising" is intended to include the term "consisting of."

[0085] The term "about" as used herein in connection with a particular value (e.g., temperature, concentration, time, etc.) is intended to refer to a + / -1% variation of the particular value to which the term "about" refers.

[0086] For purposes herein, an "acceptor human framework" is a framework that comprises the 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 may comprise the same amino acid sequence or may contain amino acid sequence changes. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0087] "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, as used herein, "binding affinity" 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 represented by a dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative explanations and exemplary embodiments for measuring binding affinity are described below.

[0088] An "affinity matured" antibody refers to an antibody that has one or more modifications in one or more hypervariable regions (HVRs) compared to a parent antibody that does not have such modifications, which improve the affinity of the antibody for its antigen.

[0089] The terms "anti-Tie2 antibody," "antibody that binds to Tie2," and "antibody that specifically binds to Tie2" refer to an antibody that is capable of binding to Tie2 with sufficient affinity such that the antibody is useful as a therapeutic and / or diagnostic agent in targeting Tie2. In one embodiment, the extent to which an anti-Tie2 antibody binds to an unrelated, non-Tie2 protein is less than about 10% of the antibody's binding to Tie2, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an antibody that binds to Tie2 has an affinity of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10 -8 M or less, e.g. 10 -8 ~10 -13 M, e.g. 10 -9 M~10 -13 Dissociation constant (K D In certain embodiments, the anti-Tie2 antibody binds to an epitope of Tie2 that is conserved among Tie2 from different species.

[0090] The term "antibody" is used herein in the broadest sense and encompasses a variety of 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.

[0091] An "antibody fragment" is a molecule other than an intact antibody that contains 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 antibody fragments.

[0092] The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a heavy chain that has a structure substantially similar to a native antibody structure or that contains an Fc region as defined herein.

[0093] As used herein, "Fab" refers to an antibody comprising a heavy chain constant region that includes a CH1 domain or a portion of the CH1 domain sufficient to form a disulfide bond with the light chain constant region, but does not include the CH2 or CH3 domains. As used herein, a Fab may include one or more amino acids of the hinge region. Thus, as used herein, the term "Fab" encompasses Fab' antibodies. A Fab may also include an additional non-natural amino acid, such as a C-terminal cysteine, in which case it may be referred to as a Fab-C. As discussed below, the term Fab-C also encompasses Fabs that include natural amino acids in the hinge region, including a natural cysteine ​​at the C-terminus. In some embodiments, a Fab includes an engineered cysteine ​​(i.e., the Fab can be THIOMAB). In some embodiments, the engineered cysteine ​​is a cysteine ​​amino acid residue in the Fab HC and / or LC polypeptide sequence that has been replaced with a non-cysteine ​​amino acid residue.

[0094] "Fab-C" refers to a Fab that includes a C-terminal cysteine, which may be a naturally occurring cysteine ​​occurring at that residue position (such as a cysteine ​​from the hinge region) or may be a cysteine ​​added to the C-terminus that does not correspond to a naturally occurring cysteine.

[0095] "Fab-SH" refers to a Fab bearing a free thiol group. In some embodiments, the free thiol group is located in the last 10 amino acids at the C-terminus of the Fab. Fab-C antibodies are also typically Fab-SH antibodies.

[0096] An "antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks the reference antibody from binding to its antigen by 50% or more in a competition assay, and conversely, a reference antibody that blocks the antibody from binding to its antigen by 50% or more in a competition assay. Exemplary competition assays are provided herein.

[0097] 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.

[0098] The "class" of an antibody refers to the type of constant domain or constant region carried by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0099] The term "conjugate" is used herein in its broadest definition, meaning joined or linked together. Molecules are "conjugated" if they act or function as if they are joined. A "conjugate" is an antibody (e.g., a Fab) conjugated to one or more heterologous molecule(s), including, but not limited to, a polyol. In certain embodiments, a "conjugate" refers to an antibody (e.g., an antibody fragment, as detailed herein) covalently attached to a multi-arm moiety. In certain embodiments, the multi-arm moiety is a polyol, an IgM molecule, or a peptide in a multimeric (e.g., hexameric) format.

[0100] As used herein, the term "Tie2," unless otherwise specified, refers to any native Tie2 of any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses "full-length," unprocessed Tie2 as well as any form of Tie2 that results from processing within a cell. The term also encompasses naturally occurring variants of Tie2, such as splice variants or allelic variants. The amino acid sequence of an exemplary human Tie2 protein has NCBI Reference Number: NP_000450 (SEQ ID NO: 1).

[0101] The term "cytotoxic agent," as used herein, refers to a substance that inhibits or prevents the function of cells and / or causes cell death or destruction. Cytotoxic agents include radioisotopes (e.g., At 211 , I 131 , I 125 , Y 90 ,Re 186 ,Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 , and radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof, e.g., 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); and various anti-tumor or anti-cancer agents disclosed below.

[0102] "Effector functions" refer to biological activities attributable to the Fc region of an antibody and vary depending on 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 receptors); and B cell activation.

[0103] An "effective amount" of an agent, eg, a pharmaceutical formulation, refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic or prophylactic result.

[0104] As used herein, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system (also referred to as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0105] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain typically consists of four FR domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences typically appear in VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0106] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, and to cells, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived from the host cell regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included in the present invention.

[0107] A "human antibody" is an antibody having an amino acid sequence corresponding to an antibody produced by a human or human cell, or an antibody of non-human origin that utilizes human antibody-encoding sequences, such as the human antibody repertoire. This definition of human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

[0108] A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda, MD (1991), vols. 1-3. In one embodiment, for VL, the subgroup is subgroup kappa I in Kabat et al. (supra). In one embodiment, for VH, the subgroup is subgroup III in Kabat et al. (supra).

[0109] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues derived from non-human HVRs and human FRs. In certain embodiments, a humanized antibody comprises substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody may optionally 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.

[0110] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains (VH and VL, respectively) of natural antibodies generally have similar structures, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). (See, e.g., Kindt et al., Kuby Immunology, 6 th ed., W.H. Freeman and Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Moreover, antibodies that bind to a specific antigen may be isolated by using the VH or VL domain of an antibody that binds the antigen to screen a library 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).

[0111] As used herein, the term "hypervariable region," "HVR," or "HV" refers to a region of an antibody variable domain that is hypervariable in sequence (also referred to herein as a "complementarity-determining region" or "CDR") and / or forms structurally defined loops. Antibodies generally contain six HVRs: three in the VH (H1, H2, and H3) and three in the VL (L1, L2, and L3). In natural antibodies, H3 and L3 exhibit the highest diversity among the six HVRs, and H3 in particular is thought to play a unique role in conferring superior specificity to 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, naturally occurring camelid antibodies consisting only of heavy chains are functional and stable in the absence of light chains. See, e.g., Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).

[0112] Several HVR delineations are used and encompassed herein. Kabat Complementarity Determining Regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia, instead, refers to the location of structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). AbM HVRs represent a compromise between Kabat HVRs and Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software. "Contact" HVRs are based on analysis of available complex crystal structures. Residues from each of these HVRs are shown below. JPEG2025138630000005.jpg43170

[0113] HVRs may include the following "extended HVRs": 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. The variable domain residues are numbered according to Kabat et al., supra, for each of these definitions.

[0114] 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 or light chain variable domains of the compilation of antibodies 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, the FRs or HVRs of the variable domain. For example, a heavy chain variable domain may contain 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 according to Kabat, etc.). The Kabat numbering of residues can be determined for a given antibody by alignment of the antibody's sequence with the "standard" Kabat-numbered sequence at the regions of homology.

[0115] The Kabat numbering system is commonly used when referring to residues in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., supra). The "EU numbering system" or "EU index" is commonly used when referring to residues in the immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra). The "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody. Unless otherwise indicated herein, references to residue numbers in the variable domain of an antibody refer to residue numbering according to the Kabat numbering system. Unless otherwise indicated herein, references to residue numbers in the constant domain of an antibody refer to residue numbering according to the EU numbering system (e.g., U.S. Patent Application Publication No. 2008 / 0181888, see figures for EU numbering).

[0116] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules, including but not limited to, cytotoxic agents.

[0117] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is human.

[0118] An "isolated" antibody is one that has been separated from a component of its natural environment. In some embodiments, the antibody is purified to greater than 95% or greater than 99% purity, for example, as determined by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0119] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. Isolated nucleic acid includes a nucleic acid molecule contained within a cell that normally contains the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0120] An "isolated nucleic acid encoding an anti-Tie2 antibody" refers to one or more nucleic acid molecules encoding the antibody heavy and light chains (or fragments thereof), including such nucleic acid molecule(s) in a single vector or separate vectors, and including such nucleic acid molecule(s) present in one or more locations within a host cell.

[0121] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind to the same epitope, except for possible variant antibodies that contain, for example, naturally occurring mutations or that arise during production of the monoclonal antibody preparation, and such variants are generally present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention can be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci; such methods and other exemplary methods for producing monoclonal antibodies are described herein.

[0122] A "naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The naked antibody may be present in a pharmaceutical composition.

[0123] "Native antibodies" refer to naturally occurring immunoglobulin molecules with diverse structures. For example, native IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, comprising two identical light chains and two identical heavy chains disulfide-linked. From the N-terminus to the C-terminus, each heavy chain contains a variable region (VH), also called a variable heavy domain or 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 contains a variable region (VL), also called a variable light domain or light chain variable domain, followed by a constant light (CL) domain. Based on the amino acid sequence of its constant domain, the light chain of an antibody may be assigned to one of two types, called κ (kappa) or λ (lambda).

[0124] The term "package insert" is used to refer to instructions typically included in commercial packaging for a therapeutic product, including information regarding the indications, uses, dosage, administration, concomitant therapy, contraindications and / or warnings regarding such therapeutic product.

[0125] "Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, after aligning the sequences and introducing gaps as necessary to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for determining percent amino acid sequence identity can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for sequence alignment, including any algorithms required to achieve maximum alignment across the entire length of the sequences being compared. However, for purposes herein, percent amino acid sequence identity values ​​are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and the source code, together with user documentation, has been filed with the U.S. Copyright Office, Washington, DC 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc. (South San Francisco, California), or can be compiled from its 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 do not vary.

[0126] In situations where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with, or relative to a given amino acid sequence B (alternatively, it may be written as a given amino acid sequence A having or comprising a certain % amino acid sequence identity to, with, or relative to a given amino acid sequence B) is calculated as follows: 100 x fraction X / Y where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is different from the length of amino acid sequence B, the % amino acid sequence identity of A to B will differ from the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

[0127] The term "pharmaceutical formulation" refers to a formulation that is in a form that allows the biological activity of the active ingredient contained therein to be effective and that does not contain additional ingredients that have unacceptable toxicity to the subject to whom the formulation will be administered.

[0128] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0129] The term "polyol" broadly refers to polyhydric alcohol compounds. Polyols can be, for example, any water-soluble poly(alkylene oxide) polymer and can have a linear or branched chain. Preferred polyols include those in which one or more hydroxyl positions are substituted with a chemical group, e.g., an alkyl group having 1 to 4 carbons. Typically, the polyol is a poly(alkylene glycol), preferably polyethylene glycol (PEG). However, those skilled in the art will understand that other polyols, such as poly(propylene glycol) and polyethylene-polypropylene glycol copolymers, can be used using the conjugation techniques described herein for PEG. Polyols of the present disclosure include those known in the art and publicly available, e.g., from commercially available sources.

[0130] As used herein, "treatment" (and grammatical variations thereof, e.g., "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either prophylactically or during the course of clinical pathology. Desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, remission or palliation of disease symptoms, and recovery or improved prognosis. In some embodiments, the antibodies of the invention are used to delay the onset of disease or to slow the progression of disease.

[0131] The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as autonomously replicating nucleic acid structures as well as vectors that are integrated into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0132] A "port delivery system" or "PDS" is an implantable device for the eye that uses a refillable reservoir to deliver a therapeutic agent over an extended period of time. The implant has a refill port that communicates with the reservoir and a release control element that determines the rate of drug release into the eye. See, for example, U.S. Patent Publication Nos. 20100174272, 8,277,830, 8,399,006, 8,795,712, and 8,808,727.

[0133] "Small bore needle" refers to a needle for injecting liquid components that is about 30, 29, 28, 27, 26, 25, 24, 23, or 22 gauge or larger, such as a 30 gauge needle. In some embodiments, the small bore needle has a standard size wall. In other embodiments, the small bore needle has a thin wall, which may be preferable for viscous solutions.

[0134] II. Compositions and Methods For example, novel Tie2 agonists are provided herein that activate Tie2 function, as evidenced by Tie2 phosphorylation. Direct agonists may be therapeutically advantageous due to their stronger activation of Tie2 signaling, promoting improved vision when endogenous agonist Ang1 is low or absent. Tie2 activators may be more advantageous than Ang2 inhibitors because they block Ang2 binding (blocking Ang2 antagonist function) and can directly bind to Tie2 to activate Tie2 activity, e.g., increase Tie2 and / or Akt phosphorylation. Tie2 activation has been shown to require Tie2 clustering upon ligand binding; therefore, the Tie2 agonists provided herein are multimeric, preferably hexamers or octamers. The multimeric Tie2 agonists described herein may have the additional unexpected advantage of not significantly reducing cellular Tie2 levels in vitro or in vivo. Furthermore, the multimeric Tie2 agonists described herein can be formulated for intravitreal injection and have molecular sizes that confer favorable pharmacokinetics and therefore pharmacodynamics. Data are provided showing that multimeric Tie2 agonists reduce endothelial cell membrane permeability and enhance cell-cell junctions (see, e.g., Example 10).

[0135] 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 two or more anti-Tie2 antibodies or antigen-binding fragments thereof, such as (but not limited to) anti-Tie2 Fabs. It may be preferable for the binding agent to comprise more than four (e.g., including 5, 6, 7, 8, 9, or 10) anti-Tie2 antibodies, e.g., more than four anti-Tie2 Fabs, such that binding of the Tie2 binding agent to Tie2 located on the cell surface is associated with Tie2 activation. Tie2 clustering can also occur upon binding of the binding agent to Tie2.

[0136] In some embodiments, it is advantageous to have a Tie2-binding agent that, upon binding to Tie2 on a protein surface, does not significantly reduce Tie2 protein levels on the cell surface. In some embodiments, a Tie2-binding agent 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.

[0137] Activation of the Tie2 protein can be measured in vitro using materials and methods readily known to those skilled in the art by measuring increased phosphorylation of the Tie2 protein (e.g., by Western blot) or by measuring increased phosphorylation of the related Akt protein (AKT serine / threonine kinase 1, e.g., GenBank accession number NP_001014431). In certain embodiments, antibodies that bind to Tie2, as well as compositions comprising two or more antibodies that bind to Tie2, are provided. Antibodies and compositions comprising multiple antibodies of the present invention are useful, for example, for diagnosing or treating vascular permeability disorders, particularly in the eye, associated with Tie2 function.

[0138] A. Exemplary Anti-Tie2 Binding Agents In one aspect, the present invention provides a Tie2-binding agent comprising an isolated antibody that binds to Tie2. In some embodiments, the Tie2-binding agent comprises a multi-arm moiety, each of the arms of which 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 moieties 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-binding agent comprising two or more anti-Tie2 antibodies or fragments thereof is provided, wherein the anti-Tie2 antibodies (optionally formatted as full-length antibodies, rather than as part of a Tie2-binding agent) have a K of less than 100 μM, or less than 50 μM, or less than 10 μM. D and / or a K greater than 1 μM Dand binds to Tie2 with an affinity such as 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, or 85%. It is understood that affinity is measured using an antibody, rather than a Tie2-binding agent comprising two or more anti-Tie2 antibodies. In some embodiments, affinity is monovalent. Additionally, Tie2-binding agents and Tie2 antibodies that activate Tie2 activity (e.g., function as Tie2 agonists) are provided. Tie2 activation is measured, for example, by measuring an increase in Tie2 phosphorylation and / or an increase in AKT phosphorylation in an in vitro assay. In some embodiments, the Tie2-binding agent does not down-regulate (reduce) intracellular Tie2 protein levels by more than about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, or 85%. In alternative embodiments, the Tie2-binding agent reduces intracellular Tie2 protein levels by less than about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, or 75%. In some embodiments, the Tie2-binding agent reduces vascular permeability. Reductions in vascular endothelial cell permeability can be measured in vitro or in vivo. In some embodiments, the Tie2-binding agent can increase the translocation of Tie2 to cell-cell junctions and / or promote the structural organization of actin and / or cadherin. In a preferred embodiment, the Tie2-binding agent comprises a hexameric PEG molecule, each of six arms of which is conjugated to an anti-Tie2 Fab. Alternatively, the PEG molecule comprises eight arms. In some embodiments, the Tie2-binding agent comprises two or more anti-Tie2 antibodies or fragments 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 (eg, to an epitope located at least in part within amino acid residues 23-120 of SEQ ID NO:1).

[0139] In one aspect, the present invention provides antibodies that specifically bind to Tie2, or an antigen-binding fragment thereof, comprising at least one, two, three, four, five, or six CDRs selected from: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 3; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 4; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 5; SEQ ID NO: 6; or SEQ ID NO: 7, where 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; (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; or (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 10. In a specific embodiment, CDR-H3 comprises SEQ ID NO: 7.

[0140] 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 (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 3, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 4, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 5, wherein 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, CDR-H3 comprises the amino acid sequence of SEQ ID NO: 6. In another embodiment, CDR-H3 comprises the amino acid sequence of SEQ ID NO: 7.

[0141] In other embodiments, the invention provides antibodies comprising at least one, at least two, or all three VL domain CDR sequences selected from (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. 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.

[0142] In another embodiment, 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 (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 an amino acid sequence selected from SEQ ID NO: 5; SEQ ID NO: 6; and SEQ ID NO: 7, 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 (b) a VL domain comprising at least one, at least two, or all three VL CDR sequences selected from (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.

[0143] In another aspect, the present invention provides an antibody comprising a VH domain comprising: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 3; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 4; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7, 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 VL domain comprising: (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9; and (f) CDR-L3 comprising 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 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 21.

[0144] In any of the above embodiments, the anti-Tie2 antibody may be humanized. In one embodiment, the anti-Tie2 antibody comprises the CDRs as in any of the preceding embodiments and further comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework. In another embodiment, the anti-Tie2 antibody comprises the CDRs of 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 another embodiment, 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.

[0145] In other aspects, an anti-Tie2 antibody or antigen-binding fragment thereof comprises a heavy chain variable domain (VH) 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. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but an anti-Tie2 antibody comprising that sequence retains the ability to bind to Tie2. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 20. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the anti-Tie2 antibody comprises the VH sequence of SEQ ID NO: 20, including post-translational modifications of that sequence. In certain embodiments, the VH comprises one, two, or three CDRs selected from the following: (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: 7.

[0146] In other aspects, anti-Tie2 antibodies are provided, wherein the antibodies comprise a light chain variable domain (VL) 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, VL sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contain substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but anti-Tie2 antibodies comprising such sequences retain 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: 21. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., within the FRs). Optionally, the anti-Tie2 antibody comprises the VL sequence of SEQ ID NO: 21, including post-translational modifications of that sequence. In certain embodiments, the VL comprises one, two, or three HVRs selected from (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.

[0147] In other aspects, an anti-Tie2 antibody is provided, wherein the antibody comprises a VH as in any of the embodiments provided above, and a VL as in any of the embodiments provided above. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO:20 and SEQ ID NO:21, respectively, including post-translational modifications of those sequences.

[0148] In a further aspect, the present invention provides antibodies that bind to the same epitope as the anti-Tie2 antibodies provided herein. For example, in certain embodiments, an antibody is provided that binds to the same epitope as an anti-Tie2 antibody comprising the VH sequence of SEQ ID NO: 20 and the VL sequence of SEQ ID NO: 21. In certain embodiments, an antibody is provided that binds to an epitope within a fragment of Tie2, for example, the Ig1 domain of Tie2, wherein the Ig1 domain comprises amino acids 23 to 120 of SEQ ID NO: 1.

[0149] In a further aspect of the 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, diabody, or F(ab')2 fragment. In other embodiments, the antibody is a full-length antibody, such as an intact IgG1 antibody or other antibody class or isotype as defined herein.

[0150] In further aspects, anti-Tie2 antibodies according to any of the foregoing embodiments may incorporate any of the features, either alone or in combination, as described in Sections 1-7 below.

[0151] 1. Antibody affinity In certain embodiments, the antibodies provided herein have a cytotoxicity of 10 μM or less, 100 μM or less, 10 μM or less, 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less, and / or 0.01 μM, 0.1 μM, or 1 μM or more (e.g., 10 -5 M or less, 10 -6 M or less, 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~10 -9 M, e.g. 10 -8 M~10 -13 M, e.g. 10 -9 M~10 -13It has a dissociation constant (Kd) of 1 M.

[0152] In one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA). In one embodiment, the RIA is performed using a Fab version of the antibody of interest and its antigen. For example, the solution binding affinity of the Fab for the antigen is measured in the presence of a titration series of unlabeled antigen at the lowest concentration ( 125 I) Fab is equilibrated with labeled antigen, followed by capturing the bound antigen on a plate coated with an anti-Fab antibody (see, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). To establish assay conditions, MICROTITER® multiwell plates (Thermo Scientific) are coated overnight with 5 μg / mL of capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), followed by blocking with 2% (w / v) bovine serum albumin in PBS for 2-5 hours at room temperature (approximately 23°C). In non-adsorbent plates (Nunc #269620), 100 pM or 26 pM [ 125 [I]-antigen is mixed with serial dilutions of the Fab of interest (e.g., consistent with the evaluation of the anti-VEGF antibody Fab-12 in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then incubated overnight, although incubation can be continued for a longer period (e.g., approximately 65 hours) to ensure equilibrium is reached. The mixture is then transferred to a capture plate for incubation at room temperature (e.g., 1 hour). The solution is then removed, and the plate is washed eight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. When the plate has dried, 150 μl / well of scintillant (MICROSCINT-20™; Packard) is added, and the plate is counted for 10 minutes in a TOPCOUNT™ gamma counter (Packard). The concentration of each Fab that results in 20% or less of maximum binding is selected for use in the competitive binding assay.

[0153] According to another embodiment, Kd is measured using a BIACORE® surface plasmon resonance assay. For example, assays using a BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) are performed at 25°C using an immobilized antigen CM5 chip at ∼10 response units (RU). In one embodiment, a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to instructions of one skilled in the art. The antigen is diluted in 10 mM sodium acetate, pH 4.8, to 5 μg / mL (approximately 0.2 μM) before injection at a flow rate of 5 μL / min to achieve approximately 10 response units (RU) of binding protein. After antigen injection, 1 M ethanolamine is injected to block unreactive groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25°C at a flow rate of approximately 25 μL / min. Association rates (k) and dissociation rates (k) are calculated by simultaneously fitting the association and dissociation sensorgrams using a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software version 3.2). The equilibrium dissociation constant (K) is calculated as the ratio k / k. See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999).If the on-rate exceeds 10 M s by the surface plasmon resonance assay described above, the on-rate can be determined by using a fluorescence quenching technique to measure 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) at 25°C in the presence of increasing concentrations of antigen as measured with a spectrophotometer such as a stopped-flow equipped spectrophotometer (Aviv Instruments) or an 8000 series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) with a stirred cuvette.

[0154] 2. Antibody fragment 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 specific antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a reference to scFv fragments, see, for example, Pluckthuen, in *The Pharmacology of Monoclonal Antibodies*, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93 / 16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. See US Pat. No. 5,869,046 for a description of Fab and F(ab')2 fragments that contain salvage receptor binding epitope residues and have increased in vivo half-lives.

[0155] In some embodiments, the C-terminus of the heavy chain of the Fab fragment terminates with 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 terminates with the sequence CDKTHX (SEQ ID NO:79), where X is any amino acid except T. Truncations and / or mutations at the C-terminus may reduce or eliminate AHA reactivity to the Fab without compromising thermostability or expression. In some embodiments, the C-terminus of the heavy chain of the Fab fragment terminates with the amino acid "CDKTHTC" (SEQ ID NO:80), "CDKTHTCPPC" (SEQ ID NO:81), "CDKTHTCPPS" (SEQ ID NO:82), "CDKTHTSPPC" (SEQ ID NO:83), "CDKTHTAPPC" (SEQ ID NO:84), "CDKTHTSGGC" (SEQ ID NO:85), or "CYGPPC" (SEQ ID NO:86). In some such embodiments, the free cysteine ​​in the C-terminal amino acid may be amenable to conjugation, for example, to a polymer such as PEG.

[0156] Diabodies are antibody fragments that have two antigen binding sites, and can be bivalent or bispecific.See, for example, European Patent Application Publication No. 404,097, International Publication No. 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).

[0157] Single-domain antibodies are antibody fragments that contain all or part of the heavy chain variable domain 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).

[0158] Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (e.g., E. coli or phage), as described herein.

[0159] 3. Chimeric and humanized antibodies In certain embodiments, the antibody provided herein is a chimeric antibody. 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 a further example, a chimeric antibody is a "class-switched" antibody whose class or subclass is changed from that of the parent antibody. A chimeric antibody includes an antigen-binding fragment thereof.

[0160] In certain embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which the HVRs, e.g., CDRs (or portions thereof), are derived from a non-human antibody and the FRs (or portions thereof) are derived from a human antibody sequence. Optionally, the humanized antibody also comprises at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

[0161] Humanized antibodies and methods for their production are reviewed by Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described 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) (describing specificity-determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing resurfacing); Dall'Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing a "guided selection" approach to FR shuffling).

[0162] Human 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)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

[0163] 4. Human antibodies 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).

[0164] Human antibodies may be prepared by administering immunogens to transgenic animals that have been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or part of a human immunoglobulin locus that replaces the endogenous immunoglobulin locus or is present extrachromosomally or randomly integrated into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin locus is generally inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584, which describe XENOMOUSE™ technology; U.S. Patent No. 5,770,429, which describes HuMab® technology; U.S. Patent No. 7,041,870, which describes KM MOUSE® technology; and U.S. Patent Application Publication No. 2007 / 0061900, which describes VelociMouse® technology. The human variable regions from intact antibodies produced by such animals may be further modified, for example, by combining with different human constant regions.

[0165] Human antibodies can also be produced by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for producing 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 via human B cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Further methods include, for example, U.S. Patent No. 7,189,826 (which describes the production of monoclonal human IgM antibodies from hybridoma cell lines), and Ni, Xiandai Mianyixue, 26(4):265-268(2006) (which describes 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).

[0166] Human antibodies can also be produced by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences can then be combined with desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

[0167] 5. Library-derived antibodies Antibodies of the invention can be isolated by screening combinatorial libraries for antibodies with one or more desired activities. For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies with desired binding characteristics. Such methods are reviewed, for example, by Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al. 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).

[0168] In a specific phage display method, repertoires of VH and VL genes are cloned separately by polymerase chain reaction (PCR) and randomly recombined in a phage library, followed by screening for antigen-binding phage, as described by Winter et al., Ann. Rev. Immunol., 12:433-455 (1994). Phages typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immune sources provide high-affinity antibodies against immunogens without the need for hybridoma construction. Alternatively, natural repertoires can be cloned (e.g., from humans) to provide a single source of antibodies against a wide range of non-self antigens and also self antigens, without immunization, as described by Griffiths et al., EMBO J., 12:725-734 (1993). Finally, natural libraries can also be generated synthetically by cloning unrearranged V gene segments from stem cells, using PCR primers containing random sequences to encode highly variable CDR3 regions, and achieving rearrangement in vitro as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example: U.S. Pat. No. 5,750,373, and U.S. Patent Application Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0169] Antibodies or antibody fragments isolated from a human antibody library are considered human antibodies or human antibody fragments herein.

[0170] 6. Multispecific antibodies 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 embodiments, one of the binding specificities is for Tie2 and the other is for any other antigen. In certain embodiments, bispecific antibodies can bind to two different epitopes of Tie2. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing Tie2. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0171] Techniques for producing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537 (1983)), International Publication No. WO 93 / 08829, and Traunecker et al., EMBO J. 10:3655 (1991)), and "knob-into-hole" engineering (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can also be produced by manipulating electrostatic steering effects to create antibody Fc heterodimeric molecules (WO 2009 / 089004 A1); cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science, 229:81 (1985)); using leucine zippers to create bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); using "diabody" technology to create bispecific 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., 148(5):1547-1553 (1992)). al., J. Immunol., 152:5368 (1994)); and, for example, by the preparation of trispecific antibodies as described in Tutt et al. J. Immunol. 147:60 (1991).

[0172] Engineered antibodies with three or more functional antigen-binding sites, including "octopus antibodies," are also included herein (see, e.g., US2006 / 0025576A1).

[0173] The antibodies or fragments herein also include "Dual Acting FAbs" or "DAFs" that contain antigen binding sites that bind to Tie2 and other different antigens (see, e.g., U.S. Patent Application Publication No. 2008 / 0069820).

[0174] 7. Antibody Variants In certain embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of antibodies may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of, residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, as long as the final construct possesses the desired characteristics (e.g., antigen binding).

[0175] a) Substitution, insertion, and deletion variants In certain embodiments, antibody variants with one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include HVRs and FRs. Conservative substitutions are shown in Table 1 under the heading of "Preferred Substitutions." More substantial changes are provided in Table 1 under the heading of "Exemplary Substitutions," and as further described below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into the antibody of interest, and the products screened for the desired activity, e.g., retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC. [Table 1]

[0176] Amino acids can be grouped according to common side chain properties. (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu, (4) Basic: His, Lys, Arg, (5) Residues that affect chain orientation: Gly, Pro, (6) Aromatic: Trp, Tyr, Phe.

[0177] Non-conservative substitutions would involve exchanging a member of one of these classes for another class.

[0178] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further testing have modified (e.g., improved) specific biological properties (e.g., increased affinity, reduced immunogenicity) compared to the parent antibody and / or have substantially retained specific biological properties of the parent antibody. An exemplary substitutional variant is an affinity-matured antibody, which can be conveniently generated using, for example, phage-display-based affinity maturation techniques as described herein. Briefly, one or more HVR residues are mutated, and the variant antibodies are displayed on phage and screened for a specific biological activity (e.g., binding affinity).

[0179] To improve antibody affinity, modifications (e.g., substitutions) may be made, for example, in CDRs. Such modifications may be made in CDR "hot spots," i.e., residues encoded by codons that undergo frequent mutation during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or within residues that contact the antigen, and the resulting variant VH or VL is tested for binding affinity. Affinity maturation by construction of and reselection from a secondary library is described, for example, 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 generated. This library is then screened to identify antibody variants with the desired affinity. Another method for introducing diversity involves CDR-directed approaches, in which several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 are often targeted in particular.

[0180] In certain embodiments, substitutions, insertions, or deletions may occur within one or more CDRs, as long as such modifications do not substantially reduce the antibody's ability to bind to the antigen. For example, conservative modifications (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in the CDRs. Such modifications may, for example, be outside the antigen-contacting residues in the CDRs. In certain embodiments of the variant VH and VL sequences provided above, each CDR is unaltered or does not contain one or more, two or more, or three or more amino acid substitutions.

[0181] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells (1989) Science, 244:1081-1085. Alternatively, or in addition, a crystal structure of an antigen-antibody complex can be used to identify contact points between the antibody and the antigen. Such contact and adjacent residues can be targeted or eliminated as candidates for substitution. Variants can be screened to determine whether they have the desired properties.

[0182] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of antibody molecules include the fusion to the N- or C-terminus of the antibody of an enzyme (e.g., in the case of ADEPT) or a polypeptide which increases the serum half-life of the antibody.

[0183] b) Glycosylation variants In certain embodiments, the antibodies provided herein are altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody can be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

[0184] If the antibody contains an Fc region, the carbohydrate attached thereto can be modified. Natural antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides, which are commonly attached to Asn297 in the CH2 domain of the Fc region by an N-linkage. See, e.g., Wright et al., TIBTECH 15:26-32 (1997). The oligosaccharides can include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharides in the antibodies of the invention can be performed to generate antibody variants with specific improved properties.

[0185] In one embodiment, antibody variants are provided that have carbohydrate structures lacking fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 relative to the sum of all glycans (e.g., complex, hybrid, and high-mannose structures) attached to Asn297, as measured, for example, by MALDI-TOF mass spectrometry as described in WO 2008 / 077546. Asn297 refers to an asparagine residue located approximately at position 297 (EU numbering of Fc region residues) within the Fc region; however, due to slight antibody sequence variations, Asn297 may also be located ±3 amino acids upstream or downstream from position 297, i.e., between positions 294 and 300. Such fucosylation variants may have improved ADCC function. See, for example, U.S. Patent Application Publication Nos. 2003 / 0157108 (Presta, L.); 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.). Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include: U.S. Patent Application Publication Nos. 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; U.S. Patent Application Publication Nos. 2003 / 0115614; 2002 / 0164328; 2004 / 0093621; 2004 / 0132140; 2004 / 0110704; and 2004 / 0110282. ; International Publication No. 2004 / 0109865; International Publication No. 2003 / 085119; International Publication No. 2003 / 084570; International Publication No. 2005 / 035586; International Publication No. 2005 / 035778; International Publication No. 2005 / 053742; International Publication No. 2002 / 031140; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004).Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells, which are deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application Publication No. 2003 / 0157108 A1, Presta, L; and WO 2004 / 056312 A1, Adams et al., especially Example 11), and knockout cell lines, such as α-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO 2003 / 085107).

[0186] Additionally, antibody variants are provided that have biantennary oligosaccharides, for example, biantennary oligosaccharides attached to the Fc region of the antibody, bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003 / 011878 (Jean-Mairet et al.); U.S. Pat. No. 6,602,684 (Umana et al.); and U.S. Patent Application Publication No. 2005 / 0123546 (Umana et al.). Antibody variants are also provided that have at least one galactose residue in the oligosaccharide attached to the Fc region. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0187] c) Fc region variants In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0188] In certain embodiments, the present invention contemplates antibody variants that possess some, but not all, effector functions, making them desirable candidates for uses in which in vivo antibody half-life is important but certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / absent CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to confirm that the antibody lacks FcγR binding (and thus likely lacks ADCC activity) but retains FcRn binding ability. NK cells, the primary cells for mediating ADCC, express Fc(RIII) only, whereas monocytes express Fc(RI), Fc(RII, and Fc(RIII). FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)), and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M et al. J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods can be used (e.g., ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA) and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells.Alternatively or additionally, the ADCC activity of a molecule of interest can be assessed in vivo, for example, in an animal model such as that disclosed in Clynes et al. Proc. Nat'l 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 lacks CDC activity. See, for example, the 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 determinations can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0189] Antibodies with reduced effector function include those containing one or more substitutions at residues 238, 265, 269, 270, 297, 327, and 329 in the Fc region (U.S. Patent No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant in which residues 265 and 297 are substituted with alanine (U.S. Patent No. 7,332,581). Certain antibody variants are described that have improved or diminished binding to FcRs. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004 / 056312; and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001).)

[0190] In certain embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, for example, substitutions at positions 298, 333, and / or 334 (EU numbering of residues) of the Fc region.

[0191] In some embodiments, modifications are made in the Fc region that result in altered (i.e., either improved or decreased) C1q binding and / or complement-dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0192] Antibodies with increased half-lives and improved binding to fetal Fc receptors (FcRn) that are responsible for transferring maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) are described in U.S. Patent Application Publication No. 2005 / 0014934 (Hinton et al.). These antibodies comprise an Fc region with one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include variants having substitutions at one or more of the following Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, e.g., a substitution at Fc region residue 434 (U.S. Patent No. 7,371,826).

[0193] For other examples of Fc region variants, 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.

[0194] d) Cysteine ​​Engineered Antibody Variants In certain embodiments, it may be desirable to create cysteine ​​engineered antibodies, e.g., "thioMAbs," in which one or more residues of an antibody are substituted with cysteine ​​residues. In certain embodiments, the substituted residues occur at accessible sites of the antibody. By substituting these residues with cysteine, reactive thiol groups are placed at accessible sites of the antibody, which can be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create immunoconjugates, as further described herein. In certain embodiments, any one or more of the following residues may be substituted with 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 generated, for example, as described in U.S. Pat. No. 7,521,541.

[0195] e) Antibody derivative In certain embodiments, the antibodies provided herein may be further modified to contain additional nonproteinaceous moieties known in the art and readily available. Suitable sites for derivatization of 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 copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone), polyethylene glycol, polypropylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous during manufacturing due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody can vary, and when multiple polymers are attached, they can be the same molecule or different molecules. Generally, the number and / or type of polymers used for derivatization can be determined based on considerations such as, but not limited to, the particular property or function of the antibody to be improved and whether the antibody derivative will be used therapeutically under defined conditions.

[0196] In another embodiment, a conjugate of an antibody and a non-protective moiety is provided that can be selectively heated by exposure to radiation. In one embodiment, the non-protective moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102:11600-11605 (2005)). The radiation may be of any wavelength, including, but not limited to, wavelengths that do not harm normal cells but heat the non-protective moiety to a temperature that kills cells proximal to the antibody non-protective moiety.

[0197] B. Recombinant Methods and Compositions Antibodies may be produced using recombinant methods and compositions described, for example, in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid encoding an anti-Tie2 antibody described herein is provided. Such a nucleic acid may encode an amino acid sequence comprising the VL of the antibody and / or an amino acid sequence constituting the VH of the antibody (e.g., the light chain and / or heavy chain of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In a further embodiment, a host cell comprising such nucleic acids is provided. In one such embodiment, the host cell comprises (e.g., transformed): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and a nucleic acid encoding an amino acid sequence comprising the VH of the antibody, or (2) a vector comprising a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the 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., a Y0, NS0, or Sp20 cell). In one embodiment, a method of producing an anti-Tie2 antibody is provided, wherein the method comprises culturing a host cell containing nucleic acid encoding the antibody under conditions suitable for expression of the antibody, as described above, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0198] For recombinant production of an anti-Tie2 antibody, nucleic acid encoding the antibody, such as those described above, is isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acid may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of binding specifically to genes encoding the antibody heavy and light chains).

[0199] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies may be produced in bacteria, particularly if glycosylation and effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli.) Following expression, antibodies of the invention may be isolated in a soluble fraction from bacterial cell paste or further purified.

[0200] In addition to prokaryotes, eukaryotic organisms such as filamentous fungi and yeast are suitable cloning or expression hosts for antibody-encoding vectors, including bacterial and yeast strains that have been "humanized" in their glycosylation pathways to produce antibodies 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).

[0201] Additionally, suitable host cells for expressing glycosylated antibodies are derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains have been identified and can be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.

[0202] Plant cell cultures can also be used as hosts. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES™ technology for producing antibodies in transgenic plants).

[0203] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in suspension can be useful. Other examples of useful mammalian host cell lines include the SV40 (COS-7) transformed monkey kidney CV1 line; human embryonic kidney lines (e.g., 293 or 293 cells described in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells described in Mather, Biol. Reprod. 23:243-251 (1980) Error! Bookmark not defined.); 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 described, for example, in Mather et al., Annals NYAcad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include DHFR - and myeloma cell lines such as Y0, NS0, and Sp2 / 0. For a review of specific mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0204] C. Assay The anti-Tie2 antibodies provided herein may be identified, screened, or characterized for their physical / chemical properties and / or biological activity by various assays known in the art.

[0205] 1. Binding and Other Assays In one aspect, an antibody of the invention is tested for its antigen binding activity by known methods such as, for example, ELISA, BIACore®, FACS, or Western blot.

[0206] In other aspects, competition assays can be used to identify antibodies that compete with an 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 (referred to herein as Tie2.1M100cF) for binding to Tie2. In certain embodiments, such competing antibodies bind to the same epitope (e.g., a linear epitope or a conformational epitope) bound by an 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 antibody-binding epitopes are provided in Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular Biology, vol. 66 (Humana Press, Totowa, NJ).

[0207] In an exemplary competitive assay, immobilized TIe2 is incubated in a solution containing a first labeled antibody (e.g., Tie2.1M100cF) that binds to Tie2 and a second unlabeled antibody being tested for its ability to compete with the first antibody for binding to TIe2. The second antibody may be present in 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 immobilized TIe2 is measured. If the amount of label associated with immobilized TIe2 is substantially reduced in the test sample compared to the control sample, this indicates that the second antibody competes 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).

[0208] 2. Activity Assay In one aspect, an assay is provided for identifying an anti-TIe2 antibody having a desired biological activity. Biological activity can include, for example, binding to Tie2 or a fragment thereof, competing with Ang1 and / or Ang2 for binding to Tie2, activating phosphorylation of Tie2 protein, activating phosphorylation of Akt, and / or reducing vascular endothelial cell permeability (either in vivo, in vitro, or ex vivo). Antibodies having such biological activity in vivo and / or in vitro are also provided. In certain embodiments, the antibodies of the present invention are tested for such biological activity.

[0209] In some embodiments, assays are provided for determining Tie2 activating (anti-Tie2 conjugate) activity, e.g., by a phosphorylated-AKT (pAKT) assay, wherein activation of AKT phosphorylation by the anti-Tie2 conjugate indicates that the conjugate is activating (agonist) active. As will be readily known to those of skill in the art, and as described in Example 3 below, activation of AKT can be demonstrated by a variety of methods, including, for example, Western blot detection of phosphorylated AKT using an antibody specific for phosphorylated AKT or a FRET assay.

[0210] In some embodiments, assays for determining the stability (e.g., thermal stability) of an anti-Tie2 antibody or its antibody conjugate, fusion protein, or polymeric formulation are provided. For example, the stability of an antibody, its antibody conjugate, fusion protein, or polymeric formulation 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).

[0211] In some embodiments, an assay is provided for determining the stability of an anti-Tie2 conjugate. The stability of the assay can be determined as described herein, for example, using capillary electrophoresis laser-induced fluorescence (CE-LIF), e.g., as described in Example 13.

[0212] D. Immunoconjugates The present invention also provides immunoconjugates comprising an anti-Tie2 antibody herein conjugated to one or more cytotoxic agents, such as a chemotherapeutic agent or drug, a growth inhibitory agent, a toxin (e.g., a protein toxin, an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof), or a radioactive isotope.

[0213] E. Conjugates The present invention also provides conjugates comprising any of the anti-Tie2 antibodies or Tie2-binding fragments thereof provided herein conjugated to one or more heterologous molecules, such as a polyol.

[0214] 1. Multi-arm polymers 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 with a multi-armed polymer. It will be understood that any multi-armed polymer that provides a conjugate with a desired size or has a selected average molecular weight as described herein is suitable for use in constructing the antibody-polymer conjugates of the present invention.

[0215] Many polymers are suitable for use in pharmaceuticals. 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-protein polymers are used to form the conjugates of the present disclosure. Non-protein polymers are usually hydrophilic synthetic polymers, i.e., polymers not found in nature. However, polymers that exist in nature and are produced by recombinant or in vitro methods can also be useful, as can polymers isolated from natural sources.

[0216] In some embodiments, an anti-Tie2 antibody is derivatized by conjugating (e.g., covalently linking) a Fab or variant thereof to a multi-arm polyol. Accordingly, in some embodiments, the present disclosure relates to a conjugate comprising one or more anti-Tie2 Fabs or variants thereof disclosed herein covalently linked to one or more multi-arm polyols, preferably six-arm polyols. The polyol used can be any water-soluble poly(alkylene oxide) polymer and can have a linear or branched chain. Suitable polyols include those in which one or more hydroxyl positions are substituted with a chemical group, e.g., an alkyl group having 1 to 4 carbon atoms. Typically, the polyol is a poly(alkylene glycol), e.g., polyethylene glycol (PEG); therefore, for ease of exposition, the remainder of the discussion will relate to exemplary embodiments in which the polyol used is PEG and the process of conjugating a polyol to a polypeptide is referred to as "PEGylation." However, one of ordinary skill in the art will understand that other polyols, such as poly(propylene glycol) and polyethylene-polypropylene glycol copolymers, can be used using techniques for conjugation similar to those described herein for PEG.

[0217] 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 linking at least two arms. The multi-arm polyol can be, for example, a dimer (2 arms), a tetramer (4 arms), a hexamer (6 arms), an octamer (8 arms), etc. In some aspects, the multi-arm polyol is a multi-arm PEG.

[0218] The weight-average molecular weight of the multi-arm PEG used in PEGylation of anti-Tie2 antibodies and antibody variants can vary and typically ranges from about 500 to about 300,000 Daltons (D). In some embodiments, the weight-average molecular weight of the multi-arm PEG is 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, PEGylation is performed using a multi-arm PEG having a weight-average molecular weight of about 6,000 D.

[0219] Various methods for PEGylating proteins are known in the art. Specific methods for producing PEG-conjugated proteins include 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, proteins are covalently bound to the terminal reactive groups on the polymer via one or more of the amino acid residues of the protein. Polymers with reactive groups (multiple) are referred to herein as activated or functionalized polymers (e.g., functionalized PEG). The reactive groups selectively react with free sulfhydryl, amino, or other reactive groups on antibodies or antibody variants. Multi-armed PEG polymers can be bound to sulfhydryl, amino, or other reactive groups on antibodies or antibody variants in either a random or site-specific manner. However, it will be understood that for optimal results, the type and amount of reactive groups selected, as well as the type and amount of polymer used, will vary depending on the particular antibody or antibody variant being used to limit, and preferably substantially avoid, having reactive groups react with too many active groups on the antibody. In some cases, this may not be possible to fully limit or avoid, so typically, 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, depending on the antibody concentration, may be used. The final amount of functionalized polymer per mole of antibody is a balance to maintain optimal activity while, if possible, simultaneously optimizing the vitreous, retinal, and / or aqueous humor half-life of the antibody.

[0220] The residue can be any reactive amino acid on the antibody or antibody variant, such as the N-terminal amino acid group, but in some embodiments the reactive amino acid is cysteine, which is linked via its free thiol group to a reactive group on the functionalized polymer, as shown, for example, 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 entireties. In such embodiments, the polymer can contain at least one terminal reactive group that can specifically react with free sulfhydryl or thiol group(s) on the parent antibody. Such groups include, but are not limited to, maleimide, sulfhydryl, thiol, trifluoromethanesulfonate, tosylate, aziridine, epoxide, pyridyl disulfide, succinimidyl ester, -NH2, aldehyde, haloacetate, haloacetamide, and para-nitrophenyl carbonate, among others. The polymer can be coupled to the parent antibody using any protocol appropriate for the chemistry of the chosen coupling system, for example, the protocols and systems described in U.S. Pat. No. 4,179,337, U.S. Pat. No. 7,122,636, and Jevsevar et al., Biotech J., Vol. 5, pp. 113-128 (2010). Alternatively, the reactive amino acid can be lysine (linked via its free epsilon-amino group to the reactive group of the functionalized polymer) (see, e.g., WO 93 / 00109, incorporated herein by reference), or glutamic acid or aspartic acid (linked to the polymer via an amide bond). The reactive group of the polymer can then react with, for example, the α (alpha) and ε (epsilon) amine or sulfhydryl groups of the protein to form a covalent bond. It will be understood that the present disclosure is not limited to conjugates using any particular type of linkage between the antibody or antibody fragment and the polymer.

[0221] Functionalized multi-arm PEGs suitable for use in preparing the conjugates of the present disclosure can be produced by a number of conventional reactions. For example, N-hydroxysuccinimide 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. Pathol., Vol. 39, Issue 182, pp. 1379-1384 (1981). In addition, the terminal hydroxy groups of PEG can be converted to amino groups, for example, by reaction with thionyl bromide to form PEG-Br, followed by aminolysis with excess ammonia to form PEG-NH2. PEG-NH2 can then be conjugated to the antibody or antibody variant of interest using standard coupling reagents such as Woodward's reagent K. Additionally, PEG terminal -CHOH groups can be converted to aldehyde groups by oxidation, for example, with MnO 2. The aldehyde groups can be conjugated to antibodies or antibody variants by reductive alkylation using reagents such as cyanoborohydride.

[0222] In some embodiments, the multi-armed PEG used to prepare the conjugates of the present disclosure has the general formula (I): JPEG2025138630000007.jpg43170 (wherein PEG is the same or different -(CH2CHO)m-, where each m represents the length or size of a particular arm of the polyol (PEG) and is independently an integer of about 45 to about 1000, about 3 to about 250, about 50 to about 200, or about 100 to about 150; and l is an integer of 2 or greater, preferably 2 or 3).

[0223] In some embodiments, the multi-arm PEG has the structure of general formula (I), l is 2, and the multi-arm PEG is a hexamer. In other embodiments, the multi-arm PEG has the structure of general formula (I), l is 3, and the multi-arm PEG is an octamer.

[0224] Multi-armed PEGs having the structure of general formula (I) can be functionalized to attach terminal reactive groups suitable for reacting with or conjugating to antibodies (e.g., antibody fragments), e.g., using any of the techniques described above to generate functionalized multi-armed PEGs. However, in other embodiments, the multi-armed PEG can be covalently attached to an anti-Tie2 antibody via a multifunctional crosslinker that reacts with PEG and one or more amino acid residues of the antibody or antibody variant to be linked, e.g., as described in U.S. Patent No. 7,122,636, which is incorporated herein by reference in its entirety.

[0225] In other aspects, the multi-armed PEG used to prepare the conjugates of the present disclosure is a functionalized multi-armed 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-armed PEG has the general formula (Ia): JPEG2025138630000008.jpg42170 (wherein each m represents the length or size of a particular arm of the polyol (PEG) and is independently an integer of about 45 to about 1000, about 3 to about 250, or about 50 to about 200, or about 20 to 30, or about 100 to about 150; n is an integer of about 1 to about 10; and each R 1 is independently either absent or a linking group, and each R 2 are independently either hydrogen or a terminal reactive group, and at least one R 2 is a terminal reactive group. In some embodiments, R 2 is independently selected from thiol-reactive groups, amino-reactive groups, and combinations thereof.

[0226] In some embodiments, the functionalized multi-arm PEG has a structure of general formula (Ia), where n is an integer from 2 to 3. In a preferred embodiment, the functionalized multi-arm PEG has a structure of general formula (Ia), where n is 2, and the multi-arm PEG is a hexamer. In other embodiments, the functionalized multi-arm PEG has a structure of general formula (Ia), where n is 3, and the multi-arm PEG is an octamer. In a preferred embodiment, the functionalized multi-arm PEG has a structure of general formula (Ia), where n is 2, as shown in Figure 39.

[0227] In other embodiments, the multi-armed PEG used to prepare the conjugates of the present disclosure has the general formula (II): JPEG2025138630000009.jpg30170 (wherein each m represents the length or size of a particular arm of the polyol (PEG) and is independently an integer of about 45 to about 1000, about 3 to about 250, about 50 to about 200, or about 100 to about 150, and n is an integer of about 1 to about 10).

[0228] In some embodiments, the multi-arm PEG has the structure of general formula (II), n is 2, and the multi-arm PEG is a tetramer. In other embodiments, the multi-arm PEG has the structure of general formula (II), n is 4, and the multi-arm PEG is a hexamer. In other embodiments, the multi-arm PEG has the structure of general formula (II), n is 6, and the multi-arm PEG is an octamer.

[0229] In other aspects, the multi-armed PEG used to prepare the conjugates of the present disclosure has the general formula (II): JPEG2025138630000010.jpg42170 (wherein each m represents the length or size of a particular arm of the polyol (PEG) and is independently an integer of about 45 to about 1000, about 3 to about 250, about 50 to about 200, or about 100 to about 150, and n is an integer of about 1 to about 10).

[0230] In some embodiments, the multi-arm PEG has the structure of general formula (III), n is 2, and the multi-arm PEG is a tetramer. In other embodiments, the multi-arm PEG has the structure of general formula (III), n is 4, and the multi-arm PEG is a hexamer. In other embodiments, the multi-arm PEG has the structure of general formula (III), n is 6, and the multi-arm PEG is an octamer.

[0231] In other embodiments, the multi-armed PEG used to prepare the conjugates of the present disclosure has the general formula (IV): JPEG2025138630000011.jpg42170 (wherein each m represents the length or size of a particular arm of the polyol (PEG) and is independently an integer of about 45 to about 1000, about 3 to about 250, about 50 to about 200, or about 100 to about 150).

[0232] Multi-armed PEGs having the structure of any of general formulas (I)-(IV) can be functionalized to attach terminal reactive groups suitable for reacting with or conjugating to antibodies (e.g., antibody fragments), e.g., using any of the techniques described above to generate functionalized multi-armed PEGs. However, in other embodiments, the multi-armed PEG can be covalently attached to an anti-Tie2 antibody via a multifunctional crosslinker that reacts with PEG and one or more amino acid residues of the antibody or antibody variant to be linked, e.g., as described in U.S. Patent No. 7,122,636, which is incorporated herein by reference in its entirety.

[0233] In other aspects, the multi-armed PEG used to prepare the conjugates of the present disclosure is a functionalized multi-armed 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-armed PEG has the general formula (Ia): JPEG2025138630000012.jpg42170 (wherein each m represents the length or size of a particular arm of the polyol (PEG) and is independently an integer of about 45 to about 1000, about 3 to about 250, or about 50 to about 200, or about 100 to about 150; n is an integer of about 1 to about 10; and each R 1 is independently either absent or a linking group, and each R 2 are independently either hydrogen or a terminal reactive group, and at least one R 2 is a terminal reactive group. In some embodiments, R 2 is independently selected from thiol-reactive groups, amino-reactive groups, and combinations thereof.

[0234] In some embodiments, the functionalized multi-arm PEG has a structure of general formula (Ia), where n is an integer from 1 to 3. In some embodiments, the functionalized multi-arm PEG has a structure of general formula (Ia), where n is 1, and the multi-arm PEG is a tetramer. In other embodiments, the functionalized multi-arm PEG has a structure of general formula (Ia), where n is 2, and the multi-arm PEG is a hexamer. In other embodiments, the functionalized multi-arm PEG has a structure of general formula (Ia), where n is 3, and the multi-arm PEG is an octamer. In such embodiments, the octamer has a structure of general formula (Ib): JPEG2025138630000013.jpg46170 (where m, R 1 , and R 2 is as defined above).

[0235] Multi-arm PEGs having the structure of general formula (Ib) have a dipentaerythritol (DP) core structure and are also referred to herein as DP hexamers.

[0236] In some embodiments, the functionalized multi-arm PEG has a structure of general formula (Ib) or (Ic), wherein each R 1 are the same or different, if present, and R 1and R 2 When they are together, JPEG2025138630000014.jpg41170JPEG2025138630000015.jpg57170 (wherein each i is independently an integer from 0 to 10; j is an integer from 0 to 10; R 2 and combinations thereof. In some embodiments, each R 1 is a linking group.

[0237] In some embodiments, the functionalized multi-arm PEG has a structure of general formula (Ib) or (Ic), wherein R 1 and R 2 When they are together, JPEG2025138630000016.jpg17170, where i, j, and R 2 is as defined herein. In some embodiments, R 1 and R 2 When they are together, JPEG2025138630000017.jpg16170, where i is 2; j is 2 or 3; R 2 is as defined herein.

[0238] In some embodiments, the functionalized multi-arm PEG has a structure of general formula (Ib), wherein each R 2 is independently selected from maleimide, sulfhydryl, thiol, trifluoromethanesulfonate, tosylate, aziridine, epoxide, pyridyl disulfide, succinimidyl ester, —NH, aldehyde, haloacetate, haloacetamide, and para-nitrophenyl carbonate. 2 is independently a haloacetate selected from bromoacetic acid, iodoacetic acid, chloroacetic acid, and combinations thereof. In some embodiments, each R 2is independently a haloacetamide selected from bromoacetic acid, iodoacetic acid, chloroacetic acid, and combinations thereof. In some embodiments, R 2 is maleimide.

[0239] In some embodiments, the functionalized multi-arm PEG has a structure of general formula (Ia) or (Ib), wherein each R 2 is maleimide. In some embodiments, the functionalized multi-arm PEG has a structure of general formula (Ia) or (Ib), wherein R 1 and R 2 When they are together, JPEG2025138630000018.jpg37170, where i and j are as defined above. In some embodiments, the functionalized multi-arm PEG has a structure of general formula (Ia) or (Ib), wherein R 1 and R 2 When they are together, JPEG2025138630000019.jpg37170, where i is 2 and j is 2.

[0240] In other aspects, the functionalized multi-arm PEG used to prepare the conjugates of the present disclosure has the general formula (IIa): JPEG2025138630000020.jpg32170 (wherein each m represents the length or size of a particular arm of the polyol (PEG) and is independently an integer of about 45 to about 1000, about 3 to about 250, or about 50 to about 200, or about 100 to about 150; n is an integer of about 1 to about 10; and each R 1 is independently either absent or a linking group, and each R 2 are independently either hydrogen or a terminal reactive group, and at least one R 2 is a terminal reactive group. In some embodiments, R 2 is independently selected from thiol-reactive groups, amino-reactive groups, and combinations thereof.

[0241] In some embodiments, the functionalized multi-arm PEG has the structure of general formula (IIa), where n is an integer from 2 to 6. In some embodiments, the functionalized multi-arm PEG has the structure of general formula (IIa), where n is 2, and the multi-arm PEG is a tetramer. In some embodiments, the functionalized multi-arm PEG has the structure of general formula (IIa), where n is 3. In another embodiment, the functionalized multi-arm PEG has the structure of general formula (IIa), where n is 4, and the multi-arm PEG is a hexamer. In another embodiment, the functionalized multi-arm PEG has the structure of general formula (IIa), where n is 6, and the multi-arm PEG is an octamer. The octamer having the structure of general formula (IIa) has a hexaglycerol (HG) core structure and is also referred to herein as an HG octamer.

[0242] In some embodiments, the functionalized multi-arm PEG has a structure of general formula (IIa), where each R 1 are the same or different, if present, and R 1 and R 2 When they are together, JPEG2025138630000021.jpg40170JPEG2025138630000022.jpg57170 (wherein each i is independently an integer from 0 to 10; j is an integer from 0 to 10; R 2 and combinations thereof. In some embodiments, each R 1 is a linking group.

[0243] In some embodiments, the functionalized multi-arm PEG has the structure of general formula (IIa), where R 1 and R 2 When they are together, JPEG2025138630000023.jpg16170, where i, j, and R 2 is as defined herein. In some embodiments, R 1 and R 2 When they are together, JPEG2025138630000024.jpg17170, where i is 2; j is 2 or 3; R 2 is as defined herein.

[0244] In some embodiments, the functionalized multi-arm PEG has a structure of general formula (IIa), where each R 2 is independently selected from maleimide, sulfhydryl, thiol, trifluoromethanesulfonate, tosylate, aziridine, epoxide, pyridyl disulfide, succinimidyl ester, —NH, aldehyde, haloacetate, haloacetamide, and para-nitrophenyl carbonate. 2 is independently a haloacetate selected from bromoacetic acid, iodoacetic acid, chloroacetic acid, and combinations thereof. In some embodiments, each R 2 is independently a haloacetamide selected from bromoacetic acid, iodoacetic acid, chloroacetic acid, and combinations thereof. In some embodiments, R 2 is maleimide.

[0245] In some embodiments, the functionalized multi-arm PEG has a structure of general formula (IIa), where each R 2 is maleimide. In some embodiments, the functionalized multi-arm PEG has the structure of general formula (IIa), where R 1 and R 2 When they are together, JPEG2025138630000025.jpg37170, where i and j are as defined above. In some embodiments, the functionalized multi-arm PEG has a structure of general formula (IIa): 1 and R 2 When they are together, JPEG2025138630000026.jpg37170, where i is 2 and j is 2.

[0246] In another aspect, the functionalized multi-arm PEG has the general formula (IIIa): JPEG2025138630000027.jpg36170 (wherein each m represents the length or size of a particular arm of the polyol (PEG) and is independently an integer of about 45 to about 1000, or about 3 to about 250, or about 50 to about 200, or about 100 to about 150; n is an integer of about 1 to about 10; and each R 1 is independently either absent or a linking group, and each R 2 are independently either hydrogen or a terminal reactive group, and at least one R 2 is a terminal reactive group. In some embodiments, R 2 is independently selected from thiol-reactive groups, amino-reactive groups, and combinations thereof.

[0247] In some embodiments, the functionalized multi-arm PEG has a structure of general formula (IIIa), where n is an integer from 2 to 6. In some embodiments, the functionalized multi-arm PEG has a structure of general formula (IIIa), where n is 2, and the multi-arm PEG is a tetramer. In other embodiments, the functionalized multi-arm PEG has a structure of general formula (IIIa), where n is 4, and the multi-arm PEG is a hexamer. In other embodiments, the functionalized multi-arm PEG has a structure of general formula (IIIa), where n is 6, and the multi-arm PEG is an octamer. The octamer having the structure of general formula (IIIa) has a hexaglycerol (HGEO) core structure and is also referred to herein as an HGEO octamer.

[0248] In some embodiments, the functionalized multi-arm PEG has a structure of general formula (IIIa), where each R 1 are the same or different, if present, and R 1 and R 2 When they are together, JPEG2025138630000028.jpg43170JPEG2025138630000029.jpg57170 (wherein each i is independently an integer from 0 to 10; j is an integer from 0 to 10; R 2 and combinations thereof. In some embodiments, each R 1 is a linking group.

[0249] In some embodiments, the functionalized multi-arm PEG has the structure of general formula (IIIa), where R 1 and R 2 When they are together, JPEG2025138630000030.jpg16170, where i, j, and R 2 is as defined herein. In some embodiments, R 1 and R 2 When they are together, JPEG2025138630000031.jpg15170, where i is 2; j is 2 or 3; R 2 is as defined herein.

[0250] In some embodiments, the functionalized multi-arm PEG has a structure of general formula (IIIa), where each R 2 is independently selected from maleimide, sulfhydryl, thiol, trifluoromethanesulfonate, tosylate, aziridine, epoxide, pyridyl disulfide, succinimidyl ester, —NH, aldehyde, haloacetate, haloacetamide, and para-nitrophenyl carbonate. 2 is independently a haloacetate selected from bromoacetic acid, iodoacetic acid, chloroacetic acid, and combinations thereof. In some embodiments, each R 2 is independently a haloacetamide selected from bromoacetic acid, iodoacetic acid, chloroacetic acid, and combinations thereof. In some embodiments, R 2 is maleimide.

[0251] In some embodiments, the functionalized multi-arm PEG has a structure of general formula (IIIa), where each R 2 is maleimide. In some embodiments, the functionalized multi-arm PEG has the structure of general formula (IIIa), where R 1 and R 2 When they are together, JPEG2025138630000032.jpg36170, where i and j are as defined herein. In some embodiments, the functionalized multi-arm PEG has a structure of general formula (IIIa): 1 and R 2 When they are together, JPEG2025138630000033.jpg37170, where i is 3 and j is 2.

[0252] In another aspect, the functionalized multi-arm PEG has the general formula (IVa): JPEG2025138630000034.jpg42170 (wherein each m represents the length or size of a particular arm of the polyol (PEG) and is independently an integer of about 45 to about 1000, or about 3 to about 250, or about 50 to about 200, or about 100 to about 150; and each R 1 is independently either absent or a linking group, and each R 2 are independently either hydrogen or a terminal reactive group, and at least one R 2 is a terminal reactive group. In some embodiments, R 2 is independently selected from thiol-reactive groups, amino-reactive groups, and combinations thereof.

[0253] Multi-arm PEGs having the structure of general formula (IVa) have a butanediol core structure and are also referred to herein as DX octamers.

[0254] In some embodiments, the functionalized multi-arm PEG has a structure of general formula (IVa), where each R 1 are the same or different, if present, and R 1 and R 2 When they are together, JPEG2025138630000035.jpg94170 (wherein each i is independently an integer from 0 to 10; j is an integer from 0 to 10; R 2 and combinations thereof. In some embodiments, each R 1 is a linking group.

[0255] In some embodiments, the functionalized multi-arm PEG has the structure of general formula (IVa), where R 1 and R 2 When they are together, JPEG2025138630000036.jpg18170, where i, j, and R 2 is as defined herein. In some embodiments, R 1 and R 2 When they are together, JPEG2025138630000037.jpg17170, where i is 2; j is 2 or 3; R 2 is as defined herein.

[0256] In some embodiments, each R 2 is independently selected from maleimide, sulfhydryl, thiol, trifluoromethanesulfonate, tosylate, aziridine, epoxide, pyridyl disulfide, succinimidyl ester, —NH, aldehyde, haloacetate, haloacetamide, and para-nitrophenyl carbonate. 2 is independently a haloacetate selected from bromoacetic acid, iodoacetic acid, chloroacetic acid, and combinations thereof. In some embodiments, each R 2is independently a haloacetamide selected from bromoacetic acid, iodoacetic acid, chloroacetic acid, and combinations thereof. In some embodiments, R 2 is maleimide.

[0257] In some embodiments, the functionalized multi-arm PEG has a structure of general formula (IVa), where each R 2 is maleimide. In some embodiments, the functionalized multi-arm PEG has the structure of general formula (IVa), where R 1 and R 2 When they are together, JPEG2025138630000038.jpg37170, where i and j are as defined above. In some embodiments, the functionalized multi-arm PEG has the structure of general formula (IVa): 1 and R 2 When they are together, JPEG2025138630000039.jpg37170, where i is 3 and j is 2.

[0258] Other functionalized multi-arm PEGs suitable for use in the present disclosure are described in U.S. Patent Application Publication No. 2011 / 0286956 and U.S. Patent Application Publication No. 2015 / 0073155, both of which are incorporated by reference in their entireties.

[0259] Functionalized multi-arm PEGs suitable for use in the present disclosure can also be purchased from a number of vendors. For example, JenKem Technology, USA, sells maleimide-functionalized PEG hexamers and octamers (e.g., 6-arm (DP)-PEG-MAL and 8-arm (TP)-PEG-MAL). NOF America Corp. also sells maleimide-functionalized PEG octamers (e.g., Sunbright® HGEO-400MA, Sunbright® DX-400MA) and tetramers (e.g., Sunbright® PTE-400MA).

[0260] In certain embodiments, activated derivatives of the described multi-arm PEGs have the following general formula (IV): JPEG2025138630000040.jpg36170, where R is dipentaerythritol.

[0261] 1. Polyol conjugates In some embodiments, the present disclosure relates to a conjugate (e.g., a Tie2 binding agent) comprising one or more anti-Tie2 antibodies or antibody variants disclosed herein and one or more multi-armed polyols, wherein the conjugate is prepared by covalently attaching at least one anti-Tie2 Fab or Fab variant to the polyol. In some embodiments, the multi-armed 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 general formula (Ia):

[0262] Conjugates of the present disclosure can be characterized by the number of anti-Tie2 antibodies (Fabs) conjugated to each multi-armed PEG. This is referred to herein as "fabrication" or "degree of fabification." The number of anti-Tie2 antibodies conjugated to each PEG can vary depending on various 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 highly polydisperse multi-armed PEGs used to prepare conjugates can, in some cases, complicate analysis of the final conjugate, making precise determination of the number of Fabs per PEG even more difficult and unclear. Thus, the PEG used to form the conjugate typically has a polydispersity (determined using methods known in the art) within the range of about 1 to about 1.35, and in various embodiments has 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.

[0263] In some embodiments, the conjugates of the present disclosure comprise a 6-arm PEG, wherein at least one anti-Tie2 antibody or variant is covalently attached to the PEG. In other embodiments, the conjugates of the present disclosure comprise a 6-arm PEG, wherein at least 2, at least 3, at least 4, at least 5, or at least 6 anti-Tie2 Fabs are covalently attached to the PEG. In other embodiments, the conjugates of the present disclosure comprise an 8-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 attached to the PEG. In other embodiments, the conjugates of the present disclosure comprise a 6-arm PEG, wherein at least 4, at least 5, or at least 6 anti-Tie2 Fabs are covalently attached to the PEG. In some embodiments, the conjugates of the present disclosure comprise a 6-arm PEG, wherein at least 4-6 or 5-6 anti-Tie2 Fabs are covalently attached to the PEG. In other embodiments, the conjugates of the present disclosure comprise a 6-arm PEG, wherein at least 4-6 anti-Tie2 Fabs are covalently attached to the PEG. In other embodiments, the conjugates of the present disclosure comprise a 6-arm PEG, wherein at least 4-6 or 5 anti-Tie2 Fabs are covalently attached to the PEG.

[0264] In some embodiments, the conjugates of the present disclosure comprise a multi-arm PEG having a structure according to any one of general formulas (Ia), (IIa), (IIIa), or (IVa). In such embodiments, at least one R 2 is covalently attached to an anti-Tie2 antibody or variant described herein. In some embodiments, the multi-arm PEG having the structure of any one of general formulas (Ia), (IIa), (IIIa), or (IVa) is a hexamer, and at least two, at least three, at least four, at least five, or all six R 2 The group is covalently attached to an anti-Tie2 Fab or variant described herein.

[0265] In some embodiments, conjugates of the present disclosure include species in which a multi-arm polyol is covalently attached to specific site(s) on the parent antibody, i.e., polymer attachment is targeted to a specific region or specific amino acid residue(s) in the parent antibody or antibody fragment. Standard mutagenesis techniques can be used to vary 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 fewer number of potential PEGylation sites than native-sequence anti-Tie2.

[0266] As discussed above, site-specific conjugation of the polymer is most commonly achieved by linkage to cysteine ​​residues in the parent antibody or antibody fragment. In such embodiments, the coupling chemistry may utilize, for example, the free sulfhydryl groups of cysteine ​​residues that are not in disulfide bridges in the parent antibody.

[0267] In some embodiments, one or more cysteine ​​residues naturally occurring in the parent Fab are used as attachment site(s) for polymer conjugation. In other embodiments, free amino groups on the Fab or variant can be thiolated with 2-iminothiolane (Traut's reagent) and then attached to, for example, maleimide-functionalized PEG, as described in Pedley et al., Br. J. Cancer, Vol. 70, pp. 1126-1130 (1994). In other embodiments, one or more cysteine ​​residue(s) are engineered into selected site(s) in the parent Fab for the purpose of providing specific attachment site(s) for the polymer.

[0268] Cysteine ​​engineered antibodies have been previously described (U.S. Patent Application Publication No. 2007 / 0092940 and Junutula, JR et al., J. Immunol Methods, Vol. 332(1-2), pp. 41-52 (2008), all of which are incorporated herein by reference in their entirety). In some embodiments, a cysteine ​​engineered antibody can be a parent antibody. These may contain cysteine ​​engineered antibodies at specific positions, typically within the constant region, e.g., C L or C H In one embodiment, this method is useful for generating antibody fragments with a free cysteine. A parent antibody engineered to contain a cysteine ​​is referred to herein as a "ThioMab," and Fab fragments produced from such cysteine-engineered antibodies, regardless of the method of production, are referred to herein as "ThioFabs." As previously described (e.g., U.S. Patent Application Publication No. 2007 / 0092940 and Junutula, JR et al., J. Immunol Methods, Vol. 332(1-2), pp. 41-52 (2008)), mutants with a substituted ("engineered") cysteine ​​(Cys) residue are evaluated for the reactivity of the newly introduced, engineered cysteine ​​thiol group. The thiol reactivity value is a relative numerical value ranging from 0 to 1.0 and can be measured for any cysteine-engineered antibody. In addition to possessing a reactive thiol group, 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, cysteine ​​engineered antibodies retain the antigen-binding ability of their wild-type parent antibody counterparts and are therefore capable of specifically binding to antigens.

[0269] In some embodiments, the present disclosure relates to antibody fragment-polymer conjugates, wherein the antigen fragment is a Fab and the polymer is attached to one or more cysteine ​​residues within the light or heavy chain of the Fab fragment that would normally form interchain disulfide bonds linking the light and heavy chains.

[0270] In another aspect, the present disclosure relates to an antibody fragment-polymer conjugate, wherein the antigen fragment is a Fab-C and polymer attachment is targeted to the hinge region of the Fab-C fragment. In some embodiments, one or more cysteine ​​residues naturally present in the hinge region of the antibody fragment are used to attach the polymer. In other embodiments, one or more cysteine ​​residues are engineered into the hinge region of the Fab-C fragment to provide specific attachment site(s) for the polymer. In some embodiments, an anti-Tie2 Fab disclosed herein is modified by adding a cysteine ​​to the C-terminus to provide one attachment site for polymer conjugation. In other embodiments, an anti-Tie2 antibody Fab described herein is modified by adding four additional residues, CPPC (SEQ ID NO: 87), to the C-terminus to provide two attachment sites for polymer conjugation. In yet another embodiment, the anti-TIe2 antibody Fab described herein is modified by adding four additional residues, SPPC (SEQ ID NO: 88), at the C-terminus to provide one attachment site for polymer conjugation.

[0271] The degree and site of PEGylation can also be manipulated by adjusting reaction conditions such as the concentration of functionalized PEG and protein and pH. Conditions suitable for the desired degree of PEGylation can be determined experimentally by varying the parameters of standard PEGylation reactions.

[0272] PEGylation of anti-Tie2 Fabs and variants is carried out by any convenient method. Suitable PEGylation conditions are described in WO 2011 / 069104 and WO 03 / 029420, both of which are incorporated herein by reference in their entireties.

[0273] 3. Characterization of Polyol Conjugates PEGylated proteins can be characterized by SDS-PAGE, gel filtration, NMR, peptide mapping, liquid chromatography-mass spectrometry, and in vitro biological assays. The degree of fabylation is typically first demonstrated by SDS-PAGE. Polyacrylamide gel electrophoresis in 10% SDS is performed in 10 mM Tris-HCl pH 8.0, 100 mM NaCl as the elution buffer. Peptide mapping using proteases such as trypsin and Lys-C protease can be performed to identify which residues are PEGylated. Thus, samples of PEGylated and non-PEGylated antibodies can be digested with proteases such as Lys-C protease, and the resulting peptides can be separated by techniques such as reverse-phase HPLC. The chromatographic patterns of the resulting peptides can be compared to peptide maps previously determined for anti-Tie2 polypeptides.

[0274] Each peak can then be analyzed by mass spectrometry to verify the size of the conjugate in the peak. Depending on the PEG used for conjugation and the size of the conjugate in the peak, the number of antibodies or their variants conjugated to PEG can be estimated. The fragment(s) conjugated to the PEG group are usually not retained on the HPLC column after injection and disappear from the chromatograph. Such disappearance from the chromatograph indicates PEGylation of a specific fragment that must contain at least one PEGylatable amino acid residue. The PEGylated anti-Tie Fab can be further assayed for its ability to interact with Tie2 and other biological activities using methods known in the art.

[0275] PEGylation alters the physical and chemical properties of antibody drugs and can result in improved pharmacokinetic behavior, such as improved stability, reduced immunogenicity, extended circulatory life, and increased ocular residence time.

[0276] In some embodiments, a conjugate of the present disclosure has an increased half-life following administration into a mammalian eye (e.g., a human) via a single intravitreal injection compared to the corresponding unconjugated anti-Tie2 Fab, hi some embodiments, the increase in half-life is at least 1.4-fold, or at least 1.8-fold, or at least 2-fold the half-life of the corresponding unconjugated anti-Tie2 Fab.

[0277] 3. IgM multimers as conjugates

[0278] In some embodiments, the Tie2-binding agents of the present disclosure are, for example, IgM C H1 Two or more anti-Tie2 antibodies described herein can be expressed as C-terminal fragments of an IgM molecule using recombinant expression methods to fuse (via a peptide bond) the C-terminus of the domain to the N-terminus of an anti-Tie2 antibody described herein. H1 IgM has proven to be a viable format for antibody therapeutics (see, e.g., Hanala, 2012, MAbs, 4:555-561). The "monomeric" component of IgM consists of two light chains (LC), each containing two Ig domains, and two heavy chains (HC), each containing five Ig domains and a short, unstructured C-terminal tail. These four chains assemble to form a homodimer of HC-LC heterodimers. The homodimers then covalently link into a ring structure containing five homodimers and a J chain (JC) (pentamer) or six homodimers (hexamer), each containing 10 and 12 binding sites, respectively. Without being bound by theory, thermal binding of multiple variable fragments (Fv) allows IgM to bind targets without substantial affinity maturation, thereby functioning as a sentinel adaptive immune receptor.

[0279] In certain embodiments, the anti-Tie2 antibody is a Fab. The IgM protein (multimer) may or may not contain a J chain, such that in the presence of a J chain, a pentamer is formed (which can contain up to five anti-Tie2 antibodies), and in the absence of a J chain, a hexamer is formed (which can contain up to five anti-Tie2 antibodies). In some embodiments, hexamers are generated by varying ratios of IgM heavy and light chains (to form hexamers) or ratios of heavy and light chains to J chains (to form pentamers). Thus, in some embodiments, the Tie2-binding agent is a multimer comprising an IgM protein and at least two, at least three, at least four, at least five, or at least six anti-Tie2 antibodies described herein to form a multimer capable of activating Tie2. Anti-Tie2 IgM molecules have been designed and shown to activate Tie2 activity (see Example 7).

[0280] Anti-Tie2 multimeric conjugates constructed using the recombinant IgM format described herein may be useful for ocular therapy due to their relatively large molecular radius compared to a single Fab, potentially slowing diffusion of the molecule from the vitreous humor to the aqueous humor and blood. As described in Example 7 below, they exhibit a hydrodynamic radius (R) of approximately 12 nM as assessed by light scattering. h ) was found, and the predicted molecular weight of the hexamer (approximately 1050 kD) slightly exceeded that of the pentamer (approximately 950 kD).

[0281] Because it may be desirable to have relatively rapid systemic clearance to limit the ocular activity of ocular therapeutic agents, the systemic half-life of recombinant IgM molecules was also investigated. As shown in Example 7, recombinantly expressed IgM pentamers and hexamers were cleared more rapidly after intravenous injection than IgM isolated from human serum. These recombinant IgM molecules were further determined to have a lower percentage of sialic acid relative to N-linked glycans than IgM isolated from serum, suggesting that the clearance rate of recombinant anti-Tie2 IgM molecules can be controlled by designing an expression system that alters the level of sialic acid in N-linked glycosylation.

[0282] IgM has previously been reported to potently recruit C1q and induce target cell killing via complement-dependent cytotoxicity (CDC). Such activity may be undesirable for ocular therapeutics. Therefore, as described in Example 7, an IgM variant, P434G (EU numbering), was designed and shown to eliminate all detectable complement activity.

[0283] 3. Hexameric Peptide Multimers as Conjugates Conjugates containing multiple antigen-binding agents (e.g., antibodies or antigen-binding fragments thereof) are also encompassed by the present disclosure, where each antigen-binding agent is linked to a peptide that naturally forms multimers, such as an NDK peptide. In some embodiments, the Tie2-binding agents of the present disclosure can be generated by linking two or more anti-Tie2 Fabs described herein to a peptide multimer. The peptide multimer is composed of at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight peptides, which, when expressed in a recombinant expression system, naturally fold to form a single multi-arm structure. Routine molecular engineering techniques and materials are used to express multimerizing peptides as fusion proteins in which an antigen-binding protein (e.g., an antibody or fragment thereof) is expressed at either the N- or C-terminus of the multimerizing peptide. In certain embodiments, the peptides in the multimer are identical, and an expression vector is constructed as taught in the present disclosure to link the C- or N-terminus of the peptide to the N- or C-terminus of the anti-Tie2 antibody or fragment thereof (via a peptide bond), respectively.

[0284] In certain embodiments, the peptides of the multimeric peptide are portions of eukaryotic nucleoside diphosphate kinase (NDK) enzymes having a homohexameric quaternary structure. There are several NDK enzymes that can be used to design multimers, including NDK1 (e.g., SEQ ID NO: 69), NDK2 (e.g., SEQ ID NO: 70), NDK3 (e.g., SEQ ID NO: 71), NDK4 (e.g., SEQ ID NO: 72), and NDK5 (e.g., SEQ ID NO: 73). In a preferred embodiment, the NDK peptide is the NDK3 peptide, e.g., from UniProt accession 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, e.g., Example 8 below.)

[0285] E. Methods and Compositions for Diagnostics and Detection In certain embodiments, any of the anti-Tie2 antibodies provided herein is useful for detecting 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 contains cells or tissues such as retinal tissue (photoreceptors and the underlying retinal pigment epithelium (RPE) and choriocapillaris).

[0286] In one embodiment, an anti-Tie2 antibody is provided for use in a method of diagnosis or detection. In a further aspect, a method of detecting the presence of Tie2 in a biological sample is provided. In certain embodiments, the method comprises contacting a biological sample with an anti-Tie2 antibody described herein under conditions that allow binding of the anti-Tie2 antibody to Tie2, and detecting whether a complex is formed between the anti-Tie2 antibody and Tie2. Such a method may be an in vitro method or an in vivo method. In one embodiment, for example, when Tie2 is a biomarker for patient selection, the anti-Tie2 antibody is used to select subjects eligible for treatment with the anti-Tie2 antibody.

[0287] In certain embodiments, labeled anti-Tie2 antibodies are provided. Labels include, but are not limited to, labels or moieties that are directly detected (e.g., fluorescent labels, chromogenic labels, electron-dense labels, chemiluminescent labels, radioactive labels, etc.) and moieties that are indirectly detected via enzymatic reactions or molecular interactions (e.g., enzymes or ligands, etc.). Exemplary labels include radioisotopes. 32 P, 14 C. 125 I, 3 H and 131I, rare earth chelates or fluorophores such as fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferase, luciferases such as firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidases such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, enzymes that use hydrogen peroxide to oxidize dye precursors such as HRP, lactoperoxidase, or microperoxidase, biotin / avidin, spin labels, bacteriophage labels, stable free radicals, and conjugates of the like.

[0288] F. Pharmaceutical Preparations

[0289] Pharmaceutical formulations of the anti-Tie2 antibodies or Tie2 conjugates described herein are prepared by mixing such anti-Tie2 antibodies or Tie2 conjugates having the desired purity, in the form of a lyophilized formulation or an aqueous solution, with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed (1980)). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl, or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; blood-binding proteins; and the like. Examples of suitable pharmaceutically acceptable carriers include, but are not limited to, 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 dextrins, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose, or sorbitol, salt-forming counterions such as sodium, metal complexes (e.g., Zn-protein complexes), and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include intercalating drug dispersants, such as soluble neutral active hyaluronidase glycoproteins (sHASEGPs), e.g., human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Application Publication Nos. 2005 / 0260186 and 2006 / 0104968.In one aspect, the sHASEGP is combined with one or more additional glycosaminoglycanases (eg, chondroitinases).

[0290] Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and WO 2006 / 044908, the latter formulation containing a histidine acetate buffer.

[0291] The formulations herein may also contain more than one active ingredient as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, it may be desirable to further provide a second biological 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. Additionally or alternatively, the second biological molecule is an antibody or fragment thereof that specifically binds to a 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, or antigen-binding fragment thereof, that binds to VEGF and / or Ang2 and / or IL-1β. Such active ingredients are suitably present in combination in amounts effective for the intended purpose.

[0292] The active ingredient may be encapsulated in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively), colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980).

[0293] Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

[0294] Formulations to be used for in vivo administration are generally sterile, which may be readily accomplished, for example, by filtration through sterile filtration membranes.

[0295] The conjugates described herein for the prevention or treatment of ocular diseases or conditions are typically administered via ocular, intraocular, and / or intravitreal injection, and / or juxtascleral injection, and / or sub-Tenon injection, and / or superchoroidal injection, and / or topical administration in the form of eye drops and / or ointments. Such compositions of the present disclosure can be delivered intravitreally in a variety of ways, including as devices and / or depots that allow for the slow release of the compound into the vitreous humor, including those described in references such as "Intraocular Drug Delivery," Jaffe, Jaffe, Ashton, and Pearson, editors, Taylor & Francis (March 2006). In one example, the device can be in the form of a minipump and / or a matrix and / or a passive diffusion system and / or encapsulated cells 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.

[0296] Formulations for ocular, intraocular, or intravitreal administration can be prepared using known ingredients by methods known in the art. The primary requirement for effective treatment is adequate penetration through the eye. Unlike diseases of the anterior ocular surface, where drugs can be delivered topically, retinal diseases require a more site-specific approach. Eye drops and ointments hardly penetrate the posterior ocular surface, and the blood-ocular barrier prevents systemically administered drugs from penetrating into ocular tissues. Therefore, direct intravitreal injection is usually the optimal method of drug delivery for treating retinal diseases such as DME and AMD. Intravitreal injections are usually repeated at intervals that depend on the patient's symptoms and the properties and half-life of the drug being delivered. For intraocular (e.g., intravitreal) penetration, smaller molecules are usually preferred.

[0297] In some embodiments, the antibodies and conjugates described herein can be formulated for delivery using an implantable port delivery system (PDS). As described above, a PDS is a refillable device in which release into the vitreous is controlled by a porous metal membrane containing a titanium frit. Because the reservoir has a low volume, in some embodiments, a high protein concentration is required for effective delivery using the PDS. Therefore, in some embodiments, the antibodies and conjugates described herein are formulated at a high concentration. In some embodiments, the antibodies and conjugates described herein may 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 may be formulated at a concentration of 150 mg / ml to 350 mg / ml, 150 mg / ml to 300 mg / ml, 170 mg / ml to 300 mg / ml, 200 mg / ml to 300 mg / ml, or 170 mg / ml to 220 mg / ml.

[0298] G. Therapeutic Methods and Compositions

[0299] Any of the anti-Tie2 antibodies or conjugates provided herein can be used in therapeutic methods. An "individual," "patient," or "subject" according to any of the embodiments herein can be a human.

[0300] The anti-Tie2 conjugates of the present disclosure can be used to treat mammals. In some embodiments, the anti-Tie2 antibody or conjugate is administered to a non-human mammal, for example, for the purpose of obtaining preclinical data. Exemplary non-human mammals to be treated include pre-human primates, dogs, cats, pigs, rodents, and other mammals undergoing clinical trials. Such mammals may be established animal models of diseases to be treated with the antibody or may be used to test the toxicity of the antibody of interest. In each of these embodiments, dose escalation studies may be performed on the mammals.

[0301] The anti-Tie2 conjugates can be administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intravitreal, intrapulmonary, and intranasal, and, if desired for local immunosuppressive treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, intravitreal, and subcutaneous administration. In addition, the conjugates are suitably administered by pulse infusion, particularly with declining doses of the antibody or antibody variant thereof or fragment thereof (e.g., antigen-binding fragment). In some embodiments, dosing is administered by injection, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic.

[0302] The appropriate dosage of anti-Tie2 antibody or conjugate for the prevention or treatment of disease will vary depending on the type of disease being treated, the severity and course of the disease, whether the antibody is being administered for prophylactic or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the judgment of the attending physician.

[0303] Depending on the type and severity of the disease, approximately 1 to 25 mg of antibody per eye (0.015 mg / kg to 0.36 mg / kg per eye) is a suggested initial dosage for administration to a patient, for example, by one or more separate administrations or by continuous infusion. For repeated administration over several days or longer, depending on the condition, treatment is sustained until a desired suppression of disease symptoms occurs. However, other dosing regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays. Exemplary dosing regimens are disclosed in WO 94 / 04188.

[0304] In one aspect, an anti-Tie2 antibody or conjugate is provided for use as a pharmaceutical. In a further aspect, an anti-Tie2 antibody or conjugate is provided for use in treating an ocular disease or disorder. In certain embodiments, an anti-Tie2 antibody or conjugate is provided for use in a therapeutic method. In certain embodiments, the present invention provides an anti-Tie2 antibody or conjugate for use in a method of treating an individual with an ocular disease or disorder, comprising administering to the individual an effective amount of an anti-Tie2 antibody or conjugate. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below. In a further embodiment, the present invention provides an anti-Tie2 antibody or conjugate for use in increasing vascular endothelial cell membrane integrity and / or reducing vascular leakage. In certain embodiments, the 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 an individual, comprising administering to the individual an anti-Tie2 antibody or conjugate effective to increase vascular endothelial cell membrane integrity and / or reduce vascular leakage. An "individual" according to any of the above embodiments is preferably a human.

[0305] As used herein, the term "ocular disorder" includes any ocular disorder (also interchangeably referred to herein as "ocular condition") associated with pathological neovascularization and / or atrophy. Ocular disorders can be characterized by altered or unregulated proliferation and / or infiltration of new blood vessels into structures of ocular tissues, such as the retina or cornea. Ocular disorders can be characterized by atrophy of retinal tissues (photoreceptors and the underlying retinal pigment epithelium (RPE) and choriocapillaris).

[0306] Diabetic macular edema (DME) is caused by a diabetic complication called diabetic retinopathy (DR). This eye condition can occur in people diagnosed with type 1 or type 2 diabetes. DME is defined as retinal thickening within 2 disc diameters central to the fovea and can be either focal or diffuse. DME is associated with retinal microvascular changes that compromise the blood-retinal barrier, causing leakage of plasma components into the surrounding retina and resulting in retinal edema.

[0307] Non-limiting ocular disorders include, for example, diabetic macular edema (DME) (e.g., focal, non-central DME, and diffuse centrally involved DME), diabetic retinopathy (DR) (e.g., proliferative DR (PDR), non-proliferative DR (NPDR), and high altitude DR), retinopathy in the absence of edema, other ischemia-related retinopathies, AMD (e.g., wet AMD, dry AMD, intermediate AMD, advanced AMD, and geographic atrophy (GA)), macular degeneration, corneal edema, and retinal edema. Macular edema, retinopathy, ROP, retinal vein occlusion (RVO) (e.g., central (CRVO) and branch (BRVO) forms), CNV (e.g., myopic CNV), corneal neovascularization, diseases associated with corneal neovascularization, corneal neovascularization, retinal neovascularization, diseases associated with retinal / choroidal neovascularization, central serous retinopathy (CSR), pathological myopia, von Hippel-Lindau disease, ocular histoplasmosis, FEVR, Coats' disease, Norrie's disease, osteoporosis-pseudogliosis Retinal abnormalities associated with Optic Nerve Posterior Proliferative Glandular Glandular Syndrome (OPPG), subconjunctival hemorrhage, rubeosis, ocular neovascular disease, neovascular glaucoma, retinitis pigmentosa (RP), hypertensive retinopathy, retinal angiomatous proliferation, telangiectasia, iris neovascularization, intraocular neovascularization, retinal degeneration, cystoid macular edema (CME), vasculitis, papilledema, retinitis, including, but not limited to, CMV retinitis, ocular melanoma, retinoblastoma, conjunctivitis (e.g., infectious conjunctivitis) and non-infectious (e.g., allergic These eye disorders include, but are not limited to, ocular conditions such as ocular malformation, ...

[0308] Exemplary diseases associated with corneal neovascularization include, but are not limited to, epidemic keratoconjunctivitis, vitamin A deficiency, contact lens overwear, atopic keratitis, superior limbal keratitis, pterygium, keratitis sicca, Sjogren's syndrome, acne rosacea, phylectenulosis, syphilis, mycobacterial infections, steatosis, chemical burns, bacterial ulcers, fungal ulcers, herpes simplex infections, herpes zoster infections, protozoal infections, Kaposi's sarcoma, Mooren's ulcer, Therrien's peripheral corneal degeneration, peripheral keratolysis, rheumatoid arthritis, generalized erythema, polyarteritis nodosa, trauma, Wegener's sarcoidosis, scleritis, Steven-Johnson syndrome, pemphigoid, radial keratotomy, and post-corneal transplant rejection.

[0309] Exemplary diseases associated with choroidal neovascularization and defects in the retinal vasculature, including increased vascular leakage, aneurysms, and capillary dropout, include, but are not limited to, diabetic retinopathy, macular degeneration, sickle cell anemia, sarcoid, syphilis, pseudoxanthoma elasticum, Paget's disease, venous occlusion, arterial occlusion, carotid occlusive disease, chronic uveitis / vitreous inflammation, mycobacterial infections, Lyme disease, systemic lupus erythematosus, retinopathy of prematurity, retinal edema (including macular edema), Eales' disease, Behcet's disease, infections causing retinitis or choroiditis (e.g., multifocal choroid), presumed ocular histoplasmosis, Best's disease (vitreomacular degeneration), myopia, optic disc, pars planitis, retinal detachment (e.g., chronic retinal detachment), hyperviscosity syndrome, toxoplasmosis, trauma, and post-laser complications.

[0310] Exemplary diseases associated with atrophy of retinal tissue (photoreceptors and the underlying RPE) include, but are not limited to, atrophic or non-exudative AMD (e.g., geographic atrophy or advanced dry AMD), macular atrophy (e.g., atrophy associated with neovascularization and / or geographic atrophy), diabetic retinopathy, Stargardt's disease, Sorsby Fundus dystrophy, retinoschisis, and retinitis pigmentosa.

[0311] For example, in certain embodiments, any of the aforementioned methods further comprises administering one or more additional compounds. In certain embodiments, the Tie2-binding agent, or conjugate or polymer formulation thereof, is administered simultaneously with the additional compounds. In certain embodiments, the Tie2-binding agent, or conjugate or polymer formulation thereof, is administered before or after the additional compounds. In certain embodiments, the additional compound binds to a second biological molecule 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 an antigen-binding fragment thereof. In certain embodiments according to (or applied to) any of the above embodiments, the ocular 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 ischemia-related retinopathies, diabetic macular edema, pathological myopia, von 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 instances, the additional compound is a bispecific antibody (e.g., an anti-VEGF / anti-Ang2 bispecific antibody, e.g., RG-7716, or any bispecific anti-VEGF / anti-Ang2 bispecific antibody or variant thereof disclosed in WO 2010 / 069532 or WO 2016 / 073157).In other examples, optionally, the additional compound is an anti-IL-6 antibody, such as EBI-031 (Eleven Biotherapeutics; see, e.g., WO 2016 / 073890), siltuximab (SYLVANT®), olokizumab, clazakizumab, sirukumab, elcilimomab, OPR-003, MEDI5117, PF-04236921, or a variant thereof. In yet further examples, optionally, the additional compound is an anti-IL-6R antibody, such as tocilizumab (ACTEMRA®) (see, e.g., WO 1992 / 019579), sarilumab, ALX-0061, SA237, or a variant thereof.

[0312] In some examples, the Tie2-binding agents or conjugates, and / or polymer formulations thereof, of the present disclosure can be administered in combination with at least one additional therapeutic agent for the treatment of an ocular disorder, such as an ocular disorder described herein (e.g., DME, DR, AMD (e.g., wet AMD), RVO, or GA). Exemplary additional therapeutic agents for combination therapy to treat ocular disorders include, but are not limited to, anti-angiogenic agents, such as anti-VEGF antibodies (e.g., anti-VEGF Fab LUCENTIS® (ranibizumab)), soluble receptor fusion proteins (e.g., recombinant soluble receptor fusion protein EYLEA® (aflibercept, also known as VEGF Trap Eye); Regeneron / Aventis)), aptamers (e.g., anti-VEGF PEGylated aptamer MACUGEN® (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 (AZD 2171), vatalanib (PTK787), semaxaminib (SU5416; SUGEN), and SUTENT® (sunitinib); tryptophanyl-tRNA synthetase (TrpRS); squalamine; RETAANE® (acanortabe acetate for depot suspension; Alcon, Inc); combretastatin A4 prodrug (CA4P); MIFEPREX® (mifepristone-ru486); subtenon triamcinolone acetonide; intravitreal crystal triamcinolone acetonide; matrix metalloproteinase inhibitors (e.g., Prinomast (AG3340; Pfizer)); fluocinolone acetonide (including fluocinolone intraocular implants; Bausch & Lomb / Control Delivery Systems); linomide; inhibitors of integrin β3 function; angiostatin, and combinations thereof.These and other therapeutic agents that can be administered in combination with the Tie2-binding agents or conjugates of the invention are described, for example, in U.S. Patent Application Publication No. 2014 / 0017244, which is incorporated herein by reference in its entirety.

[0313] Further examples of additional therapeutic agents that can be used in combination with a Tie2 binding agent or conjugate and / or polymer formulation thereof for the treatment of ocular disorders (e.g., DME, DR, AMD, RVO, or GA) include, but are not limited to, VISUDYNE® (verteporfin; a light-activated drug typically used in conjunction with photodynamic therapy using a non-thermal laser), PKC412, Endovion (NS3728; NeuroSearch A / S), neurotrophic factors (e.g., glial-derived neurotrophic factor (GDNF) and ciliary neurotrophic factor (CNTF)), diltiazem, dorzolamide, PHOTOTROP®, 9-cis-retinal, eye drops (e.g., phospholipid, echothiophate, or carbonic anhydrase inhibitors), beovastat (AE-941; AEterna Laboratories, Inc.), Sirna-027 (AGF-745; Sima Therapeutics, Inc.), neurotrophins (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., EntreMed, Inc.used by the University of Michigan), cardiotrophin-1 (Genentech), 2-methoxyestradiol (Organ / 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., from Allergan, SUGEN, or Pfizer), NX-278-L (NeXstar Pharmaceuticals / Gilead Sciences), Opt-24 (OPTIS France SA), retinal ganglion neuroprotectants (Cogent Neurosciences), N-nitropyrazole derivatives (Texas A&M University System), KP-102 (Krenitsky Pharmaceuticals), cyclosporine A, therapeutic agents used in photodynamic therapy (e.g., VISUDYNE®; receptor-targeted PDT, Bristol-Myers Squibb, Co.; PDT and rostaporfin, Miravent Medical Technologies; PDT and talaporfin sodium, Nippon Petroleum; and motexafin lutetium, Pharmacyclics, Inc.), antisense oligonucleotides (e.g., Novagali Pharma SA and ISIS-13650, a product tested by Ionis Pharmaceuticals), and combinations thereof.

[0314] Tie2 binders or conjugates, and / or polymeric formulations thereof, may be used in, for example, laser photocoagulation (e.g., panretinal photocoagulation (PRP)), drusen lasering, macular hole surgery, macular translocation surgery, implantable miniscopes, PHI motion angiography (also known as microlaser therapy and feeder vessel procedures), proton therapy, microstimulation therapy, retinal detachment and vitreous surgery, scleral buckling, submacular surgery, transpapillary thermotherapy, photosystem I therapy, use of RNA interference (RNAi), extracorporeal rheopheresis (also known as membrane fractional filtration and rheotherapy), microchip transplantation, stem cell therapy, gene replacement therapy, ribozyme gene therapy (including hypoxia response element gene therapy, Oxford Biomedica; Lentipak, Genetix; and PDEF gene therapy, GenVec), photoreceptor / retinal cell transplantation (including transplantable retinal epithelial cells, Diacrin, Inc.); retinal cell transplantation, e.g., Astellas Pharma US, Inc., ReNeuron, CHA Biotech), paracentesis, and combinations thereof.

[0315] In some examples, the Tie2-binding agents or conjugates of the invention, and / or polymer formulations thereof, can be administered in combination with an anti-angiogenic agent for the treatment of ocular disorders (e.g., DME, DR, AMD, RVO, or GA). Any suitable anti-angiogenic agent can be used in combination with the Tie2-binding agents or conjugates of the invention, including, but not limited to, those listed by Carmeliet et al., Nature 407:249-257, 2000. In some embodiments, the anti-angiogenic agent is selected from the group consisting of, but not limited to, anti-VEGF antibodies (e.g., anti-VEGF Fab LUCENTIS® (ranibizumab), RTH-258 (formerly ESB-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., EYLEA® (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 DARPins® (e.g., abysciperpegol, Molecular Partners), and the like. AG / Allergan), small interfering RNAs that inhibit VEGF or VEGFR expression, 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 SUTENT® (sunitinib)), and combinations thereof.In some examples, the anti-Tie2 antibody to a fragment thereof is selected from the group consisting of IL-1β; IL-6; IL-6R; PDGF (e.g., PDGF-BB); angiopoietin; angiopoietin 2; Tie2; S1P; integrins αvβ3, αvβ5, and α5β1; betacellulin; apelin / APJ; erythropoietin; complement factor D; TNFα; HtrA1; VEGF receptors (e.g., VEGFR1, VEGFR2, VEGFR3, mbVEGFR, or sVEGFR); ST-2 receptors and proteins genetically associated with age-related macular degeneration (AMD) risk, such as complement pathway components C2, factor B, factor H, CFHR3, C3b, C5, C5a, and C3a; HtrA1; ARMS2; TIMP3; HLA; IL-8; CX3CR1; TLR3; TLR4; CETP; LIPC; COL10A1; and TNFRSF10A. For example, in some instances, the additional compound is a bispecific antibody (e.g., an anti-VEGF / anti-Ang2 bispecific antibody, such as RG-7716 or any bispecific anti-VEGF / anti-Ang2 bispecific antibody or variant thereof disclosed in WO2010 / 069532 or WO2016 / 073157).

[0316] Other suitable anti-angiogenic agents that may be administered in combination with the anti-Tie2 conjugates and / or polymeric formulations thereof for the treatment of ocular disorders (e.g., DME, DR, AMD, RVO, or GA) include corticosteroids, antiangiogenic steroids, acancolturbine acetate, angiostatin, 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), gamma-secretase, Delta-like ligand 4, integrin antagonists, hypoxia-inducible factor (HIF)-1α antagonists, protein kinase CK2 antagonists, agents that inhibit stem cells (e.g., endothelial progenitor cells) homing to sites of neovascularization (e.g., anti-vascular endothelial cadherin (CD-144) antibodies and / or anti-SDF-1 antibodies), and combinations thereof.

[0317] In further examples, in some instances, the Tie2-binding conjugate and / or polymer formulation thereof can be administered in combination with an agent active against neovascularization for the treatment of ocular disorders (e.g., DME, DR, AMD, RVO, or GA), such as an anti-inflammatory agent, a mammalian target of rapamycin (mTOR) inhibitor (e.g., rapamycin, AFINITOR® (everolimus) and TORISEL® (temsirolimus)), cyclosporine, a tumor necrosis factor (TNF) antagonist (e.g., an anti-TNFα antibody or antigen-binding fragment thereof (e.g., infliximab, adalimumab, certolizumab pegol, and golimumab) or a soluble receptor fusion protein (e.g., etanercept)), an anti-complement agent, a nonsteroidal anti-inflammatory drug (NSAID), or a combination thereof.

[0318] In yet a further example, the Tie2-binding conjugate and / or polymer formulation thereof can optionally be administered in combination with agents that are neuroprotective and can potentially reduce the progression of dry AMD to wet AMD, such as a class of drugs called "neurosteroids," which includes drugs such as dehydroepiandrosterone (DHEA) (trade names: PRASTERA™ and FIDELIN®), dehydroepiandrosterone sulfate, and pregnenolone sulfate.

[0319] Any suitable AMD therapeutic agent can be administered as an additional therapeutic agent in combination with the Tie2-binding conjugates and / or polymer formulations thereof of the present invention to treat ocular disorders (e.g., DME, DR, AMD, RVO, or GA), including, but not limited to, VEGF antagonists, such as anti-VEGF antibodies (e.g., LUCENTIS® (ranibizumab), RTH-258 (formerly ESB-1008, an anti-VEGF single-chain antibody fragment; Novartis), or bispecific anti-VEGF antibodies (e.g., anti-VEGF / anti-angiopoietin 2 bispecific antibodies, e.g., RG-7716; Roche)), soluble VEGF receptor fusion proteins (e.g., EYLEA® (aflibercept)), anti-VEGF DARPins (e.g., abicipar pegol; Molecular Partners), and the like. AG / Allergan), or anti-VEGF aptamers (e.g., MACUGEN® (pegaptanib sodium); platelet-derived growth factor (PDGF) antagonists, such as anti-PDGF antibodies, anti-PDGFR antibodies (e.g., REGN2176-3), anti-PDGF-BB pegylated aptamers (e.g., FOVISTA®; Ophthotech / Novartis), soluble PDGFR receptor fusion proteins, or dual PDGF / VEGF antagonists (e.g., small molecule inhibitors (e.g., DE-120 (Santen) or X-82 (TyrogeneX)) or bispecific anti-PDGF / anti-VEGF antibodies)); in combination with photodynamic therapy VISUDYNE® (verteporfin); antioxidants; complement system antagonists, such as 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 regulators (e.g., emixustat hydrochloride); squalamine (e.g., OHR-102;Ohr Pharmaceutical; vitamin and mineral supplements (e.g., those described in Age-Related Eye Disease Study 1 (AREDS1; zinc and / or antioxidants) and Study 2 (AREDS2; zinc, antioxidants, lutein, zeaxanthin, and / or omega-3 fatty acids)); cell-based therapies, e.g., NT-501 (Renexus); PH-05206388 (Pfizer), huCNS-SC cell 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); α-adrenergic receptor agonists (e.g., brimonidine tartrate; Allergan); peptide vaccines (e.g., S-646240; Shionogi); amyloid-β antagonists (e.g., anti-β amyloid monoclonal antibodies, e.g., GSK-933776); S1P antagonists (e.g., anti-S1P antibodies, e.g., iSONEP™; Lpath Inc); ROBO4 antagonists (e.g., anti-ROBO4 antibodies, e.g., DS-7080a; Daiichi Sankyo); lentiviral vectors expressing endostatin and angiostatin (e.g., RetinoStat); and any combination thereof. In some examples, AMD therapeutic agents (including any of the aforementioned AMD therapeutic agents) can be co-formulated. For example, the anti-PDGFR antibody REGN2176-3 can be co-formulated with aflibercept (EYLEA®). In some instances, such co-formulations can be administered in combination with the Tie2-binding agents or conjugates of the present invention. In some instances, the ocular disorder is DME and / or DR. In some instances, the ocular disorder is AMD (e.g., wet AMD).

[0320] The Tie2-binding conjugate and / or polymer formulation thereof can be administered in combination with LUCENTIS® (ranibizumab) for the treatment of ocular disorders (e.g., DME, DR, AMD, RVO, or GA). In some examples, the ocular disorder is DME and / or DR. In some examples, the ocular disorder is AMD (e.g., wet AMD). In some examples, the ocular disorder is GA.

[0321] The Tie2-binding conjugate and / or polymer formulation thereof can be administered in combination with EYLEA® (aflibercept) for the treatment of ocular disorders (e.g., DME, DR, AMD, RVO, or GA). In some examples, the ocular disorder is DME and / or DR. In some examples, the ocular disorder is AMD (e.g., wet AMD). In some examples, the ocular disorder is GA.

[0322] The Tie2-binding conjugate and / or polymer formulation thereof can be administered in combination with MACUGEN® (pegaptanib sodium) for the treatment of ocular disorders (e.g., DME, DR, AMD, RVO, or GA). In some examples, the ocular disorder is DME and / or DR. In some examples, the ocular disorder is AMD (e.g., wet AMD). In some examples, the ocular disorder is GA.

[0323] The Tie2-binding conjugate and / or polymer formulation thereof can be administered in combination with VISUDYNE® (verteporfin) in combination with photodynamic therapy for the treatment of ocular disorders (e.g., DME, DR, AMD, RVO, or GA). In some examples, the ocular disorder is DME and / or DR. In some examples, the ocular disorder is AMD (e.g., wet AMD). In some examples, the ocular disorder is GA.

[0324] The Tie2-binding conjugates and / or polymeric formulations thereof can be administered in combination with a PDGF antagonist for the treatment of ocular disorders (e.g., DME, DR, AMD, RVO, or GA). Exemplary PDGF antagonists that can be used in combination with the Tie2-binding conjugates of the invention include anti-PDGF antibodies, anti-PDGFR antibodies, small molecule inhibitors (e.g., squalamine), anti-PDGF-B PEGylated aptamers such as FOVISTA® (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, FOVISTA® can be administered as adjunctive therapy to the Tie2-binding agents or conjugates of the invention. OHR-102 can be administered in combination with a VEGF antagonist, such as LUCENTIS® or EYLEA®. In some embodiments, the Tie2-binding agent or conjugate of the invention can be administered in combination with OHR-102, LUCENTIS® and / or EYLEA®. In some examples, the ocular disorder is DME and / or DR. In some examples, the ocular disorder is AMD (e.g., wet AMD). In some examples, the ocular disorder is GA.

[0325] The Tie2-binding conjugate and / or its polymer formulation can be administered in combination with RTH-258 for the treatment of ocular disorders (e.g., DME, DR, AMD, RVO, or GA). RTH-258 can be administered, for example, by intravitreal injection or ocular infusion. In some examples, the ocular disorder is DME and / or DR. In some examples, the ocular disorder is AMD (e.g., wet AMD). In some examples, the ocular disorder is GA.

[0326] The Tie2-binding conjugate and / or polymer formulation thereof can be administered in combination with abicipar pegol for the treatment of an ocular disorder (e.g., DME, DR, AMD, RVO, or GA). In some examples, the ocular disorder is DME and / or DR. In some examples, the ocular disorder is AMD (e.g., wet AMD). In some examples, the ocular disorder is GA.

[0327] The Tie2-binding conjugate and / or its polymer formulation can be administered in combination with abyssiparpegol for the treatment of ocular disorders (e.g., DME, DR, AMD, RVO, or GA). In some examples, the ocular disorder is DME and / or DR. In some examples, the ocular disorder is AMD (e.g., wet AMD). In some examples, the ocular disorder is GA.

[0328] Any suitable DME and / or DR therapeutic agent, including but not limited to a VEGF antagonist (e.g., LUCENTIS® or EYLEA®), a corticosteroid (e.g., a corticosteroid implant (e.g., OZURDEX® (dexamethasone intravitreal implant) or ILUVIEN® (fluocinolone acetonide intravitreal implant)) or a corticosteroid formulated for administration by intravitreal injection (e.g., triamcinolone acetonide)), or a combination thereof, can be administered in combination with a Tie2-binding conjugate and / or a polymer formulation thereof for the treatment of an ocular disorder (e.g., AMD, DME, DR, RVO, or GA). In some examples, the ocular disorder is DME and / or DR.

[0329] The Tie2-binding conjugate and / or polymeric formulation thereof can be administered in combination with LUCENTIS® (ranibizumab) for the treatment of DME and / or DR.

[0330] The Tie2-binding conjugate and / or polymeric formulation thereof can be administered in combination with EYLEA® (aflibercept) for the treatment of DME and / or DR.

[0331] The Tie2-binding conjugate and / or polymer formulation thereof can be administered in combination with OZURDEX® (dexamethasone intravitreal implant) for the treatment of DME and / or DR.

[0332] The Tie2-binding conjugate and / or polymer formulation thereof can be administered in combination with ILUVIEN® (dexamethasone intravitreal implant) for the treatment of DME and / or DR.

[0333] In some cases, TAO / PRN treatment regimen or TAE treatment regimen can be used to administer AMD therapeutic agent (for example, ranibizumab or aflibercept) in combination with Tie2 binding conjugate and / or its polymer formulation.In some examples, eye disorder is DME and / or DR.In some examples, eye disorder is AMD (for example, wet AMD).In some examples, eye disorder is GA.

[0334] Such combination therapy, as described above, encompasses combined administration (wherein two or more therapeutic agents are contained in the same or separate formulations) and separate administration, where administration of a Tie2-binding conjugate of the invention can precede, be concurrent with, and / or follow administration of the additional therapeutic agent or agent. In one embodiment, administration of the Tie2-binding conjugate or polymer 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.

[0335] The Tie2-binding conjugate and / or its polymer formulation is further intended for the treatment of glaucoma. Glaucoma is a group of eye diseases characterized by progressive damage to the eye, at least in part due to elevated intraocular pressure (IOP) (Merck Manual of Diagnosis and Therapy (1999)). Furthermore, glaucoma is characterized by retinal ganglion cell (RGC) death, axon loss, and an excavated 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 acuity testing and ophthalmoscopic examination of the optic nerve to detect "cupping." The average IOP in normal adults is 15 mmHg to 16 mmHg; the normal range is 10 to 21 mmHg. One form of glaucoma management is based on lowering IOP using topically applied drugs ("Glaucoma" Lancet, 1999;354:1803-1810).

[0336] Currently, there are five major classes of medications used to lower IOP: beta-adrenergic antagonists, adrenergic agonists, parasympathomimetics, prostaglandin-like analogs, and carbonic anhydrase inhibitors. Most medications are applied topically to the eye, but they can cause severe systemic side effects that negatively impact the patient's quality of life. If further IOP reduction is indicated, or if medication fails to sufficiently lower IOP, laser trabeculoplasty is usually the next step. If IOP is still not adequately controlled, incisional glaucoma surgery is indicated. Despite significantly reducing the degree of neuronal loss, lowering IOP does not guarantee cessation of the disease process, as RGC loss may continue. Recent studies of the relationship between IOP control and visual field loss after medical or surgical intervention have shown that low IOP can reduce ongoing neuronal loss as reflected in visual field tests. Glaucomatous optic neuropathy is thought to result from specific pathophysiological changes and the subsequent death of RGCs and their axons. The fate of RGC death is thought to be biphasic: a primary insult involved in the initiation of damage, followed by a slower secondary degeneration due to the harsh environment surrounding the degenerating cells.

[0337] In a further aspect, the present invention provides use of an anti-Tie2 conjugate in the manufacture or preparation of a medicament. In one embodiment, the medicament is for the treatment of an ocular disorder (e.g., DME, DR, AMD, RVO, or GA). In a preferred embodiment, the medicament is for the treatment of DME and / or DR. In a further embodiment, the medicament is for use in a method of treating an ocular disorder (e.g., DME, DR, AMD, RVO, or GA), comprising administering an effective amount of the medicament to an individual having the ocular disorder. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below. In a further embodiment, the medicament is for reducing vascular permeability, particularly in the eye. In a further embodiment, the medicament is for use in a method of reducing vascular permeability in an individual, particularly in the eye, comprising administering to the individual an effective amount of the medicament, particularly to reduce vascular permeability in the eye. An "individual" according to any of the above embodiments may be a human.

[0338] In a further aspect, the present invention provides a method for treating an ocular disorder (e.g., DME, DR, AMD, RVO, or GA). In one embodiment, the method comprises administering to an individual having such an ocular disorder (e.g., DME, DR, AMD, RVO, or GA) an effective amount of a Tie2-binding conjugate of the present invention. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, as described below. An "individual" according to any of the above embodiments may be a human.

[0339] In a further aspect, the present invention provides a method for reducing vascular permeability, particularly in the eye of an individual. In one embodiment, the method comprises administering to the individual an effective amount of a Tie2-binding conjugate of the invention to reduce vascular permeability, particularly in the eye. In one embodiment, the "individual" is a human.

[0340] In a further aspect, the present invention provides pharmaceutical formulations comprising any of the Tie2-binding conjugates of the present invention provided herein, for use in, for example, any of the above-described therapeutic methods. In one embodiment, the pharmaceutical formulation comprises any of the Tie2-binding conjugates of the present invention provided herein and a pharmaceutically acceptable carrier. In other embodiments, the pharmaceutical formulation comprises any of the Tie2-binding conjugates of the present invention provided herein and at least one additional therapeutic agent, for example, as described above.

[0341] Such combination therapy, as described above, encompasses combined administration (wherein two or more therapeutic agents are contained in the same or separate formulations) and separate administration, where administration of an antibody of the invention can precede, be concurrent with, and / or follow administration of the additional therapeutic agent or agent. In one embodiment, administration of the anti-Tie2 binding conjugate and administration of the additional therapeutic agent occur within about one month, or within about one, two, or three weeks, or within about one, two, three, four, five, or six days of each other.

[0342] The Tie2-binding conjugates of the present invention (and any additional therapeutic agents) can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal administration, as well as intralesional administration if desired for localized treatment. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing may be by any suitable route, for example, by injection, e.g., intravenous or subcutaneous, depending in part on whether administration is temporary or chronic. Various dosing schedules are contemplated herein, including, but not limited to, single or multiple doses over various time points, bolus administration, and pulse infusion.

[0343] The Tie2-binding conjugates of the present invention will be formulated, administered, and administered in a manner consistent with good medical practice. Factors to consider in this regard include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the administration schedule, and other factors known to medical professionals. The antibody is optionally, but need not be, formulated with one or more drugs currently used to prevent or treat the disorder in question. The effective amount of such other agents will depend on the amount of antibody present in the formulation, the type of disorder or treatment, and the other factors discussed above. These will generally be used in the same dosages and by any route of administration as described herein, or about 1-99% of the dosages described herein, or at any dosage and via any route empirically / clinically determined to be appropriate.

[0344] For the prevention or treatment of disease, the appropriate dosage of the conjugate of the invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease being treated, the type of antibody, the severity and course of the disease, whether the antibody is being administered for prophylactic or therapeutic purposes, previous therapy, the patient's medical history and response to the antibody, and the discretion of the attending physician. The conjugate is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg to 10 mg / kg) of the conjugate may be an initial candidate dosage for administration to the patient, whether by one or more separate administrations or by continuous infusion, for example. A typical daily dosage may range from about 1 μg / kg to 100 mg / kg, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, treatment is usually continued until a desired suppression of disease symptoms occurs. One exemplary dosage of the conjugate ranges from about 0.05 mg / kg to about 10 mg / kg. Thus, 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) may be administered to the patient. 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 larger dose may be administered, followed by one or more smaller doses. The progress of this therapy is easily monitored by conventional techniques and assays.

[0345] It will be understood that any of the above formulations or methods of treatment may be practiced using an immunoconjugate of the invention in place of, or in addition to, a Tie2-binding conjugate of the invention.

[0346] H.Product

[0347] Another aspect of the present invention provides an article of manufacture containing materials useful for the treatment, prevention, and / or diagnosis of the aforementioned disorders. The article of manufacture includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The container may be formed from a variety of materials, such as glass or plastic. The container holds a composition to be used alone or in combination with another composition effective in treating, preventing, and / or diagnosing a condition and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic needle). At least one active agent in the composition is a Tie2 binding agent or conjugate of the present invention. The label or package insert indicates that the composition is used to treat a selected condition. Additionally, the article of manufacture may include (a) a first container containing a composition contained in the article of manufacture and including a Tie2 binding agent or conjugate of the present invention, and (b) a second container containing a composition contained in the article of manufacture and including an additional cytotoxic or other therapeutic agent. The article of manufacture, in this embodiment of the invention, may further comprise a package insert indicating that the composition can be used to treat a particular condition. Alternatively, or in addition, the article of manufacture may further comprise a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, such as other buffers, diluents, filters, needles, syringes, etc.

[0348] It will be understood that any of the above products may include an immunoconjugate of the invention instead of, or in addition to, a Tie2-binding conjugate.

[0349] I. EMBODIMENTS OF THE INVENTION

[0350] Specific embodiments of the present invention are listed below.

[0351] 1. An isolated antibody or fragment thereof that binds to Tie2, 1. An isolated antibody or antigen-binding fragment thereof, comprising: a heavy chain variable domain (VH) comprising: (a) a CDR-H1 comprising the amino acid sequence NTDIS (SEQ ID NO: 3), (b) a CDR-H2 comprising the amino acid sequence RISPSDGNTYYADSVKG (SEQ ID NO: 4), and (c) a CDR-H3 comprising the amino acid sequence 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) a CDR-L1 comprising the amino acid sequence RASQDVSTAVA (SEQ ID NO: 8), (e) a CDR-L2 comprising the amino acid sequence SASFLYS (SEQ ID NO: 9), and (f) a CDR-L3 comprising the amino acid sequence QQSYTTPPT (SEQ ID NO: 10).

[0352] 2. The antibody of the preceding embodiment, wherein CDR-H3 comprises the amino acid sequence RTRWASWAMDY (SEQ ID NO: 6).

[0353] 3. The antibody of embodiment 1, wherein CDR-H3 comprises the amino acid sequence RTRWASWAFDY (SEQ ID NO: 7).

[0354] 4. The antibody of any one of the preceding embodiments, which is a monoclonal antibody.

[0355] 5. The antibody of any one of the preceding embodiments, which is a humanized or chimeric antibody.

[0356] 6. The antibody of any one of the preceding embodiments, which is an antibody fragment that binds to Tie2.

[0357] 7. The antibody of any one of the preceding embodiments, which is a Fab fragment.

[0358] 10. An antibody described in any one of embodiments 1 to 7, comprising a VL domain comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 21; and a VH domain comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 20.

[0359] 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 with the amino acid sequence of SEQ ID NO: 21; and a VH domain comprising an amino acid sequence having at least 96% sequence identity with the amino acid sequence of SEQ ID NO: 20.

[0360] 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 with the amino acid sequence of SEQ ID NO: 21; and a VH domain comprising an amino acid sequence having at least 97% sequence identity with the amino acid sequence of SEQ ID NO: 20.

[0361] 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.

[0362] 14. The antibody of any one of embodiments 1 to 7, comprising a VL domain comprising an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 21; and a VH domain comprising an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 20.

[0363] 15. The antibody of any one of the preceding embodiments, comprising 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.

[0364] 16. The antibody of any one of the preceding embodiments, comprising an engineered cysteine.

[0365] 17. The antibody of embodiment 16, an antibody wherein the engineered cysteines are selected from T120C, G166C, G178C, T187C and T209C in the HC, or the engineered cysteines are selected from Q124C, R142C, Q155C, L201C, T206C, K107C, K126C and K149C in the LC, and the residue numbers of the engineered cysteines are according to EU numbering.

[0366] 18. The antibody of embodiment 16 or 17, wherein the engineered cysteines are selected from T209C in the HC and T206C in the LC.

[0367] 19. The antibody of any one of embodiments 16 to 18, wherein the engineered cysteine ​​is T206C in LC.

[0368] 20. The antibody of any one of embodiments 16-18, wherein the engineered cysteine ​​is T209C in HC.

[0369] 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.

[0370] 22. The antibody of any one of the preceding embodiments, comprising: a LC comprising an amino acid sequence having at least 96% sequence identity to the amino acid sequence of SEQ ID NO: 25; and a HC comprising an amino acid sequence having at least 96% sequence identity to the amino acid sequence of SEQ ID NO: 55.

[0371] 23. The antibody of any one of the preceding embodiments, comprising: a LC comprising an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO: 25; and a HC comprising an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO: 55.

[0372] 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.

[0373] 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.

[0374] 26. The antibody of any one of the preceding embodiments, comprising a LC comprising the sequence of SEQ ID NO: 25 and a HC comprising the sequence of SEQ ID NO: 55.

[0375] 27. The antibody of any one of embodiments 1 to 25, comprising an LC comprising the sequence of SEQ ID NO: 25 and an HC comprising a sequence selected from SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91 and SEQ ID NO: 92.

[0376] 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 with the amino acid sequence of SEQ ID NO: 56; and an HC comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 23.

[0377] 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.

[0378] 30. An antibody described in any one of embodiments 1 to 20, comprising: an LC comprising an amino acid sequence having at least 97% sequence identity with the amino acid sequence of SEQ ID NO: 56; and an HC comprising an amino acid sequence having at least 97% sequence identity with the amino acid sequence of SEQ ID NO: 23.

[0379] 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.

[0380] 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.

[0381] 33. The antibody of any one of embodiments 1 to 20, comprising 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.

[0382] 34. An antibody that specifically binds to Tie-2, comprising an HC comprising the sequence of SEQ ID NO: 55 and an LC comprising the sequence of SEQ ID NO: 25.

[0383] 35. An isolated nucleic acid encoding the antibody of any one of the preceding embodiments.

[0384] 36. A host cell comprising a nucleic acid according to embodiment 38.

[0385] 37. A method for producing an antibody that binds to Tie-2, comprising culturing a host cell according to embodiment 39 under conditions suitable for expression of the antibody.

[0386] 38. A conjugate that binds to Tie2, comprising at least two, at least three, at least four, at least five, at least six, at least seven or at least eight antibodies according to any one of embodiments 1 to 34, wherein each of the antibodies is linked to a multimerization moiety.

[0387] 39. The conjugate of embodiment 38, which activates the phosphorylation of Tie2 in an in vitro or in vivo cell assay.

[0388] 40. The conjugate of embodiment 38 or 39, which reduces Tie-2 protein levels by less than 25%, less than 50% or less than 75% in an in vitro assay, or does not reduce Tie-2 protein levels by more than 25%, more than 50% or more than 75% in an in vitro assay.

[0389] 41. A conjugate according to any one of embodiments 38 to 40, which reduces vascular permeability as determined by an in vitro barrier function assay.

[0390] 42. The conjugate according to any one of embodiments 38 to 42, wherein the multimerization moiety comprises a polyol, a polypeptide and / or a peptide.

[0391] 43. The conjugate of embodiment 42, wherein the polyol is a multi-arm polyol selected from dimers, tetramers, hexamers, and octamers.

[0392] 44. The conjugate of embodiment 43, wherein the multi-arm polyol is a hexamer.

[0393] 45. The conjugate of embodiment 43 or 44, wherein the multi-arm polyol is an octamer.

[0394] 46. ​​The conjugate of any one of embodiments 42 to 45, wherein the polyol is polyethylene glycol (PEG).

[0395] 47. The conjugate of any one of embodiments 42 to 45, wherein the polyol is covalently bound to at least two antibodies via free sulfhydryl groups of cysteine ​​amino acids.

[0396] 48. The conjugate of embodiment 47, wherein the cysteine ​​amino acid is an engineered cysteine.

[0397] 49. The conjugate of embodiment 48, wherein the engineered cysteine ​​is in the HC and / or LC constant region of the antibody. 50. The engineered cysteines are selected from the group consisting of T120C, G166C, G178C, T187C and T209C in HC; or 50. The conjugate of embodiment 48 or 49, wherein the engineered cysteines are selected from the group consisting of Q124C, R142C, Q155C, L201C, T206C, K107C, K126C, and K149C in LC; and residue numbers are according to EU numbering.

[0398] 51. The conjugate according to any one of embodiments 48-50, wherein the engineered cysteine ​​is T206C in LC, and the residue numbers are according to EU numbering.

[0399] 52. The conjugate according to any one of embodiments 48 to 50, wherein the engineered cysteine ​​is T209C in HC, the residue numbers being according to EU numbering.

[0400] 53. The conjugate according to any one of embodiments 42 to 45, wherein the polyol is covalently attached to at least one antibody via a free amino group of a lysine amino acid.

[0401] 54. The conjugate of embodiment 53, wherein the lysine amino acid is in the HC or LC constant region of the antibody, and / or the lysine amino acid is at the C-terminus of the heavy or light chain of the antibody.

[0402] 55. A conjugate according to any one of embodiments 46 to 54, wherein the PEG has a weight-average molecular weight of from about 500 Daltons (Da) to about 300,000 Da, or from about 500 Da to about 20,000 Da.

[0403] 56. A conjugate according to any one of embodiments 46 to 55, wherein the PEG has a weight average molecular weight of about 6000 Da.

[0404] 57. The conjugate of any one of embodiments 46-56, wherein the PEG comprises a dipentaerythritol hexameric or octameric core. 58. PEG is represented by the general formula (Ia): JPEG2025138630000041.jpg42170 (wherein each n is an integer from 1 to 10 and each m is independently an integer from 3 to 250; each R 1 is independently either absent or a linking group; and each R 2 is independently either hydrogen or a terminal reactive group; At least one R 2 is a terminal reactive group and is covalently bound to the antibody according to any one of claims 1 to 14. 58. The conjugate of any one of embodiments 46 to 57, having the structure: 59. PEG is represented by the general formula (Ib): JPEG2025138630000042.jpg45170 (wherein each m is independently an integer from 3 to 250; each R 1 is independently either absent or a linking group; and each R 2 is independently either hydrogen or a terminal reactive group; At least one R 2 is a terminal reactive group and is covalently bound to the antibody according to any one of claims 1 to 14. 58. The conjugate of any one of embodiments 46 to 57, having the structure:

[0405] 60. The conjugate of embodiment 59, wherein each m is independently an integer between 15 and 35, preferably between about 20 and 30.

[0406] 61. A conjugate according to any one of embodiments 43 to 60, prepared by covalently linking at least one antibody according to any one of embodiments 1 to 34 to a multi-arm polyol.

[0407] 62. A conjugate comprising an antibody that specifically binds to Tie-2, wherein the antibody comprises a VH sequence of SEQ ID NO: 20 and a VL sequence of SEQ ID NO: 21, and wherein the antibody has the general formula (Ib): JPEG2025138630000043.jpg48170 (wherein each m is independently an integer from 3 to 250; each R 1 is independently either absent or a linking group; and each R 2 is independently either hydrogen or a terminal reactive group; At least one R 2 is covalently attached to the antibody) The conjugate is covalently attached to polyethylene glycol having the formula:

[0408] 63. The conjugate of embodiment 62, wherein m is an integer from 10 to 200.

[0409] 64. The conjugate of embodiment 62 or 63, wherein m is an integer from 20 to 30.

[0410] 65.R 1 does not exist or R 1 but, JPEG2025138630000044.jpg93170 (wherein each i is independently an integer from 0 to 10; j is an integer from 0 to 10; R 2 is a terminal reactive group selected from the group consisting of a thiol-reactive group, an amino-reactive group, and combinations thereof); and combinations thereof 65. The conjugate of any one of embodiments 58 to 64, selected from the group consisting of:

[0411] 66.Each R 2 is a reactive group selected from maleimide, sulfhydryl, thiol, trifluoromethanesulfonate, tosylate, aziridine, epoxide, pyridyl disulfide, succinimidyl ester, -NH, aldehyde, haloacetate, haloacetamide, and para-nitrophenyl carbonate.

[0412] 67.R 2 67. The conjugate of any one of embodiments 58-66, wherein is maleimide.

[0413] 68. A conjugate according to any one of embodiments 43 to 67, prepared by covalently linking at least one antibody according to any one of claims 1 to 34 to a multi-arm polyol.

[0414] 69. A pharmaceutical composition comprising a conjugate according to any one of embodiments 38 to 68 and a pharmaceutically acceptable carrier.

[0415] 70. The pharmaceutical formulation of embodiment 69, wherein the concentration of the conjugate is from about 50 mg / ml to about 300 mg / ml.

[0416] 71. The pharmaceutical composition according to embodiment 69 or 70, further comprising an additional therapeutic agent.

[0417] 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.

[0418] 73. The pharmaceutical composition according to embodiment 72, wherein the VEGF antagonist is selected from the group consisting of a VEGF trap and an anti-VEGF antibody.

[0419] 74. A long-acting delivery device for ocular delivery, comprising a pharmaceutical composition according to any one of embodiments 79 to 73 and a means for delivering the composition into the vitreous of a patient, wherein the composition remains effective in situ for an extended period of time.

[0420] 75. A method for treating a Tie2 pathway-mediated disorder in a subject, comprising administering to the subject an effective amount of an antibody described in any one of embodiments 1 to 34, a conjugate described in any one of embodiments 38 to 68, or a pharmaceutical composition described in any one of embodiments 69 to 73.

[0421] 76. The method of embodiment 75, wherein the Tie2 pathway-mediated disorder is a vascular permeability disorder.

[0422] 77. The method of embodiment 75 or 76, wherein the Tie2 pathway-mediated disorder is an ocular condition.

[0423] 78. The method of embodiment 77, wherein the ocular condition is selected from diabetic macular edema (DME), diabetic retinopathy, age-related macular degeneration (AMD), including dry and wet (non-exudative and exudative) forms, choroidal neovascularization (CNV), uveitis, ischemia-related retinopathy, pathological myopia, von Hippel-Lindau disease, ocular histoplasmosis, central retinal vein occlusion (CRVO), corneal neovascularization, glaucoma, and retinal neovascularization.

[0424] 79. The method of any one of embodiments 75-78, wherein the ocular condition is DME.

[0425] 80. The method of any one of embodiments 75-79, comprising administering the antibody, conjugate or pharmaceutical formulation using an implantable port delivery system.

[0426] 81. The method of any one of embodiments 75-79, comprising administering the antibody, conjugate or pharmaceutical preparation by intravitreal administration.

[0427] 82. The method of embodiment 81, wherein the intravitreal administration is through a narrow bore needle.

[0428] 83. The method of embodiment 82, wherein the narrow bore needle is about 30, 29, 28, 27, 26, 25, 24, 23, or 22 gauge.

[0429] 84. The method of any one of embodiments 75-83, further comprising administering an additional therapeutic agent. [Example]

[0430] III. Working Examples The following are examples of methods and compositions of the present invention. Given the general description provided above, it will be understood that various other embodiments may be practiced.

[0431] Example 1 - Generation of anti-Tie2 antibodies from a naive phage library Antibodies that bind to the extracellular domain (ECD) of Tie2 were initially selected from a phage-displayed synthetic antibody library built on a single human framework by introducing synthetic diversity into solvent-exposed positions within the heavy chain CDRs as described below. Phages were screened against the ECD as well as various subdomains of the ECD.

[0432] Phagemid vectors for library construction Phagemids pV0350-2b and pV0350-4 were designed to display Fab templates monovalently or bivalently on the surface of M13 phage particles, respectively. The Fab template was based on the h4D5 antibody, a humanized antibody that recognizes a cancer-associated antigen known as Her-2 (erbB2). The h4D5 sequence was obtained by polymerase chain reaction using the humAb4D5 version 8 ("humAb4D5-8") sequence (Carter et al., (1992) PNAS 89:4285-4289). The h4D5 nucleic acid sequence encodes modified CDR regions from a mouse monoclonal antibody specific for Her-2 within 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 producing anti-Her-2 antibodies and the identity of the variable domain sequences are set forth in US Pat. Nos. 5,821,337 and 6,054,297.

[0433] The vector pV0350-2b was constructed by modifying a previously described phagemid (pHGHam-gIII) used for phage display of human growth hormone (hGH) under the control of the phoA promoter. The open reading frame of phGHam-gIII, encoding hGH fused to the StII secretion signal sequence and the C-terminal domain of the M13 minor coat protein P3 (cP3), was replaced with a DNA fragment containing two open reading frames. The first open reading frame encodes the h4D5 light chain (version 8), and the second encodes the variable (VH) domain and first constant (CH1) domain of the h4D5 heavy chain fused to cP3; each protein is targeted for secretion by the N-terminal StII signal sequence. The amber stop codon 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 bivalent display vector (pV0350-4) was identical to pV0350-2b, except that a DNA fragment encoding the GCN4 leucine zipper was inserted between the heavy chain CH1 domain and cP3, as described. The light chain gene was further modified in both phagemids at three positions to encode the most commonly found amino acids in the Kabat database of natural antibody sequences: specifically, Arg 66 was changed to Gly, and Asn 30 and His 91 were changed to Ser. These changes were found to increase Fab expression and display on phage. Site-directed mutagenesis was performed using the method of Kunkel et al. (Kunkel, JD et al. (1987) Methods Enzymol 154:367-82).

[0434] Phage display libraries were generated using oligonucleotide-directed mutagenesis and "stop template" versions of pV0350-2b or pV0350-4 as described (Lee, CV, et al., (2004) J. Immunol. Methods 284:119-132; Lee, CV, et al., (2004) JMB 340:1073-1093). Stop codons (TAA) were embedded in all three heavy chain CDRs. These were repaired during the mutagenesis reaction by a mixture of degenerate oligonucleotides that annealed across the sequences encoding CDRs H1, -H2, and -H3, replacing the codons at positions selected for randomization with tailored degenerate codons. The mutagenesis reaction was electroporated into E. coli SS320 cells, and cultures were grown overnight at 30°C in 2YT broth supplemented with KO7 helper phage, 50 μg / ml carbenicillin, and 50 μg / ml kanamycin. Phages were recovered from the culture medium by PEG / NaCl precipitation as described (Sidhu, SS et al. (2000), Methods Enzymol. 328:333-363). Each electroporation reaction used approximately 10 E. coli cells and approximately 10 μg of DNA, resulting in 1 × 10–5 × 10 transformants.

[0435] A separate library was created using the Fab.zip template with degenerate oligonucleotides tailored to mimic the natural diversity of CDR-H1 and CDR-H2 (see Table 1 in Lee, C.V. et al. (2004), supra): Library 3 (Lib-3). See Lib-3 described in Lee, C.V. et al. (2004), supra. 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 (2:1 ratio) and H2a–c (1:2:01 ratio) for CDR-H1 and CDR-H2, respectively (see Table 1 in Lee, C.V. et al. (2004), supra, for a description of the oligonucleotides).

[0436] For positions 95-100 of the CDR-H3, Lib-3 consisted of a set of libraries with extended CDR-H3 lengths containing either the NNS codon (or NNK codon) or a modified version of the NNS codon (XYZ codon) with unequal nucleotide ratios at each position of the codon triplet. The NNS codon encompassed 32 codons, encoding all 20 amino acids. X contained 38% G, 19% A, 26% T, and 17% C; Y contained 31% G, 34% A, 17% T, and 18% C; and Z contained 24% G and 76% C. The CDR-H3 design of Lib-3 is listed in Table 5 of Lee, CV et al. (2004) supra. Separate mutagenesis reactions were performed and electroporated for each CDR-H3 length, except for the 7- and 8-residue lengths, which were electroporated together.

[0437] The phage display level of intact Fabs in each library was examined by measuring the binding of 48 randomly selected clones to anti-gD antibodies. For uLib-3, similar levels of display were observed for different CDR-H3 lengths, except that the library incorporating the longest CDR-H3 (15-19 residues) had a lower percentage of Fab-displaying clones (15-30%). This may reflect a decrease in mutagenesis efficiency when using very long synthetic oligonucleotides.

[0438] Phage selection Lib-3, described above, was screened against various 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 Figure 1 for an example). Constructs encoding the complete extracellular domain (ECD), the membrane-proximal FN3 domain, or an ECD without the FN3 domain (termed ECD5) were generated. Thus, the ligand-binding domain Ig2 is encoded by both the ECD and ECD5 constructs but is absent from the FN3 construct. The encoded proteins were C-terminally fused to either the Fc region of hIgG1 for human and cynomolgus monkey proteins, mIgG2a for mouse and rat proteins, or a C-terminal Flag tag for all species. Figure 1 shows the proteins used for panning. Complete ECD constructs for the human, mouse and rat Tie1 receptor were also generated with both C-terminal Fc fusions or Flag tags.

[0439] Expression and purification of Tie2 ECD protein Flag-tagged Tie2 ECD was expressed and purified from Chinese hamster ovary (CHO) cell-conditioned medium. After 11–14 days, the conditioned medium was collected and concentrated approximately 10-fold. The concentrate was loaded onto an anti-Flag tag column and washed with binding buffer (25 mM TRIS, 150 mM NaCl, 1 mM EDTA, pH 7.5, containing 0.1% Triton X-114 and 0.1% Triton X-110). The Flag-tagged protein was then eluted with 50 mM N-citrate, 150 mM NaCl, pH 3.0, and then neutralized to pH 5.0 using 1 M arginine, 400 mM succinate, pH 9.0. The eluted protein was loaded onto a size-exclusion column (either Superdex 200 or Superdex 75) in phosphate-buffered saline, and fractions were collected; the monomer peak fractions were pooled, concentrated, and filtered through a 0.2 μm filter.

[0440] For panning, 96-well Nunc Maxisorp plates were coated overnight with 100 ul / well of target Tie2 antigen (5 ug / ml) in PBS at 4°C. Plates were blocked with 65 ul of 1% blocking protein for 30 minutes (min) and 40 ul of 1% Tween 20 for an additional 30 minutes (blocking proteins: 1st round: bovine serum albumin (BSA), 2nd round: casein, 3rd round: bovine serum albumin (BSA), 4th round: casein). The phage library was then plated in 1% BSA containing 0.1% Tween 20 (1 OD = 1.13 x 10 13The phage library was diluted to approximately 3-5 OD / ml (using 100 μL of phage / ml). Typically, the phage input was as follows: 1st round 3-5 OD / ml, 2nd round 3 OD / ml, 3rd round approximately 0.5-1 OD / ml, and 4th round approximately 0.1-0.5 OD / ml. The diluted phage was incubated at room temperature for 30 minutes. The wells were washed at least five times consecutively with PBS and 0.05% Tween 20. The blocked phage library was added at 100 μL / well to eight target antigen-coated wells and two uncoated wells for 2 hours (hr) at room temperature. The plate was washed at least 10 times consecutively with PBS and 0.05% Tween 20. Starting from the third panning, 1 μM of omalizumab, Tie2.ECD5, or the antibody anti-Tie2.20 (ligand blocking) as an irrelevant Fc-containing protein was added to the phage library for 1 h, after which the mixture was applied to Tie2-coated wells where binding occurred for 2 h. Phage were eluted with 100 μl / well of 100 mM HCl for 20 min at room temperature. Eluted phage (from coated wells) and background phage (from uncoated wells) were collected in separate tubes. The eluted collection was neutralized by adding 1 / 10 volume of 1 M Tris pH 11.0 to both tubes. BSA was added to the eluted phage tube to a final concentration of 0.1%. To titer the phage, 90 μl of log-phase XL-1 cells (OD600nm approximately 0.1-0.3) was infected with 10 μl of eluted phage or background phage for 30 min at 37°C. The infected cells were then serially diluted 10-fold in 90 ul of 2 YT. 10 ul aliquots of infected cells were plated onto carbenicillin plates.

[0441] To propagate phage between rounds of panning, approximately 400 μl of eluted phage was used to infect approximately 4 ml of log-phase XL-1 (OD 0.1-0.3) for 30-45 minutes at 37°C. Helper phage KO7 and carbenicillin were added at 1 × 10 10A final concentration of 100 pfu / ml KO7 and 50 μg / ml carbenicillin was added to the infection for an additional hour at 37°C. The culture was grown in 2YT medium containing 50 μg / ml carbenicillin and 50 μg / ml kanamycin at 37°C for 4 hours and at 30°C overnight (or at least 18 hours) to a final volume of 20-25 ml. The next day, library phage was purified by spinning down the cells at 8000 rpm for 10 minutes. The supernatant was collected. 20% PEG / 2.5M NaCl was added at 1 / 5 the supernatant volume, mixed, and placed on ice for 5 minutes. The phage was pelleted at 12000 rpm for 15 minutes. The pellet was spun again at 5000 rpm for 5 minutes. The pellet was resuspended in 1 ml of PBS, spun down at 12000 rpm for 15 minutes to remove debris, and precipitated with PEG / NaCl. The phage pellet was resuspended in PBS. The OD of the regrowth phage pellet was read at 268 nm.

[0442] Screening ELISA assay Clones from the fourth round were screened for Tie2 binding and specificity by ELISA. Screening ELISAs were performed by coating wells of a 96-well microtiter plate with Tie2 protein or an irrelevant protein at 65 μl / well (1 μg / ml in coating buffer) overnight at 4°C. Colonies from the fourth round were grown in 96-tube plates in 400 μl of 2YT medium containing 50 μg / ml carbenicillin and helper phage KO7 at 37°C overnight. Plates were spun down at 3000 rpm for 10 minutes. Thirty μl of culture supernatant was added to the Tie2-coated plate along with 60 μl of ELISA buffer (PBS containing 0.5% BSA and 0.05% Tween 20) and incubated at room temperature for 1 hour. Plates were washed with PBS-0.05% Tween 20 and 100 μl / well of horseradish peroxidase (HRP)-conjugated anti-M13 antibody (1 / 5000 dilution in PBS supplemented with 0.5% BSA and 0.05% Tween 20) for 30 minutes at room temperature (Sidhu et al., supra). After washing the wells with PBS-0.05% Tween 20, 100 μl / well of a 1:1 ratio of 3,3',5,5'-tetramethylbenzidine (TMB) peroxidase substrate and peroxidase solution B (H2O2) (Kirkegaard-Perry Laboratories, Gaithersburg, MD) was added and incubated for 5 minutes at room temperature. The reaction was stopped by adding 100 μl of 1 M phosphoric acid (H3PO4) per well, and the OD of the wells was determined at 450 nm using a standard ELISA plate reader.

[0443] Clones with above background Tie2 binding and low binding to Tie2-binding species specificity and an unrelated Fc-containing protein (omalizumab) were further analyzed. Tables 2-5 below summarize the binding data. None of the clones in Tables 2-5 showed binding to omalizumab. [Table 2] [Table 3] [Table 4] [Table 5]

[0444] Expression and purification of anti-Tie2 IgG The positive binders identified above, with the desired species specificity and low irrelevant protein binding, were sequenced, and the variable domains of the anti-Tie2 heavy chains were cloned into a vector predesigned for transient human IgG1 expression in mammalian cells (Lee et al., 2004a).

[0445] The resulting anti-Tie2 human IgG was expressed using 293 transient transfection with the heavy chain encoded by the construct generated above and the 4D5 light chain (SEQ ID NO: 25). IgG was purified from transfection supernatants by protein A affinity chromatography and screened by ELISA for Tie2 binding confirmation, Tie1 binding, and epitope mapping. Eight antibodies were not further analyzed because hIgG protein was not expressed or expressed poorly.

[0446] Example 2 - Characterization of anti-Tie2 antibodies derived from a phage library Tie2 join To confirm Tie2 binding by anti-Tie2 antibodies, an ELISA format was used in which Tie2 extracellular constructs, prepared as described above in IgG1 format, were immobilized to Maxisorp immunoplates at 2 μg / ml in 65 μl of PBS overnight at 4°C. Serial dilutions of anti-Tie2 IgG were applied to plates with immobilized Tie2, pre-blocked with 1% BSA in PBS, and incubated for 20 minutes at room temperature. Plates were washed and detected with an anti-huFC-conjugated HRP secondary antibody using the method described above. Figures 2A and 2B show the binding of various clones to the human Tie2 ECD.

[0447] Tie1 binding was assessed using a binding ELISA with immobilized Tie1 protein. The results, presented in Figures 3A and 3B, show a lack of Tie1 binding by several anti-Tie2 antibodies, demonstrating that these anti-Tie2 antibodies specifically bind to Tie2.

[0448] Ligand blockade 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 a Maxisorp immunoplate (2 μg / ml), and biotinylated huTie2ECD.Fc was equilibrated in solution with serial dilutions of anti-Tie2 antibody. Unbound biotin-Tie2.ECD.Fc was then captured by immobilized hAng1 or hAng2 and detected with streptavidin-conjugated HRP. These results demonstrate blockade of both Ang1 and Ang2 by at least the anti-Tie2 antibodies, Tie2.1, Tie2.12, and Tie2.20 (see Figures 4A and 4B).

[0449] Example 3 - Functional analysis of anti-Tie2 antibodies The anti-Tie2 antibodies identified above as capable of specifically binding to Tie2 were further analyzed to identify those that could function as Tie2 agonists. The functional activity of the anti-Tie2 IgGs confirmed to bind to Tie2 was evaluated using human umbilical vein endothelial cells (HUVECs) and rat aortic endothelial cells (RAECs), both of which are known to express Tie2 (see Figures 5A and 5B).

[0450] Stimulation of phosphorylated AKT (pAKT) Experiments were conducted to evaluate the function of anti-Tie2 antibodies in Tie2 activation. As a Tie2 agonist, the Tie2 conjugate of the present disclosure is expected to bind to and activate Tie2, resulting in downstream activation of AKT enzyme. AKT activation can be demonstrated by the phosphorylation of AKT protein to produce pAKT, as described below. AKT phosphorylation was determined by Western blot analysis using an antibody specific for phosphorylated AKT, or by FRET assay as described below.

[0451] Cells and antibodies: HUVECs were purchased from Lonza (catalog number CC-2517; Lonza, Ltd., Basel, Switzerland). RAECs were purchased from VEC Technologies and cultured in growth medium (catalog number MCDB-131 10; VEC Technologies, Inc., Rensselaer, NY). Anti-Tie2 antibody was 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).

[0452] Preparation of HUVECs: HUVECs (human vascular endothelial cells) were trypsinized and plated in sterile 96-well plates (Corning® Costa®, Cat. No. 3997) at 0.4 × 10 5Cells were seeded in 100 μl of culture medium per well, and the plate was incubated overnight in a 37°C, 5% CO2 incubator. The culture medium was removed, and 100 μL of prewarmed serum starvation medium (Basal Medium EndoGRO™; Catalog No. SCME-BM, MilliporeSigma) was added to each well of the plate. The plate was incubated for 3 hours in a 37°C, 5% CO2 incubator before being incubated with anti-Tie2 antibody.

[0453] Preparation of RAECs: RAECs (rat aortic endothelial cells) were seeded at a density of 12,000 cells / well in a 96-well cell culture plate and cultured overnight in 100 μl of EGM2 MV medium (Lonza, Ltd.) at 5% CO2 and 37°C. 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.

[0454] 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 of anti-Tie2 bivalent antibody and incubated at room temperature for 1 hour. After incubation, the mixture of hIgG1 and anti-Tie2-IgG antibody was subjected to 3-fold serial dilutions.

[0455] To test other Tie2 antibodies, the molecules were first diluted in assay buffer to a high stock concentration (typically 30-1000 μg / ml) and then serially diluted (typically 2-10-fold). After removing the serum starvation medium, these dilutions (50 μl) were added to each well, and the plate was incubated for 15 minutes at 37°C and 5% CO2. The solution was removed, and 50 μl of lysis buffer containing blocking buffer from the Phospho-AKT1 / 2 / 3 Ser473 Cellular Kit (catalog no. 64 AKSPEH; Cisbio, Codolet, France) was added to the cells. The plate was incubated at room temperature for approximately 30-45 minutes with gentle shaking and then kept at -80°C until use, or used directly in the FRET assay.

[0456] Western blot assay: HUVEC cells 1 x 10 per well 6 Cells were seeded in Endogro medium and cultured at 37°C for 16–18 hours. Four to five hours before stimulation, the culture medium was replaced with 0.1% BSA Endogro basal medium. Cells were incubated with the relevant Tie-2 agonist for 30 minutes at 37°C and washed three times with cold phosphate-buffered saline, pH 7.4. Cells were placed on ice and incubated for 5 minutes with 100 μl / well of RIPA buffer (Sigma, #20-188) containing Roche Complete Protease and Phosphatase Inhibitor (Thermo Scientific, #1861281). Lysates were collected from the wells using a cell scraper and centrifuged at 17,800 × g for 10 minutes. The supernatant was evaluated by SDS-PAGE (8% NuPAGE Bis-Tris (Invitrogen, NW 800085)) followed by transfer 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, #9271 S) 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. The blot was then exposed to film.

[0457] FRET assay: Cell lysates (15 μl) were 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 (catalog no. 784080; Greiner Bio-One North America, Inc., Monroe, NC). The plate was incubated at room temperature for 4 hours or at 4°C overnight and read at 620 nm and 665 nm on a CLARIOstar (BMG LABTECH, software version: 5.01 R2). Data were calculated as the ratio of acceptor and donor emission signals × 10 for each individual well.

[0458] To identify anti-Tie-2 antibodies capable of activating Tie2 activity, we formatted the Tie-2-binding antibodies identified as detailed above as human IgG1 (hIgG1) antibodies and tested their ability to stimulate AKT phosphorylation (generating pAKT), which is downstream of Tie2 activation. RAECs were treated with recombinant anti-Tie2 antibodies (hIgG1): Tie2.1, Tie2.4, Tie2.5, Tie2.16, and Tie2.20, for 10 minutes. Cell lysates were subjected to Western blot (WB) analysis of pAKT. Figure 6A shows that incubation of the anti-Tie2 antibodies Tie2.1 and Tie2.20 increased AKT phosphorylation in this in vitro cell-based assay, with Tie2.1 having a stronger Tie2 agonist effect than Tie2.20.

[0459] Next, we performed experiments to evaluate the effect of cross-linking on the agonistic activity of anti-Tie2 antibodies. Anti-hIgG1 cross-linking antibodies were incubated with Tie2.1 or Tie2.20 in a pAKT assay. As shown in Figure 6B, cross-linking of Tie2.1 further increased its ability to activate Tie2, as determined by increased AKT phosphorylation.

[0460] To identify additional agonistic anti-Tie2 antibodies, we also used an HTRF-based assay (Cisbio). RAECs were treated with recombinant anti-Tie2 antibody (hIgG1) for 10 minutes, and cell lysates were subjected to HTRF analysis of pAKT (Cisbio). As shown in Figure 7A, antibodies found to significantly increase pAKT levels (acting as Tie2 agonists) included at least Tie2.24, Tie2.31, Tie2.32, T2.33, Tie2.38, and Tie2.1, with Tie2.1 exhibiting potent activity.

[0461] It was thought that the level of Tie2 activation upon binding by anti-Tie2 IgG (full-length) antibodies could be partially attributed to nonspecific aggregation of IgG molecules in the reaction mixture. As shown in Figure 7B, Tie2.1 antibody preparations with a higher percentage of aggregates (e.g., 3.5%) exhibited stronger agonistic activity compared to Tie2.1 antibody preparations with a lower percentage of aggregates (e.g., 0.15%). Figure 7B further shows that cross-linking anti-hIgG1 enhanced the agonistic activity of Tie2.1. Without being bound by theory, it is believed that Tie2 activation by anti-Tie2 agonist antibodies may be promoted by cross-linking Tie2-binding antibodies.

[0462] Example 4 - Binning of anti-Tie2 antibodies derived from a naive phage library To further characterize the binding of anti-Tie2 antibodies to the Tie2 receptor protein and identify antibodies with shared epitopes, we performed binning of anti-Tie2 antibodies using both phage ELISA and Octet measurements.

[0463] For phage ELISA, huTie2ECD.Fc protein was immobilized on Maxisorp immunoplates at 2 μg / ml in 65 μl of PBS overnight at 4°C. 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 plates containing immobilized huTie2ECD.Fc pre-blocked with 1% BSA in PBS and incubated for 1 hour at room temperature. Anti-Tie2 phage was added at an OD of 0.1 for 15 minutes at room temperature. Plates were then washed and detected with an anti-M13-conjugated HRP secondary antibody using the method described above. An increase in signal with decreasing serially diluted antibodies indicates that the test phage antibody competes with the serially diluted antibodies, indicating that they bind to the same site or epitope.

[0464] Epitope binning, as determined by the above ELISA, was confirmed by an Octet epitope binning assay using Octet RED384 (Pall Forte Bio Corporation, Menlo Park, CA) and a standard sandwich-format binning assay. Specifically, a streptavidin biosensor (Pall Forte Bio Corporation) was coated with biotinylated hTie2.ECD.Fc protein (10 μg / mL) and then exposed to benchmark anti-Tie2 hIgG1 (50 μg / mL Ab1, Ab20, 13H10), followed by a second (test) anti-Tie2 hIgG1. Data were processed using Forte Bio's data analysis software. Further binding by the second (test) antibody indicates an unoccupied epitope (non-competitor), while a lack of binding indicates epitope blocking (competitor). An illustration of the experiment using data obtained for Ab20 (Tie2.20) is provided in Figures 8A and 8B. No data are presented for other anti-Tie2 antibodies.

[0465] The results of the ELISA and Octet binning assay analyses are summarized in Figure 9, which indicates which anti-Tie2 antibodies likely compete with each other and bind to the same or similar epitopes. Specifically, the binding assays described above demonstrated that the phage-derived antibodies Tie2.1 (Ab1), Tie2.12 (Ab12), Tie2.24 (Ab24), and Tie2.33 (Ab33) all bind to the Tie2 IgG2 domain, block Ang1 and Ang2 binding to Tie2, and are Tie2 agonists (e.g., enhance AKT and / or Tie2 phosphorylation and / or increase vascular endothelial membrane integrity).

[0466] Taken together, the results indicate that there are at least three classes of epitopes bound by the anti-Tie2 antibodies described herein. These at least three epitope classes are shown in Figure 9.

[0467] Affinity determination of anti-Tie2 IgG antibodies The monovalent affinity of selected anti-Tie2 hIgG was determined using a Biacore T200 instrument (GE Life Sciences). Anti-human Fc was covalently immobilized onto a Series S CM5 Biacore sensor chip to enable noncovalent 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 experimental format. Between cycles, the surface was regenerated with 3 M MgCl2. Monovalent affinity was 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. [Table 6]

[0468] Example 5 - In vivo generation of anti-Tie2 antibodies In addition to phage display, the generation of anti-Tie2 antibodies that may be useful for therapeutic applications has been achieved by animal immunization.

[0469] 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 based on published literature, see, e.g., Seeber et al., PLoS ONE 9(2), 2014. B cell culture supernatants were assayed for binding to human, rat, and cynomolgus monkey Tie2 and an unrelated control protein by ELISA, and for binding to HUVEC cells by FACS. Tie2-specific B cells were lysed and immediately frozen at -80°C for storage until molecular cloning. The variable regions (VH and VL) of each monoclonal antibody from rabbit B cells were cloned into expression vectors derived from extracted mRNA, as previously described, see, e.g., Seeber et al., PLoS ONE 9(2), 2014. Individual recombinant rabbit antibodies were expressed in Expi 293 cells and subsequently purified with protein A. The purified anti-Tie2 antibodies were subjected to functional activity assays and kinetic screening.

[0470] Rats were immunized in a similar manner, and hybridomas were generated using modified fusion partners (see, e.g., Price et al., J Immunol Methods 31;343(1):28-41 (2009)). Various conditions were optimized to allow for the sorting of individual IgG+huTie2+ hybridomas into single wells, followed by further culture after sorting. The resulting hybridoma supernatants were assayed by ELISA for binding to human, mouse, and cynomolgus monkey Tie2 and an unrelated control protein, and by FACS for binding to HUVEC cells, and positive samples were purified using Protein A for subsequent functional and kinetic characterization.

[0471] Functional screening of rat and rabbit clones using anti-IgG cross-linkers. Antibodies generated from immunization of rats and rabbits as described above were characterized with respect to pAKT induction downstream of Tie2 agonism.

[0472] Cells and antibodies: Human umbilical vein endothelial (HUVEC) cells were purchased from Lonza (catalog number CC-2517; lot number 0000321046), and rat aortic endothelial cells (RAECs) were purchased from VEC Technologies and cultured in growth medium (VEC Technologies, INC, CAT# MCDB-131 10). Anti-Tie2 antibody was prepared at Genentech. Species-specific antibodies used as crosslinkers were purchased from Jackson ImmunoResearch Laboratories Inc.

[0473] Preparation of HUVECs: HUVECs were trypsinized and plated in sterile 96-well plates (Costar catalog number 3997) at 0.4 × 10 5 Cells were seeded in 100 μL of culture medium per well, and the plates were incubated overnight in a 37°C, 5% CO2 incubator. The culture medium was removed, and 100 μL of prewarmed serum starvation medium (Basal Medium EndoGRO™; Catalog No. SCME-BM) was added to each well of the plate. The plates were incubated in a 37°C, 5% CO2 incubator for 3 hours before incubation with Tie-2 agonists.

[0474] 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 in 5% CO. After overnight culture, the cells were starved for 3 hours in EBM2 basal medium containing 0.1% BSA and then incubated with Tie-2 agonists.

[0475] To test the effect of cross-linking anti-Tie2 antibodies, 20 μg / ml of cross-linker in assay buffer (basal medium from the GNE medium preparation facility + 0.2% BSA) was mixed with an equal volume of 60 μg / ml of anti-Tie2 bivalent antibody and incubated at room temperature for 1 hour. After incubation, the antibody was subjected to 3-fold serial dilutions.

[0476] To test other Tie2 agonists, the molecules were first diluted in assay buffer to a high stock concentration (typically 30-1000 μg / ml), followed by serial dilutions (typically 2-10 fold). After removing the serum starvation medium, these dilutions (50 μl) were added to each well, and the plate was incubated for 15 min at 37 °C, 5% CO2. The solution was removed, and 50 μl of lysis buffer containing blocking buffer from the pAKT Ser473 kit (Cisbio, reference number 64 AKSPEH) was added to the cells. The plate was incubated at room temperature for approximately 30-45 min with gentle shaking and then kept in a -80 °C freezer until use, or used directly in the FRET assay.

[0477] FRET assay: Cell lysates (15 μl) were 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., Cat. No. 784080). The plate was incubated at room temperature for 4 hours or in a 4°C refrigerator overnight, and read at 620 nm and 665 nm on a CLARIOstar (BMG LABTECH, software version: 5.01 R2). Data were calculated as the ratio of acceptor and donor emission signals × 10 for each individual well. 4 It was calculated as:

[0478] Generation of recombinant antibodies DNA encoding the antibody heavy and light chain variable domains was generated by gene synthesis and inserted into mammalian vectors for expression of the heavy and light chains of the IgG1 antibody, respectively. Some variable domain sequences were edited to remove obvious unpaired cysteine ​​residues and NX[S / T]N-glycosylation motifs. Recombinant antibodies were produced by transient transfection of Expi293 cells with mammalian expression vectors encoding the antibody heavy and light chains. 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. The antibodies were purified from cell culture supernatants by affinity chromatography. In some cases, the antibodies underwent an additional purification step based on SEC.

[0479] Antibodies were screened for binding to recombinant human and cynomolgus monkey Tie2 using a Biacore T200 instrument (GE Life Sciences). Briefly, human antibody capture chips were prepared using a Series S 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 monkey 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 3 M MgCl2 injected for 30 seconds at 30 μl / min. 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 0. Selected antibodies (Tables 7A-7B; ch indicates rabbit Ab; TEK indicates rat Ab) were advanced for secondary activity screening in the Fab-NDK format (see Example 8 below). [Table 7A] [Table 7B]

[0480] HTP epitope binning Antibodies generated by animal immunization and selected phage-derived antibodies described in Example 1 were reformatted into a hIgG1 backbone, and binning was performed using a CFM 2 / MX 96 SPR system (Wasatch Microfluidics, now Carterra) equipped with DAv6.19.3, IBIS SUIT, SprintX, and Carterra Epitope Tool software. Antibodies were immobilized on an SPR sensor prism CMD 200M (Xantec Bioanalytics) by amine coupling using a 10 mM sodium acetate pH 4.5 immobilization buffer. Immobilization was performed using a CFM 2 instrument, and the sensor prism was then transferred to an IBIS MX96 instrument for SPR-based competitive analysis. The immobilized antibodies were first exposed to 1 μM recombinant human Tie2 extracellular domain and then to 20 μg / ml of antibody in solution using HBS-EP running buffer (10 mM HEPES, 150 mM NaCl, 0.05% Tween 20, pH 7.4, 1 mM EDTA). The results are shown in Figures 11A-11B and demonstrate several distinct Tie2 sandwich profiles. The results in Figures 11A-11B are typically interpreted as demonstrating the presence of multiple (e.g., at least 8) distinct Tie2 epitopes within the analyzed antibody panel. Interestingly, the data indicate that Tie2.1, Tie2.1M100cF (described below in Example 11), Tie2.12, and Tie2.24 can all function as Tie2 agonists and binned together.

[0481] Example 6 - Generation of PEG-conjugated anti-Tie2 Fab multimers As shown above in Example 3, activation of Tie2 by anti-Tie2 antibodies is facilitated by cross-linking of bound anti-Tie2 antibodies. Therefore, multimeric Tie2 binding compositions were designed and generated to determine the multimeric configuration with optimal therapeutic efficacy.

[0482] One multimerization method used was the use of a multi-arm polyethylene glycol (PEG) molecule as a core, with each arm linked or conjugated to a single anti-Tie2 Fab molecule. Various multimer configurations were tested using Tie2.1 Fab, which has been shown to specifically bind to Tie2 with high affinity and function as a Tie2 agonist (activating AKT phosphorylation upon interaction with Tie2). Specifically, the C-terminus of the heavy chain of Tie2.1 Fab was modified to contain the amino acid residue SPPC (SEQ ID NO: 89) to provide a linker and cysteine ​​to which a PEG moiety could be conjugated. This "Tie2.1-SPPC" Fab was conjugated to a PEG-maleimide backbone with various numbers of arms and arm lengths (multi-arm PEG-maleimide was obtained from JenKem Technology USA, Plano, TX).

[0483] Fab purification and deblocking All chromatography resins were supplied by GE Healthcare.

[0484] Fab was expressed in E. coli. The E. coli pellet was resuspended at 1.5 L / kg in 25 mM TrisHCl, 150 mM NaCl, 5 mM EDTA, pH 7.5 (EQ) 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 minutes, and the supernatant was 0.22 μm filtered. The E. coli filtrate was then loaded onto a 1.2 L Gammabind Plus (GBP) column equilibrated with EQ at 30 ml / min, followed by washing with 2 CV of EQ containing 0.1% TX114 and 0.1% TX100 at 30 ml / min and 4 CV of EQ at 5 ml / min. This was followed by elution with 2 CV of EQ at 30 ml / min, then 2 CV of 25 mM succinate pH 6.0 at 30 ml / min and 0.15 M acetic acid at 30 ml / min. The eluate was neutralized from the column with 1 M Tris pH 9.0 to pH 5.0.

[0485] Alternatively, the E. coli filtrate was loaded onto a 1.7 L Capto L column equilibrated with EQ at 35 ml / min, then washed with 2 CV EQ at 50 ml / min, washed with 5 CV EQ containing 0.1% (v / v) Triton X114 and 0.1% (v / v) Triton X100 at 4 ml / min, then washed with 2 CV EQ at 50 ml / min, then washed with 25 mM succinate pH 6.0 at 50 ml / min, and eluted with 0.15 M acetic acid at 50 ml / min. The eluate was neutralized from the column with 1 M Tris pH 9.0 to pH 5.0.

[0486] The neutralized GBP or Capto L eluate was diluted 1-3 times with 20 mM sodium acetate, pH 5.0 (A) and loaded onto a cation exchange resin (SPHP). The column was washed with 2 CV (A) containing 0.1% Triton X114 and 0.1% Triton X100, followed by 5 CV (A) and 2 CV (A). It was then gradient eluted with a 10 CV gradient of 0-20% (A) containing 1 M NaCl (B), with fraction collection. Fractions containing the Fab peak were pooled.

[0487] The purified Fab pool was adjusted to pH 8.0 with 1 M Tris pH 8.5 and EDTA was added to a final concentration of 2 mM. To reduce the Fab, a 50-fold molar excess of DTT was added to the solution, which was then incubated overnight at 22°C and additive removal was confirmed by mass spectrometry.

[0488] 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 reoxidation proceeded using a 15-fold molar excess of DHAA. After 1.5 h, the Fab was checked for reoxidation by mass spectrometry; if necessary, an additional 10-fold molar excess of DHAA was added, and the sample was incubated for 1 h and retested. The reoxidized Fab was then brought to pH 5 with 10% acetic acid and purified on an SPHP column as above, but eluted with a 20 CV 10-60% gradient (B = 25 mM sodium acetate, 300 mM sodium chloride, pH 5.0); the major peak was concentrated to 10 kDa and 0.2 μm filtered. Other Fabs can be purified and deblocked in essentially the same way.

[0489] Hexameric conjugates and purification Tie2.1-SPPC was conjugated to a 6KDa PEG hexamer (JenKem) at a concentration of approximately 10 mg / mL in 25 mM NaOAC, pH 5.0, 150 mM NaCl, and 2 mM EDTA. After equilibration to room temperature, the 6KDa PEG hexamer (JenKem) was resuspended in 25 mM N-acetate (pH 5.0) to a concentration of 3 mM. The pH was maintained below pH 6 to avoid maleimide ring-opening. PEG was solubilized and added to the Tie2.1-SPPC deblocked Fab at a molar ratio of 9:1 (Fab:PEG). The mixture was then left overnight at room temperature with gentle stirring. After conjugation, the Tie2.1 Fab-PEG hexamer was purified using size-exclusion chromatography (SEC) on a Superdex-200 column in 20 mM His-acetate, pH 5.5, and 150 mM NaCl. This purification step removed excess Fab and aggregates from the conjugate mixture. Reducing and non-reducing NuPage 4-12% Bis-Tris gels were run to determine which conjugate fractions to further purify and 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, enriching for the six Fab / PEGs. The CEX step was performed in 25 mM sodium acetate, pH 5.0, and eluted using a 20% to 33.4% gradient in Buffer B (Buffer B is 25 mM NaOAC, pH 5.0, 300 mM NaCl) over 44.5 CV, followed by a 33.4% to 50% B gradient in 10 min, and finally elution at 100% B. A few different minipools of different fractions were run on the gel as above, and the data was used to determine which fractions to pool. Minipool 3 was pooled and then formulated at 40 mg / mL in PBS, pH 7.2.

[0490] The final sample was then run on analytical SEC using a TSKgel G 3000 SW x1 column in 0.2 M KPO, 0.25 M KCl, pH 6.2, 15% isopropyl alcohol as buffer to determine the percentage of aggregate present, which was also run on a gel as previously described.

[0491] FabIgG purification CHO conditioned medium was purified on a MabSelect Sure affinity column (GE Healthcare) followed by size-exclusion chromatography (SEC) on a S200 column. Alternatively, the affinity elution was diluted and loaded onto a cation exchange column (SPHP), which was then washed and elut...

Claims

1. A pharmaceutical composition for treating a Tie2 pathway-mediated disorder, comprising a conjugate that binds to Tie2, the conjugate comprises 2, 3, 4, 5, 6, 7, or 8 antibodies or antigen-binding fragments thereof conjugated to a multimerization moiety; each of the antibodies or antigen-binding fragments thereof binds to Tie2; and Each of the antibodies or antigen-binding fragments thereof comprises a heavy chain variable domain (VH) comprising (a) a CDR-H1 comprising the amino acid sequence NTDIS (SEQ ID NO: 3), (b) a CDR-H2 comprising the amino acid sequence RISPSDGNTYYADSVKG (SEQ ID NO: 4), and (c) a CDR-H3 comprising the amino acid sequence RTRWASWAFDY (SEQ ID NO: 7), and a light chain variable domain (VL) comprising (d) a CDR-L1 comprising the amino acid sequence RASQDVSTAVA (SEQ ID NO: 8), (e) a CDR-L2 comprising the amino acid sequence SASFLYS (SEQ ID NO: 9), and (f) a CDR-L3 comprising the amino acid sequence QQSYTTPPT (SEQ ID NO: 10); Pharmaceutical compositions.

2. The multimerization moiety comprises a polyol, 2. The pharmaceutical composition of claim 1, wherein the polyol is a multi-arm polyol selected from a dimer, a tetramer, a hexamer, and an octamer.

3. The pharmaceutical composition described in claim 2, wherein the multi-arm polyol is a hexamer.

4. The pharmaceutical composition of claim 2, wherein the multi-arm polyol is a multi-arm polyethylene glycol (PEG).

5. The method of claim 1, wherein each of the antibodies or antigen-binding fragments thereof is a Fab, and the polyol is conjugated to the Fab via the free sulfhydryl group of an engineered cysteine ​​amino acid; the engineered cysteines are selected from the group consisting of T120C, G166C, G178C, T187C, and T209C in the heavy chain (HC); or the engineered cysteines are selected from the group consisting of Q124C, R142C, Q155C, L201C, T206C, K107C, K126C, and K149C in the light chain (LC); The pharmaceutical composition according to any one of claims 2 to 4, wherein residue numbers are according to EU numbering.

6. The pharmaceutical composition described in claim 5, wherein the engineered cysteine ​​is T209C in the heavy chain (HC) and the residue number is according to EU numbering.

7. The pharmaceutical composition of claim 4, wherein the multi-arm PEG has a weight average molecular weight of about 500 Daltons (Da) to about 300,000 Da, and optionally, the multi-arm PEG has a weight average molecular weight of about 1,000 Da to about 10,000 Da, and optionally, the multi-arm PEG has a weight average molecular weight of about 6,000 Da.

8. The multi-arm PEG is represented by the general formula (Ib): wherein each m is independently an integer from 3 to 250; each R 1 is independently either absent or a linking group; each R 2 is independently either hydrogen or a terminal reactive group; and at least one R 2 is a terminal reactive group and is conjugated to an antibody or antigen-binding fragment thereof. The pharmaceutical composition according to any one of claims 4 to 7, having the structure:

9. The pharmaceutical composition of claim 8, wherein each m is independently an integer from 10 to 30.

10. At least one R 1 is a linking group, and R 1 and R 2 together form (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 amino-reactive group, and combinations thereof); and combinations thereof.

11. The pharmaceutical composition according to any one of claims 8 to 10, wherein each R 2 is maleimide.

12. A pharmaceutical composition described in any one of claims 1 to 11, wherein the VH of each of the antibodies or antigen-binding fragments thereof comprises the sequence of SEQ ID NO: 20, and the VL of each of the antibodies or antigen-binding fragments thereof comprises the sequence of SEQ ID NO:

21.

13. The pharmaceutical composition of any one of claims 1 to 12, wherein each of the antibodies or antigen-binding fragments thereof is a Fab comprising a Fab heavy chain (HC) sequence of SEQ ID NO:55 and a Fab light chain (LC) sequence of SEQ ID NO:

25.

14. A pharmaceutical composition for treating a Tie2 pathway-mediated disorder, comprising a conjugate that binds to Tie2, the conjugate comprises a Fab that binds to Tie2, the Fab comprising a heavy chain (HC) having SEQ ID NO: 55 and a light chain (LC) having SEQ ID NO: 25; and Fab has the general formula (Ib): wherein each m is independently an integer from 10 to 30, and R 1 and R 2 together form the structure wherein R 2 is maleimide and the polyol is conjugated to the Fab heavy chain (HC) at residue C209 (EU numbering), Pharmaceutical compositions.

15. A pharmaceutical composition for treating a Tie2 pathway-mediated disorder, comprising a conjugate that binds to Tie2, the conjugate comprises six Fabs that bind to Tie2, Each of the six Fabs is conjugated to a multi-arm polyethylene glycol (PEG), and Each of the six Fabs comprises a heavy chain variable domain (VH) comprising (a) a CDR-H1 comprising the amino acid sequence NTDIS (SEQ ID NO: 3), (b) a CDR-H2 comprising the amino acid sequence RISPSDGNTYYADSVKG (SEQ ID NO: 4), and (c) a CDR-H3 comprising the amino acid sequence RTRWASWAFDY (SEQ ID NO: 7), and a light chain variable domain (VL) comprising (d) a CDR-L1 comprising the amino acid sequence RASQDVSTAVA (SEQ ID NO: 8), (e) a CDR-L2 comprising the amino acid sequence SASFLYS (SEQ ID NO: 9), and (f) a CDR-L3 comprising the amino acid sequence QQSYTTPPT (SEQ ID NO: 10); Pharmaceutical compositions.

16. Each of the six Fabs is conjugated to a multi-arm PEG via the free sulfhydryl group of an engineered cysteine ​​amino acid, wherein the engineered cysteine ​​is selected from the group consisting of T120C, G166C, G178C, T187C and T209C in the Fab heavy chain (HC); or the engineered cysteine ​​is selected from the group consisting of Q124C, R142C, Q155C, L201C, T206C, K107C, K126C and K149C in the Fab light chain (LC); Residue numbers follow EU numbering.

16. The pharmaceutical composition of claim 15.

17. The pharmaceutical composition described in claim 16, wherein the engineered cysteine ​​is T209C in the heavy chain (HC) and the residue number is according to EU numbering.

18. The pharmaceutical composition of any one of claims 15 to 17, wherein the multi-arm PEG has a weight average molecular weight of about 500 Daltons (Da) to about 300,000 Da, optionally, the multi-arm PEG has a weight average molecular weight of about 1,000 Da to about 10,000 Da, and optionally, the multi-arm PEG has a weight average molecular weight of about 6,000 Da.

19. The multi-arm PEG of claim 18, wherein the multi-arm PEG is represented by the general formula (Ib): wherein each m is independently an integer from 3 to 250; each R 1 is independently either absent or a linking group; each R 2 is independently either hydrogen or a terminal reactive group; At least one R 2 is a terminal reactive group. The pharmaceutical composition according to any one of claims 15 to 18, having the structure:

20. The pharmaceutical composition of claim 19, wherein each R 2 is a terminal reactive group.

21. The pharmaceutical composition of claim 20, wherein each R 2 is maleimide.

22. The VH of each of the six Fabs comprises the sequence of SEQ ID NO: 20; The pharmaceutical composition according to any one of claims 15 to 21, wherein the VL of each of the six Fabs comprises the sequence of SEQ ID NO:

21.

23. The method of claim 22, wherein the Fab heavy chain (HC) of each of the six Fabs comprises the sequence of SEQ ID NO: 55; The Fab light chain (LC) of each of the six Fabs comprises the sequence of SEQ ID NO:

25. The pharmaceutical composition according to any one of claims 15 to 22.

24. A pharmaceutical composition for treating a Tie2 pathway-mediated disorder, comprising a conjugate that binds to Tie2, the conjugate comprises six Fabs that bind to Tie2, Each of the six Fabs is conjugated to a multi-arm polyethylene glycol (PEG), Each of the six Fabs comprises a Fab heavy chain (HC) comprising the sequence of SEQ ID NO: 55 and a Fab light chain (LC) comprising the sequence of SEQ ID NO: 25; and The multi-arm PEG has the general formula (Ib): wherein each m is independently an integer from 10 to 30; R 1 and R 2 together form the structure and i and j are 2, R 2 is maleimide, each of the maleimides being conjugated to one of six Fabs via the free sulfhydryl group of an engineered cysteine ​​amino acid T209C (EU numbering) of the Fab heavy chain (HC), Pharmaceutical compositions.

25. A pharmaceutical composition according to any one of claims 1 to 24, further comprising a pharmaceutically acceptable carrier.

26. A pharmaceutical composition according to any one of claims 1 to 25, wherein the conjugate is contained within a delivery device for ocular delivery.

27. A pharmaceutical composition for treating a Tie2 pathway-mediated disorder, comprising a conjugate that binds to Tie2, The conjugate has the formula: wherein each n is an integer from 10 to 30; each Fab comprises a Fab heavy chain (HC) comprising the sequence of SEQ ID NO:55 and a Fab light chain (LC) comprising the sequence of SEQ ID NO:25; each Fab heavy chain (HC) comprises a cysteine ​​residue at amino acid residue number 209 (C209), where the amino acid residue numbers are according to EU numbering and the S atom linked to the succinimide moiety is from the thiol group at C209; Pharmaceutical compositions.

28. Structure 28. The pharmaceutical composition of claim 27, wherein the weight average molecular weight of the portion of the conjugate lacking the Fab portion having

29. Structure 29. The pharmaceutical composition of claim 28, wherein the weight average molecular weight of the portion of the conjugate lacking the Fab portion having 30. A pharmaceutical composition for treating a Tie2 pathway-mediated disorder, comprising a conjugate that binds to Tie2, The conjugate has the general formula (Ib): wherein each m is independently an integer from 10 to 30; Each R 1 and R 2 taken together represents the structure and each i and j is 2; each R 2 is a maleimide group), each of the Fabs comprises a Fab heavy chain (HC) comprising the sequence of SEQ ID NO:55 and a Fab light chain (LC) comprising the sequence of SEQ ID NO:25; each of the Fab heavy chains (HC) comprises a cysteine ​​residue at amino acid residue number 209 (C209); Amino acid residue numbers are based on EU numbering, conjugating comprises reacting the C209 residue of each of the six Fab heavy chains (HC) with R2 of the hexameric molecule; Pharmaceutical compositions.

31. The pharmaceutical composition described in claim 30, wherein the weight average molecular weight of the hexameric molecule is about 1,000 to about 10,000 daltons.

32. The pharmaceutical composition of claim 30, wherein the weight-average molecular weight of the hexameric molecule is about 6,000 daltons.

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