TIE2 binder and method of use

Abstract

This invention provides Tie-2 antibodies, their fragments, and conjugates, as well as methods for using them. [Solution] An isolated antibody or antigen-binding fragment thereof that specifically binds to Tie2, comprising a heavy chain variable domain in which 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 in which CDR-L1 is RASQDVSTAVA (SEQ ID NO: 8), CDR-L2 is SASFLYS (SEQ ID NO: 9), and CDR-L3 is QQSYTTPPT (SEQ ID NO: 10).

Description

Cross-reference of related applications

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

[0002] Sequence List This application includes a sequence listing submitted electronically in ASCII format, the entirety of which is incorporated herein by reference. The ASCII copy, created on March 17, 2021, is named P35891-WO_SeqList.txt and has a size of 107,354 bytes. [Technical Field]

[0003] Field of Invention The subject matter of this disclosure is Tie2 conjugates and anti-Tie2 antibodies, as well as methods of using them. [Background technology]

[0004] background Tie2 is a promising therapeutic target for the treatment of various eye disorders (see, for example, Campochiaro and Peters, 2016, Curr Diab Rep, 16:126, Whitehead et al., 2019, J Diabetes Res, 2019:5140521, Hussain et al., 2019, Expert Opin Investig Drug, 28:861-869). Tie2 is a receptor tyrosine kinase 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 eye 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 disruption of the blood-retinal barrier (see, e.g., Nambu et al., 2004, Gene Therapy, 11:865-873). Ang2 is a context-dependent antagonist of Tie2, and its expression is increased in association with several ocular disorders, including DME, exudative AMD, DR, metastasis, sepsis, and inflammation. In particular, Ang2 competitively binds to Tie2, inhibiting Ang1 signaling and leading to endothelial and vascular destabilization, disruption of the blood-retinal barrier, and inflammation (Klaassen et al., 2013, Prog Retin Eye Res, 34:19-48, Saharinen et al., 2017, Nat Rev Drug Discov, 16:635-661).

[0006] The Tie receptor, including Tie1 and Tie2, is a type 1 transmembrane protein receptor tyrosine kinase (RTK) (Ramsauer, M. & D'Amore, PAJClin.Invest. (2002); 110:1615-1617). Tie represents a tyrosine kinase receptor with immunoglobulin and EGF homologous domains. Tie2 is located in the endothelial cells of all forming blood vessels and in the endocardium of mouse embryos (Korhonen et al., Blood (1992); 80:2548-2555). The external 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 region 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, angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2), have been identified. Ang1, a Tie2 agonist, binds to and induces tyrosine phosphorylation of Tie2, and its in vivo expression is associated with proximity to developing blood vessels (Davis et al., Cell (1996); 87:1161-1169). Mice lacking Ang1 exhibit angiogenesis defects very similar to those seen in mice lacking Tie2, supporting the idea that Ang1 is the primary physiological ligand for Tie2 and that Tie2 has important in vivo angiogenic activity (Suri et al., Cell (1996); 87:1171-1180). Ang1 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 and can disrupt EC quiescence and increase vascular permeability. Together, studies support that the Ang1 / Ang2 / Tie2 system plays a crucial role 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 increase vascular integrity have great potential as therapeutic agents, particularly for the treatment of ocular disorders. [Overview of the project]

[0008] overview The present invention provides an anti-Tie2 antibody, a composition (e.g., a conjugate) comprising an anti-Tie2 antibody or a fragment thereof, and methods for using the same.

[0009] The subject matter of this disclosure provides isolated antibodies or antigen-binding fragments that specifically bind to Tie2, compositions comprising at least one anti-Tie2 antibody or antigen-binding fragment, and methods for using the same. In exemplary embodiments, the anti-Tie2 antibody is Fab.

[0010] In one aspect, an antibody or antigen-binding fragment that specifically binds to Tie2 is provided, where the anti-Tie2 antibody is CDR-H1 containing the amino acid sequence NTDIS (SEQ ID NO: 3), CDR-H2 containing the amino acid sequence RISPSDGNTYYADSVKG (SEQ ID NO: 4), and amino acid sequence (a)RTRWASX1AX2DY (SEQ ID NO: 5, 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). The CDR-H3 comprises a heavy chain (HC) variable domain (VH domain) including (b)RTRWASWAMDY (SEQ ID NO: 6), or (c)RTRWASWAFDY (SEQ ID NO: 7), and a light chain (LC) variable domain (VL domain) including CDR-L1 containing the amino acid sequence RASQDVSTAVA (SEQ ID NO: 8), CDR-L2 containing the amino acid sequence SASFLYS (SEQ ID NO: 9), and CDR-L3 containing the amino acid sequence QQSYTTPPT (SEQ ID NO: 10). In some embodiments, CDR-H3 comprises SEQ ID NO: 6 or SEQ ID NO: 7. In certain embodiments, CDR-H3 comprises SEQ ID NO: 7.

[0011] In some embodiments, the anti-Tie2 antibody comprises the VH framework FR1 sequence of SEQ ID NO: 11, the VH framework FR2 sequence of SEQ ID NO: 12, the VH framework FR3 sequence of SEQ ID NO: 13, and / or the VH framework FR4 sequence of SEQ ID NO: 14. In other embodiments, the anti-Tie2 antibody comprises the VL framework FR1 sequence of SEQ ID NO: 15, the VL framework FR2 sequence of SEQ ID NO: 16, the VL framework FR3 sequence of SEQ ID NO: 17, and / or the VL framework FR3 sequence of SEQ ID NO: 18.

[0012] In some embodiments, the anti-Tie2 antibody comprises a VH domain comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 3, CDR-H2 containing the amino acid sequence of SEQ ID NO: 4, and a VL domain comprising CDR-H3 containing the amino acid sequence of SEQ ID NO: 5, (b) SEQ ID NO: 6, or (c) SEQ ID NO: 7, where (a) 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 CDR-L1 containing the amino acid sequence of SEQ ID NO: 8, CDR-L2 containing the amino acid sequence of SEQ ID NO: 9, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 10. In other embodiments, CDR-H3 comprises SEQ ID NO: 6 or SEQ ID NO: 7. In certain embodiments, CDR-H3 comprises SEQ ID NO: 7. In yet another embodiment, 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, an anti-Tie2 antibody or its antigen-binding fragment 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 contains the VH domain sequence of SEQ ID NO: 20. In other embodiments, the anti-Tie2 antibody contains the VL domain sequence of SEQ ID NO: 21. In other embodiments, the anti-Tie2 antibody contains 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 includes 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 includes 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 embodiments, an antibody or antigen-binding fragment that specifically binds to Tie2 is provided, comprising a VH domain comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 28, CDR-H2 containing the amino acid sequence of SEQ ID NO: 29, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 30, and a VL domain comprising CDR-L1 containing the amino acid sequence of SEQ ID NO: 8, CDR-L2 containing the amino acid sequence of SEQ ID NO: 9, and CDR-L3 containing 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 with SEQ ID NO: 31, and the VL domain comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 21. In yet another embodiment, the VH domain comprises SEQ ID NO: 31, and the VL domain comprises SEQ ID NO: 21. In yet another embodiment, HC comprises SEQ ID NO: 32, and LC comprises SEQ ID NO: 25.

[0017] In some embodiments, an antibody or antigen-binding fragment that specifically binds to Tie2 is provided, comprising a VH domain comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 33, CDR-H2 containing the amino acid sequence of SEQ ID NO: 34, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 35, and a VL domain comprising CDR-L1 containing the amino acid sequence of SEQ ID NO: 8, CDR-L2 containing the amino acid sequence of SEQ ID NO: 9, and CDR-L3 containing 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 with SEQ ID NO: 36, and the VL domain comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 21. In yet another embodiment, the VH domain comprises SEQ ID NO: 36, and the VL domain comprises SEQ ID NO: 21. In yet another embodiment, HC comprises SEQ ID NO: 37, and LC comprises SEQ ID NO: 25.

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

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

[0020] In some aspects, an antibody is provided that specifically binds to Tie2 or its antigen-binding fragment, comprising two VH domains containing CDR-H1 containing SEQ ID NO: 6, CDR-H2 containing SEQ ID NO: 8, CDR-H3 containing SEQ ID NO: 9, CDR-H1 containing SEQ ID NO: 48, CDR-H2 containing SEQ ID NO: 49, and CDR-H3 containing SEQ ID NO: 50, in the direction from the N-terminus to the C-terminus, and two VL domains containing CDR-L1 containing the amino acid sequence of SEQ ID NO: 8, CDR-L2 containing the amino acid sequence of SEQ ID NO: 9, CDR-L3 containing the amino acid sequence of SEQ ID NO: 10, CDR-L1 containing the amino acid sequence of SEQ ID NO: 8, CDR-L2 containing the amino acid sequence of SEQ ID NO: 9, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 10, in the direction from the N-terminus to the C-terminus. In some embodiments, HC contains SEQ ID NO: 54, and LC contains SEQ ID NO: 53.

[0021] In some embodiments, the anti-Tie2 antibody or its antigen-binding fragment comprises an engineered cysteine, which is located in the HC constant domain and / or 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, with residue numbers following EU numbering. In other embodiments, the anti-Tie2 antibody or its antigen-binding fragment is a Fab whose HC is terminated with the amino acid CDKTHTSPPC (SEQ ID NO: 83). In some embodiments, the Fab is terminated 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 antibody or a 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 a fragment thereof binds to Tie2, where Tie2 is a protein having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 1.

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

[0027] In some embodiments, the anti-Tie2 antibody is an antibody or Fab that competes with the 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 does not bind to the Ig1 domain of Tie2. In other embodiments, the anti-Tie2 antibody or fragment does not bind to the EGF domain of Tie2. In yet another embodiment, the anti-Tie2 antibody or fragment does not bind to the Ig3 domain of Tie2. In yet another embodiment, the anti-Tie2 antibody or fragment does not bind to the FNIII domain of Tie2.

[0029] In some embodiments, the antibody or its antigen-binding fragment binds to cynomolgus monkey Tie2. In other embodiments, 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 that binds to Tie2 is a multispecific antibody. In other embodiments, the multispecific antibody binds to Tie2 and VEGF. In yet another embodiment, the multispecific antibody binds to Tie2 and factor D. In yet another embodiment, 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 can activate Tie2. In yet another embodiment, the multispecific antibody binds to Tie2 and factor D, and the multispecific antibody can activate Tie2. In yet another embodiment, the multispecific antibody binds to Tie2 and Ang2, and the multispecific antibody can activate Tie2.

[0032] In one aspect, isolated nucleic acids encoding an anti-Tie2 antibody or its antigen-binding fragment are provided.

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

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

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

[0036] In one aspect, embodiments disclosed herein provide a conjugate comprising at least two antibodies that specifically bind to Tie2 or its antigen-binding fragment, and a multi-armed portion. In a preferred embodiment, each of the at least two anti-Tie2 antibodies is a Fab.

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

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

[0039] In some embodiments, a 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 yet another embodiment, a Tie2 conjugate activates Tie2 phosphorylation. In yet another embodiment, the activation of Tie2 phosphorylation is measured in vitro.

[0040] In some embodiments, exposure of Tie2-expressing cells to the conjugate does not reduce the Tie2 protein level in the cells. In other embodiments, exposure does not reduce the Tie2 protein level in the cells by more than 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, or 75%. In other embodiments, exposure reduces the Tie2 protein level in the cells by less than 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, or 75%. In yet another embodiment, exposure reduces the Tie2 protein level 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 blotting before and after in vitro incubation of Tie2-expressing cells with the Tie2 conjugate. In other embodiments, incubation is 10–36 hours or 12–24 hours at 37°C.

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

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

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

[0044] In some embodiments, the conjugate promotes vascular stability and / or enhances 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-armed polyol is selected from dimers, tetramers, hexamers, and octamers. In preferred embodiments, the multi-armed polyol is a hexamer or an octamer. In more preferred embodiments, the multi-armed 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 yet another embodiment, the polyol is polyethylene glycol (PEG). In some embodiments, the PEG is a functionalized multi-arm PEG. In some embodiments, PEG is based on the general formula (Ia): JPEG2026053356000001.jpg42170 (wherein each m represents the length or size of a particular arm of polyol (PEG), independently an integer between approximately 45 and approximately 1000, approximately 20 and approximately 1000, approximately 10 and approximately 1000, approximately 3 and approximately 250, approximately 3 and approximately 200, approximately 3 and approximately 100, approximately 10 and approximately 50, approximately 10 and approximately 30, approximately 20 and approximately 30, approximately 50 and approximately 200, or approximately 100 and approximately 150, and n is an integer between approximately 1 and approximately 10; each R 1 Each R either does not exist independently or is a linking group, 2 Each is independently either a hydrogen atom or a terminal reactive group, and at least one R 2 It has the structure (where is a terminal reactive group). In some embodiments, R 2 These are independently selected from thiol-reactive groups, amino-reactive groups, and combinations thereof.

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

[0051] In some embodiments, PEG has a structure of general formula (Ia), n is 1, and the multi-arm PEG is a tetramer. In some embodiments, 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 general formula (Ib): JPEG2026053356000002.jpg46170(where 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 each R 1 is either independently absent or a linking group, and each R 2 is independently either hydrogen or a terminal reactive group, where at least one R 2 is a terminal reactive group and is covalently bound to the anti-Tie2 antibody fragment or Fab as described above). In some embodiments, each m is independently an integer of about 15 to 35 or about 20 to 30. In other embodiments, each m is independently an integer of about 22.

[0052] In some embodiments, R 1 and R 2 together have the structure JPEG2026053356000003.jpg15170where 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): JPEG2026053356000004.jpg46170 (In the formula, each m is an integer between 3 and 250, and each R 1 Each R is either independently absent or a linking group, 2 Each is independently either a hydrogen atom or a terminal reactive group, and at least one R 2 The structure has a terminal reactive group that is covalently bound to the anti-Tie2 antibody described above. In some embodiments, each m is an integer between 15 and 35 independently. In other embodiments, each m is an integer of approximately 22 independently.

[0054] In some embodiments, at least two anti-Tie2 antibody fragments or Fab described herein are covalently bound to a multi-armed polyol. In other embodiments, the conjugated multi-armed polyol is covalently bound to at least two anti-Tie2 antibody fragments or Fab via free sulfhydryl groups of cysteine ​​amino acids. In other embodiments, the cysteine ​​amino acid is engineered cysteine. In yet another embodiment, the cysteine ​​amino acid is located in the anti-Tie2 constant domain. In another embodiment, the cysteine ​​amino acid is located at the C-terminus of the heavy chain (HC) or light chain (LC) of the anti-Tie2 Fab. In a preferred embodiment, 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 its antigen-binding fragment containing manipulated cysteine ​​in its HC and / or LC. In other embodiments, the manipulated cysteine ​​is selected from T120C, G166C, G178C, T187C and T209C in the HC, or the manipulated cysteine ​​is selected from Q124C, R142C, Q155C, L201C, T206C, K107C, K126C and K149C in the LC, and the residue number of the manipulated cysteine ​​follows EU numbering.

[0056] In some embodiments, the conjugate comprises a multi-armed polyol covalently bound to at least two anti-Tie2 antibody fragments or Fab via the free amino group of the lysine amino acid. In other embodiments, the lysine amino acid is located in the constant region of the anti-Tie2 antibody fragment or Fab. In yet another embodiment, the lysine amino acid is located at the C-terminus of the heavy or light chain of the anti-Tie2 antibody fragment or Fab. In an alternative embodiment, the Tie2 binder is not covalently bound to at least two anti-Tie2 Fab via the free amino group of lysine.

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

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

[0059] In one embodiment, a conjugate is provided comprising an anti-Tie2 antibody or its antigen-binding fragment and a multi-armed portion, the multi-armed portion comprising an IgM molecule. In another embodiment, the IgM molecule comprises a J chain, and the multi-armed portion comprises five anti-Tie2 Fabs, each of the five anti-Tie2 Fabs being linked to an IgM molecule. In yet another embodiment, the IgM molecule does not comprise a J chain, and the multi-armed portion comprises six anti-Tie2 Fabs, each of the six anti-Tie2 Fabs being linked to an IgM molecule.

[0060] In some embodiments, the conjugate comprises an IgM molecule in which the IgM variant has amino acid substitutions that reduce or eliminate complement-dependent cell-mediated cytotoxicity (CDC) activity. In preferred embodiments, the IgM variant comprises substitution P436G based on EU numbering.

[0061] In one embodiment, a conjugate is provided comprising an anti-Tie2 antibody or its antigen-binding fragment and a multi-armed moiety, the multi-armed moiety comprising at least 2, 4, 6, 8, or 10 peptides, approximately each of which peptides is covalently bound to the anti-Tie2 Fab. In some embodiments, the anti-Tie2 Fab sequence is terminated at residues 221, 222, 223, 224, or 225 (EU numbering). In other embodiments, each peptide is a nucleoside diphosphate kinase (NDK) peptide. In yet another embodiment, each NDK peptide comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 71.

[0062] In some embodiments, each NDK peptide is ligated 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 yet another embodiment, the linker is an amino acid linker. In yet another embodiment, the amino acid linker contains 2, 3, 4, 5, 6, 7, 8, 2-20, 5-10, or 4-10 amino acids. In yet another embodiment, the linker contains glycine.

[0063] In some embodiments, the multi-arm portion contains 6 or 8 peptides. In other embodiments, the multi-arm portion contains 6 peptides. In preferred embodiments, the multi-arm portion contains 6 NDK peptides.

[0064] In one embodiment, a conjugate is provided comprising an antibody or its antigen-binding fragment and a multi-armed portion, the multi-armed portion comprising an IgM molecule. In another embodiment, the IgM molecule comprises a J chain, and the multi-armed portion comprises five antibody fragments, Fabs, or their antigen-binding fragments, with substantially each of the five antibody fragments, Fabs, or their antigen-binding fragments being linked to the IgM molecule. In yet another embodiment, the IgM molecule does not comprise a J chain, and the multi-armed portion comprises six antibody fragments, Fabs, or their antigen-binding fragments, with each of the six antibody fragments, Fabs, or their antigen-binding fragments being linked to the IgM molecule.

[0065] In some embodiments, the conjugate comprises an IgM molecule in which the IgM variant has amino acid substitutions that reduce or eliminate complement-dependent cell-mediated cytotoxicity (CDC) activity. In preferred embodiments, the IgM variant comprises substitution P436G based on EU numbering.

[0066] In one embodiment, a conjugate is provided comprising an antibody, an antibody fragment, Fab or its antigen-binding fragment, and a multi-armed portion, the multi-armed portion comprising at least 2, 4, 6, 8, or 10 peptides, approximately each of the peptides covalently bound to the anti-Tie2 antibody, antibody fragment, Fab or its antigen-binding fragment. In some embodiments, the sequence of the antibody, antibody fragment, Fab, or its antigen-binding fragment is terminated at residues 221, 222, 223, 224, or 225 (EU numbering). In other embodiments, each peptide is a nucleoside diphosphate kinase (NDK) peptide. In yet another embodiment, each NDK peptide comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 71.

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

[0068] In some embodiments, the multi-arm portion contains 6 or 8 peptides. In other embodiments, the multi-arm portion contains 6 peptides. In preferred embodiments, the multi-arm portion contains 6 NDK peptides.

[0069] In one aspect, a pharmaceutical composition is provided comprising an anti-Tie2 antibody or a 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 conjugate comprising a plurality of anti-Tie2 Fabs linked to the multi-arm portion described herein, and a pharmaceutically acceptable carrier.

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

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

[0072] In some embodiments, any of the aforementioned pharmaceutical compositions can be used in the manufacture of a pharmaceutical for treating a target eye disorder.

[0073] In some embodiments, any of the aforementioned pharmaceutical compositions can be used to reduce or inhibit pathological vascular permeability in subjects with eye disorders.

[0074] In other contexts, any of the aforementioned pharmaceutical compositions can be used to treat the eye disorder in question.

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

[0076] In some embodiments, the individual is diagnosed with a vascular disorder. In other embodiments, the individual is diagnosed with an ocular vascular disorder.

[0077] In other aspects, the present invention provides a method for inhibiting vascular permeability in a subject suffering from an undesirable impairment related to vascular permeability, comprising administering an effective amount of one of the aforementioned antibodies or conjugates to the subject, thereby inhibiting vascular permeability in the subject.

[0078] In other aspects, the present invention is characterized by a method for treating an undesirable impairment related to vascular permeability, comprising administering an effective amount of one of the aforementioned antibodies or conjugates to a subject requiring such treatment.

[0079] In some embodiments, individuals are diagnosed with disorders related to the Tie2 pathway.

[0080] In some embodiments, individuals are diagnosed with an ocular disorder selected from the group consisting of diabetic macular edema (DME), age-related macular degeneration (AMD) including dry and wet types (non-exudative and exudative), choroidal neovascularization (CNV), uveitis, diabetic retinopathy, ischemia-associated 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, individuals have been treated with anti-VEGF antibodies. In other embodiments, individuals did not experience therapeutic efficacy of anti-VEGF antibodies, experienced a decrease in therapeutic efficacy of anti-VEGF antibodies, and / or ceased experiencing therapeutic efficacy of anti-VEGF antibodies.

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

[0083] [Figure 1] Figure 1 provides a schematic diagram of the Tie2 domain used for panning in the VH library. [Figure 2A] Figures 2A and 2B show the results of assays evaluating the binding of anti-Tie2 antibodies Tie2.1, Tie2.10, Tie2.11, and Tie2.12 to the mouse Tie2 ECD5 domain (Figure 2A) and the human Tie2 ECD5 domain (Figure 2B). The ECD5 domain contains the Ig1, Ig2, EGF, and Ig3 domains of Tie2. [Figure 2B]Figures 2A and 2B show the results of assays evaluating the binding of anti-Tie2 antibodies Tie2.1, Tie2.10, Tie2.11, and Tie2.12 to the mouse Tie2 ECD5 domain (Figure 2A) and the human Tie2 ECD5 domain (Figure 2B). The ECD5 domain contains the Ig1, Ig2, EGF, and Ig3 domains of Tie2. [Figure 3A] Figures 3A and 3B show the results of assays to evaluate the binding of anti-Tie2 antibodies Tie2.1, Tie2.10, Tie2.11, and Tie2.12 (Figure 3A) and Tie2.2, Tie2.3, Tie2.4, Tie2.5, Tie2.7, Tie2.9, Tie2.15, Tie2.16, Tie2.17, and Tie2.20 (Figure 3B) to human Tie1. [Figure 3B] Figures 3A and 3B show the results of assays to evaluate the binding of anti-Tie2 antibodies Tie2.1, Tie2.10, Tie2.11, and Tie2.12 (Figure 3A) and Tie2.2, Tie2.3, Tie2.4, Tie2.5, Tie2.7, Tie2.9, Tie2.15, Tie2.16, Tie2.17, and Tie2.20 (Figure 3B) to human Tie1. [Figure 4A] Figures 4A and 4B show the results of assays to evaluate the blockade 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 and 4B show the results of assays to evaluate the blockade 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] Figures 5A and 5B show the results of assays to evaluate the binding of anti-Tie2 antibodies Tie2.1, Tie2.10, Tie2.11, and Tie2.12 to HUVEC (Figure 5A) and RAEC (Figure 5B). [Figure 5B]Figures 5A and 5B show the results of assays to evaluate the binding of anti-Tie2 antibodies Tie2.1, Tie2.10, Tie2.11, and Tie2.12 to HUVEC (Figure 5A) and RAEC (Figure 5B). [Figure 6A] Figures 6A and 6B show the results of assays to evaluate the activation of AKT phosphorylation by anti-Tie2 antibodies Tie2.1, Tie2.4, Tie2.5, and Tie2.20 in RAEC (Figure 6A), and to evaluate the anti-IgG crosslinking effect of anti-Tie2 antibodies on AKT phosphorylation (Figure 6B). The level of phosphorylated AKT was determined by Western blot analysis. [Figure 6B] Figures 6A and 6B show the results of assays to evaluate the activation of AKT phosphorylation by anti-Tie2 antibodies Tie2.1, Tie2.4, Tie2.5, and Tie2.20 in RAEC (Figure 6A), and to evaluate the anti-IgG crosslinking effect of anti-Tie2 antibodies on AKT phosphorylation (Figure 6B). The level of phosphorylated AKT was determined by Western blot analysis. [Figure 7A] Figures 7A and 7B show the results of assays to evaluate the activation of AKT phosphorylation by anti-Tie2 antibodies Tie2.1, Tie2.22, Tie2.23, Tie2.24, Tie2.27, Tie2.28, Tie2.31, Tie2.33, Tie2.34, Tie2.34, Tie2.38, and Tie2.1 (Figure 7A). Figure 7B shows the effect of anti-Tie2 antibody aggregation or anti-IgG crosslinking on the activity of anti-Tie2 antibody Tie2.1 inducing AKT phosphorylation. The level of phosphorylated AKT was determined by FRET analysis. [Figure 7B]Figures 7A and 7B show the results of assays to evaluate the activation of AKT phosphorylation by anti-Tie2 antibodies Tie2.1, Tie2.22, Tie2.23, Tie2.24, Tie2.27, Tie2.28, Tie2.31, Tie2.33, Tie2.34, Tie2.34, Tie2.38, and Tie2.1 (Figure 7A). Figure 7B shows the effect of anti-Tie2 antibody aggregation or anti-IgG crosslinking on the activity of anti-Tie2 antibody Tie2.1 inducing AKT phosphorylation. The level of phosphorylated AKT was determined by FRET analysis. [Figure 8A] Figures 8A and 8B show the results from binning studies of anti-Tie2 antibodies using methods (Figure 8A) and ELISA (Figure 8B). [Figure 8B] Figures 8A and 8B show the results from binning studies of anti-Tie2 antibodies using methods (Figure 8A) and ELISA (Figure 8B). [Figure 9] Figure 9 is a schematic diagram showing the epitope groups on Tie2 to which the anti-Tie2 antibody of this disclosure binds. [Figure 10A] Figures 10A and 10B show the sequence alignment of the amino acid sequences of the heavy chain variable region (VH) of the anti-Tie2 antibodies Tie2.1 (SEQ ID NO: 22), Tie2.1.M100cF (SEQ ID NO: 20), Tie2.12 (SEQ ID NO: 31), Tie2.24 (SEQ ID NO: 36), Tie2.33 (SEQ ID NO: 41), and Tie2.38 (SEQ ID NO: 51) (Figure 10A), and the light chain variable region (VL) of the anti-Tie2 antibodies Tie2.1 (SEQ ID NO: 21), Tie2.1.M100cF (SEQ ID NO: 21), Tie2.12 (SEQ ID NO: 21), Tie2.24 (SEQ ID NO: 21), Tie2.33 (SEQ ID NO: 21), and Tie2.38 (SEQ ID NO: 21) (Figure 10B). [Figure 10B]Figures 10A and 10B show the sequence alignment of the amino acid sequences of the heavy chain variable region (VH) of the anti-Tie2 antibodies Tie2.1 (SEQ ID NO: 22), Tie2.1.M100cF (SEQ ID NO: 20), Tie2.12 (SEQ ID NO: 31), Tie2.24 (SEQ ID NO: 36), Tie2.33 (SEQ ID NO: 41), and Tie2.38 (SEQ ID NO: 51) (Figure 10A), and the light chain variable region (VL) of the anti-Tie2 antibodies Tie2.1 (SEQ ID NO: 21), Tie2.1.M100cF (SEQ ID NO: 21), Tie2.12 (SEQ ID NO: 21), Tie2.24 (SEQ ID NO: 21), Tie2.33 (SEQ ID NO: 21), and Tie2.38 (SEQ ID NO: 21) (Figure 10B). [Figure 11A] Figures 11A and 11B show the results of binning assays using various anti-Tie2.1 antibodies generated by phage presentation 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 and 11B show the results of binning assays using various anti-Tie2.1 antibodies generated by phage presentation 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 and 12B show the results 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 the biepitope FabIgG(1.38). Figure 12B compares the multimeric formats of anti-Tie2 antibodies. [Figure 12B] Figures 12A and 12B show the results 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 the biepitope FabIgG(1.38). Figure 12B compares the multimeric formats of anti-Tie2 antibodies. [Figure 13A]Figures 13A and 13B show the effect of various huIgM heavy-to-light chain ratios, with and without J chains, on the total yield of proteins isolated from affinity columns, as measured by A280 (Figure 13A) and SEC profiles (Figure 13B) of total protein. [Figure 13B] Figures 13A and 13B show the effect of various huIgM heavy-to-light chain ratios, with and without J chains, on the total yield of proteins isolated from affinity columns, as measured by A280 (Figure 13A) and SEC profiles (Figure 13B) of total protein. [Figure 13C] Figure 13C shows rheological measurements of IgM compared to a multivalent PEG format targeting factor D(fD) in the eye. [Figure 13D] Figure 13D shows the intravitreous pharmacokinetic analysis of unbound IgM and unbound Fab. [Figure 13E] Figure 13E shows the systemic pharmacokinetic analysis of IgM, comparing unbound recombinant hIgM pentamers and recombinant hIgM hexamers with IgM isolated from human serum injected intravenously into female SCID mice. Figure 13E shows serum IgM levels. [Figure 13F] Figure 13F shows the LC-MS analysis of the overall N-linked glycan profile from the serum sample. [Figure 13G] Figures 13G to 13H show the results of assays characterizing anti-Tie2 antibodies in IgM multimer format. Figure 13G shows the results of a complement 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 to 13H show the results of assays characterizing anti-Tie2 antibodies in IgM multimer format. Figure 13G shows the results of a complement 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 and 14B show the design and analysis of hexameric anti-Tie2 antibodies via the peptide moiety. Figure 14A is a schematic diagram of the multimer design. Figure 14B shows the results of an AKT phosphorylation assay comparing hexameric anti-Tie2 antibodies via NDK peptide, IgM, and multi-armed PEG. [Figure 14B] Figures 14A and 14B show the design and analysis of hexameric anti-Tie2 antibodies via the peptide moiety. Figure 14A is a schematic diagram of the multimer design. Figure 14B shows the results of an AKT phosphorylation assay comparing hexameric anti-Tie2 antibodies via NDK peptide, IgM, and multi-armed PEG. [Figure 15A] Figures 15A and 15B show the results of AKT phosphorylation assays to evaluate the agonist activity of various anti-Tie2 antibodies in hexamer format. The 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 and 15B show the results of AKT phosphorylation assays to evaluate the agonist activity of various anti-Tie2 antibodies in hexamer format. The 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 the cellular level of the Tie2 protein in in vitro assays. Figures 16A, 16B, and 16C each compare Tie2 levels upon exposure of HUVECs to anti-Tie2 antibodies generated via phage presentation and animal immunization. All assays in Figures 16A, 16B, and 16C were analyzed by Western blotting. [Figure 16B]Figures 16A–16C show the results of assays to evaluate the effect of anti-Tie2 antibodies on the cellular level of the Tie2 protein in in vitro assays. Figures 16A, 16B, and 16C each compare Tie2 levels upon exposure of HUVECs to anti-Tie2 antibodies generated via phage presentation and animal immunization. All assays in Figures 16A, 16B, and 16C were analyzed by Western blotting. [Figure 16C] Figures 16A–16C show the results of assays to evaluate the effect of anti-Tie2 antibodies on the cellular level of the Tie2 protein in in vitro assays. Figures 16A, 16B, and 16C each compare Tie2 levels upon exposure of HUVECs to anti-Tie2 antibodies generated via phage presentation and animal immunization. All assays in Figures 16A, 16B, and 16C were analyzed by Western blotting. [Figure 17] Figure 17 shows the results of an in vivo assay to evaluate the effect of anti-Tie2 antibodies on the cellular level at the Tie2 protein level. [Figure 18A] This shows the assay method (Figure 18A) and results (Figure 18B) of an in vitro endothelial cell assay to study the effect of an anti-Tie2.1 antibody on endothelial cell barrier permeability. [Figure 18B] This shows the assay method (Figure 18A) and results (Figure 18B) of an in vitro endothelial cell assay to study the effect of an anti-Tie2.1 antibody on endothelial cell barrier permeability. [Figure 19A] Figures 19A and 19B show the assay method (Figure 19A) and results (Figure 19B) of an in vivo vascular permeability assay to study the effect of an anti-Tie2.1 antibody on VEGF-induced vascular leakage. [Figure 19B] Figures 19A and 19B show the assay method (Figure 19A) and results (Figure 19B) of an in vivo vascular permeability assay to study the effect of an anti-Tie2.1 antibody on VEGF-induced vascular leakage. [Figure 20A]Figures 20A and 20B show the assay method (Figure 20A) and results (Figure 20B) of an in vivo vascular permeability assay to study the effect of anti-Tie2.1 antibodies or anti-VEGF antibodies on VEGF-induced vascular leakage. [Figure 20B] Figures 20A and 20B show the assay method (Figure 20A) and results (Figure 20B) of an in vivo vascular permeability assay to study the effect of anti-Tie2.1 antibodies or anti-VEGF antibodies on VEGF-induced vascular leakage. [Figure 21A] Figures 21A and 21B show the in vivo effect of a biepitope anti-Tie2 agonist (anti-Tie2 Fab-IgG 1.38) 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-IgG 1.38 on VEGF-induced vascular leakage (Figure 21B). [Figure 21B] Figures 21A and 21B show the in vivo effect of a biepitope anti-Tie2 agonist (anti-Tie2 Fab-IgG 1.38) 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-IgG 1.38 on VEGF-induced vascular leakage (Figure 21B). [Figure 22] Figure 22 shows the effects of anti-Tie2 agonists on cell organization by VE-cadherin (upper panel) and F-actin (lower panel) in cultured HUVECs. [Figure 23] Figure 23 shows the results of an AKT phosphorylation assay to evaluate the agonist activity of variants of the IG1-formatted Tie2.1 anti-Tie2 antibody. [Figure 24] Figure 24 shows the results of an AKT phosphorylation assay to evaluate the agonist activity of a variant of the hexameric Tie2.1 anti-Tie2 conjugate compared to a non-PEG conjugate Tie2.1 Fab (without hexamers). [Figure 25]Figure 25 shows the pharmacokinetics of the anti-Tie2 Fab hexamer conjugate after ocular injection in cynomolgus monkeys. [Figure 26] Figures 26 and 27 show electrophoretic analyses of anti-Tie2.1 PEG conjugates with monomer peaks considered for relative quantification (Figure 26) and without (Figure 27). [Figure 27] Figures 26 and 27 show electrophoretic analyses of anti-Tie2.1 PEG conjugates with monomer peaks considered for relative quantification (Figure 26) and without (Figure 27). [Figure 28] Figure 28 shows the electrophoretic analysis of anti-Tie2.1 PEG conjugate in a sample collected from the vitreous body fluid of a cynomolgus monkey. [Figure 29] Figure 29 shows the relative amounts of anti-Tie2 PEG conjugate hexamers and pentamers. [Figure 30A] Figures 30A and 30B provide data showing the oxidation of M100c (Figure 30A) in samples taken from the eyes of cynomolgus monkeys over a period of 21 days (Figure 30B). [Figure 30B] Figures 30A and 30B provide data showing the oxidation of M100c (Figure 30A) in samples taken from the eyes of cynomolgus monkeys over a period of 21 days (Figure 30B). [Figure 31] Figure 31 shows the effect of oxidation on pAKT activity. [Figure 32] Figures 32 and 33 show the effect of deconjugation on pAKT activity (Figure 32), using the normalized values ​​graphed in Figure 33. [Figure 33] Figures 32 and 33 show the effect of deconjugation on pAKT activity (Figure 32), using the normalized values ​​graphed in Figure 33. [Figure 34] Figure 34 shows the effect of the PEG conjugation site location on deconjugation. [Figure 35A] Figure 35A shows the effect of the PEG conjugation site location on activity. [Figure 35B] Figure 35B shows the effect of the PEG conjugation site location on activity. [Figure 36]Figure 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 and 38B compare the activity of different Tie2.1M 100cF PEG conjugates. Figure 38A shows pAKT activity, and Figure 38B shows total conjugate levels determined by ELISA. [Figure 38B] Figures 38A and 38B compare the activity of different Tie2.1M 100cF PEG conjugates. Figure 38A shows pAKT activity, and Figure 38B shows total conjugate levels determined by ELISA. [Figure 39] Figure 39 shows the structure of the PEG-conjugated hexamer core molecule conjugated to the anti-Tie2 antibody according to this disclosure. [Modes for carrying out the invention]

[0084] Detailed description of embodiments of the invention I. Definition Unless otherwise defined herein, the term “including” includes the term “consisting of.”

[0085] As used herein in relation to a specific value (e.g., temperature, concentration, time, etc.), the term "approximately" refers to a variation of + / - 1% of the specific value to which the term "approximately" refers.

[0086] For the purposes of this specification, “acceptor human framework” means a framework comprising 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 contain the same amino acid sequence or may contain a modification of the amino acid sequence. In some embodiments, the number of amino acid modifications 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 sequence-identical to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0087] "Affinity" refers to the sum of the 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 the 1:1 interaction between the members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for its partner Y can generally be expressed by a dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative descriptions and exemplary embodiments for measuring binding affinity are given below.

[0088] An "affinity-mature" antibody refers to an antibody that, compared to an unmodified parent antibody, has one or more modifications in one or more hypervariable regions (HVRs), and in which such modifications improve the antibody's affinity for the 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 to be useful as a therapeutic and / or diagnostic agent in targeting Tie2. In one embodiment, the degree 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 a particular embodiment, the antibody that binds to Tie2 has concentrations of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 -8 M or less, for example, 10 -8 ~10 -13 M, for example 10 -9 M~10 -13 The dissociation constant (K) of M D ) has. In certain embodiments, the anti-Tie2 antibody binds to Tie2 epitopes that are conserved among different species of Tie2.

[0090] The term "antibody" is used herein in its broadest sense and is not limited thereto, but encompasses a variety of antibody structures, including monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as 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 an 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 herein synonymously to refer to antibodies having a structure substantially similar to that of a natural antibody or having a heavy chain containing an Fc region as defined herein.

[0093] As used herein, “Fab” refers to an antibody containing a heavy chain constant region that includes a CH1 domain, or a portion of the CH1 domain sufficient to form a disulfide bond in the light chain constant region, but does not contain a CH2 domain or CH3 domain. As used herein, Fab may contain one or more amino acids in the hinge region. Therefore, as used herein, the term “Fab” encompasses Fab' antibodies. Fab may also contain further non-natural amino acids, such as a C-terminal cysteine, in which case it may be referred to as Fab-C. As will be discussed below, the term Fab-C also encompasses Fabs containing natural amino acids in the hinge region that include a natural cysteine ​​at the C-terminus. In some embodiments, Fab contains an engineered cysteine ​​(i.e., Fab may 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 substituted with a non-cysteine ​​amino acid residue.

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

[0095] "Fab-SH" refers to a Fab antibody that has a free thiol group. In some embodiments, the free thiol group is located in the last 10 amino acids of the C-terminus of the Fab antibody. A Fab-C antibody is also typically a Fab-SH antibody.

[0096] A reference antibody and an antibody that "binds to the same epitope" refer to an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competitive assay, and conversely, a reference antibody that blocks the binding of an antibody to its antigen by 50% or more in a competitive assay. Exemplary competitive assays are provided herein.

[0097] The term "chimeric" antibody refers to an antibody in which part of the heavy chain and / or light chain originates from a particular source or species, while the rest of the heavy chain and / or light chain originates from a different source or species.

[0098] The "class" of an antibody refers to the type of constant domain or constant region held by its heavy chain. Antibodies have five main classes: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0099] The term “conjugate” is used herein in its broadest sense, meaning that two molecules are joined or linked together. A molecule is “conjugated” if it acts or functions as if it were joined. A “conjugate” is an antibody (e.g., Fab) conjugated to one or more heterogeneous molecules, including but not limited to polyols. In certain embodiments, a “conjugate” refers to an antibody (e.g., an antibody fragment, as detailed herein) covalently bound to a multi-armed portion. In certain embodiments, the multi-armed portion is a polyol, an IgM molecule, or a peptide in a multimer (e.g., hexamer) format.

[0100] As used herein, the term "Tie2" refers to any natural Tie2 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. This term encompasses both "full-length" untreated Tie2 and any form of Tie2 resulting from intracellular processing. This term also encompasses naturally occurring variants of Tie2, such as splice variants or allele variants. The amino acid sequence of an exemplary human Tie2 protein has NCBI reference number: NP_000450 (SEQ ID NO: 1).

[0101] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cellular function and / or causes cell death or destruction. Examples of cytotoxic agents include radioactive isotopes (e.g., At). 211 , I 131 , I 125 , Y 90 Re 186 Re 188 Sm 153 , Bi 212 , P 32 Pb 212 , and radioisotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other inserts); growth inhibitors; enzymes and their fragments, e.g., nucleases; antibiotics; toxins such as low molecular weight toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin (including their fragments and / or variants); and various antitumor or anticancer agents disclosed below, but not limited to these.

[0102] "Effector function" refers to the biological activity resulting from the Fc region of an antibody, which varies depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cell-mediated 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] The “effective dose” of a drug, for example, a pharmaceutical preparation, refers to the amount that is effective in the dosage and duration required to achieve the desired therapeutic or preventive outcome.

[0104] In this specification, the term “Fc region” is used to define the C-terminal region of an immunoglobulin heavy chain, including at least a portion of the constant region. This term includes both native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) 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 known 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] The "framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The variable domain FR generally consists of four FR domains: FR1, FR2, FR3, and FR4. Therefore, the HVR and FR sequences generally appear in the following sequence in VH (or VL): 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 acids have been introduced, and cells including the offspring of such cells. Host cells include “transformed organisms” and “transformed cells,” which include primary transformed cells and offspring derived from host cells regardless of the number of passages. Offspring may contain mutations, but their nucleic acid content may not be exactly the same as that of the parent cells. Offspring of mutants having the same function or biological activity as those screened or selected for the original transformed cells are included in the present invention.

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

[0108] The "Human Consensus Framework" is a framework representing the most commonly occurring amino acid residues in the selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from subgroups of variable domain sequences. Generally, the sequence subgroups are those described 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. (above). In one embodiment, for VH, the subgroup is subgroup III in Kabat et al. (above).

[0109] A “humanized” antibody refers to a chimeric antibody containing amino acid residues derived from non-human HVR and amino acid residues derived from human FR. In certain embodiments, a humanized antibody contains substantially all of at least one, typically two, variable domains, where all or substantially all of the HVR (e.g., CDR) corresponds to a non-human antibody and all or substantially all of the FR corresponds to a human antibody. The humanized antibody may optionally contain at least a portion of the antibody constant region derived from a human antibody. The “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 domains in the heavy or light chain of an antibody that are involved in the binding of the antibody to the antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, and each domain contains four conserved framework regions (FRs) and three hypervariable regions (HVRs). (e.g., Kindt et al. Kuby Immunology, 6) th See ed., WH Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen may be isolated by screening a library of complementary VL or VH domains using the VH or VL domain of the antibody that binds to the antigen, respectively. See, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0111] As used herein, the terms “hypervariable region,” “HVR,” or “HV” refer to regions of antibody variable domains whose sequences are hypervariable (also referred herein as “complementarity-determining regions” or “CDR”) and / or which form structurally defined loops. Generally, antibodies contain six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, 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 camel antibodies consisting only of heavy chains are functional and stable in the absence of light chains. For example, see Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).

[0112] Several HVR descriptions are used and incorporated herein. The Kabat Complementarity Determining Region (CDR) is based on sequence variability and is 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)). Alternatively, Chothia refers to the location of the structural loop (Chothia and Lesk J. Mol. Biol. 196:901~917 (1987)). AbM HVR represents a compromise between Kabat HVR and Chothia structural loop and is used by Oxford Molecular's AbM antibody modeling software. "Contact" HVR is based on the analysis of available complex crystal structures. The residues derived from each of these HVRs are shown below. JPEG2026053356000005.jpg43170

[0113] HVR may include the following “extended HVRs”: in VL, 24–36 or 24–34 (L1), 46–56 or 50–56 (L2), and 89–97 or 89–96 (L3); and in VH, 26–35 (H1), 50–65 or 49–65 (H2), and 93–102, 94–102, or 95–102 (H3). Variable domain residues are numbered according to Kabat et al. above for each of these definitions.

[0114] The terms “Kabat-like variable domain residue numbering” or “Kabat-like amino acid position numbering,” and their variations, refer to the numbering system used in the heavy-chain or light-chain variable domains of antibody edits by Kabat et al. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to the shortening or insertion of FR or HVR in the variable domain. For example, a heavy-chain variable domain may contain a single amino acid insertion after H2 residue 52 (Kabat-like residue 52a) and a residue inserted after heavy-chain FR residue 82 (e.g., Kabat-like residues 82a, 82b, and 82c). The Kabat numbering of residues can be determined for a given antibody by the alignment of the antibody sequence with the homologous region of the sequence numbered by “standard” Kabat.

[0115] The Kabat numbering system is generally 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.). The "EU numbering system" or "EU index" is generally used when referring to residues in the constant region of the immunoglobulin heavy chain (e.g., the EU index reported by Kabat et al.). "EU index as in Kabat" refers to the residue numbering of human IgG1 EU antibodies. Unless otherwise specified herein, references to residue numbers in the variable domain of an antibody mean residue numbering according to the Kabat numbering system. Unless otherwise specified herein, references to residue numbers in the constant domain of an antibody mean residue numbering according to the EU numbering system (e.g., see U.S. Patent Application Publication 2008 / 0181888, diagram relating to EU numbering).

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

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

[0118] An “isolated” antibody is an antibody that has been separated from its components in its natural environment. In some embodiments, the antibody is purified to a purity of over 95% or over 99%, as determined, for example, 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 evaluating antibody purity, see, for example, Flatman et al., J.Chromatogr.B 848:79-87 (2007).

[0119] "Isolated" nucleic acids refer to nucleic acid molecules that have been separated from their natural environment. Isolated nucleic acids include nucleic acid molecules that are normally found inside cells containing nucleic acid molecules, but the nucleic acid molecules are located outside of chromosomes or at chromosomal locations different from their natural chromosomal locations.

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

[0121] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies constituting that population are identical and / or bind to the same epitope, with the exception of possible mutant antibodies, such as those containing spontaneous mutations or arising during the production of a monoclonal antibody preparation, which are generally present in small amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies directed toward different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed toward a single determinant on an antigen. Therefore, the modifier “monoclonal” indicates the characteristic of an antibody obtained from a substantially homogeneous collection of antibodies and should not be interpreted as requiring antibody production 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 presentation methods, and methods utilizing transgenic animals containing all or part of a human immunoglobulin locus, and such and other exemplary methods for producing monoclonal antibodies are described herein.

[0122] A "naked antibody" refers to an antibody that is not bound to a heterogeneous site (e.g., a cytotoxic site) or a radioactive label. Naked antibodies may be present in pharmaceutical compositions.

[0123] "Natural antibodies" refer to naturally occurring immunoglobulin molecules with various structures. For example, a natural IgG antibody is a heterotetrameric glycoprotein with approximately 150,000 daltons, containing two identical light chains and two identical heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has 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 has a variable region (VL), also called a variable light domain or light chain variable domain, followed by a constant light (CL) domain. The light chains of an antibody may be assigned to one of two types, called κ (kappa) or λ (lambda), based on the amino acid sequence of their constant domains.

[0124] The term “package insert” is used to refer to the instructions typically included on the market packaging of a therapeutic product, including information relating to indications, use, dosage, administration, combination therapy, contraindications, and / or warnings for such therapeutic product.

[0125] The "amino acid sequence identity percentage (%)" for a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps as necessary to achieve the maximum possible sequence identity percentage, without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining the amino acid sequence identity percentage can be achieved in various methods within the scope 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 algorithm necessary to achieve the maximum alignment over the entire length of the sequences being compared. However, for the purposes of this specification, the amino acid sequence identity % values ​​are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and its source code, along with user documentation, has been filed with the U.S. Copyright Office, Washington DC, 20559, where it is registered under U.S. Copyright Registration Number 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 with UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and remain unchanged.

[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 (or, a given amino acid sequence A may be described as having or containing 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 In the formula, X is the number of amino acid residues scored as identical matches in the alignment of A and B by the sequence alignment program ALIGN-2, 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 be different from the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraph.

[0127] The term "pharmaceutical preparation" refers to a preparation that is in a form that enables the biological activity of the active ingredient contained herein to be effective, and that does not contain any additional ingredients that are toxic to the subject to whom the preparation will be administered.

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

[0129] The term “polyol” broadly refers to polyhydric alcohol compounds. A polyol can be, for example, any water-soluble poly(alkylene oxide) polymer and may have a linear or branched chain. Preferred polyols include those in which one or more hydroxyl positions are substituted with chemical groups, such as alkyl groups having 1 to 4 carbon atoms. Typically, polyols are 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 with respect to PEG using the conjugate techniques described herein. The polyols of this disclosure include those known in the art and those publicly available, such as from commercially available sources.

[0130] As used herein, “treatment” (and its grammatical variations, e.g., “to treat” or “to treat”) refers to a clinical intervention in an attempt to alter the natural course of the individual being treated, and may be performed either for prevention or during the course of a clinicopathological condition. Desired effects of treatment include, but are not limited to, preventing the onset or recurrence of the disease, reducing symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, achieving remission or mitigation of the condition, and improving recovery or prognosis. In some embodiments, the antibodies of the present invention are used to delay the onset of the disease or to delay the progression of the disease.

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

[0132] A “port delivery system” or “PDS” is an implantable ocular device that uses a refillable reservoir to enable the delivery of therapeutic agents over a long period of time. The implantation involves 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 Publications 20100174272, 8,277,830, 8,399,006, 8,795,712, and 8,808,727.

[0133] "Small-hole needle" refers to injection needles for liquid components of approximately 30, 29, 28, 27, 26, 25, 24, 23, or 22 gauge, such as a 30 gauge needle. In some embodiments, the small-hole needle has a standard-sized wall. In other embodiments, the small-hole needle has a thin wall, which may be preferable for viscous solutions.

[0134] II. Compositions and Methods Novel Tie2 agonists that activate Tie2 function, as demonstrated by Tie2 phosphorylation, are provided herein. Direct agonists may be therapeutically advantageous in more potent activation of Tie2 signaling and promote visual acuity improvement when the endogenous agonist Ang1 is scarce or absent. Tie2 activators may be more advantageous than Ang2 inhibitors because they can block Ang2 binding (blocking Ang2 antagonist function) and directly bind to Tie2 to activate Tie2 activity, for example, by increasing Tie2 and / or Akt phosphorylation. Tie2 activation has been shown to require Tie2 clustering upon ligand binding, and therefore the Tie2 agonists provided herein are multimers, preferably hexamers or octamers. The multimer Tie2 agonists described herein may have the additional unexpected advantage of not significantly reducing cellular Tie2 levels in vitro or in vivo. Furthermore, the multimerized Tie2 agonists described herein can be formulated for intravitreous injection and possess molecular sizes that confer favorable pharmacokinetics and thus pharmacodynamics. Data are provided demonstrating that multimerized Tie2 agonists reduce endothelial cell membrane permeability and enhance cell-cell junctions (see, for example, Example 10).

[0135] In one aspect, the present invention is partially based on antibodies that bind to Tie2. In particular, the present invention provides conjugates (also referred to herein) comprising two or more anti-Tie2 antibodies or their antigen-binding fragments, for example (but not limited to) anti-Tie2 Fab. The conjugate may preferably comprise more than four (e.g., including 5, 6, 7, 8, 9, or 10) anti-Tie2 antibodies, for example more than four anti-Tie2 Fab, such that the binding of the Tie2 conjugate to Tie2 located on the cell surface is associated with Tie2 activation. Tie2 clustering may also occur upon binding of the conjugate to Tie2.

[0136] In some embodiments, it is advantageous to have a Tie2 conjugate that, when bound to Tie2 on a protein surface, does not significantly reduce the level of Tie2 protein on the cell surface. In some embodiments, the Tie2 conjugate that activates Tie2 comprises an anti-Tie2 antibody (Fab) having an affinity for Tie2 in the range of approximately 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 an increase in phosphorylation of the Tie2 protein (e.g., by Western blotting) or by measuring an increase in phosphorylation of the associated 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. The antibodies and compositions comprising multiple antibodies of the present invention are useful, for example, in the diagnosis or treatment of impaired vascular permeability of the eye, particularly those related to Tie2 function.

[0138] A. Exemplary anti-Tie2 conjugates In one aspect, the present invention provides a Tie2 conjugate comprising an isolated antibody that binds to Tie2. In some embodiments, the Tie2 conjugate comprises a multi-armed portion in which each arm is conjugated or linked to an anti-Tie2 antibody or a fragment thereof. In a preferred embodiment, the anti-Tie2 antibody or fragment thereof is an anti-Tie2 Fab. Examples of multi-armed portions include, but are not limited to, multi-armed polyols (e.g., polyethylene glycol (PEG)), IgM, and polymers, such as hexameric peptides. In a particular embodiment, a Tie2 conjugate comprising two or more anti-Tie2 antibodies or fragments thereof is provided, where the anti-Tie2 antibody (if optionally formatted as a full-length antibody rather than being part of the Tie2 conjugate) is less than 100 μM, or less than 50 μM, or less than 10 μM of K D and / or K greater than 1 μM DThe Tie2 conjugate binds to Tie2 with affinity such as 1, 2, 3, 4, 5, 60%, 75%, or 85%. It is understood that affinity is measured using antibodies rather than Tie2 conjugates containing two or more anti-Tie2 antibodies. In some embodiments, affinity is monovalent affinity. Furthermore, Tie2 conjugates and Tie2 antibodies that activate Tie2 activity (e.g., function as Tie2 agonists) are provided. Tie2 activation is measured, for example, by measuring increased phosphorylation of Tie2 and / or AKT in an in vitro assay. In some embodiments, the Tie2 conjugate does not downregulate (reduce) intracellular Tie2 protein levels beyond approximately 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, or 85%. In alternative embodiments, the Tie2 conjugate reduces intracellular Tie2 protein levels by approximately 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, or less than 75%. In some embodiments, the Tie2 conjugate reduces vascular permeability. The reduction in vascular endothelial cell permeability can be measured in vitro or in vivo. In some embodiments, the Tie2 conjugate may increase the migration of Tie2 to cell-cell junctions and / or promote the structural organization of actin and / or cadherins. In preferred embodiments, the Tie2 conjugate comprises a hexameric PEG molecule in which each of the six arms is conjugated with anti-Tie2 Fab. Alternatively, the PEG molecule comprises eight arms. In some embodiments, the Tie2 conjugate comprises two or more anti-Tie2 antibodies or fragments thereof as described herein. The present invention also provides anti-Tie2 antibodies or fragments thereof that bind to Tie2. In some embodiments, an anti-Tie2 antibody or fragment thereof binds to the Ig2 domain of Tie2 (for example, to an epitope that is at least partially present within amino acid residues 23-120 of SEQ ID NO: 1).

[0139] In one aspect, the present invention provides an antibody that specifically binds to Tie2 or its antigen-binding fragment, 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; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 10. In a particular 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, 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. All possible combinations of the above substitutions are encompassed in 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 present invention provides an antibody comprising at least one, at least two, or all three VL domain CDR sequences selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 8, (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10. In one embodiment, the VL domain comprises (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10.

[0142] In another embodiment, the antibody of the present invention comprises a VH domain comprising at least one, at least two or three all VH CDR sequences selected from (a) (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 3, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 4, and (iii) CDR-H3 comprising an amino acid sequence selected from SEQ ID NO: 5; SEQ ID NO: 6; and 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; and a VL domain comprising at least one, at least two or three all VL CDR sequences selected from (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8, (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10.

[0143] In another embodiment, the present invention provides an antibody comprising a VH domain including (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, 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; 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 includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 20, and the VL domain includes a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 21.

[0144] In any of the embodiments described above, the anti-Tie2 antibody may be humanized. In one embodiment, the anti-Tie2 antibody comprises a CDR, similar to any of the embodiments described above, and further comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework. In another embodiment, the anti-Tie2 antibody comprises a CDR from any of the embodiments described above, and further comprises the VH framework FR1 sequence of SEQ ID NO: 11, the VH framework FR2 sequence of SEQ ID NO: 12, the VH framework FR3 sequence of SEQ ID NO: 13, and / or the VH framework FR4 sequence of SEQ ID NO: 14. In yet another embodiment, the anti-Tie2 antibody comprises the VL framework FR1 sequence of SEQ ID NO: 15, the VL framework FR2 sequence of SEQ ID NO: 16, the VL framework FR3 sequence of SEQ ID NO: 17, and / or the VL framework FR3 sequence of SEQ ID NO: 18.

[0145] In other aspects, the anti-Tie2 antibody or its antigen-binding fragment includes a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 20. In certain embodiments, the 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 the anti-Tie2 antibody containing that sequence retains its 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 the region outside the CDR (i.e., in the FR). Optionally, the anti-Tie2 antibody includes the VH sequence of SEQ ID NO: 20, which includes post-translational modifications of that sequence. In certain embodiments, the VH includes one, two, or three CDRs selected from: (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 3, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 4, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 7.

[0146] In other contexts, an anti-Tie2 antibody is provided, wherein the antibody comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 21. In certain embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but the anti-Tie2 antibody containing 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: 21. In certain embodiments, the substitutions, insertions, or deletions occur in the region outside the HVR (i.e., within the FR). Optionally, the anti-Tie2 antibody includes the VL sequence of SEQ ID NO: 21, which includes post-translational modifications of that sequence. In certain embodiments, the VL includes one, two, or three HVRs selected from (a) HVR-L1 containing the amino acid sequence of SEQ ID NO: 8, (b) HVR-L2 containing the amino acid sequence of SEQ ID NO: 9, and (c) HVR-L3 containing 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 sequence as in any of the embodiments provided above, and a VL sequence as in any of the embodiments provided above. In one embodiment, the antibody comprises the VH sequence and VL sequence 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 antibody 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, where the Ig1 domain comprises amino acids 23-120 of SEQ ID NO: 1.

[0149] In a further embodiment of the present invention, the anti-TIe2 antibody according to any of the above embodiments is a monoclonal antibody comprising a chimeric antibody, a humanized antibody, or a human antibody. In one embodiment, the anti-TIe2 antibody is an antibody fragment, e.g., Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment. In another embodiment, the antibody is a full-length antibody, e.g., an intact IgG1 antibody or other antibody class or isotype as defined herein.

[0150] In further embodiments, anti-Tie2 antibodies according to any of the above embodiments may incorporate any of the features described below, either alone or in combination.

[0151] 1. Antibody affinity In certain embodiments, the antibodies provided herein are 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 (for example, 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, for example 10 -8 M~10 -13 M, for example 10 -9 M~10 -13It has a dissociation constant (Kd) of 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 Fab to the antigen is measured in the presence of a titration series of the unlabeled antigen at a minimum concentration. 125 I) Fab is equilibrated with labeled antigen, and then measured by capturing the bound antigen with a plate coated with anti-Fab antibody (see, for example, Chen et al., "J.Mol.Biol." Vol. 293, pp. 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), and then blocked with 2% (w / v) bovine serum albumin in PBS for 2-5 hours at room temperature (approximately 23°C). In a non-adsorbent plate (Nunc#269620), 100 pM or 26 pM [ 125 Mix the [I]-antigen 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.", Vol. 57, pp. 4593-4599 (1997)). Then incubate the Fab of interest overnight, but incubation can be extended for a longer period (e.g., about 65 hours) to ensure equilibrium is reached. Then transfer the mixture to a capture plate for incubation at room temperature (e.g., 1 hour). Next, remove the solution and wash the plate eight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. When the plate is dry, add 150 μl / well of scintillant (MICROSCINT-20®; Packard) and count the plate with a TOPCOUNT® gamma counter (Packard) for 10 minutes. Select the concentration of each Fab that yields less than 20% of maximum binding for use in competitive binding assays.

[0153] In another embodiment, Kd is measured using a BIACORE® surface plasmon resonance assay. For example, assays using BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) are performed at 25°C to ~10 response units (RUs) using an immobilized antigen CM5 chip. 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 the instructions of those skilled in the art. The antigen is diluted in 10 mM sodium acetate, pH 4.8, and then diluted to 5 μg / mL (approximately 0.2 μM) before injection at a flow rate of 5 μL / min to achieve approximately 10 response units (RUs) of the binding protein. After antigen injection, 1 M ethanolamine is injected to block unreactive groups. For dynamic measurement, Fab's 2-fold serial dilutions (0.78 nM to 500 nM) are injected at a flow rate of approximately 25 μL / min into PBS containing 0.05% polysorbate 20 (TWEEN-20®) surfactant (PBST) at 25°C. The association rate (kon) and dissociation rate (koff) are calculated by simultaneously fitting the association sensogram and dissociation sensogram using a simple 1:1 Langmuir coupling model (BIACORE® Evaluation Software version 3.2). The equilibrium dissociation constant (Kd) is calculated as the koff / kon ratio. See, for example, Chen et al. J.Mol.Biol.293:865-881 (1999).If the on-rate exceeds 10⁶ M⁻¹ s⁻¹ by the surface plasmon resonance assay described above, the on-rate can be determined by using a fluorescence quenching technique, which measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm, emission = 340 nm, 16 nm band passthrough) of 20 nM anti-antigen antibody (Fab form) in PBS (pH 7.2) at 25°C in the presence of an increased concentration of antigen, measured with a spectrophotometer such as an Aviv Instruments spectrophotometer equipped with a stop flow or an 8000 series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) with a stirring cuvette.

[0154] 2. Antibody fragment In certain embodiments, the antibodies provided herein are antibody fragments. Examples of antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, and scFv fragments, and other fragments described below. For a review of specific antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For references 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); also see International Publication No. 93 / 16185; and U.S. Patents No. 5,571,894 and 5,587,458. For a description of the Fab and F(ab')2 fragments, which contain salvage receptor-binding epitope residues and have a longer in vivo half-life, see U.S. Patent No. 5,869,046.

[0155] In some embodiments, the C-terminus of the Fab fragment's heavy chain is terminated 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 Fab fragment's heavy chain is terminated with the sequence CDKTHX (SEQ ID NO: 79), where X is any amino acid except T. C-terminus cleavage and / or mutation can reduce or eliminate AHA reactivity to Fab without impairing thermal stability or expression. In some embodiments, the C-terminus of the Fab fragment's heavy chain is terminated 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 affected by conjugations, such as polymers like PEG.

[0156] A diabody is an antibody fragment having two antigen-binding sites, which may 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] A single-domain antibody is an antibody fragment that contains 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, the single-domain antibody is a human single-domain antibody (see, for example, Domantis, Inc., Waltham, MA; 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., Escherichia coli or phages), as described herein.

[0159] 3. Chimeric antibodies and humanized antibodies In certain embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567 and in Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In one example, a chimeric antibody includes a non-human variable region (e.g., a variable region derived from mouse, rat, hamster, rabbit, or non-human primate, e.g., monkey) and a human constant region. In further examples, a chimeric antibody is a “class-switched” antibody in which the class or subclass is modified from that of the parent antibody. A chimeric antibody includes its antigen-binding fragment.

[0160] In certain embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce its immunogenicity against humans while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody contains one or more variable domains in which the HVR, e.g., CDR (or part thereof), is derived from a non-human antibody and the FR (or part thereof) is derived from a human antibody sequence. The humanized antibody also optionally contains at least a portion of the human constant region. In some embodiments, some FR residues in the humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., an antibody from which the HVR residues are derived) to restore or improve antibody specificity or affinity, for example.

[0161] Humanized antibodies and their production methods are reviewed in 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. Patents No. 5,821,337, No. 7,527,791, No. 6,982,321, and No. 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (Describes specificity-determining region (SDR) grafts); Padlan, Mol.Immunol.28:489-498 (1991) (Describes resurfacing); Dall'Acqua et al., Methods 36:43-60 (2005) (Describes "FR shuffling"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br.J.Cancer,83:252-260 (2000) (Describes the "guided selection approach" to FR shuffling).

[0162] Human framework regions that may be used for humanization include, but are not limited to, the following: framework regions selected using the “best fit” method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from consensus sequences of human antibodies of specific subgroups of light chain 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 maturation (somatic mutation) framework regions or human germ cell framework regions (e.g., Almagro and See Fransson, Front. Biosci. 13:1619-1633 (2008); as well as framework areas derived from screening of FR libraries (see, for example, 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 prepared using a variety of 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 an immunogen to a transgenic animal modified to produce intact human antibodies or intact antibodies with a human variable region in response to antigen challenge. Such animals typically contain all or part of a human immunoglobulin locus that replaces the endogenous immunoglobulin locus, is located extrachromosomally, or is randomly incorporated into the animal's chromosome. 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, for example, U.S. Patent Nos. 6,075,181 and 6,150,584 describing XENOMOUSE® technology; U.S. Patent No. 5,770,429 describing HuMab® technology; U.S. Patent No. 7,041,870 describing KM MOUSE® technology; and U.S. Patent Application Publication 2007 / 0061900 describing VelociMouse® technology. The human variable region from intact antibodies produced by such animals may be further modified, for example, by combining it with a different human constant region.

[0165] Human antibodies can also be produced by hybridoma-based methods. Human myeloma and mouse-human xenomyeloma 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 have also been described by Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Further methods include, for example, U.S. Patent No. 7,189,826 (describes the production of monoclonal human IgM antibodies derived from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (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-presenting libraries. Such variable domain sequences may then be combined with desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

[0167] 5. Antibodies derived from libraries The antibodies of the present invention can be isolated by screening a combinatorial library for one or more antibodies having desired activity. For example, various methods are known in the art for generating phage-presenting libraries and screening such libraries for antibodies having desired binding properties. Such methods are outlined, for example, in 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. Further details are found in 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 certain phage presentation methods, the VH and VL gene repertoires can be cloned separately by polymerase chain reaction (PCR), randomly recombined in a phage library, and then screened for antigen-binding phages as described in Winter et al., Ann. Rev. Immunol., 12:433-455 (1994). The phages typically present antibody fragments either as single-stranded Fv (scFv) fragments or as Fab fragments. Libraries from immunogens provide high-affinity antibodies against immunogens without the need to construct hybridomas. Alternatively, the natural repertoire can be cloned (e.g., from humans) without immunization to provide a single source of antibodies against a wide range of non-self and also self-antigens, as described by Griffiths et al., EMBO J, 12:725-734 (1993). Finally, natural libraries can also be constructed synthetically by cloning an unrearranged V gene segment from stem cells, encoding a highly variable CDR3 region using PCR primers containing random sequences, and achieving rearrangement in vitro, as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). Examples of patent publications describing human antibody phage libraries include U.S. Patent No. 5,750,373, and U.S. Patent Application Publications 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 in this specification.

[0170] 6. Multispecific antibodies In certain embodiments, the antibodies provided herein are multispecific antibodies, for example, bispecific antibodies. A multispecific antibody is a monoclonal antibody having binding specificity to at least two different sites. In certain embodiments, one binding specificity is to Tie2 and the other is to any other antigen. In certain embodiments, a bispecific antibody may bind to two different epitopes of Tie2. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing Tie2. Bispecific antibodies may be prepared as full-length antibodies or antibody fragments.

[0171] Techniques for producing multispecific antibodies include, but are not limited to, the recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537 (1983)), International Publication No. WO93 / 08829, and Traunecker et al., EMBO J.10:3655 (1991)), and the "knob-into-hole" operation (see, for example, U.S. Patent No. 5,731,168). Multispecific antibodies also utilize techniques such as: manipulating the electrostatic steering effect to produce antibody Fc heterodimer molecules (International Publication No. 2009 / 089004A1); crosslinking two or more antibodies or fragments (see, e.g., U.S. Patent No. 4,676,980 and Brennan et al., Science, 229:81 (1985)); using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny et al., J.Immunol., 148(5):1547-1553 (1992)); using "diabody" techniques to produce bispecific antibody fragments (see, e.g., Hollinger et al., Proc.Natl.Acad.Sci.USA, 90:6444-6448 (1993)); and using single-stranded Fv(sFv) dimers (see, e.g., Gruber et al. See al., J.Immunol., 152:5368 (1994); and it can also be prepared by the preparation of a triplicate antibody as described, for example, Tutt et al. J.Immunol. 147:60 (1991).

[0172] This also includes antibodies designed to have three or more functional antigen-binding sites, such as "octopus antibodies." (See, for example, US2006 / 0025576A1)

[0173] The antibodies or fragments described herein also include “Dual Acting FAb” or “DAF” which include antigen-binding sites that bind to Tie2 and other different antigens (see, for example, U.S. Patent Application Publication No. 2008 / 0069820).

[0174] 7. Antibody variants In certain embodiments, amino acid sequence variants of antibodies provided herein are intended. 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 residues in the amino acid sequence of the antibody, and / or insertions into residues in the amino acid sequence of the antibody, and / or substitutions of residues in the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be carried out so as to reach the final construct, insofar as the final construct has the desired characteristics (e.g., antigen binding).

[0175] a) Substitution, insertion, and deletion variants In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Sites of interest with respect to mutagenesis by substitution include HVR and FR. Conservative substitutions are shown in Table 1 under the heading "Preferred Substitutions." More substantial variations are provided in Table 1 under the heading "Exemplary Substitutions" and are further described below with reference to amino acid side chain classes. Amino acid substitutions are introduced into the antibody of interest, and the product can be screened for desired activity, e.g., retention / improvement of antigen binding, reduction of immunogenicity, or improvement of ADCC or CDC. [Table 1]

[0176] Amino acids can be grouped according to their 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) Basicity: His, Lys, Arg, (5) Residues that affect chain orientation: Gly, Pro, (6) Aromatic: Trp, Tyr, Phe.

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

[0178] One type of substitution variant involves substitution of 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 a modification (e.g., improvement) of a particular biological property (e.g., increased affinity, reduced immunogenicity) compared to the parent antibody and / or have a particular biological property of the parent antibody that is substantially retained. Exemplary substitution variants are affinity matured antibodies and can be readily generated, for example, using phage display-based affinity maturation techniques as described herein. Briefly, one or more HVR residues are mutated, the variant antibodies are displayed on phage, and screened for a particular biological activity (e.g., binding affinity).

[0179] To improve antibody affinity, modifications (e.g., substitutions) may be made in the CDR, for example. Such modifications may be made in CDR "hot spots," i.e., residues encoded by codons that frequently undergo mutations during the somatic cell maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)) and / or residues that come into contact with the antigen, and the resulting variant VH or VL is tested for binding affinity. Affinity maturation by construction of a secondary library and re-selection therefrom 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, variability is introduced into the variable genes selected for maturation by one of various methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then constructed. Next, this library is screened to identify antibody variants with the desired affinity. Another method for introducing diversity involves CDR-directed methods 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 particularly often targeted.

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

[0181] A useful method for identifying residues or regions of an antibody 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, the crystal structure of an antigen-antibody complex to identify the contact points between an antibody and an antigen. Such contact residues and adjacent residues may be targeted or excluded as candidates for substitution. Variants may be screened to determine whether they have the desired properties.

[0182] Amino acid sequence insertions include amino-terminal fusions and / or carboxyl-terminal fusions that span from 1 residue to over 100 residues in length in a polypeptide, as well as in-sequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies having an N-terminal methionyl residue. Other insertion variants of an antibody molecule include fusions of the N-terminal or C-terminal of the antibody with an enzyme (e.g., in the case of ADEPT) or a polypeptide that 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 degree to which the antibody is glycosylated. Addition or deletion of glycosylation sites to the antibody can be conveniently achieved by modifying the amino acid sequence such that one or more glycosylation sites are created or removed.

[0184] If an antibody contains an Fc region, the carbohydrate attached to it may be modified. Natural antibodies produced by mammalian cells typically contain branched oligosaccharides that are commonly attached to Asn297 of the CH2 domain of the Fc region by an N-bond. See, for example, Wright et al. TIBTECH 15:26-32 (1997). Oligosaccharides may 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 branched oligosaccharide structure. In some embodiments, modification of the oligosaccharide in the antibody of the present invention may be performed to create antibody variants having specific improved properties.

[0185] In one embodiment, an antibody variant is provided having a carbohydrate structure lacking fucose (directly or indirectly) bound to the Fc region. For example, the amount of fucose in such an antibody may be 1%–80%, 1%–65%, 5%–65%, or 20%–40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 relative to the total of all sugar structures (e.g., complexes, hybrids, and high-mannose structures) bound to Asn297, measured by MALDI-TOF mass spectrometry, for example, as described in International Publication No. 2008 / 077546. Asn297 refers to the asparagine residue located approximately at position 297 (EU numbering of Fc region residues) within the Fc region, although Asn297 may also be located ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to slight sequence variations in the antibody. Such fucosylated variants may have improved ADCC function. See, for example, U.S. Patent Application Publication No. 2003 / 0157108 (Presta, L.); and No. 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include: U.S. Patent Application Publication No. 2003 / 0157108; International Publication No. 2000 / 61739; International Publication No. 2001 / 29246; U.S. Patent Application Publication No. 2003 / 0115614; International Publication No. 2002 / 0164328; International Publication No. 2004 / 0093621; International Publication No. 2004 / 0132140; International Publication No. 2004 / 0110704; International Publication No. 2004 / 0110282 ;Ibid. No. 2004 / 0109865; International Publication No. 2003 / 085119; Ibid. No. 2003 / 084570; Ibid. No. 2005 / 035586; Ibid. No. 2005 / 035778; Ibid. No. 2005 / 053742; Ibid. 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 lacking protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application Publication No. 2003 / 0157108 A1, Presta, L; and International Publication No. 2004 / 056312 A1, Adams et al., particularly Example 11), and knockout cell lines, e.g., 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 International Publication No. 2003 / 085107).

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

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

[0188] In certain embodiments, the present invention aims to create antibody variants that, by possessing some, but not all, effector functions, are desirable candidate candidates for applications where the in vivo half-life of the antibody is important, but certain effector functions (such as complement and ADCC) are unnecessary or harmful. In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / loss of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to confirm that the antibody lacks FcγR binding (and therefore is likely to lack ADCC activity) but retains FcRn binding ability. NK cells, the primary cells for mediating ADCC, express only Fc(RIII), while monocytes express Fc(RI), Fc(RII, and Fc(RIII). FcR expression in 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 for evaluating the ADCC activity of target molecules are found in U.S. Patent No. 5,500,362 (see, for example, Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)), and Hellstrom, I et al., Proc. Nat'l This is described in Acad.Sci.USA82:1499~1502(1985);5,821,337 (see Bruggemann, M et al. J.Exp.Med.166:1351-1361(1987)). Alternatively, non-radioactive assay methods may 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 (PBMCs) and natural killer (NK) cells.Alternatively or additionally, the ADCC activity of the molecule of interest can be evaluated in vivo in animal models, such as those disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay may also be performed to confirm that the antibody is unable to bind to C1q and therefore lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in International Publication Nos. 2006 / 029879 and International Publication Nos. 2005 / 100402. To evaluate 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 determination 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 those with two or more substitutions at amino acid positions 265, 269, 270, 297, and 327, and include the so-called "DANA" Fc mutant in which residues 265 and 297 are substituted with alanine (U.S. Patent No. 7,332,581). A specific antibody variant exhibiting improved or reduced binding to FcR is described. (See, for example, U.S. Patent No. 6,737,056; International Publication No. 2004 / 056312; and Shields et al., J. Biol. Chem. 9(2):6591-6604(2001).)

[0190] In certain embodiments, the antibody variant includes an Fc region having 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 results in modified (i.e., improved or reduced) C1q binding and / or complement-dependent cytotoxicity (CDC), as described, for example, in U.S. Patent No. 6,194,551, International Publication No. 99 / 51642, Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0192] Antibodies that have increased half-lives and improved binding to the embryonic Fc receptor (FcRn) and play a role in 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 contain an Fc region having one or more substitutions therein that improve the binding of the Fc region to FcRn. Such Fc variants include variants having substitutions in one or more 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, for example, a variant having a substitution in 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 Nos. 5,648,260, 5,624,821, and International Publication No. 94 / 29351.

[0194] d) Cysteine-modified antibody variant In certain embodiments, it may be desirable to create a cysteine-manipulated antibody, e.g., "thioMAb," in which one or more residues of the 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, a reactive thiol group is positioned at an accessible site of the antibody, which can be used to create an immunoconjugate by conjugating the antibody to other parts, such as a drug moiety or a linker-drug moiety, 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-manipulated antibodies can be produced, for example, as described in U.S. Patent No. 7,521,541.

[0195] e) Antibody derivative In certain embodiments, the antibodies provided herein may be further modified to include further non-proteinoid moieties known and readily available in the art. Suitable sites for antibody derivatization 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 homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous in production due to its stability in water. The polymers may have any molecular weight and may be branched or unbranched. The number of polymers attached to an antibody varies, and if multiple polymers are attached, they may be the same molecule or different molecules. Generally, the number and / or type of polymers used for derivatization is not limiting, but can be determined based on considerations such as the specific properties or functions of the antibody being improved, and whether the antibody derivative will be used for therapeutic purposes under defined conditions.

[0196] In other embodiments, conjugates of an antibody and an unprotected site that can be selectively heated by exposure to radiation are provided. In one embodiment, the unprotected site 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 unprotected site to a temperature that kills cells proximal to the antibody unprotected site.

[0197] B. Recombination methods and compositions The antibody may be produced, for example, using a recombinant method and composition described in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid encoding an anti-Tie2 antibody as described herein is provided. Such nucleic acid may encode an amino acid sequence comprising the VL of the antibody and / or 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., an expression vector) comprising such nucleic acid are provided. In a further embodiment, a host cell comprising such nucleic acid is provided. In one such embodiment, the host cell comprises (e.g., transformed) a vector comprising: (1) 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, e.g., a Chinese hamster ovary (CHO) cell, or a lymphoid cell (e.g., Y0, NS0, Sp20 cells). In one embodiment, a method for producing an anti-Tie2 antibody is provided, which includes culturing host cells containing nucleic acids encoding the antibody under conditions suitable for antibody expression, and optionally recovering the antibody from the host cells (or host cell culture medium).

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

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

[0200] In addition to prokaryotes, eukaryotes such as filamentous fungi and yeast are suitable as cloning or expression hosts for vectors encoding antibodies, including strains and yeast strains in which the glycosylation pathway has been "humanized," resulting in the production of antibodies having a partially or fully human glycosylation pattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006).

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

[0202] Plant cell cultures can also be used as hosts. See, for example, U.S. Patents 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe PLANTIBODIES® technology for antibody production in transgenic plants).

[0203] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in suspensions may be useful. Other examples of useful mammalian host cell lines include the CV1 monkey kidney cell line transformed with SV40 (COS-7); human embryonic kidney cells (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)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor cells (MMT 060562); TRI cells described in Mather et al., Annals NYAcad. Sci. 383:44-68 (1982); MRC These are 5 cells and FS4 cells. Other useful mammalian host cell lines include DHFR - Examples include Chinese hamster ovary (CHO) cells, including CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0, and Sp2 / 0. For a review of specific mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKCLo, 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 assays and other assays In one aspect, the antibody of the present invention is tested for its antigen-binding activity by known methods such as ELISA, BIACore®, FACS, or Western blotting.

[0206] In other contexts, competitive assays can be used to identify antibodies that compete with an anti-Tie2 antibody (referred to herein as Tie2.1M100cF) containing a VH sequence with sequence number 20 and a VL sequence with sequence number 21 for binding to Tie2. In certain embodiments, such competing antibodies bind to the same epitope (e.g., a linear epitope or conformational epitope) bound by the anti-Tie2 antibody containing a VH sequence with sequence number 20 and a VL sequence with sequence number 21. Detailed illustrative methods for mapping the epitopes to which antibodies bind 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 that binds to Tie2 (e.g., Tie2.1M100cF) and a second unlabeled antibody being tested for its ability to compete with the first antibody for binding to TIe2. The second antibody may be present in the hybridoma supernatant. As a control, immobilized Tie2 is incubated in a solution containing the first labeled antibody but not in a solution containing the second unlabeled antibody. After incubation under conditions that allow the first antibody to bind to Tie2, excess unbound antibody is removed and the amount of labeling associated with the immobilized TIe2 is measured. If the amount of labeling associated with the immobilized TIe2 is substantially reduced in the test sample compared to the control sample, it indicates that the second antibody is competing with the first antibody for binding to TIe2. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0208] 2. Activity assay In one aspect, an assay is provided for identifying an anti-TIe2 antibody having a desired biological activity. Biological activity may include, for example, binding to Tie2 or its fragments, competing with Ang1 and / or Ang2 for binding to Tie2, activating phosphorylation of the 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, antibodies of the present invention are tested for such biological activity.

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

[0210] In some embodiments, assays are provided for determining the stability (e.g., thermal stability) of an anti-Tie2 antibody or its antibody conjugate, fusion protein, or polymer formulation. For example, the stability of an antibody, its antibody conjugate, fusion protein, or polymer formulation can be determined using any method known in the art, such as differential scanning fluorescence (DSF), circular dichroism (CD), endogenous protein fluorescence, differential scanning calorimetry, spectroscopy, light scattering (e.g., dynamic light scattering (DLS) and static light scattering (SLS), and self-interacting chromatography (SIC).

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

[0212] D. Immunoconjugate The present invention also provides an immune complex comprising one or more cytotoxic agents, such as a chemotherapeutic agent or chemotherapeutic drug, a growth inhibitor, a toxin (e.g., a protein toxin, a bacterial or fungal enzyme-active toxin of animal origin, or a fragment thereof), or a radioisotope, and an anti-Tie2 antibody as defined herein.

[0213] E. conjugate The present invention also provides a conjugate comprising any anti-Tie2 antibody or its Tie2-binding fragment provided herein, which is conjugated to one or more heterogeneous molecules such as polyols.

[0214] 1. Multi-arm polymer In some embodiments, the conjugates of the present disclosure may be constructed by derivatizing the anti-Tie2 antibody described herein by conjugating a multi-armed polymer with anti-Tie2 Fab or a variant thereof. Any multi-armed polymer that provides a conjugate of a desired size or has a selected average molecular weight as described herein will be understood to be 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 this disclosure, non-protein polymers are used to form the conjugates of this disclosure. Non-protein polymers are typically hydrophilic synthetic polymers, i.e., polymers that are not actually found. However, polymers that do exist and are produced by recombinant or in vitro methods can also be useful, as they are polymers isolated from natural sources.

[0216] In some embodiments, anti-Tie2 antibodies are derivatized by conjugating (e.g., covalently bonding) Fab or a variant thereof to a multi-armed polyol. Thus, in some embodiments, the disclosure relates to a conjugate comprising one or more anti-Tie2 Fab or variants thereof disclosed herein, covalently bonded to one or more multi-armed polyols, preferably 6-armed polyols. The polyol used may be any water-soluble poly(alkylene oxide) polymer and may have linear or branched chains. Suitable polyols include those in which one or more hydroxyl positions are substituted with chemical groups, e.g., alkyl groups having 1 to 4 carbon atoms. Typically, the polyol is poly(alkylene glycol), e.g., polyethylene glycol (PEG), and therefore, for the sake of brevity of the explanation, the remainder of the discussion relates to exemplary embodiments, where the polyol used is PEG, and the process of conjugating the polyol to a polypeptide is referred to as "PEGylation." 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 conjugate techniques similar to those described herein for PEG.

[0217] The polyols used to form the conjugates of this disclosure are multi-armed polyols. As used herein, “multi-armed polyol” refers to a polyol comprising a core structure that connects at least two arms. Multi-armed polyols may be, for example, dimers (2-armed), tetramers (4-armed), hexamers (6-armed), octamers (8-armed), etc. In some aspects, multi-armed polyols are multi-armed PEGs.

[0218] The weight-average molecular weight of multi-armed PEGs used for PEGylation of anti-Tie2 antibodies and antibody variants may vary, typically ranging from about 500 to about 300,000 daltons (D). In some embodiments, the weight-average molecular weight of the multi-armed PEGs 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 carried out using a multi-armed PEG having a weight-average molecular weight of about 6,000 D.

[0219] Various methods for PEGylation of proteins are known in the art. Specific methods for producing PEG-conjugated proteins include U.S. Patents 4,179,337, 4,935,465, and 5,849,535, all of which are incorporated herein by reference in their entirety. Typically, a protein is covalently bonded to a terminal reactive group on a polymer via one or more amino acid residues of the protein. Polymers having reactive groups are shown herein as activated or functionalized polymers (e.g., functionalized PEGs). The reactive groups selectively react with free sulfhydryl or amino or other reactive groups on an antibody or antibody variant. Multi-armed PEG polymers can conjugate to sulfhydryl or amino or other reactive groups on an antibody or antibody variant in either a random or site-specific manner. However, it will be understood that, in order to obtain 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 specific antibody or antibody variant used to limit, and preferably substantially avoid, the presence of reactive groups that react with too many reactive groups on the antibody. In some cases, it may not be possible to adequately limit or avoid this, so typically, about 0.05 to about 1000 moles of functionalized polymer, or in some embodiments, about 0.05 to about 200 moles per mole of antibody, depending on the antibody concentration, may be used. The final amount of functionalized polymer per mole of antibody is the equilibrium for maintaining optimal activity, but, if possible, simultaneously optimizes the vitreous, retinal, and / or aqueous humor half-lives of the antibody.

[0220] The residue may be any reactive amino acid on the antibody or antibody variant, such as an N-terminal amino acid group, but in some embodiments the reactive amino acid is cysteine, which is linked to the reactive group of the functionalized polymer via its free thiol group, as shown, for example, in International Publication Nos. 99 / 03887, 94 / 12219, 94 / 22466, U.S. Patent Nos. 5,206,344, 5,166,322 and 5,206,344, all of which are incorporated herein by reference in their entirety. In such embodiments the polymer may contain at least one terminal reactive group that can react specifically with a free sulfhydryl or thiol group(s) on the parent antibody. Such groups include, but are not limited to, maleimides, sulfhydryls, thiols, trifluoromethanesulfonates, tosylates, aziridines, epoxides, pyridyl disulfides, succinimidyl esters, -NH2, aldehydes, haloacetates, haloacetamides, and para-nitrophenyl carbonates. The polymers can be coupled to parent antibodies using any protocol suitable for the chemistry of the selected coupling system, e.g., U.S. Patent No. 4,179,337, U.S. Patent No. 7,122,636, and the protocols and systems described in Jevsevar et al., Biotech J., Vol. 5, pp. 113-128 (2010). Alternatively, the reactive amino acid may be lysine (linked to the reactive group of the functionalized polymer via its free epsilon-amino group) (see, for example, International Publication No. 93 / 00109, which is 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 be reacted with the alpha- and epsilon-amine or sulfhydryl groups of the protein, for example, to form a covalent bond. It will be understood that this disclosure is not limited to conjugates using any particular type of linkage between an antibody or antibody fragment and a polymer.

[0221] Suitable functionalized multi-armed PEGs for use in preparing the conjugates of this disclosure can be produced by many conventional reactions. For example, N-hydroxysuccinimide ester 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 PEG-terminal hydroxyl group can be converted to an amino group by, for example, reacting it with thionyl bromide to form PEG-Br, which is then aminolytically decomposed with excess ammonia to form PEG-NH2. PEG-NH2 can then be conjugated to the antibody or antibody variant of interest using a standard coupling reagent such as Woodward's reagent K. Furthermore, the PEG-terminal CH2OH group can be converted to an aldehyde group, for example, by oxidation with MnO2. The aldehyde group can then be conjugated into an antibody or antibody variant by reductive alkylation using reagents such as cyanoboron hydride.

[0222] In some embodiments, the multi-arm PEG used to prepare the conjugate of the present disclosure is general formula (I): JPEG2026053356000007.jpg43170 (wherein PEG is the same or different -(CH2CH2O)m-, where each m represents the length or size of a particular arm of polyol (PEG), independently being 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; l is an integer of 2 or more, preferably 2 or 3).

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

[0224] A multi-armed PEG having the structure of general formula (I) may be functionalized to react with an antibody (e.g., an antibody fragment) or to bind to a terminal reactive group suitable for conjugation thereto, for example, by using any of the techniques described above to produce a functionalized multi-armed PEG. However, in other embodiments, for example, as described in U.S. Patent No. 7,122,636, which is incorporated herein by reference in whole, a multi-armed PEG may be covalently bound to an anti-Tie2 antibody via a polyfunctional crosslinking agent that reacts with the PEG and one or more amino acid residues of the antibody or antibody variant to be conjugated.

[0225] In other contexts, 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 may be directly conjugated to an anti-Tie2 antibody to form the conjugates of the present disclosure. In some embodiments, the functionalized multi-armed PEG is given by general formula (Ia): JPEG2026053356000008.jpg42170 (wherein each m represents the length or size of a particular arm of polyol (PEG), independently an integer between approximately 45 and approximately 1000, approximately 3 and approximately 250, or approximately 50 and approximately 200, or approximately 20 and 30, or approximately 100 and approximately 150, and n is an integer between approximately 1 and approximately 10, and each R 1 Each R is either independently absent or a linking group, 2 Each is independently either a hydrogen atom or a terminal reactive group, and has at least one R 2 It has the structure of (where is a terminal reactive group). In some embodiments, R 2 These are independently selected from thiol-reactive groups, amino-reactive groups, and combinations thereof.

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

[0227] In other embodiments, the multi-arm PEG used to prepare the conjugate of the present disclosure is general formula (II): JPEG2026053356000009.jpg30170 has the structure of (wherein each m represents the length or size of a particular arm of polyol (PEG), independently being an integer between approximately 45 and approximately 1000, approximately 3 and approximately 250, or approximately 50 and approximately 200, or approximately 100 and approximately 150, and n is an integer between approximately 1 and approximately 10).

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

[0229] In other contexts, the multi-arm PEG used to prepare the conjugate of this disclosure is general formula (II): JPEG2026053356000010.jpg42170 has the structure of (wherein each m represents the length or size of a particular arm of polyol (PEG), independently being an integer between approximately 45 and approximately 1000, approximately 3 and approximately 250, or approximately 50 and approximately 200, or approximately 100 and approximately 150, and n is an integer between approximately 1 and approximately 10).

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

[0231] In other embodiments, the multi-arm PEG used to prepare the conjugate of the present disclosure is general formula (IV): JPEG2026053356000011.jpg42170 has the structure of (wherein each m represents the length or size of a particular arm of polyol (PEG), which is independently an integer between approximately 45 and approximately 1000, approximately 3 and approximately 250, or approximately 50 and approximately 200, or approximately 100 and approximately 150).

[0232] A multi-armed PEG having any of the structures of general formulas (I) to (IV) may be functionalized to react with or conjugate to an antibody (e.g., an antibody fragment) using any of the techniques described above to produce a functionalized multi-armed PEG. However, in other embodiments, for example, as described in U.S. Patent No. 7,122,636, which is incorporated herein by reference in whole, a multi-armed PEG may be covalently conjugated to an anti-Tie2 antibody via a polyfunctional crosslinking agent that reacts with the PEG and one or more amino acid residues of the antibody or antibody variant to be conjugated.

[0233] In other contexts, 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 may be directly conjugated to an anti-Tie2 antibody to form the conjugates of the present disclosure. In some embodiments, the functionalized multi-armed PEG is given by general formula (Ia): JPEG2026053356000012.jpg42170 (wherein each m represents the length or size of a particular arm of polyol (PEG), independently an integer between approximately 45 and approximately 1000, approximately 3 and approximately 250, or approximately 50 and approximately 200, or approximately 100 and approximately 150, and n is an integer between approximately 1 and approximately 10, and each R 1 Each R is either independently absent or a linking group, 2 Each is independently either a hydrogen atom or a terminal reactive group, and has at least one R 2 It has the structure of (where is a terminal reactive group). In some embodiments, R 2 These are independently selected from thiol-reactive groups, amino-reactive groups, and combinations thereof.

[0234] In some embodiments, the functionalized multi-armed PEG has the structure of general formula (Ia), where n is an integer from 1 to 3. In some embodiments, the functionalized multi-armed PEG has the structure of general formula (Ia), where n is 1, and the multi-armed PEG is a tetramer. In other embodiments, the functionalized multi-armed PEG has the structure of general formula (Ia), where n is 2, and the multi-armed PEG is a hexamer. In other embodiments, the functionalized multi-armed PEG has the structure of general formula (Ia), where n is 3, and the multi-armed PEG is an octamer. In such embodiments, the octamer is general formula (Ib): JPEG2026053356000013.jpg46170 (where m, R 1 , and R 2 It has the structure (as defined above).

[0235] A multi-armed PEG having the structure of general formula (Ib) has a dipentaerythritol (DP) core structure and is also referred to herein as a DP hexamer.

[0236] In some embodiments, the functionalized multi-arm PEG has a structure of general formula (Ib) or (Ic), and each R 1 If present, they are identical or different, R 1and R 2 When they come together, JPEG2026053356000014.jpg41170JPEG2026053356000015.jpg57170(wherein each i is an independent integer between 0 and 10; j is an integer between 0 and 10; R 2 R is as defined herein); as well as selected from combinations thereof. In some embodiments, each R 1 It is a linking group.

[0237] In some embodiments, the functionalized multi-arm PEG has a structure of general formula (Ib) or (Ic), and R 1 and R 2 When they come together, The image is JPEG2026053356000016.jpg17170, where i, j, and R are in the formula. 2 R is defined herein. In some embodiments, R 1 and R 2 When they come together, The filename is JPEG2026053356000017.jpg16170, where i is 2; j is 2 or 3; R 2 This is defined herein.

[0238] In some embodiments, the functionalized multi-arm PEG has the structure of general formula (Ib), and each R 2 R is independently selected from maleimide, sulfhydryl, thiol, trifluoromethanesulfonate, tosylate, aziridine, epoxide, pyridyl disulfide, succinimidyl ester, -NH2, aldehyde, haloacetate, haloacetamide, and para-nitrophenyl carbonate. In some embodiments, each R 2 R is independently selected from bromoacetic acid, iodoacetic acid, chloroacetic acid, and combinations thereof, and is a haloacetate. In some embodiments, each R 2R is independently selected from bromoacetic acid, iodoacetic acid, chloroacetic acid, and combinations thereof, and is a haloacetamide. In some embodiments, R 2 It is maleimide.

[0239] In some embodiments, the functionalized multi-arm PEG has a structure of general formula (Ia) or (Ib), and each R 2 is maleimide. In some embodiments, the functionalized multi-arm PEG has the structure of general formula (Ia) or (Ib), and R 1 and R 2 When they come together, The image is JPEG2026053356000018.jpg37170, where i and j are as defined above. In some embodiments, the functionalized multi-arm PEG has the structure of general formula (Ia) or (Ib), and R 1 and R 2 When they come together, The image is JPEG2026053356000019.jpg37170, where i is 2 and j is 2.

[0240] In other contexts, the functionalized multi-arm PEG used to prepare the conjugates of this disclosure is general formula (IIa): JPEG2026053356000020.jpg32170 (wherein each m represents the length or size of a particular arm of polyol (PEG), independently an integer between approximately 45 and approximately 1000, approximately 3 and approximately 250, or approximately 50 and approximately 200, or approximately 100 and approximately 150, and n is an integer between approximately 1 and approximately 10, and each R 1 Each R is either independently absent or a linking group, 2 Each is independently either a hydrogen atom or a terminal reactive group, and has at least one R 2 It has the structure of (where is a terminal reactive group). In some embodiments, R 2 These are independently selected from thiol-reactive groups, amino-reactive groups, and combinations thereof.

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

[0242] In some embodiments, the functionalized multi-arm PEG has the structure of general formula (IIa), and each R 1 If present, they are identical or different, R 1 and R 2 When they come together, JPEG2026053356000021.jpg40170JPEG2026053356000022.jpg57170(wherein each i is an independent integer between 0 and 10; j is an integer between 0 and 10; R 2 R is as defined herein); as well as selected from combinations thereof. In some embodiments, each R 1 It is a linking group.

[0243] In some embodiments, the functionalized multi-arm PEG has the structure of general formula (IIa), and R 1 and R 2 When they come together, The image is JPEG2026053356000023.jpg16170, where i, j, and R are in the formula. 2 R is defined herein. In some embodiments, R 1 and R 2 When they come together, is JPEG2026053356000024.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), and each R 2 is independently selected from maleimide, sulfhydryl, thiol, trifluoromethanesulfonate, tosylate, aziridine, epoxide, pyridyldisulfide, succinimidyl ester, -NH2, aldehyde, haloacetate, haloacetamide, and para-nitrophenyl carbonate. In some embodiments, each R 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), and each R 2 is maleimide. In some embodiments, the functionalized multi-arm PEG has a structure of general formula (IIa), and R 1 and R 2 when combined together, is JPEG2026053356000025.jpg37170, where i and j are as defined above. In some embodiments, the functionalized multi-arm PEG has a structure of general formula (IIa), and R 1 and R 2 when combined together, <0001​​​​In other contexts, functionalized multi-arm PEG is given by general formula (IIIa): JPEG2026053356000027.jpg36170 (wherein each m represents the length or size of a particular arm of polyol (PEG), independently an integer between approximately 45 and approximately 1000, or approximately 3 and approximately 250, or approximately 50 and approximately 200, or approximately 100 and approximately 150, and n is an integer between approximately 1 and approximately 10, and each R 1 Each R is either independently absent or a linking group, 2 Each is independently either a hydrogen atom or a terminal reactive group, and has at least one R 2 It has the structure of (where is a terminal reactive group). In some embodiments, R 2 These are independently selected from thiol-reactive groups, amino-reactive groups, and combinations thereof.

[0247] In some embodiments, the functionalized multi-armed PEG has the structure of general formula (IIIa), where n is an integer from 2 to 6. In some embodiments, the functionalized multi-armed PEG has the structure of general formula (IIIa), where n is 2, and the multi-armed PEG is a tetramer. In other embodiments, the functionalized multi-armed PEG has the structure of general formula (IIIa), where n is 4, and the multi-armed PEG is a hexamer. In other embodiments, the functionalized multi-armed PEG has the structure of general formula (IIIa), where n is 6, and the multi-armed 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), and each R 1 If present, they are identical or different, R 1 and R 2 When they come together, JPEG2026053356000028.jpg43170JPEG2026053356000029.jpg57170(wherein each i is an independent integer between 0 and 10; j is an integer between 0 and 10; R 2 R is as defined herein); as well as selected from combinations thereof. In some embodiments, each R 1 It is a linking group.

[0249] In some embodiments, the functionalized multi-arm PEG has the structure of general formula (IIIa), R 1 and R 2 When they come together, The filename is JPEG2026053356000030.jpg16170, where i, j, and R are in the formula. 2 R is defined herein. In some embodiments, R 1 and R 2 When they come together, The filename is JPEG2026053356000031.jpg15170, where i is 2; j is 2 or 3; R 2 This is defined herein.

[0250] In some embodiments, the functionalized multi-arm PEG has a structure of general formula (IIIa), and each R 2 R is independently selected from maleimide, sulfhydryl, thiol, trifluoromethanesulfonate, tosylate, aziridine, epoxide, pyridyl disulfide, succinimidyl ester, -NH2, aldehyde, haloacetate, haloacetamide, and para-nitrophenyl carbonate. In some embodiments, each R 2 R is independently selected from bromoacetic acid, iodoacetic acid, chloroacetic acid, and combinations thereof, and is a haloacetate. In some embodiments, each R 2 R is independently selected from bromoacetic acid, iodoacetic acid, chloroacetic acid, and combinations thereof, and is a haloacetamide. In some embodiments, R 2 It is maleimide.

[0251] In some embodiments, the functionalized multi-arm PEG has a structure of general formula (IIIa), and each R 2 is maleimide. In some embodiments, the functionalized multi-arm PEG has the structure of general formula (IIIa), and R 1 and R 2 When they come together, JPEG2026053356000032.jpg36170, where i and j are as defined herein. In some embodiments, the functionalized multi-arm PEG has the structure of general formula (IIIa), R 1 and R 2 When they come together, The image is JPEG2026053356000033.jpg37170, where i is 3 and j is 2.

[0252] In other contexts, functionalized multi-arm PEG is given by general formula (IVa): JPEG2026053356000034.jpg42170 (wherein each m represents the length or size of a particular arm of polyol (PEG), independently being an integer between approximately 45 and approximately 1000, or approximately 3 and approximately 250, or approximately 50 and approximately 200, or approximately 100 and approximately 150, and each R 1 Each R is either independently absent or a linking group, 2 Each is independently either a hydrogen atom or a terminal reactive group, and has at least one R 2 It has the structure of (where is a terminal reactive group). In some embodiments, R 2 These are independently selected from thiol-reactive groups, amino-reactive groups, and combinations thereof.

[0253] A multi-armed PEG having the structure of general formula (IVa) has a butanediol core structure and is also referred to herein as a DX octamer.

[0254] In some embodiments, the functionalized multi-arm PEG has a structure of general formula (IVa), and each R 1 If present, they are identical or different, R 1 and R 2 When they come together, JPEG2026053356000035.jpg94170(where i is an independent integer between 0 and 10; j is an integer between 0 and 10; R 2 R is as defined herein); as well as selected from combinations thereof. In some embodiments, each R 1 It is a linking group.

[0255] In some embodiments, the functionalized multi-arm PEG has the structure of general formula (IVa), R 1 and R 2 When they come together, The image is JPEG2026053356000036.jpg18170, where i, j, and R are in the formula. 2 R is defined herein. In some embodiments, R 1 and R 2 When they come together, The filename is JPEG2026053356000037.jpg17170, where i is 2; j is 2 or 3; R 2 This is defined herein.

[0256] In some embodiments, each R 2 R is independently selected from maleimide, sulfhydryl, thiol, trifluoromethanesulfonate, tosylate, aziridine, epoxide, pyridyl disulfide, succinimidyl ester, -NH2, aldehyde, haloacetate, haloacetamide, and para-nitrophenyl carbonate. In some embodiments, each R 2 R is independently selected from bromoacetic acid, iodoacetic acid, chloroacetic acid, and combinations thereof, and is a haloacetate. In some embodiments, each R 2R is independently selected from bromoacetic acid, iodoacetic acid, chloroacetic acid, and combinations thereof, and is a haloacetamide. In some embodiments, R 2 It is maleimide.

[0257] In some embodiments, the functionalized multi-arm PEG has a structure of general formula (IVa), and each R 2 is maleimide. In some embodiments, the functionalized multi-arm PEG has the structure of general formula (IVa), and R 1 and R 2 When they come together, The image is JPEG2026053356000038.jpg37170, where i and j are as defined above. In some embodiments, the functionalized multi-arm PEG has the structure of general formula (IVa), and R 1 and R 2 When they come together, The image is JPEG2026053356000039.jpg37170, where i is 3 and j is 2.

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

[0259] Functionalized multi-arm PEGs suitable for use in this disclosure can also be purchased from many manufacturers and suppliers. 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 a particular embodiment, the active derivative of the multi-armed PEG described is the following general formula (IV): This is an active NHS ester derivative of a multi-armed PEG having the structure shown in JPEG2026053356000040.jpg36170, where R is dipentaerythritol.

[0261] 1. Polyol conjugate In some embodiments, the disclosure relates to a conjugate (e.g., a Tie2 binder) 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 bonding at least one anti-Tie2 Fab or Fab variant to a polyol. In some embodiments, the multi-armed polyol is PEG. In preferred embodiments, PEG is a hexamer. In other embodiments, PEG is an octamer. In some embodiments, PEG has the structure of general formula (Ia).

[0262] The conjugates of this disclosure may be characterized by the number of anti-Tie2 antibodies (Fabs) conjugated to each multi-armed PEG. This is referred to herein as “fabing” or “degree of fabing.” The number of anti-Tie2 antibodies conjugated to each PEG may vary depending on a variety of factors, including: 1) the number of arms in the PEG, 2) the number and / or reactivity of terminal reactive groups on the PEG, 3) the core structure of the PEG, and / or 4) the pegylation reaction conditions. Highly polydispersible multi-armed PEGs used to prepare the conjugates may, in some cases, complicate the analysis of the final conjugate, specifically making precise measurement of the number of Fabs per PEG even more difficult and obscured. Therefore, the PEGs used to form the conjugates typically have polydispersity in the range of about 1 to about 1.35 (determined using methods known in the art), and in various embodiments, they have 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 further about 1.

[0263] In some embodiments, the conjugate of the present disclosure comprises a 6-arm PEG, wherein at least one anti-Tie2 antibody or variant is covalently bound to the PEG. In other embodiments, the conjugate of the present disclosure comprises 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 bound to the PEG. In other embodiments, the conjugate of the present disclosure comprises 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 bound to the PEG. In other embodiments, the conjugate of the present disclosure comprises a 6-arm PEG, wherein at least 4, at least 5, or at least 6 anti-Tie2 Fabs are covalently bound to the PEG. In some embodiments, the conjugate of the present disclosure comprises a 6-arm PEG, wherein at least 4-6 or 5-6 anti-Tie2 Fabs are covalently bound to the PEG. In other embodiments, the conjugate of the present disclosure comprises a 6-arm PEG, wherein at least 4 to 6 anti-Tie2 Fabs are covalently bonded to the PEG.

[0264] In some embodiments, the conjugate of the present disclosure includes a multi-arm PEG having a structure of any one of the general formulas (Ia), (IIa), (IIIa), or (IVa). In such embodiments, at least one R 2 It is covalently bound to the anti-Tie2 antibody or variant described herein. In some embodiments, the multi-armed PEG having any one structure of general formula (Ia), (IIa), (IIIa), or (IVa) is a hexamer and has at least 2, at least 3, at least 4, at least 5, or all 6 R 2 The group is covalently bonded to the anti-Tie2 Fab or variant described herein.

[0265] In some embodiments, the conjugates of the present disclosure include species in which a multi-armed polyol is covalently bound to a specific site(s) on the parent antibody, i.e., the polymer bond targets a specific region or specific amino acid residue(s) in the parent antibody or antibody fragment. Standard mutagenesis techniques can be used to alter the number and / or location of potential PEGylation sites in the parent antibody or antibody fragment. Thus, to the extent that amino acid substitutions introduce or replace amino acids such as cysteine ​​and lysine, the anti-Tie2 antibodies and their variants of the present disclosure may contain more or fewer potential PEGylation sites than the natural sequence anti-Tie2.

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

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

[0268] Cysteine-modified antibodies have been previously described (all of which are incorporated herein by reference in their entirety, U.S. Patent Application Publication No. 2007 / 0092940 and Junutula, JR et al., J. Immunol Methods, Vol. 332(1-2), pp. 41-52 (2008)). In some embodiments, the cysteine-modified antibody may be the parent antibody. These are located at specific positions, typically within a constant region, for example, C L or C H In step 1, it is useful for generating antibody fragments containing free cysteine. The parent antibody manipulated to contain cysteine ​​is referred to herein as “thioMab,” and the Fab fragment produced from such cysteine-manipulated antibody, regardless of the method of production, is referred to herein as “thioFab.” As previously described (e.g., U.S. Patent Application Publication 2007 / 0092940 and Junutula, JR et al., J. Immunol Methods, Vol. 332(l-2), pp. 41-52 (2008)), variants having substituted (“manipulated”) cysteine ​​(Cys) residues are evaluated for the reactivity of the newly introduced manipulated cysteine ​​thiol group. The thiol reactivity value is a relative number in the range of 0 to 1.0 and can be measured for any cysteine-manipulated antibody. In addition to having a reactive thiol group, thioMab should be selected so that they retain their antigen-binding ability. The design, selection, and preparation of cysteine-modified antibodies have been previously described in detail (see, for example, International Publication No. 2011 / 069104, which is incorporated herein by reference). In some embodiments, the modified cysteine ​​is introduced into the constant domain of the heavy or light chain. Thus, the cysteine-modified antibodies retain the antigen-binding ability of their wild-type parental antibody counterparts and are therefore capable of specifically binding to antigens.

[0269] In some embodiments, the disclosure relates to an antibody fragment-polymer conjugate, wherein the antigen fragment is a Fab, and the polymer is bound to one or more cysteine ​​residues in the light chain or heavy chain of the Fab fragment, which can typically form interchain disulfide bonds linking the light and heavy chains.

[0270] In other aspects, this disclosure relates to an antibody fragment-polymer conjugate, wherein the antigen fragment is Fab-C, and the polymer binding targets the hinge region of the Fab-C fragment. In some embodiments, one or more naturally occurring cysteine ​​residues in the hinge region of the antibody fragment are used to bind the polymer. In other embodiments, one or more cysteine ​​residues are manipulated in the hinge region of the Fab-C fragment to provide a specific binding site for the polymer. In some embodiments, the anti-Tie2 Fab disclosed herein is modified by adding one cysteine ​​to the C-terminus for the purpose of providing one binding site for polymer conjugation. In other embodiments, the anti-TIe2 antibody Fab described herein is modified by adding four further residues, CPPC (SEQ ID NO: 87), to the C-terminus for the purpose of providing two binding 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), to the C-terminus for the purpose of providing a binding site for polymer conjugation.

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

[0272] PEGylation of anti-Tie2 Fab and variants is carried out by any convenient method. Preferred PEGylation conditions are described in International Publication No. 2011 / 069104 and International Publication No. 03 / 029420, both of which are incorporated herein by reference in their entirety.

[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 fabicization is typically first indicated by SDS-PAGE. Polyacrylamide gel electrophoresis in 10% SDS is performed with 10 mM Tris-HC1 pH 8.0 and 100 mM NaCl as elution buffer. Peptide mapping can be performed using trypsin and proteases such as Lys-C protease to indicate 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 produced peptides can be compared with peptide maps previously determined for anti-Tie2 polypeptides.

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

[0275] PEGylation can alter the physical and chemical properties of antibody drugs, potentially leading to improved pharmacokinetic behavior such as enhanced stability, reduced immunogenicity, extended circulating life, and increased ocular residence time.

[0276] In some embodiments, the conjugates of the present disclosure have an increased half-life after administration to a mammalian eye (e.g., human) via a single intravitreal injection, compared to the corresponding unconjugated anti-Tie2 Fab. In some embodiments, the increased half-life is at least 1.4 times, or at least 1.8 times, or at least 2 times the half-life of the corresponding unconjugated anti-Tie2 Fab.

[0277] 3. IgM polymers as conjugates

[0278] In some embodiments, the Tie2 binder of the present disclosure is, for example, IgM C H1 Using a recombinant expression method for fusing the C-terminus of a domain to the N-terminus of an anti-Tie2 antibody described herein (via a peptide bond), two or more anti-Tie2 antibodies described herein are expressed in the C-terminus of an IgM molecule. H1 It can be constructed by linking to domains. IgM has proven to be a viable format for antibody therapeutics (see, e.g., Hanala, 2012, MAbs, 4:555-561). The "monomer" components of IgM consist of two light chains (LCs), each containing two Ig domains, and two heavy chains (HCs), each containing five Ig domains and a short, unstructured C-terminal tail fragment. These four chains assemble to form a homodimer of an HC-LC heterodimer. The homodimer then covalently bonds to a cyclic structure containing five homodimers and a J chain (JC) (pentamer) or six homodimers (hexamer), each containing 10 and 12 binding sites, respectively. While not theoretically bound, the thermal bonding of multiple variable fragments (Fv) allows IgM to bind to targets without substantial affinity maturation, thereby functioning as a sentinel adaptive immune receptor.

[0279] In certain embodiments, the anti-Tie2 antibody is Fab. The IgM protein (multimer) may or may not contain a J chain, such that a pentamer is formed in the presence of the J chain (which may contain up to 5 anti-Tie2 antibodies) and a hexamer is formed in the absence of the J chain (which may contain up to 5 anti-Tie2 antibodies). In some embodiments, the hexamer is produced by various ratios of IgM heavy chain to light chain (for hexamer formation) or by the ratio of heavy chain to light chain to J chain (for pentamer formation). Thus, in some embodiments, the Tie2 binder is a multimer comprising the IgM protein and at least 2, at least 3, at least 4, at least 5, or at least 6 anti-Tie2 antibodies as described herein, in order to form a multimer that can activate Tie2. An anti-Tie2 IgM molecule has been designed and shown to activate Tie2 activity (see Example 7).

[0280] Anti-Tie2 multimer conjugates constructed using the recombinant IgM format described herein may be useful for ophthalmic treatment because they have a relatively large molecular radius compared to single Fabs, which may result in slower molecular diffusion from vitreous fluid to aqueous humor and blood. As described in Example 7 below, evaluation by light scattering revealed a hydrodynamic radius of approximately 12 nM (R h A result was found, and the predicted molecular weight of the hexamer (approximately 1050 kD) was slightly higher than that of the pentamer (approximately 950 kD).

[0281] Since relatively rapid systemic clearance may be desirable to limit the activity of ophthalmic therapeutics in the eye, the systemic half-life of recombinant IgM molecules was also investigated. As shown in Example 7, recombinantly expressed IgM pentamers and hexamers were removed more rapidly after intravenous injection than IgM isolated from human serum. It was further determined that these recombinant IgM molecules had 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 modifies the level of sialic acid in N-linked glycosylation.

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

[0283] 3. Hexameric peptide polymers as conjugates By linking each antigen-binding agent to a naturally multimerizing peptide, such as an NDK peptide, conjugates containing multiple antigen-binding agents (e.g., an antibody or its antigen-binding fragment) are also included in this disclosure. In some embodiments, the Tie2 binding agents of this disclosure can be prepared by linking two or more anti-Tie2 Fabs described herein to a peptide multimer. The peptide multimer consists 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, spontaneously fold to form a single multi-arm structure. Routine molecular engineering techniques and materials are used to express the multimerizing peptide as a fusion protein in which an antigen-binding protein (e.g., an antibody or its fragment) is expressed at either the N-terminus or C-terminus of the multimerizing peptide. In certain embodiments, the multiple peptides in the multimer are identical, and the expression vector is constructed to link the C-terminus or N-terminus of the peptide to the N-terminus or C-terminus of an anti-Tie2 antibody or its fragment, respectively (via peptide bonds), as taught in this disclosure.

[0284] In certain embodiments, the peptide of the multimer peptide is part of a eukaryotic nucleoside diphosphate kinase (NDK) enzyme having a homohexameric quaternary structure. Several NDK enzymes exist that can be used to design the multimer, 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, for example, the NDK3 peptide derived from UniProt accession P22887. SEQ ID NO: 74 provides the sequence of Tie2.1.M100cF ligated at its C-terminus to the N-terminus of NDK3. In this preferred embodiment, the Fab light chain includes SEQ ID NO: 21. (See, for example, Example 8 below.)

[0285] E. Methods and compositions for diagnosis and detection In certain embodiments, any of the anti-Tie2 antibodies provided herein are useful for detecting the presence of Tie2 in a biological sample. As used herein, the term “detection” includes quantitative or qualitative detection. In certain embodiments, the biological sample includes cells or tissues such as retinal tissue (photoreceptors and the underlying retinal pigment epithelium (RPE) and choroidal capillary plate).

[0286] In one embodiment, an anti-Tie2 antibody is provided for use in a diagnostic or detection method. In a further aspect, a method for detecting the presence of Tie2 in a biological sample is provided. In a particular embodiment, the method includes contacting a biological sample with the anti-Tie2 antibody described herein under conditions that allow the 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 in vitro or in vivo. In one embodiment, for example, if 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, directly detectable labels or sites (e.g., fluorescent labels, chromogenic labels, electron density labels, chemiluminescent labels, radioactive labels, etc.) and sites indirectly detectable via enzymatic reactions or intermolecular interactions (e.g., enzymes or ligands, etc.). Exemplary labels include: radioactive isotopes. 32 P, 14 C, 125 I, 3 H and 131I, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferase, luciferase such as firefly luciferase and bacterial luciferase (U.S. Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazinedione, wasabi peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidase such as glucose oxidase, galactose oxidase, and heterocyclic oxidases such as glucose-6-phosphate dehydrogenase, uricase and xanthine oxidase, enzymes that use hydrogen peroxide to oxidize pigment precursors such as HRP, lactoperoxidase, or microperoxidase, biotin / avidin, spin-labeled, bacteriophage-labeled, stable free radicals, and those conjugated with such.

[0288] F. Pharmaceutical preparations

[0289] Pharmaceutical formulations of anti-Tie2 antibodies or Tie2 conjugates described herein are prepared by mixing such anti-Tie2 antibodies or Tie2 conjugates of desired purity with one or more optionally selected pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed (1980)) in the form of lyophilized formulations or aqueous solutions. The pharmaceutically acceptable carriers are generally non-toxic to the recipient at the dosage and concentration used and include buffers such as phosphates, citrates, 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 approximately 10 residues) polypeptides, blood Examples of pharmaceutically acceptable carriers herein include, but are not limited to, proteins such as clear albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include intervening drug dispersants such as soluble neutrally active hyaluronidase glycoproteins (sHASEGP), e.g., human soluble PH-20 hyaluronidase glycoproteins such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Specific exemplary sHASEGP and methods of use, including rHuPH20, are described in U.S. Patent Application Publications 2005 / 0260186 and 2006 / 0104968.In one phase, sHASEGP is combined with one or more additional glycosaminoglycansases (e.g., chondroitinase).

[0290] An example of a lyophilized antibody preparation is described in U.S. Patent No. 6,267,958. Examples of aqueous antibody preparations are described in U.S. Patent No. 6,171,586 and International Publication No. 2006 / 044908, the latter of which contains histidine acetate buffer.

[0291] The formulations described herein may also contain two or more active ingredients necessary for the specific indication being treated, preferably having 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, B factor, H factor, 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, further compounds include antibodies that bind to VEGF and / or Ang2 and / or IL-1β, or antigen-binding fragments thereof. Such active ingredients are preferably present in combination in amounts effective for the intended purpose.

[0292] The active ingredient may be encapsulated in microcapsules prepared, for example, by coacervation technology or interfacial polymerization (e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively), or encapsulated in 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 formulations may be prepared. Preferred examples of sustained-release preparations include a semipermeable matrix of a solid hydrophobic polymer containing an antibody, where these matrices are in the form of molded articles, such as films or microcapsules.

[0294] Preparations used for in vivo administration are generally sterilized. Sterilization can be easily achieved, for example, by filtration using a sterile filtration membrane.

[0295] Conjugates described herein for the prevention or treatment of ocular diseases or conditions are typically administered by ocular, intraocular, and / or intravitreal injection, and / or near-scleral injection, and / or sub-Tenon's capsule injection, and / or superchoroidal injection, and / or topical administration in the form of eye drops and / or ointments. Such compositions of this disclosure may be delivered intravitreously as devices and / or depots that allow for slow release of the compound into the vitreous fluid, including by various methods, such as those described in reference literature, *Intraocular Drug Delivery*, Jaffe, Ashton, and Pearson, editors, Taylor & Francis (March 2006). In one example, the device may be in the form of a minipump and / or matrix and / or passive diffusion system and / or encapsulated cells that release the compound over a long period of time (Intraocular Drug Delivery, Jaffe, Ashton, and Pearson, editors, Taylor & Francis (March 2006). Other methods of administration may be used, including but not limited to topical, parenteral, subcutaneous, intraperitoneal, intrapulmonary, intranasal, and intrafocal administration. Parenteral administration includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration.

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

[0297] In some embodiments, the antibodies and conjugates described herein may 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 humor is controlled by a porous metal membrane containing titanium frit. Because its reservoir has a low capacity, in some embodiments, high protein concentrations are required for effective delivery using the PDS. Therefore, in some embodiments, the antibodies and conjugates described herein are formulated at high concentrations. In some embodiments, the antibodies and conjugates described herein may be formulated at concentrations 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 concentrations 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. Treatment methods and compositions

[0299] Any anti-Tie2 antibody or conjugate provided herein can be used in a therapeutic method. In any embodiment herein, “individual,” “patient,” or “subject” may be a human.

[0300] The anti-Tie2 conjugates of this disclosure may be used to treat mammals. In some embodiments, the anti-Tie2 antibody or conjugate is administered to a non-human mammal, for example, to obtain preclinical data. Exemplary non-human mammals to be treated include pre-non-human primates, dogs, cats, pigs, rodents, and other mammalian animals in which clinical trials are conducted. Such mammalian animals may be established animal models of diseases 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 mammalian animals.

[0301] Anti-Tie2 conjugates may be administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intravitreous, intrapulmonary, and intranasal, and, if desired for topical immunosuppressive treatment, intrafocal administration. Parenteral infusions include intramuscular, intravenous, intra-arterial, intraperitoneal, intravitreous, and subcutaneous administration. In addition, conjugates are preferably administered by pulse infusion, particularly while reducing the dose of the antibody or its antibody variant or fragments (e.g., antigen-binding fragments). In some embodiments, administration is carried out by injection, such as intravenous or subcutaneous injection, depending in part whether the administration is short-term or chronic.

[0302] For the prevention or treatment of a disease, the appropriate dosage of anti-Tie2 antibodies or conjugates varies depending on the type of disease being treated, its severity and course, whether the antibody is administered for preventive or therapeutic purposes, previous treatments, the patient's clinical history and response to the antibody, and the attending physician's judgment.

[0303] Depending on the type and severity of the disease, for example, approximately 1–25 mg / eye (0.015 mg / kg–0.36 mg / kg per eye) of antibody is the initial candidate dose for administration to the patient, either by one or more separate doses or by continuous infusion. In cases of repeated administration over several days or more, depending on the condition, treatment is continued until the desired suppression of disease symptoms occurs. However, other drug regimens may be useful. The progress of this therapy is readily monitored by conventional techniques and assays. Exemplary drug regimens are disclosed in International Publication No. 94 / 04188.

[0304] In one aspect, an anti-Tie2 antibody or conjugate for use as a pharmaceutical is provided. In a further aspect, an anti-Tie2 antibody or conjugate for use in the treatment of an eye disease or disorder is provided. In a particular embodiment, an anti-Tie2 antibody or conjugate for use in a therapeutic method is provided. In a particular embodiment, the present invention provides an anti-Tie2 antibody or conjugate for use in a method of treating an individual having an eye disease or disorder, comprising administering an effective amount of the anti-Tie2 antibody or conjugate to the individual. In such one embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent to the individual, such as described below. In a further embodiment, the present invention provides an anti-Tie2 antibody or conjugate for use in increasing the integrity of vascular endothelial cell membranes and / or reducing vascular leakage. In certain embodiments, the present invention provides an anti-Tie2 antibody or conjugate for use in a method for increasing the integrity of vascular endothelial cell membranes and / or reducing vascular leakage in an individual, comprising administering the individual an anti-Tie2 antibody or conjugate effective in increasing the integrity of vascular endothelial cell membranes and / or reducing vascular leakage. The “individual” in 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 unaccommodated proliferation and / or infiltration of neovascularization into the structures of ocular tissues such as the retina or cornea. Ocular disorders can also be characterized by atrophy of retinal tissue (photoreceptors and the underlying retinal pigment epithelium (RPE) and choroidal capillary tubules).

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

[0307] Non-limited ocular disorders include, for example, diabetic macular edema (DME) (e.g., focal, non-central DME, and diffuse central involvement 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 retinopathy, AMD (e.g., exudative AMD, dry AMD, intermediate AMD, progressive AMD, and geographic atrophy (GA)), macular degeneration, and yellowing. Spotted edema, retinopathy, ROP, retinal vein occlusion (RVO) (e.g., central (CRVO) and bifurcation (BRVO) morphology), 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, Norley's disease, osteoporosis-pseudogliosis Retinal abnormalities associated with ocular vascular plaque syndrome (OPPG), subconjunctival hemorrhage, rubeosis, ocular neovascularization, neovascular glaucoma, retinitis pigmentosa (RP), hypertensive retinopathy, retinal hemangioma proliferation, telangiectasia, iris neovascularization, intraocular neovascularization, retinal degeneration, cystic macular edema (CME), vasculitis, papilledema, retinitis, e.g., non-limiting CMV retinitis, ocular melanoma, retinoblastoma, conjunctivitis (e.g., infectious conjunctivitis and non-infectious conjunctivitis (e.g., allergic conjunctivitis) - Conjunctivitis, Leber's congenital amaurosis (also known as Leber's congenital amaurosis or LCA), uveitis (infectious and non-infectious uveitis), choroiditis (e.g., multifocal choroiditis), ocular histoplasmosis, blepharitis, dry eye, traumatic eye injury, Sjögren's disease, and other eye diseases in which the disease or disorder is associated with ocular neovascularization, vascular leakage, and / or retinal edema or retinal atrophy. Further exemplary eye disorders include retinal schizophrenia (abnormal splitting of the retinal nerve sensory layer), diseases associated with rubeosis (angiovascular neovascularization), and diseases caused by abnormal proliferation of fibrovascular tissue or fibrous tissue (including all forms of proliferative vitreoretinopathy).

[0308] Exemplary conditions associated with corneal neovascularization include, but are not limited to, epidemic keratoconjunctivitis, vitamin A deficiency, contact lens overwear, atopic keratitis, limbal keratitis, pterygium, keratitis sicca, Sjögren's syndrome, acne rosacea, phylectenulosis, syphilis, microbacterial infections, steatosis, chemical burns, bacterial ulcers, fungal ulcers, herpes simplex infections, herpes zoster, protozoal infections, Kaposi's sarcoma, Mohren's ulcer, pericorneal degeneration of Terien, peripheral keratolysis, rheumatoid arthritis, generalized erythema, polyarteritis, trauma, Wegener's sarcoidosis, scleritis, Stevens-Johnson syndrome, bullous pemphigoid, radial corneal incision, and corneal transplant rejection.

[0309] Exemplary diseases associated with choroidal neovascularization and defects of the retinal vascular system, including increased vascular leakage, aneurysms, and capillary detachment, include, but are not limited to, diabetic retinopathy, macular degeneration, sickle cell anemia, sarcoid, syphilis, pseudoxanthoma elastica, Paget's disease, venous occlusion, arterial occlusion, carotid artery occlusive disease, chronic uveitis / vitritis, mycobacterial infections, Lyme disease, systemic lupus erythematosus, retinopathy of prematurity, retinal edema (including macular edema), Eels' disease, Behçet's disease, infections causing retinitis or choroiditis (e.g., multifocal choroid), presumptive ocular histoplasmosis, Best's disease (vitreomacular degeneration), myopia, optic disc, squamous cellulitis, 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 progressive dry AMD), macular atrophy (e.g., atrophy associated with neovascularization and / or geographic atrophy), diabetic retinopathy, Stargardt disease, Sorsby Fundus dystrophy, retinoschisis, and retinitis pigmentosa.

[0311] For example, in certain embodiments, any of the methods described above further includes administering one or more additional compounds. In certain embodiments, the Tie2 binder or its conjugate or polymer formulation is administered simultaneously with the additional compounds. In certain embodiments, the Tie2 binder or its conjugate or polymer formulation 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 its antigen-binding fragment. In certain embodiments following (or to which are applied) any of the embodiments described above, 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 ischemic retinopathy, 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 examples, 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 International Publication No. 2010 / 069532 or International Publication No. 2016 / 073157).In other examples, the additional compound may be an anti-IL-6 antibody, e.g., EBI-031 (Eleven Biotherapeutics; see, e.g., International Publication No. 2016 / 073890), siltuximab (SYLVANT®), olokizumab, crazakizumab, silkumab, elcilimomab, OPR-003, MEDI5117, PF-04236921, or a variant thereof. In further examples, the additional compound may be an anti-IL-6R antibody, e.g., tocilizumab (ACTEMRA®) (see, e.g., International Publication No. 1992 / 019579), sarilumab, ALX-0061, SA237, or a variant thereof.

[0312] In some cases, the Tie2 conjugates or conjugates and / or polymer formulations of the present disclosure may be administered in combination with at least one additional therapeutic agent for the treatment of ocular disorders, e.g., ocular disorders described herein (e.g., DME, DR, AMD (e.g., exudative AMD), RVO, or GA). Exemplary additional therapeutic agents for combination therapy to treat ocular disorders include, but are not limited to, anti-angiogenic agents, e.g., anti-VEGF antibodies (e.g., anti-VEGF Fab LUCENTIS® (ranibizumab)), soluble receptor fusion proteins (e.g., recombinant soluble receptor fusion protein EYLEA® (also known as aflibercept (VEGF Trap Eye); Regeneron / Aventis)), and 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-methylpiperidine-4-ylmethoxy)quinazoline (ZD6474), 4-(4-fluoro-2-methylindole-5-yloxy)-6-methoxy-7-(3-pyrrolidine-1-ylpropoxy)quinazoline (AZD Examples include 2171), batalanib (PTK787), semaxaminib (SU5416; SUGEN), and SUTENT® (sunitinib); tryptophanyl-tRNA synthetase (TrpRS); squalamine; RETAANE® (acannortabe acetate for depot suspensions; Alcon, Inc); combretastatin A4 prodrug (CA4P); MIFEPREX® (mifepristone-ru486); subtenone triamcinolone acetonide; intravitreal crystalline triamcinolone acetonide; matrix metalloproteinase inhibitors (e.g., Prinomast (AG3340; Pfizer)); fluocinolone acetonide (including fluocinolone intraocular implants; Bausch & Lomb / Control Delivery Systems); linamide; integrin β3 function inhibitors; angiostatins, and combinations thereof.These and other therapeutic agents, which can be administered in combination with the Tie2 binder or conjugate of the present invention, are described, for example, in U.S. Patent Application Publication No. 2014 / 0017244, which is incorporated herein in its entirety by reference.

[0313] Further examples of additional therapeutic agents that can be used in combination with Tie2 binders or conjugates and / or polymer formulations for the treatment of eye disorders (e.g., DME, DR, AMD, RVO, or GA) include, but are not limited to, VISUDYNE® (verteporfin; a photoactivating agent typically used in conjunction with non-thermal laser photodynamic therapy), PKC412, Endovion (NS3728; NeuroSearch A / S), neurotrophic factors (e.g., glial neurotrophic factor (GDNF) and ciliary neurotrophic factor (CNTF)), diltiazem, dorzolamide, PHOTOTROP®, 9-cis-retinal, eye drops (e.g., phospholine iodide, echothiophate, or carbonic anhydrase inhibitors), beovastat (AE-941; AEterna Laboratories, Inc.), Sirna-027 (AGF-745; Sima Ark Therapeutics Ltd., Neurotrophin (for example, 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 (for example, EntreMed, Inc.)(Used by), 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 cell ganglion neuroprotective agents (Cogent Neurosciences), N-nitropyrazole derivatives (Texas A&M University System), KP-102 (Krenitsky Examples include 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., products tested by Novagali Pharma SA and ISIS-13650, Ionis Pharmaceuticals), and combinations thereof.

[0314] Tie2 conjugates or conjugates, and / or polymer formulations thereof, are used, for example, in laser photocoagulation (e.g., panretinal photocoagulation (PRP)), drusen laser oscillation, macular hole surgery, macular translocation surgery, implantable miniature telescopes, PHI motor angiography (also known as microlaser therapy and feeder vascular procedures), proton therapy, microstimulation therapy, retinal detachment and vitrectomy, scleral buckling, submacular surgery, transpapillary thermotherapy, photosystem I therapy, use of RNA interference (RNAi), extracorporeal reophagesis (also known as membrane fractionation filtration and reotherapy), microchip implantation, 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) It may be administered in combination with therapeutic or surgical procedures for the treatment of eye disorders (e.g., AMD, DME, DR, RVO, or GA), including US, Inc., ReNeuron, CHA Biotech, puncture, and combinations thereof.

[0315] In some cases, the Tie2 binder or conjugate and / or polymer formulations of the present invention can be administered in combination with anti-angiogenic agents for the treatment of eye disorders (e.g., DME, DR, AMD, RVO, or GA). Any suitable anti-angiogenic agent, including but not limited to those enumerated by Carmeliet et al., Nature 407:249-257, 2000, can be used in combination with the Tie2 binder or conjugate of the present invention. In some embodiments, the anti-angiogenic agent may include, but is not limited to, anti-VEGF antibodies (e.g., anti-VEGF Fab LUCENTIS® (ranibizumab), RTH-258 (formerly ESB-1008, 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, and anti-VEGF DARPin® (e.g., avisiperpegol, Molecular Partners) VEGF antagonists include 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-methylpiperidine-4-ylmethoxy)quinazoline (ZD6474), 4-(4-fluoro-2-methylindole-5-yloxy)-6-methoxy-7-(3-pyrrolidine-1-ylpropoxy)quinazoline (AZD2171), batalanib (PTK787), semaxaminib (SU5416; SUGEN), and SUTENT® (sunitinib)), and combinations thereof.In some cases, anti-Tie2 antibodies against the fragment include IL-1β; IL-6; IL-6R; PDGF (e.g., PDGF-BB); angiopoietin; angiopoietin 2; Tie2; S1P; integrins αvβ3, αvβ5 and α5β1; betacerlin; apelin / APJ; erythropoietin; complement factor D; TNFα; HtrA1; VEGF receptors (e.g., VEGFR1, VEGFR2, VEGFR3, mbVEGFR, or sVEGFR); and ST-2 receptors. The body and proteins genetically associated with the risk of age-related macular degeneration (AMD), 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, can be combined with antibodies or fragments thereof, or other therapeutic agents. For example, in some cases, 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 International Publication No. 2010 / 069532 or International Publication No. 2016 / 073157).

[0316] Other suitable anti-angiogenic agents that can be administered in combination with anti-Tie2 conjugates and / or polymer formulations for the treatment of eye disorders (e.g., DME, DR, AMD, RVO, or GA) include corticosteroids, anti-angiogenic steroids, akancortarb acetate, angiostatin, endostatin, tyrosine kinase inhibitors, matrix metalloproteinase (MMP) inhibitors, insulin-like growth factor-binding protein 3 (IGFBP3), and interstitial-derived factor (SDF- 1) These include antagonists (e.g., anti-SDF-1 antibodies), pigment epithelial-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 that home to neovascularization sites (e.g., endothelial progenitor cells) (e.g., anti-vascular endothelial cadherin (CD-144) antibodies and / or anti-SDF-1 antibodies), and combinations thereof.

[0317] In further examples, in some cases, Tie2-binding conjugates and / or polymer formulations can be administered in combination with agents active against neovascularization for the treatment of eye disorders (e.g., DME, DR, AMD, RVO, or GA), such as anti-inflammatory drugs, mammalian target (mTOR) inhibitors of rapamycin (e.g., rapamycin, AFINITOR® (everolimus), and TORISEL® (temsirolimus)), cyclosporine, tumor necrosis factor (TNF) antagonists (e.g., anti-TNFα antibodies or their antigen-binding fragments (e.g., infliximab, adalimumab, certolizumab pegol, and golimumab)) or soluble receptor fusion proteins (e.g., etanercept)), anti-complement agents, nonsteroidal anti-inflammatory drugs (NSAIDs), or combinations thereof.

[0318] In further examples, Tie2-binding conjugates and / or polymer formulations may be administered in combination with a class of drugs called "neurosteroids," which include drugs that are neuroprotective and can potentially reduce the progression from dry AMD to wet AMD, such as dehydroepiandrosterone (DHEA) (trade names: PRASTERA® and FIDELIN®), dehydroepiandrosterone sulfate, and pregnenolone sulfate.

[0319] Any suitable AMD therapeutic agent may be administered as an additional therapeutic agent in combination with the Tie2-binding conjugate and / or polymer formulation of the present invention to treat ocular disorders (e.g., DME, DR, AMD, RVO, or GA), but is not limited to, VEGF antagonists, such as anti-VEGF antibodies (e.g., LUCENTIS® (ranibizumab), RTH-258 (formerly ESB-1008, anti-VEGF single-chain antibody fragment; Novartis), or bispecific anti-VEGF antibodies (e.g., anti-VEGF / anti-angiopoietin 2 bispecific antibody, e.g., RG-7716; Roche)), soluble VEGF receptor fusion proteins (e.g., EYLEA® (aflibercept)), or anti-VEGF DARPin® (e.g., abiscipal pegol; Molecular Partners) AG / Allergan), or anti-VEGF aptamers (e.g., MACUGEN® (pegaptanib sodium); platelet-derived growth factor (PDGF) antagonists, e.g., anti-PDGF antibodies, anti-PDGFR antibodies (e.g., REGN2176-3), anti-PDGF-BB pegylated aptamers (e.g., FOVISTA®; Opthotech / 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, e.g., complement factor C5 antagonists (e.g., small molecule inhibitors (e.g., ARC-1905; Optotech) 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., lamparizumab; Roche)); C3 blocking peptides (e.g., APL-2, Appellis); visual cycle modulators (e.g., emixustat hydrochloride); squalamine (e.g., OHR-102;Ohr Pharmaceutical); Vitamin and mineral supplements (e.g., those listed in Age-Related Eye Disease Test 1 (AREDS1; zinc and / or antioxidants) and Test 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 (RPE cell suspension; 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-beta antagonists (e.g., anti-beta-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 endostatins and angiostatins (e.g., RetinoStat); and any combination thereof. In some cases, AMD therapeutics (including any of the aforementioned AMD therapeutics) can be co-formulated. For example, the anti-PDGFR antibody REGN2176-3 can be co-formulated with aflibercept (EYLEA®). In some cases, such co-formulations can be administered in combination with the Tie2 binder or conjugate of the present invention. In some cases, the ocular disorder is DME and / or DR. In some cases, the ocular disorder is AMD (e.g., exudative AMD).

[0320] Tie2-conjugate conjugates and / or polymer formulations 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 cases, the ocular disorder is DME and / or DR. In some cases, the ocular disorder is AMD (e.g., exudative AMD). In some cases, the ocular disorder is GA.

[0321] Tie2-conjugate conjugates and / or polymer formulations 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 cases, the ocular disorder is DME and / or DR. In some cases, the ocular disorder is AMD (e.g., exudative AMD). In some cases, the ocular disorder is GA.

[0322] Tie2-conjugate conjugates and / or polymer formulations 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 cases, the ocular disorder is DME and / or DR. In some cases, the ocular disorder is AMD (e.g., exudative AMD). In some cases, the ocular disorder is GA.

[0323] Tie2-conjugate conjugates and / or polymer formulations 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 cases, the ocular disorder is DME and / or DR. In some cases, the ocular disorder is AMD (e.g., exudative AMD). In some cases, the ocular disorder is GA.

[0324] Tie2-binding conjugates and / or polymer formulations thereof can be administered in combination with PDGF antagonists for the treatment of ocular disorders (e.g., DME, DR, AMD, RVO, or GA). Exemplary PDGF antagonists that may be used in combination with the Tie2-binding conjugates of the present invention include anti-PDGF antibodies, anti-PDGFR antibodies, small molecule inhibitors (e.g., squalamine), anti-PDGF-B PEGylated aptamers such as FOVISTA® (E10030; Opthotech / Novartis), or bispecific 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 agent or conjugate of the present invention. OHR-102 can be administered in combination with a VEGF antagonist such as LUCENTIS® or EYLEA®. In some embodiments, the Tie2 conjugate or conjugate of the present 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., exudative AMD). In some examples, the ocular disorder is GA.

[0325] Tie2-conjugate conjugates and / or polymer formulations thereof 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 injection. In some cases, the ocular disorder is DME and / or DR. In some cases, the ocular disorder is AMD (e.g., exudative AMD). In some cases, the ocular disorder is GA.

[0326] Tie2-conjugate conjugates and / or polymer formulations thereof can be administered in combination with abiscipal pegol for the treatment of ocular disorders (e.g., DME, DR, AMD, RVO, or GA). In some cases, the ocular disorder is DME and / or DR. In some cases, the ocular disorder is AMD (e.g., exudative AMD). In some cases, the ocular disorder is GA.

[0327] Tie2-conjugate conjugates and / or polymer formulations thereof can be administered in combination with abisipalpegol for the treatment of ocular disorders (e.g., DME, DR, AMD, RVO, or GA). In some cases, the ocular disorder is DME and / or DR. In some cases, the ocular disorder is AMD (e.g., exudative AMD). In some cases, the ocular disorder is GA.

[0328] Any suitable DME and / or DR treatment, including but not limited to VEGF antagonists (e.g., LUCENTIS® or EYLEA®), corticosteroids (e.g., corticosteroid implants (e.g., OZURDEX® (dexamethasone intravitreal implant) or ILUVIEN® (fluocinolone acetonide intravitreal implant)) or corticosteroids formulated for intravitreal injection (e.g., triamcinolone acetonide)), or combinations thereof, may be administered in combination with Tie2-bound conjugates and / or polymer formulations for the treatment of ocular disorders (e.g., AMD, DME, DR, RVO, or GA). In some cases, the ocular disorders are DME and / or DR.

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

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

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

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

[0333] In some cases, AMD treatments (e.g., ranibizumab or aflibercept) can be administered in combination with Tie2-conjugated conjugates and / or their polymer formulations using TAO / PRN or TAE treatment regimens. In some cases, the ocular disorder is DME and / or DR. In some cases, the ocular disorder is AMD (e.g., exudative AMD). In some cases, the ocular disorder is GA.

[0334] Such combination therapies described above encompass combined administration (where two or more therapeutic agents are contained in the same or separate formulations) and separate administrations, in which case the administration of the Tie2-conjugate of the present invention may be performed prior to, simultaneously with, and / or subsequently to the administration of the additional therapeutic agent or drug. In one embodiment, the administration of the Tie2-conjugate or polymer formulation and the administration of the additional therapeutic agent are performed within about 1, 2, 3, 4, or 5 months from each other, or within about 1, 3, or 4 weeks, or within about 1, 2, 3, 4, 5, or 6 days.

[0335] Tie2-binding conjugates and / or polymer formulations thereof are further intended for the treatment of glaucoma. Glaucoma is a group of ocular diseases characterized by progressive damage to the eye due to at least partial elevation of intraocular pressure (IOP) (Merck Manual of Diagnosis and Therapy (1999)). Furthermore, glaucoma is characterized by retinal ganglion cell (RGC) death, axonal loss, and a raked appearance of the optic disc (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 mean IOP of a normal adult is 15 mmHg–16 mmHg; the normal range is 10–21 mmHg. One form of glaucoma management is based on reducing IOP using topically applied medications ("Glaucoma," Lancet, 1999;354:1803-1810).

[0336] Currently, there are five main classes of medications used to lower IOP: β-adrenergic antagonists, adrenaline agonists, parasympathomimetic drugs, prostaglandin-like analogs, and carbonic anhydrase inhibitors. Most drugs are applied topically to the eye, but they can cause severe systemic side effects and negatively impact the patient's quality of life. If further reduction of IOP is shown, or if medication fails to sufficiently lower IOP, laser trabeculoplasty is usually the next step. If IOP is still not well controlled, incisional glaucoma surgery is indicated (Id). Despite a significant reduction in the degree of neuronal loss, reduction of RGCs does not guarantee the cessation of the disease process, as RGC loss may continue. Recent studies on the association between IOP regulation and visual field loss after medical or surgical intervention have shown that when IOP is low, ongoing neuronal loss reflected in visual field testing may be reduced. Glaucomatous optic neuropathy is thought to be caused by specific pathophysiological changes and subsequent death of RGCs and their axons. The family of RGC death is thought to be biphasic: primary injury involved in the initiation of damage, followed by slower secondary degeneration due to the harsh environment surrounding the degenerated cells.

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

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

[0340] In a further aspect, the present invention provides a pharmaceutical formulation comprising, for example, one of the Tie2-binding conjugates of the present invention provided herein for use in any of the above-described therapeutics. In one embodiment, the pharmaceutical formulation comprises one of the Tie2-binding conjugates of the present invention provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical formulation comprises one of the Tie2-binding conjugates of the present invention provided herein and, for example, one of the above-described additional therapeutic agents.

[0341] Such combination therapies described above encompass combined administration (where two or more therapeutic agents are contained in the same or separate formulations) and separate administrations, in which case the administration of the antibody of the present invention may be performed prior to, simultaneously with, and / or subsequently to the administration of the additional therapeutic agent or drug. In one embodiment, the administration of the anti-Tie2 conjugate and the administration of the additional therapeutic agent are performed within about one month of each other, or within about one, two or three weeks, or within about one, two, three, four, five or six days.

[0342] The Tie2-conjugated conjugate (and any additional therapeutic agents) of the present invention may be administered by any suitable means, including parenteral, intrapulmonary, intranasal, and, if desired for topical treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Dosage may be by any suitable route, for example, by injection, for example, intravenously or subcutaneously, depending in part whether the administration is temporary or chronic. Various dosing schedules, including but not limited to single or multiple doses at various time points, bolus administration, and pulse infusion, are contemplated herein.

[0343] The Tie2-conjugate of the present invention will be formulated, administered, and given in a manner consistent with good medical practice. Factors to be considered in this regard include the specific disorder being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of drug delivery, the method of administration, the administration schedule, and other factors known to the healthcare professional. The antibody will be formulated, optionally but not necessarily, with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other active agents will depend on the amount of antibody present in the formulation, the type of disorder or treatment, and the other factors mentioned above. These will generally be used by the same dosages and routes of administration as described herein, or at about 1–99% of the dosages described herein, or by any dosage and route as empirically / clinically deemed appropriate.

[0344] For the prevention or treatment of disease, the appropriate dose of the conjugate of the present 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 administered for preventive or therapeutic purposes, previous therapies, the patient's medical history and response to the antibody, and the discretion of the attending physician. The conjugate is administered appropriately to the patient, either in a single dose or over a series of treatments. Depending on the type and severity of the disease, a conjugate of approximately 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg to 10 mg / kg) may be the initial candidate dose for administration to the patient, whether, for example, by one or more separate doses or by continuous infusions. A typical daily dose may range from approximately 1 μg / kg to 100 mg / kg, depending on the factors mentioned above. In repeated administrations over several days or more, treatment is usually continued, depending on the condition, until the desired suppression of disease symptoms occurs. One exemplary dose of the conjugate ranges from approximately 0.05 mg / kg to approximately 10 mg / kg. Therefore, one or more doses of approximately 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 (for example, so that the patient receives approximately 2 to approximately 20 doses of the conjugate, or for example, approximately 6 doses). A larger initial dose may be administered, followed by one or more smaller doses. The progress of this treatment is readily monitored by conventional techniques and assays.

[0345] It is understood that any of the above formulations or therapeutic methods may be carried out using the immunoconjugate of the present invention in place of, or in addition to, the Tie2-binding conjugate of the present invention.

[0346] H.Product

[0347] In another aspect of the present invention, a manufactured article is provided comprising a material useful for the treatment, prevention, and / or diagnosis of the aforementioned disorders. The manufactured article comprises a container and a label or accompanying document 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 the composition to be used alone or in combination with another composition effective for treating, preventing, and / or diagnosing the condition and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial with a stopper that can be punctured by a subcutaneous injection needle). At least one activator in the composition is the Tie2 binder or conjugate of the present invention. The label or accompanying document indicates that the composition is used to treat a selected pathological condition. Furthermore, the product may comprise (a) a first container comprising a composition contained in the product and comprising the Tie2 binder or conjugate of the present invention, and (b) a second container comprising a composition contained in the product and comprising further cytotoxic or other therapeutic agents. In this embodiment of the present invention, the product may further comprise a package insert indicating that its composition is usable to treat a particular condition. Alternatively, or in addition thereto, the manufactured article may further comprise a second (or third) container containing pharmaceutically acceptable buffers, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further comprise other materials desirable from a commercial and user perspective, such as other buffers, diluents, filters, needles, syringes, etc.

[0348] It will be understood that any of the above products may contain the immunoconjugate of the present invention in place of, or in addition to, the Tie2-binding conjugate.

[0349] I. Embodiments of the Invention

[0350] The following lists specific embodiments of the present invention.

[0351] 1. An isolated antibody or fragment thereof that binds to Tie2, An isolated antibody or its antigen-binding fragment comprising (a) CDR-H1 containing the amino acid sequence NTDIS (SEQ ID NO: 3), (b) CDR-H2 containing the amino acid sequence RISPSDGNTYYADSVKG (SEQ ID NO: 4), and (c) CDR-H3 containing the amino acid sequence RTRWASX1AX2DY (SEQ ID NO: 5), with a heavy chain variable domain (VH) 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, and (d) CDR-L1 containing the amino acid sequence RASQDVSTAVA (SEQ ID NO: 8), (e) CDR-L2 containing the amino acid sequence SASFLYS (SEQ ID NO: 9), and (f) CDR-L3 containing the amino acid sequence QQSYTTPPT (SEQ ID NO: 10), and a light chain variable domain (VL).

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

[0353] 3. The antibody according to Embodiment 1, wherein CDR-H3 contains the amino acid sequence RTRWASWAFDY (SEQ ID NO: 7).

[0354] 4. An antibody described in any one of the prior embodiments, which is a monoclonal antibody.

[0355] 5. An antibody according to any one of the prior embodiments, which is a humanized antibody or a chimeric antibody.

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

[0357] 7. An antibody described in any one of the prior embodiments, which is a Fab fragment.

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

[0359] 11. An antibody according to any one of Embodiments 1 to 7, comprising a VL domain containing an amino acid sequence having at least 96% sequence identity with the amino acid sequence of SEQ ID NO: 21; and a VH domain containing an amino acid sequence having at least 96% sequence identity with the amino acid sequence of SEQ ID NO: 20.

[0360] 12. An antibody according to any one of Embodiments 1 to 7, comprising a VL domain containing an amino acid sequence having at least 97% sequence identity with the amino acid sequence of SEQ ID NO: 21; and a VH domain containing an amino acid sequence having at least 97% sequence identity with the amino acid sequence of SEQ ID NO: 20.

[0361] 13. An antibody according to any one of Embodiments 1 to 7, comprising a VL domain containing an amino acid sequence having at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 21; and a VH domain containing an amino acid sequence having at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 20.

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

[0363] 15. An antibody according to any one of the prior embodiments, comprising the VL sequence of SEQ ID NO: 21 and the VH sequence of SEQ ID NO: 19; the VL sequence of SEQ ID NO: 21 and the VH sequence of SEQ ID NO: 22; or the VL sequence of SEQ ID NO: 21 and the VH sequence of SEQ ID NO: 20.

[0364] 16. An antibody according to any one of the prior embodiments, comprising manipulated cysteine.

[0365] 17. The antibody described in Embodiment 16, An antibody in which the manipulated cysteine ​​is selected from T120C, G166C, G178C, T187C, and T209C in HC, or the manipulated cysteine ​​is selected from Q124C, R142C, Q155C, L201C, T206C, K107C, K126C, and K149C in LC, and the residue number of the manipulated cysteine ​​follows EU numbering.

[0366] 18. The antibody according to Embodiment 16 or 17, wherein the manipulated cysteine ​​is selected from T209C in HC and T206C in LC.

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

[0368] 20. The antibody according to any one of Embodiments 16 to 18, wherein the manipulated cysteine ​​is T209C in HC.

[0369] 21. An antibody according to any one of the prior embodiments, comprising an LC containing an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 25; and an HC containing an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 55.

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

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

[0372] 24. An antibody according to any one of the prior embodiments, comprising an LC containing an amino acid sequence having at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 25; and an HC containing an amino acid sequence having at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 55.

[0373] 25. An antibody according to any one of the prior embodiments, comprising an LC containing an amino acid sequence having at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 25; and an HC containing an amino acid sequence having at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 55.

[0374] 26. An antibody according to any one of the prior embodiments, comprising an LC containing the sequence of SEQ ID NO: 25 and an HC containing the sequence of SEQ ID NO: 55.

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

[0376] 28. An antibody according to any one of Embodiments 1 to 20, comprising an LC containing an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 56; and an HC containing an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 23.

[0377] 29. An antibody according to any one of Embodiments 1 to 20, comprising an LC containing an amino acid sequence having at least 96% sequence identity with the amino acid sequence of SEQ ID NO: 56; and an HC containing an amino acid sequence having at least 96% sequence identity with the amino acid sequence of SEQ ID NO: 23.

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

[0379] 31. An antibody according to any one of Embodiments 1 to 20, comprising an LC containing an amino acid sequence having at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 56, and an HC containing an amino acid sequence having at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 23.

[0380] 32. An antibody according to any one of Embodiments 1 to 20, comprising an LC containing an amino acid sequence having at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 56, and an HC containing an amino acid sequence having at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 23.

[0381] 33. An antibody according to any one of Embodiments 1 to 20, comprising an LC containing the sequence of SEQ ID NO: 56 and an HC containing 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, containing an HC containing the sequence of SEQ ID NO: 55 and an LC containing the sequence of SEQ ID NO: 25.

[0383] 35. An isolated nucleic acid encoding an antibody described in any one of the prior embodiments.

[0384] 36. A host cell comprising the nucleic acid described in Embodiment 38.

[0385] 37. A method for producing an antibody that binds to Tie-2, comprising culturing the host cells described in Embodiment 39 under conditions suitable for antibody expression.

[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 as described in any one of Embodiments 1 to 34, wherein each antibody is linked to a multimerizing portion.

[0387] 39. The conjugate according to Embodiment 38, which activates Tie2 phosphorylation in an in vitro or in vivo cell assay.

[0388] 40. The conjugate according to Embodiment 38 or 39, which causes Tie-2 protein levels to be less than 25%, less than 50%, or less than 75% in an in vitro assay, or does not cause a decrease of more than 25%, more than 50%, or more than 75% in Tie-2 protein levels in an in vitro assay.

[0389] 41. A conjugate according to any one of embodiments 38 to 40 that 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 polymerizing portion comprises a polyol, a polypeptide, and / or a peptide.

[0391] 43. The conjugate according to Embodiment 42, wherein the polyol is a multi-armed polyol selected from dimers, tetramers, hexamers, and octamers.

[0392] 44. The conjugate according to Embodiment 43, wherein the multi-armed polyol is a hexamer.

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

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

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

[0396] 48. The conjugate according to Embodiment 47, wherein the cysteine ​​amino acid is a modified cysteine.

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

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

[0399] 52. The conjugate according to any one of Embodiments 48 to 50, wherein the manipulated cysteine ​​is T209C in HC and the residue number follows EU numbering.

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

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

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

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

[0404] 57. The conjugate according to any one of embodiments 46 to 56, wherein PEG comprises a dipentaerythritol hexamer or octamer core. 58. PEG is the general formula (Ia): JPEG2026053356000041.jpg42170 (wherein each n is an integer from 1 to 10 and each m is an integer from 3 to 250 independently; each R 1 However, independently, they are either nonexistent or are linking groups; and each R 2 However, independently, it is either hydrogen or a terminal reactive group; at least one R 2 (However, it is a terminal reactive group and is covalently bound to the antibody described in any one of claims 1 to 14.) A conjugate according to any one of embodiments 46 to 57, having the structure of [the specified structure]. 59. PEG is the general formula (Ib): JPEG2026053356000042.jpg45170 (wherein each m is an integer between 3 and 250 independently; each R 1 is either independently absent or a linking group; and each R 2 However, independently, it is either hydrogen or a terminal reactive group; at least one R 2 (However, it is a terminal reactive group and is covalently bound to the antibody described in any one of claims 1 to 14.) A conjugate according to any one of embodiments 46 to 57, having the structure of [the specified structure].

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

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

[0407] 62. A conjugate comprising an antibody that specifically binds to Tie-2, wherein the antibody comprises the VH sequence of SEQ ID NO: 20 and the VL sequence of SEQ ID NO: 21, and the antibody has general formula (Ib): JPEG2026053356000043.jpg48170 (wherein each m is an integer between 3 and 250 independently; each R 1 is either independently absent or a linking group; and each R 2 However, independently, it is either hydrogen or a terminal reactive group; at least one R 2 (However, it is covalently bound to the antibody.) A conjugate covalently bonded to polyethylene glycol.

[0408] The conjugate according to embodiment 62, wherein 63.m is an integer between 10 and 200.

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

[0410] 65.R 1 Either it does not exist, or R 1 but, JPEG2026053356000044.jpg93170(where i is an independent integer between 0 and 10; j is an integer between 0 and 10; R 2 (These are terminal reactive groups selected from the group consisting of thiol reactive groups, amino reactive groups, and combinations thereof); and combinations thereof A conjugate according to any one of embodiments 58 to 64, selected from the group consisting of the following.

[0411] 66.Each R 2 The conjugate according to any one of embodiments 58 to 65, wherein the reactive group is selected from maleimide, sulfhydryl, thiol, trifluoromethanesulfonate, tosylate, aziridine, epoxide, pyridyl disulfide, succinimidyl ester, -NH2, aldehyde, haloacetate, haloacetamide, and para-nitrophenyl carbonate.

[0412] 67.R 2 A conjugate according to any one of embodiments 58 to 66, wherein is maleimide.

[0413] 68. The conjugate according to any one of embodiments 43 to 67, which is prepared by covalently binding at least one antibody according to any one of claims 1 to 34 to a multi-armed 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 according to Embodiment 69, wherein the concentration of the conjugate is approximately 50 mg / ml to approximately 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 according to Embodiment 71, wherein the additional therapeutic agent is selected from the group consisting of VEGF antagonists, Ang2 antagonists, HtrA1 antagonists and IL33 antagonists, complement component antagonists and second Tie2 agonists.

[0418] 73. The pharmaceutical composition according to Embodiment 72, wherein the VEGF antagonist is selected from the group consisting of VEGF traps and anti-VEGF antibodies.

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

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

[0421] 76. The method according to embodiment 75, wherein the Tie2 pathway-mediated impairment is impaired vascular permeability.

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

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

[0424] 79. The method according to any one of embodiments 75 to 78, wherein the eye condition is DME.

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

[0426] 81. The method according to any one of Embodiments 75 to 79, comprising administering an antibody, conjugate, or pharmaceutical preparation by intravitreal administration.

[0427] 82. The method according to Embodiment 81, wherein intravitreal administration is performed by administering through a narrow-diameter needle.

[0428] 83. The method according to Embodiment 82, wherein the narrow-bore needle is approximately 30, 29, 28, 27, 26, 25, 24, 23, or 22 gauge.

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

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

[0431] Example 1 - Generation of anti-Tie2 antibodies derived from a natural phage library Antibodies binding to the extracellular domain (ECD) of Tie2 were initially selected from a library of phage-presenting synthetic antibodies constructed on a single human framework by introducing synthetic diversity to solvent-exposed positions within the heavy chain CDR, as described below. Phages were screened against the ECD and various subdomains of the ECD.

[0432] Phagemid vectors for library construction Phagemids pV0350-2b and pV0350-4 were designed to present a Fab template on the surface of M13 phage particles in monovalent or bivalent form, 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 a modified CDR region derived from a mouse monoclonal antibody specific to Her-2 within the human consensus sequence Fab framework. Specifically, the sequence includes a kappa light chain (LC region) upstream of the VH and CH1 domains (HC region). The method for producing anti-Her-2 antibodies and the identity of the variable domain sequence are described in U.S. Patents No. 5,821,337 and No. 6,054,297.

[0433] Vector pV0350-2b was constructed by modifying a previously described phagemide (pHGHam-gIII) used for phage presentation 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 encoded the h4D5 light chain (version 8), and the second open reading frame encoded the variable (VH) domain and the first constant (CH1) domain of the h4D5 heavy chain fused to cP3; each protein was directed to secretion via the N-terminal stII signal sequence. Since this modification has been shown to increase the level of Fab presented on phages, the amber stop codon between the heavy chain fragment and cP3 was deleted. An epitope tag was added to the C-terminus of the h4D5 light chain (gD tag). As described, the bivalent presentation vector (pV0350-4) was identical to pV0350-2b except for the insertion of a DNA fragment encoding the GCN4 leucine zipper between the heavy chain CH1 domain and cP3. The light chain gene was further modified at both phagemids at three positions to encode the amino acids most commonly found in the Kabat database of natural antibody sequences; specifically, Arg 66 was changed to Gly, and Asn30 and His91 were changed to Ser. These changes were found to increase Fab expression and presentation on phages. Site-directed mutagenesis was performed using the method of Kunkel et al. (Kunkel, JD et al., (1987) Methods Enzymol 154:367-82).

[0434] Phage-presenting 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 (TAAs) were embedded in all three heavy chain CDRs. These were annealed across sequences encoding CDR-H1, -H2, and -H3, and repaired during the mutagenesis reaction by a mixture of degenerate oligonucleotides in which codons at randomized positions were replaced with adjusted degenerate codons. Mutagenic reagents were electroporated into E. coli SS320 cells, and the cultures were grown overnight at 30°C in 2YT broth supplemented with KO7 helper phage, 50 g / ml carbenicillin, and 50 g / ml kanamycin. The phages were recovered from the culture medium by precipitation with PEG / NaCl as described (Sidhu, SS et al. (2000), Methods Enzymol. 328:333-363). Each electroporation reaction used approximately 10¹¹ E. coli cells and approximately 10 ug of DNA, yielding 1 × 10⁹ to 5 × 10⁹ transformants.

[0435] A separate library was prepared using degenerate oligonucleotides adjusted to mimic the natural diversity of CDR-H1 and CDR-H2 (Lee, CV et al., (2004), JMB Table 1) using the Fab.zip template. See Lib-3 described above by Lee, CV et al. (2004). Two to four oligonucleotides from CDR-H1 and CDR-H2 were combined to increase coverage of natural diversity. Lib-3 used oligonucleotides H1a and H1b (ratio 2:1) and H2a-c (ratio 1:2:01) from CDR-H1 and CDR-H2, respectively (see Lee, CV et al., (2004), JMB Table 1 for descriptions of oligonucleotides).

[0436] For CDR-H3 positions 95–100, Lib-3 consists of a set of libraries with extended CDR-H3 lengths containing either NNS codons (or NNK codons) or modified versions of NNS codons (XYZ codons) with unequal nucleotide ratios at each position of the codon triplet. The NNS codons encompassed 32 codons and encoded 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 designs of Lib-3 are described in Table 5 of Lee, CV et al. (2004) above. Separate mutagenesis reactions were performed and each CDR-H3 length was electroporated, except for the lengths of 7 and 8 residues which were electroporated together.

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

[0438] Phage selection The above Lib-3 was sorted for various Tie2 extracellular domain (ECD) proteins. The Tie2 ECD consists of three IgG domains (Ig1 and Ig2), three EGF domains (EGF1-3), a third IgG domain (Ig3), and three fibronectin type III domains (FN3), from distal to proximal to the membrane (see, for example, Figure 1). Constructs encoding the complete extracellular domain (ECD), the proximal FN3 domain, or an ECD without the FN3 domain (referred to as ECD5) were generated. Therefore, the ligand-binding domain Ig2 is encoded by both the ECD and ECD5 constructs, but not by the FN3 construct. The encoded proteins were C-terminated 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 human, mouse, and rat Tie1 receptors were also constructed using either 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-tagged column and washed with 25 mM TRIS containing 0.1% Triton X-114 and 0.1% Triton X-110, 150 mM NaCl, 1 mM EDTA, pH 7.5, and binding buffer. Flag-tagged proteins were 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 proteins were loaded onto a phosphate-buffered saline size exclusion column (either Superdex 200 or Superdex 75), and the fractions were collected; monomer peak fractions were pooled, concentrated, and filtered through 0.2 μm.

[0440] For panning, 96-well Nunc Maxisorp plates were coated overnight with 100 ul / well of targeted Tie2 antigen (5 ug / ml) in PBS at 4°C. The plates were then blocked with 65 ul of 1% blocking protein for 30 minutes, and then with 40 ul of 1% Tween 20 for another 30 minutes (blocking protein: 1st time: bovine serum albumin (BSA), 2nd time: casein, 3rd time: bovine serum albumin (BSA), 4th time: casein). The phage library was then blocked with 1% BSA (1 OD = 1.13 × 10⁻¹) containing 0.1% Tween 20. 13The phage was diluted to approximately 3–5 OD / ml (phage / ml). Generally, the phage input was as follows: 1st time 3–5 OD / ml, 2nd time 3 OD / ml, 3rd time approximately 0.5–1 OD / ml, and 4th time approximately 0.1–0.5 OD / ml. The diluted phages were incubated at room temperature for 30 minutes. The wells were washed at least 5 times consecutively with PBS and 0.05% Tween 20. The blocked phage library was added at 100 ul / well to 8 target antigen-coated wells and 2 uncoated wells at room temperature for 2 hours (hr). The plates were washed at least 10 times consecutively with PBS and 0.05% Tween 20. Starting with the third panning, 1 μM omalizumab, Tie2.ECD5, or antibody anti-Tie2.20 (ligand blocking) as an unrelated Fc-containing protein was added to the phage library for 1 hour, and the mixture was applied to Tie2-coated wells for 2 hours of binding. The phages were eluted with 100 mM HCl at 100 μl / well for 20 minutes at room temperature. The eluted phages (from coated wells) and background phages (from uncoated wells) were collected in separate tubes. The eluted collections were neutralized by adding 1 / 10 volume 1 M Tris pH 11.0 to both tubes. BSA was added to the tubes containing the eluted phages until a final 0.1%. To titrate the phages, 90 μl of log-phase XL-1 (OD 600 nm, approximately 0.1-0.3) was used to infect 10 μl of eluted phages or background phages at 37°C for 30 minutes. Next, the infected cells were serially diluted 10-fold in 90 µl of 2 YT. A 10 µl aliquot of the infected cells was plated onto a carbenicillin plate.

[0441] To allow phage growth between multiple panning cycles, approximately 400 µl of eluted phage was used to infect approximately 4 ml of logarithmic-phase XL-1 (OD600 nm, approximately 0.1-0.3) at 37°C for 30-45 minutes. Helper phage KO7 and carbenicillin were administered at a dose of 1 × 10⁻⁶. 10The final concentrations of pfu / ml KO7 and 50ug / ml carbenicillin were added to the infectant at 37°C for an additional 1 hour. The cultures were grown in 2YT medium containing 50ug / ml carbenicillin and 50ug / ml kanamycin at 37°C for 4 hours and overnight (or at least 18 hours) at 30°C until a final volume of 20-25 ml. The following day, the library phages were 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 of the supernatant volume, mixed, and allowed to stand on ice for 5 minutes. The phages were pelletized 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 by adding PEG / NaCl. The phage pellet was resuspended in PBS. The OD of the regenerated phage pellet was read at 268 nm.

[0442] Screening ELISA assay Clones from the fourth colony onward were screened for Tie2 binding and specificity by ELISA. The screening ELISA was performed by coating the wells of a 96-well microtiter plate with 65 μl / well (1 μg / ml in coating buffer) of Tie2 protein or an unrelated protein at 4°C overnight. Colonies from the fourth colony onward were grown overnight at 37°C in 400 μl of 2YT medium containing 50 μg / ml of carbenicillin and helper phage KO7 in a 96-tube plate. The plate was spun down at 3000 rpm for 10 minutes. 30 μl of the culture supernatant was added to the Tie2-coated plate with 60 μl of ELISA buffer (PBS containing 0.5% BSA and 0.05% Tween 20) and incubated at room temperature for 1 hour. The plates were washed at room temperature for 30 minutes with PBS-0.05% Tween 20 and 100 μl / well of horseradish peroxidase (HRP) conjugate anti-M13 antibody (1 / 5000 dilution in PBS supplemented with 0.5% BSA and 0.05% Tween 20) (Sidhu et al., above). After washing the wells with PBS-0.05% Tween 20, 100 μl / well of 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 at room temperature for 5 minutes. The reaction was stopped by adding 100 μl per well of 1M phosphate (H3PO4), and the OD of the wells was determined at 450 nm using a standard ELISA plate reader.

[0443] We further analyzed clones that possessed Tie2 binding above the background level and exhibited low binding affinity to Tie2-binding species specificity and unrelated Fc-containing proteins (omalizumab). 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 conjugates identified above, possessing desired species specificity and low irrelevant protein binding, were sequenced, and the variable domains of the anti-Tie2 heavy chain were cloned into vectors pre-designed for transient human IgG1 expression in mammalian cells (Lee et al., 2004a).

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

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

[0447] Tie1 binding was evaluated using a binding ELISA with immobilized Tie1 protein. The results provided 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 A competitive ELISA format was used to evaluate the Ang1 and Ang2 ligand blocking activity of anti-Tie2 antibodies. In this assay, hAng 2 (GenBank Acc. No. NP_001137) or hAng 1 (GenBank Acc. No. NP_000450) was immobilized on Maxisorp immunosorbent plates (2 ug / ml), and biotinylated huTie2ECD.Fc was equilibrated in solution with serial dilutions of anti-Tie2 antibody. Unbound biotin-Tie2ECD.Fc was then captured with immobilized hAng1 or hAng2 and detected with streptavidin-conjugated HRP. These results demonstrate blockade of both Ang1 and Ang2 by at least anti-Tie2 antibodies, Tie2.1, Tie2.12, and Tie2.20 (see Figures 4A and 4B).

[0449] Example 3 - Functional analysis of anti-Tie2 antibody Further analysis of the anti-Tie2 antibodies identified above as being able to specifically bind to Tie2 identified those that can function as Tie2 agonists. The functional activity of anti-Tie2 IgGs that showed Tie2 binding 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 relation to Tie2 activation. The Tie2 conjugate according to this disclosure, which is a Tie2 agonist, is expected to bind to and activate Tie2, resulting in downstream activation of the AKT enzyme. AKT activation can be demonstrated by phosphorylation of the AKT protein to produce pAKT, as described below. AKT phosphorylation was determined by Western blotting analysis using an antibody specific to phosphorylated AKT, or by a FRET assay as described below.

[0451] Cells and antibodies: HUVEC cells were purchased from Lonza (catalog number CC-2517; Lonza, Ltd., Basel, Switzerland). RAEC cells were purchased from VEC Technologies and cultured in growth medium (catalog number MCDB-131 10; VEC Technologies, Inc., Rensselaer, NY). Anti-Tie2 antibodies were prepared by Genentech, Inc. (South San Francisco, CA). Polyclonal goat anti-hIgG was used as a crosslinking agent (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA).

[0452] Preparation of HUVECs: HUVECs (human vascular endothelial cells) are trypsin-treated and placed in a sterile 96-well plate (Corning® Costa®, catalog number 3997) in a 0.4 × 10⁶ arrangement. 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 preheated serum starvation medium (basic medium EndoGRO®; catalog number 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, and then incubated with anti-Tie2 antibody.

[0453] Preparation of RAEC cells: RAEC (rat aortic endothelial cells) were seeded at a density of 12,000 cells / well in 96-well cell culture plates and cultured overnight in 100 μl of EGM2 MV medium (Lonza, Ltd.) at 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 efficacy of the crosslinked anti-Tie2 antibody, 20 μg / ml of crosslinking agent (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 dilution.

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

[0456] Western blot assay: HUVEC cells 1 × 10⁶ per well 6 Cells were seeded in Endogro medium and cultured at 37°C for 16–18 hours. Four–5 hours before stimulation, the culture medium was replaced with 0.1% BSA Endogro basal medium. Cells were incubated with the relevant Tie-2 agonist at 37°C for 30 minutes and washed three times with cold phosphate-buffered saline pH 7.4. Cells were placed on ice and incubated for 5 minutes with 100 ul / well of RIPA buffer (Sigma, #20-188) containing a 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)) and subsequently transferred to a nitrocellulose membrane. The membrane was blocked with 5% BSA in TBS-T at room temperature for 1 hour 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) at RT for 1 hour. The membrane was washed three times with TBS-T, incubated with ECL reagent (Thermo Scientific, 32132) at room temperature for 5 minutes, and then the blot was exposed to film.

[0457] FRET assay: Cell lysates (15 μl) were thawed on ice and then mixed in a 384-well microplate (catalog number 784080; Greiner Bio-One North America, Inc., Monroe, NC) with 5 μl 1:40 dilutions of each phosphorylated-AKT d2 antibody and phosphorylated-AKT Cryptate antibody from the phosphorylated-AKT Ser473 kit. The plate was incubated at room temperature for 4 hours or overnight at 4°C and read at 620 nm and 665 nm using CLARIOstar (BMG LABTECH, software version: 5.01 R2). Data were calculated for each individual well as the ratio of acceptor and donor luminescence signals × 10⁴.

[0458] To identify anti-Tie-2 antibodies capable of activating Tie2 activity, the Tie-2-binding antibodies identified as detailed above were formatted as human IgG1 (hIgG1) antibodies and tested for their ability to stimulate AKT phosphorylation downstream of Tie2 activation (generating pAKT). RAEC cells 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 for pAKT. Figure 6A shows that incubation with anti-Tie2 antibodies Tie2.1 and Tie2.20 increased AKT phosphorylation in this in vitro cell-based assay, with Tie2.1 exhibiting a stronger Tie2 agonist effect than Tie2.20.

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

[0460] To identify further agonist anti-Tie2 antibodies, an HTRF-based assay (Cisbio) was also used. RAEC cells were treated with recombinant anti-Tie2 antibody (hIgG1) for 10 minutes, and the 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 strong activity.

[0461] The level of Tie2 activation upon binding with anti-Tie2 IgG (full-length) antibody was thought to be partly due to the 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%) showed stronger agonist activity compared to Tie2.1 antibody preparations with a lower percentage of aggregates (e.g., 0.15%). Figure 7B further shows that crosslinked anti-hIgG1 enhanced the agonist activity of Tie2.1. Although not bound by theory, it is thought that Tie2 activation by anti-Tie2 agonist antibodies may be promoted by crosslinking of the Tie2-binding antibody.

[0462] Example 4 - Binning of anti-Tie2 antibodies derived from a natural phage library To further characterize the binding of anti-Tie2 antibodies to the Tie2 receptor protein and identify antibodies with shared epitopes, anti-Tie2 antibodies were binned using both phage ELISA and Octet assays.

[0463] For the phage ELISA, the huTie2ECD.Fc protein was immobilized overnight in 65 µl of PBS at 2 µg / ml in Maxisorp immunosorbent plates 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 at room temperature for 1 hour. Anti-Tie2 phages were added at 0.1 µl of OD268 nm / mL at room temperature for 15 minutes. The plates were then washed and detected with anti-M13 conjugate HRP secondary antibody using the method described above. An increase in signal with decreasing serial dilution of antibody indicates that the test phage antibody competes with the serial dilution antibody, indicating that they bind to the same site or epitope.

[0464] Epitope binning, as determined by the ELISA described above, was confirmed by the Octet epitope binning assay using Octet RED384 (Pall Forte Bio Corporation, Menlo Park, CA) and a standard sandwich-type binning assay. Specifically, streptavidin biosensors (Pall Forte Bio Corporation) were coated with biotinylated hTie2.ECD.Fc protein (10 ug / mL), then exposed to benchmark anti-Tie2 hIgG1 (50 ug / mL Ab1, Ab20, 13H10), followed by exposure to 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-competitive substance), while the absence of binding indicates epitope blocking (competitive substance). An example of an experiment using the data obtained for Ab20 (Tie2.20) is provided in Figures 8A and 8B. Data on other anti-Tie2 antibodies is not provided.

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

[0466] Combined, the results indicate the presence of at least three epitope classes conjugated 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 the Biacore T200 instrument (GE Life Sciences). Anti-human Fc was covalently immobilized on a Series S CM5 Biacore sensor chip to enable non-covalent capture of the anti-Tie2 antibody, and binding of human or rat Tie2 ECD.flag was monitored in real time at 25°C using a multi-cycle kinetics experiment format. The surface was regenerated with 3 M MgCl2 between cycles. 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 antibody In addition to phage presentation, we used animal immunization to generate anti-Tie2 antibodies that may be useful for therapeutic purposes.

[0469] Rabbit-immunized New Zealand white rabbits were immunized with human and cynomolgus monkey Tie2 (SEQ ID NO: 1, residues 23-442) ECD, and single B cells were isolated using a modified protocol from published literature, e.g., Seeber et al., PLoS ONE 9(2), 2014. The B cell culture supernatant was assayed by ELISA for binding to human, rat, and cynomolgus monkey Tie2 and unrelated control proteins, and by FACS for binding to HUVEC cells. Tie2-specific B cells were lysed, immediately frozen at -80°C, and stored until molecular cloning. The variable regions (VH and VL) of each monoclonal antibody derived from rabbit B cells were cloned into expression vectors derived from extracted mRNA, as previously described, 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 antibody was subjected to functional activity assays and kinetic screening.

[0470] Rat-immunized 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 sorting of individual IgG+huTie2+ hybridomas into single wells, followed by further culture. The resulting hybridoma supernatants were assayed by ELISA for binding to human, mouse, and cynomolgus monkey Tie2, as well as unrelated control proteins, and by FACS for binding to HUVEC cells. Positive samples were purified using protein A for subsequent functional and kinetic characterization.

[0471] Functional screening of rat and rabbit clones using anti-IgG crosslinking agents. As described above, antibodies generated from rat and rabbit immunization were characterized for pAKT induction downstream of Tie2 agonism.

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

[0473] Preparation of HUVEC: HUVEC is trypsinized and placed in a sterile 96-well plate (Costar catalog number 3997) in 0.4 × 10⁶ units. 5 Cells 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 preheated serum starvation medium (basal medium EndoGRO®; catalog number SCME-BM) was added to each well of the plate. The plate was incubated for 3 hours in a 37°C, 5% CO2 incubator, and then incubated with a Tie-2 agonist.

[0474] Preparation of RAEC: RAEC cells were seeded at a density of 12,000 cells / well in 96-well cell culture plates and incubated overnight in 100 μl of EGM2 MV medium at 37°C with 5% CO2. After overnight incubation, cells were starved for 3 hours in EBM2 basal medium containing 0.1% BSA, and then incubated with a Tie-2 agonist.

[0475] To test the effectiveness of the crosslinked anti-Tie2 antibody, 20 μg / ml of crosslinking agent 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 to high stock concentrations (typically 30–1000 μg / ml) in assay buffer, followed by serial dilutions (typically 2–10-fold). After removing serum starvation medium, these dilutions (50 μl) were added to each well, and the plates were incubated at 37°C, 5% CO2 for 15 minutes. The solutions were removed, and 50 μl of lysis buffer containing blocking buffer from the pAKT Ser473 kit (Cisbio, reference no. 64 AKSPEH) was added to the cells. The plates were incubated at room temperature for approximately 30–45 minutes 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 1:40 dilutions of each phosphorylated-AKT d2 antibody and phosphorylated-AKT Cryptate antibody from the pAKT Ser473 kit in a 384-well microplate (Greiner Bio-One North America, Inc., catalog number 784080). The plate was incubated at room temperature for 4 hours or overnight in a 4°C refrigerator and read at 620 nm and 665 nm using CLARIOstar (BMG LABTECH, software version: 5.01 R2). Data were obtained for each individual well as the ratio of acceptor and donor luminescence signals × 10⁻⁶ 4 It was calculated as follows.

[0478] Recombinant antibody production DNA encoding the heavy chain and light chain variable domains of the antibody was synthesized and inserted into mammalian vectors for IgG1 antibody heavy chain and light chain expression, respectively. Several variable domain sequences were edited to remove obvious unpaired cysteine ​​residues and NX[S / T]N-glycosylation motifs. Recombinant antibodies were prepared 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. Antibodies were purified from cell culture supernatant by affinity chromatography. In some cases, the antibodies underwent further purification steps based on SEC.

[0479] Antibodies were screened for binding to recombinant human and cynomolgus monkey Tie2 using a Biacore T 200 instrument (GE Life Sciences). Specifically, human antibody capture chips were prepared using a Series S CM5 chip, human antibody capture kit, and 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 kinetics 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 chips were regenerated using 3M MgCl2 injected at 30 μl / min for 30 seconds. Data were evaluated using Biacore T200 evaluation software (GE Life Sciences). Kinetic constants were obtained using a 1:1 binding model with the parameter RI set to 0. For secondary activity screening in the Fab-NDK format, selected antibodies (Tables 7A-7B; 'ch' indicates rabbit antibodies; 'TEK' indicates rat antibodies) were advanced (see Example 8 below). [Table 7A] [Table 7B]

[0480] HTP Epitope Binning Antibodies produced by animal immunization, and selected phage-derived antibodies described in Example 1, were reformatted to the 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 10 mM sodium acetate pH 4.5 immobilization buffer. Immobilization was performed using a CFM2 instrument, and the sensor prism was then transferred to an IBIS MX96 instrument for competitive analysis based on SPR. The immobilized antibodies were first exposed to 1 μM recombinant human Tie2 extracellular domains using HBS-EP running buffer (10 mM HEPES, 150 mM NaCl, 0.05% Tween 20, pH 7.4, 1 mM EDTA), and then exposed to 20 ug / ml of antibody in solution. The results are shown in Figures 11A–11B, showing several different Tie2 sandwich profiles. The results in Figures 11A–11B are typically interpreted as demonstrating the presence of multiple (e.g., at least 8) different Tie2 epitopes within the analyzed antibody panel. Interestingly, the data show that Tie2.1, Tie2.1M100cF (described later in Example 11), Tie2.12, and Tie2.24 can all function as Tie2 agonists and bin together.

[0481] Example 6 - Production of PEG-conjugate anti-Tie2 Fab polymer As shown above in Example 3, activation of Tie2 by an anti-Tie2 antibody is promoted by crosslinking of the conjugated anti-Tie2 antibody. Therefore, to determine the optimal multimer configuration for therapeutic efficacy, multimer Tie2-conjugated compositions were designed and produced.

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

Claims

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

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

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

4. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, which is a monoclonal antibody, a humanized antibody, an antibody fragment and / or a chimeric antibody.

5. An antibody or antigen-binding fragment according to any one of claims 1 to 4, which is a Fab fragment that binds to Tie2.

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

20.

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

20.

8. An antibody or antigen-binding fragment according to any one of claims 1 to 7, comprising the VL sequence of SEQ ID NO: 21 and the VH sequence of SEQ ID NO:

20.

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

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

11. The antibody or antigen-binding fragment according to claim 9 or 10, wherein the manipulated cysteine ​​is T206C in the LC.

12. The antibody or antigen-binding fragment according to claim 9 or 10, wherein the manipulated cysteine ​​is T209C in the HC.

13. The antibody or antigen-binding fragment according to any one of claims 1 to 12, comprising an LC containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 25, and an HC containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:

55.

14. The antibody or antigen-binding fragment according to any one of claims 1 to 13, comprising an LC containing an amino acid sequence having at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 25, and an HC containing an amino acid sequence having at least 99% sequence identity with the amino acid sequence of SEQ ID NO:

55.

15. An antibody or antigen-binding fragment according to any one of claims 1 to 14, comprising an LC containing the sequence of SEQ ID NO: 25 and an HC containing the sequence of SEQ ID NO:

55.

16. An antibody or antigen-binding fragment according to any one of claims 1 to 15, wherein the HC constant region terminates at position 221 (EU numbering).

17. An antibody or its antigen-binding fragment that specifically binds to Tie2, comprising the HC sequence of SEQ ID NO: 55 and the LC sequence of SEQ ID NO:

25.

18. An antibody or its antigen-binding fragment that specifically binds to Tie2, comprising the HC sequence of SEQ ID NO: 23 and the LC sequence of SEQ ID NO:

56.

19. An isolated nucleic acid encoding an antibody or antigen-binding fragment according to any one of claims 1 to 18.

20. A host cell comprising the nucleic acid described in claim 19.

21. 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 or antigen-binding fragments as described in any one of claims 1 to 18, wherein each of the antibodies or antigen-binding fragments is linked to a polymerizing portion.

22. The conjugate according to claim 21, which causes a decrease in Tie2 protein levels of less than 25%, less than 50%, or less than 75% in an in vitro assay.

23. The conjugate according to claim 21 or 22, which does not cause a decrease in Tie2 protein levels of more than 25%, more than 50%, or more than 75% in an in vitro assay.

24. The conjugate according to any one of claims 21 to 23, wherein the polymerized portion comprises a polyol, a polypeptide, and / or a peptide.

25. The conjugate according to any one of claims 21 to 24, wherein the polymerized portion comprises a polyol, and the polyol is a multi-armed polyol selected from a dimer, a tetramer, a hexamer, and an octamer.

26. The conjugate according to claim 25, wherein the multi-armed polyol is a hexamer.

27. The conjugate according to any one of claims 24 to 26, wherein the polyol is polyethylene glycol (PEG).

28. The polyol is covalently bound to the at least two antibodies or their antigen-binding fragments via the free sulfhydryl groups of the manipulated cysteine ​​amino acids, and the manipulated cysteine ​​is Selected from the group consisting of T120C, G166C, G178C, T187C and T209C in HC; or The manipulated cysteine ​​is selected from the group consisting of Q124C, R142C, Q155C, L201C, T206C, K107C, K126C, and K149C in LC. The conjugate according to any one of claims 24 to 27, wherein the residue numbers conform to EU numbering.

29. The conjugate according to claim 28, wherein the manipulated cysteine ​​is T206C in the LC, and the residue number follows EU numbering.

30. The conjugate according to claim 28, wherein the manipulated cysteine ​​is T209C in the HC, and the residue number follows EU numbering.

31. The conjugate according to any one of claims 25 to 27, wherein the polyol is covalently bound to the at least one antibody via the free amino group of the lysine amino acid.

32. The conjugate according to any one of claims 27 to 31, wherein the PEG has a weight-average molecular weight of about 500 Daltons (Da) to about 300,000 Da.

33. The aforementioned PEG is given by the general formula (Ib): (In the formula, each m is an integer between 3 and 250 independently; each R １ Each R is either nonexistent or a linking group; ２ These are independently either hydrogen or a terminal reactive group; at least one R ２ (This is a terminal reactive group, which is covalently bound to the antibody or antigen-binding fragment described in any one of claims 1 to 14.) A conjugate according to any one of claims 27 to 32, having the structure of the conjugate.

34. at least one R １ is a linking group, R １ and R ２ However, if they were to come together, (In the formula, each i is an independent integer between 0 and 10; j is an integer between 0 and 10; R ２ (wherein a terminal reactive group is selected from the group consisting of thiol reactive groups, amino reactive groups, and combinations thereof); and combinations thereof A conjugate according to claim 33, selected from the following.

35. at least one R ２ The conjugate according to claim 33 or claim 34, wherein the maleamide is covalently bound to the antibody or its antigen-binding fragment.

36. Each R ２ The conjugate according to any one of claims 33 to 35, wherein is maleamide.

37. A conjugate that binds to Tie2, Fab includes HC having sequence number 55 and LC having sequence number 25; Formula 1(b) (In the formula, each m is an integer between 20 and 30, and R １ and R ２ They form a structure together It has R ２ (It is maleamide.) It is conjugated to a polyol having the structure, The aforementioned polyol is linked to Fab HC at residue C209 (EU numbering), forming a conjugate.

38. A pharmaceutical composition comprising a conjugate according to any one of claims 21 to 37 and a pharmaceutically acceptable carrier.

39. The pharmaceutical composition according to claim 38, further comprising a further therapeutic agent, wherein the further 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.

40. A long-acting delivery device for ocular delivery, comprising a pharmaceutical composition according to claim 38 or 39 and means for delivering the composition into the vitreous humor of a patient, wherein the composition remains effective in situ for an extended period of time.

41. A method for treating a Tie2 pathway-mediated disorder in a subject requiring treatment, comprising administering to the subject an effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 18, a conjugate according to any one of claims 21 to 36, or a pharmaceutical composition according to claim 37 or 38.

42. The method according to claim 41, wherein the Tie2 pathway-mediated impairment is a vascular permeability impairment.

43. The method according to claim 41 or 42, wherein the Tie2 pathway-mediated disorder is an ocular symptom.

44. The method according to claim 43, wherein the eye symptoms are selected from diabetic macular edema (DME), diabetic retinopathy, age-related macular degeneration (AMD) including dry and wet types (non-exudative and exudative), choroidal neovascularization (CNV), uveitis, ischemic retinopathy, pathological myopia, von Hippel-Lindau disease, ocular histoplasmosis, central retinal vein occlusion (CRVO), corneal neovascularization, glaucoma, and retinal neovascularization.

45. The method according to claim 43 or 44, wherein the eye symptom is DME.

46. An antibody according to any one of claims 1 to 18 or a conjugate according to any one of claims 21 to 39, for use as a pharmaceutical.

47. An antibody according to any one of claims 1 to 18 or a conjugate according to any one of claims 21 to 39 for use in the treatment of Tie2 pathway-mediated disorders and / or ocular disorders in subjects requiring treatment.

48. Use of an antibody according to any one of claims 1 to 18 or a conjugate according to any one of claims 21 to 39 in the manufacture of a pharmaceutical product for treating Tie2 pathway-mediated disorders and / or ocular disorders in subjects requiring treatment.

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