Antibodies against Tie-2 and VEGF and methods of use

Abstract

The present invention relates to specific constructs of Tie2 / VEGF bispecific antibodies. The invention also relates to pharmaceutical compositions of the bispecific antibodies and methods of using these pharmaceutical compositions to treat a disease in a subject. Also provided are isolated nucleic acids encoding the isolated bispecific antibodies, vectors comprising the isolated nucleic acids, and host cells comprising the vectors.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 331,656, filed on April 15, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0003] Sequence Listing

[0004] The Sequence Listing is provided herewith as a Sequence Listing XML "UNITY-032WO Seq List", created on March 24, 2023, and 46KB in size. The contents of the Sequence Listing XML are incorporated herein by reference in their entirety. Technical Field

[0005] The present invention relates to Tie2 / VEGF bispecific antibodies and methods of using the same. Background Art

[0006] Angiogenesis is a tightly regulated process by which new blood vessels are formed from pre-existing vessels. Although angiogenesis is important during development to ensure adequate blood circulation, many disorders are associated with pathological angiogenesis, such as certain eye disorders, such as retinopathy. For example, diabetic retinopathy, a microvascular complication of diabetes, occurs in approximately 30% of people aged 40 or older with diabetes. 3.8% of diabetic adults in this age group suffer from DME, a complication of diabetic retinopathy, equivalent to 746,000 people in the US population in 2010 (Varma et al. 2014). Diabetic macular edema (DME) is the leading cause of vision loss in diabetic patients, secondary to the breakdown of the blood-retinal barrier (BRB) and the subsequent leakage of plasma and lipids in the macula (Hall 2016; Das, McGuire and Rangasamy 2015). This causes swelling of the macula, impaired vision, and if untreated, many patients eventually develop legal blindness. Management of patients with diabetic retinopathy includes improving hyperglycemic control, treating and preventing cardiovascular complications, regular eye examinations, and medical / surgical ophthalmic interventions. Currently, drug therapies for the treatment of ocular angiogenesis are based on the administration of anti-VEGF agents via the intravitreal route. Molecules approved for ocular diseases include pegaptanib, ranibizumab, and aflibercept.

[0007] Despite the efficacy of anti-VEGF therapy in many patients with DR and DME, a large unmet need remains as a large proportion of such patients are refractory to anti-VEGF therapy (Hussain and Ciulla 2016). After 24 weeks of treatment with the most effective regimen (aflibercept 2 mg every 4 weeks), 32% of patients in the Diabetes Clinical Research Network Protocol T trial showed persistent macular edema as measured by optical coherence tomography (OCT) and central retinal thickness (CST) (Bressler et al. 2018). After 24 weeks of treatment, 20% of eyes showed no resolution of macular edema as measured by OCT-CST, and 11% of eyes showed resolution of macular edema as measured by OCT-CST and failed to achieve 20 / 40 visual acuity required for automobile driving. Therefore, there is an unmet medical need for new therapies to treat patients who do not adequately improve on existing anti-VEGF therapy. The claimed invention is intended to address this need. Summary of the invention

[0008] The present invention provides bispecific antibodies that specifically bind both human Tie2 and human VEGF and methods of using these bispecific antibodies for therapeutic and diagnostic purposes.The bispecific antibodies of the present invention exhibit unique properties that make them particularly suitable for use in therapy and different from known standard of care therapies.

[0009] Specifically contemplated as part of the disclosed invention are:

[0010] Example 1. An isolated bispecific antibody that specifically binds to human Tie2 and human VEGF, the isolated bispecific antibody being selected from the group consisting of:

[0011] (a) an IgG heavy chain comprising the amino acid sequence of SEQ ID NO: 4; and a light chain κ comprising the amino acid sequence of SEQ ID NO: 2;

[0012] (b) an IgG heavy chain comprising the amino acid sequence of SEQ ID NO: 3; and a light chain κ comprising the amino acid sequence of SEQ ID NO: 2;

[0013] (c) an IgG heavy chain comprising the amino acid sequence of SEQ ID NO: 1; and a fully human light chain κ comprising the amino acid sequence of SEQ ID NO: 2;

[0014] (d) an IgG heavy chain comprising the amino acid sequence of SEQ ID NO: 5; and a fully human light chain κ comprising the amino acid sequence of SEQ ID NO: 2;

[0015] (e) an IgG heavy chain comprising the amino acid sequence of SEQ ID NO: 6 and a fully human light chain κ comprising the amino acid sequence of SEQ ID NO: 2;

[0016] (f) the fully human amino acid sequence of SEQ ID NO:7;

[0017] (g) the fully human amino acid sequence of SEQ ID NO: 8;

[0018] (h) the fully human amino acid sequence of SEQ ID NO:9; and

[0019] (i) the fully human amino acid sequence of SEQ ID NO: 10;

[0020] (j) an IgG heavy chain comprising the amino acid sequence of SEQ ID NO: 19; and a light chain κ comprising the amino acid sequence of SEQ ID NO: 2;

[0021] (k) an IgG heavy chain comprising the amino acid sequence of SEQ ID NO: 20; and a light chain κ comprising the amino acid sequence of SEQ ID NO: 2;

[0022] (l) an IgG heavy chain comprising the amino acid sequence of SEQ ID NO: 21; and a light chain κ comprising the amino acid sequence of SEQ ID NO: 2;

[0023] (m) an IgG heavy chain comprising the amino acid sequence of SEQ ID NO: 22; and a light chain κ comprising the amino acid sequence of SEQ ID NO: 2;

[0024] (n) an IgG heavy chain comprising the amino acid sequence of SEQ ID NO: 23; and a light chain κ comprising the amino acid sequence of SEQ ID NO: 2;

[0025] (o) an IgG heavy chain comprising the amino acid sequence of SEQ ID NO: 24; and a light chain κ comprising the amino acid sequence of SEQ ID NO: 2;

[0026] (p) an IgG heavy chain comprising the amino acid sequence of SEQ ID NO: 25; and a light chain κ comprising the amino acid sequence of SEQ ID NO: 2;

[0027] (q) an IgG heavy chain comprising the amino acid sequence of SEQ ID NO: 26; and a light chain κ comprising the amino acid sequence of SEQ ID NO: 2;

[0028] (r) an IgG heavy chain comprising the amino acid sequence of SEQ ID NO: 27; and a light chain kappa comprising the amino acid sequence of SEQ ID NO: 2; and

[0029] (s) an IgG heavy chain comprising the amino acid sequence of SEQ ID NO:28; and a light chain κ comprising the amino acid sequence of SEQ ID NO:2.

[0030] Example 2. An isolated nucleic acid encoding the isolated bispecific antibody as described in Example 1.

[0031] Embodiment 3. A vector comprising the isolated nucleic acid as described in embodiment 2.

[0032] Embodiment 4. A host cell comprising the vector as described in embodiment 3.

[0033] Example 5. A method for producing an isolated bispecific antibody, the method comprising culturing the host cell described in Example 4 in a culture medium, and isolating the resulting bispecific antibody.

[0034] Example 6. An immunoconjugate comprising the isolated bispecific antibody as described in Example 1.

[0035] Example 7. A fusion polypeptide comprising the isolated bispecific antibody as described in Example 1.

[0036] Embodiment 8. A pharmaceutical composition comprising the isolated bispecific antibody as described in Embodiment 1, the immunoconjugate as described in Embodiment 6, or the fusion polypeptide as described in Embodiment 7.

[0037] Example 9. A method of treating a disease in a subject in need thereof, the method comprising administering to the subject the pharmaceutical composition as described in Example 8.

[0038] Embodiment 10. The method of embodiment 9, wherein the disease comprises an eye disease, an infectious disease, or an ischemic disease or injury.

[0039] Embodiment 11. The method of embodiment 10, wherein the infectious diseases include sepsis, dengue virus infection, coronavirus infection, tuberculosis or influenza.

[0040] Embodiment 12. The method of embodiment 10, wherein the ischemic diseases or injuries include diabetic nephropathy, acute kidney injury, chronic kidney disease, organ transplantation or critical limb ischemia.

[0041] Embodiment 13. The method of embodiment 10, wherein the ocular disorders include diabetic retinopathy (DR), diabetic macular edema (DME), non-proliferative diabetic retinopathy (nPDR), proliferative diabetic retinopathy (PDR), wet age-related macular degeneration (AMD), dry AMD, geographic atrophy (GA), retinopathy of prematurity (ROP), glaucoma, retinal vein occlusion (RVO), central RVO or branched RVO.

[0042] Example 14. The isolated bispecific antibody of Example 1, or the immunoconjugate of Example 6, or the fusion polypeptide of Example 7, for use in treating the diseases of Examples 10-13.

[0043] Example 15. Use of the isolated bispecific antibody described in Example 1, or the immunoconjugate described in Example 6, or the fusion polypeptide described in Example 7 for the manufacture of a medicament for treating the diseases described in Examples 10-13. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] FIG. 1 (panels AC) shows schematic diagrams of some bispecific constructs described herein based on the use of intact IgG anti-Tie2 antibody clone No. 3 and aflibercept: FIG. 1 (panel A) shows construct No. 1: anti-Tie2 intact IgG is linked to aflibercept in a forward sequence (N-terminus to C-terminus); FIG. 1 (panel B) shows construct No. 3: anti-Tie2 intact IgG is linked to aflibercept in a forward sequence, wherein n=4; FIG. 1 (panel C) shows construct No. 13: anti-Tie2 intact IgG is linked to aflibercept, but in a reverse sequence (C-terminus to N-terminus). See also Figure 7 and Table 1.

[0045] Figure 2 (panels AD) shows schematic diagrams of some bispecific constructs described herein based on the use of intact IgG anti-Tie2 antibody clone No. 3 and B20VEGF conjugate or ranibizumab: Figure 2 (panel A) shows construct No. 2: anti-Tie2 intact IgG connected to VEGF scFv clone B20; Figure 2 (panel B) shows construct No. 10: anti-Tie2 intact IgG connected to ranibizumab Fab fragment; Figure 2 (panel C) shows construct No. 8: anti-Tie2 intact IgG connected to ranibizumab Fab fragment via a long linker; Figure 2 (panel D) shows construct No. 9: similar to construct No. 8, but in which the variable heavy chain and variable light chain of the ranibizumab Fab fragment are reversed. See also Figure 8A-B and Table 1.

[0046] Figure 3 (panels AD) shows schematic diagrams of some bispecific constructs described herein based on the use of aflibercept and antibody fragments of anti-Tie2 antibody clones 3 and 2: Figure 3 (panel A) shows construct No. 5: aflibercept is linked to the scFv of anti-Tie2 clone No. 3; Figure 3 (panel B) shows construct No. 6: aflibercept is linked to the Fab fragment of anti-Tie2 clone No. 3; Figure 3 (panel C) shows construct No. 7: aflibercept is linked to the scFv of anti-Tie2 clone No. 2, which is further linked to the scFv of anti-Tie2 clone No. 3; Figure 3 (panel D) shows construct No. 12: aflibercept is linked to the scFv of anti-Tie2 clone No. 3, which is further linked to the scFv of anti-Tie2 clone No. 2. See also Figure 7 and Table 1.

[0047] FIG4 (panels AB) shows Akt activation measured by EC50 (in nM) for certain bispecific antibody constructs (Constructs No. 1 and No. 5 and Ang2 / VEGF) compared to control (ANG1, anti-Tie2 clone No. 3) ( FIG4 , panel A). Akt potency was quantified as EC50 values ​​in nM ( FIG4 , panel B). See Example 1.

[0048] Figure 5 (panels AB) shows the in vitro potency of VEGF neutralization as measured by % VEGF activity (Figure 5, panel A), and the in vitro potency of phosphorylated Tie2 (pTIE2) as measured by % Ang1 activation (Figure 5, panel B) of constructs No. 2, No. 4, and No. 5 compared to Ang1, anti-Tie2, and Aflibercept-LALA-PG. See Examples 2 and 3.

[0049] Figure 6 (panels AE) shows the in vitro phosphorylated Tie2 potency measured by % Ang1 activation of two bispecific antibody constructs of the invention No. 1 (Figure 6, panel C) and No. 5 (Figure 6, panel D) compared to Ang1 control (Figure 6, panel A) and anti-Tie2 clone No. 3 (Figure 6, panel B) with and without VEGF. The results were quantified as p-Tie2 EC50 nM with or without VEGF (Figure 6, panel E). See Example 4.

[0050] Figure 7Shown are the CMC characteristics of yield (in mg), concentration [conc] (in mg / ml), purity SEC% and stress stability (37°C, 2 weeks) by Tie2 binder format and VEGF binder format, as well as a table summary comparing certain Tie2-VEGF Trap bispecific constructs of the invention according to the in vitro potency of each bispecific antibody construct tested by EC50 (in nM) for phosphorylated Tie2 activation and VEGF neutralization in vitro potency. N / A = data not measurable, TBD = data pending, ND = not tested. See Figures 1A-1C, Figures 3A-3D, Table 1 and Examples 2, 3 and 5.

[0051] Figure 8A-B Both show the CMC characteristics of yield (in mg), concentration [conc] (in mg / ml), purity SEC% and stress stability (37°C, 2 weeks) by Tie2 binder format and VEGF binder format, as well as a table summary comparing certain Tie2-anti-VEGF monoclonal antibody bispecific constructs of the invention according to the in vitro potency of each bispecific antibody construct measured by EC50 (in nM) for phosphorylated Tie2 activation and VEGF neutralization in vitro potency. N / A = data not measurable, TBD = data pending, ND = not tested. See Figure 2 (panels AD), Table 1 and Examples 2, 3 and 5.

[0052] Figure 9 (panels AB) shows the in vitro potency of VEGF neutralization as measured by % VEGF activity inhibition (Figure 9, panel A) and phosphorylated Tie2 (pTIE2) as measured by % Ang1 activation (Figure 9, panel B) of constructs No. 3 and No. 1 compared to Ang1, anti-Tie2 clone No. 3 and aflibercept. See Examples 2 and 3. Figure 9 (panel C) shows a table of EC50 values ​​(pTIE2 EC50 (nM) and VEGF EC50 (nM)) for various constructs / controls.

[0053] FIG. 10 (panels AD) shows the in vitro phosphorylated Tie2 potency measured by % Ang1 activation of two bispecific antibody constructs of the invention, No. 1 (FIG. 10, panel B) and No. 3 (FIG. 10, panel C), compared to the anti-Tie2 clone No. 3 control (FIG. 10, panel A), with and without the addition of VEGF. The results were quantified as p-Tie2 EC50 nM with or without VEGF (FIG. 10, panel D). See Example 4.

[0054] FIG. 11 (Panels AB) shows Akt activation measured by EC50 (in nM) for certain bispecific antibody constructs (Constructs No. 1 and No. 3) compared to control (ANG1) ( FIG. 11 , Panel A and FIG. 11 , Panel B). Akt potency was quantified as EC50 values ​​in nM ( FIG. 11 , Panel B). See Example 1.

[0055] Fig.12 ERK activation measured by Western blot for bispecific construct No. 3 compared to control (ANG1) is shown. Fig.12 ). See Example 9.

[0056] Figure 13 (panels AC) shows target engagement of phosphorylated Tie2 in the primary choroidal tissue of young mice at 2.5ug / eye, or 5ug / eye, or 10ug / eye using bispecific construct No. 3 or control (NSigG) at certain time points: 2h (Figure 13, panel B) or 7d (Figure 13, panel C). See Example 8.

[0057] Figure 14 (panels AC) shows target engagement of phosphorylated Tie2 in primary retinal tissue of young mice at 2.5ug / eye, 5ug / eye or 10ug / eye using bispecific construct No. 3 or control (NSigG) at time points: 2h (Figure 14, panel B) or 7d (Figure 14, panel C). See Example 8.

[0058] Figure 15 (panels AB) shows the comparison of 5ug and 20ug of bispecific construct No. 3 with 5ug and 20ug of mouse Ang2 / VEGF antibody (FMS), 20ug of Aflibercept-LALA-PG, 20ug of In vivo efficacy studies of avascularity ( FIG. 15 , panel A) and neovascularization ( FIG. 15 , panel B) of NS-huIgG1 and the irrelevant negative control NS-huIgG1. See Example 10 and Tables 3 and 4.

[0059] FIG. 16 (panels AB) shows target engagement of pTie2 (FIG. 16, panel A) and Ang2 (FIG. 16, panel B) in hyperoxic retina using constructs No. 1, No. 3 (all compared to an irrelevant negative control NSIgG) dosed at P13 and harvested at P15 compared to murine Ang2 / VEGF antibody (FMS). For FIG. 16 (panel A), each point represents the mean amount of protein detected and the error bars represent the SD. For FIG. 16 (panel B), each bar represents the mean amount of protein detected and the error bars represent the SD. See Example 6.

[0060] FIG. 17 (panels AB) shows target engagement of pTie2 (FIG. 17, panel A) and Ang2 (FIG. 17, panel B) in hyperoxic retina using constructs No. 1, No. 3 (all compared to an irrelevant negative control NSIgG) dosed at P15 and harvested at P16 compared to murine Ang2 / VEGF antibody (FMS). For FIG. 17 (panel A), each point represents the mean amount of protein detected and the error bars represent the SD. For FIG. 17 (panel B), each bar represents the mean amount of protein detected and the error bars represent the SD. See Example 6. DETAILED DESCRIPTION

[0061] I. Definitions

[0062] "Tie2" is also known as angiopoietin-1 receptor or TEK receptor tyrosine kinase or CD202B (cluster of differentiation 202B), and is a protein encoded by the TEK gene in humans (Partanen J et al., (April 1992). A novel endothelial cell surface receptor tyrosine kinase with extracellular epidermal growth factor homology domains. Molecular and Cellular Biology. 12(4): 1698-707). The receptor has a unique extracellular domain that contains three immunoglobulin-like loops, three epidermal growth factor-like repeats, and three fibronectin type III-like repeats (see Fiedler et al., 2006. Angiopoietins: A Link between Angiogenesis and Inflammation. Trends in Immunology 27(12):552-58; Barton et al., Crystal structures of the Tie2 receptor ectodomain and the angiopoietin-2-Tie2 complex. Nature Struc. & Mol. Biology, 13, 524-532 (2006)). The contact residues of angiopoietin-1 and angiopoietin-2 mostly overlap on the Tie-2 receptor and are primarily located in the second Ig-like loop, as shown by crystal structure analysis of the Ang2 / Tie2 complex (Barton et al., Nat Str Biol [Nature Structural Biology] 2006).Other studies support the concept that the binding domains of Ang1 and Ang2 are similar or identical (Fiedler et al., Angiopoietin-1 and angiopoietin-2 share the same binding domains in the Tie-2 receptor involving the first Ig-like loop and the epidermal growth factor-like repeats. JBC [Journal of Biological Chemistry]. Vol. 278 (3): 1721-7 (2003)). The amino acid sequence of an exemplary human Tie2 can be found under UniProt Accession No. Q02763.

[0063] "Vascular endothelial growth factor (VEGF)" is a signaling protein produced by many cells to stimulate angiogenesis. VEGF belongs to a subfamily of growth factors, i.e., the platelet-derived growth factor family of cystine knot growth factors. The normal function of VEGF is to generate new blood vessels during embryonic development, generate new blood vessels after injury, generate muscles after exercise, and form new blood vessels (collateral circulation) to bypass blocked blood vessels. Abnormal expression of VEGF may also lead to disease. Without adequate blood supply, solid cancers cannot grow beyond a limited size, while cancers that can express VEGF can grow and metastasize. Overexpression of VEGF can lead to vascular diseases in the retina of the eye and other parts of the human body (e.g., kidneys). Therefore, VEGF is a clinically validated angiogenesis driver, and neutralizing VEGF can be used to treat disorders associated with pathological angiogenesis by, for example, using anti-VEGF blocking antibodies. The amino acid sequence of an exemplary human VEGF can be found under UniProtKB accession number A0A024RD33.

[0064] As used herein, the term "about" refers to the usual error range for the corresponding value that is readily known to those skilled in the art. References herein to "about" values ​​or parameters include (and describe) embodiments for the value or parameter itself.

[0065] "Acceptor human framework" for the purposes of this article is a framework comprising an amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain amino acid sequence changes. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0066] In the context of the bispecific antibodies of the invention, "active" or "activity" or "biological activity" is the ability to (a) agonize (partially or fully activate) the biological activity of human Tie2 in vitro and / or in vivo and (b) antagonize (partially or fully inhibit) the biological activity of human VEGF.

[0067] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to internal binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by a dissociation constant (KD). Affinity can be measured by conventional methods known in the art, including those described herein.

[0068] An "affinity matured" antibody is one that has one or more alterations in one or more hypervariable regions (CDRs) and / or framework regions (FRs) compared to a parent antibody, where the parent antibody does not have such alterations, which alterations improve the affinity of the antibody for antigen.

[0069] "Allosteric activation of Tie2" is the activation of Tie2 by agonistic anti-Tie2 antibodies that specifically interact with regions of Tie2 outside of the ligand binding or active site such that binding results in a change in Tie2 conformation or aggregation that enhances receptor activity.

[0070] The term "antibody" herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, bispecific antibodies, and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0071] "Antibody fragment" refers to a molecule that is not an intact antibody, which comprises a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and bispecific antibodies formed from antibody fragments.

[0072] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, and a residual "Fc" fragment (the name reflects the ability to crystallize readily). The Fab fragment consists of an entire light (L) chain as well as the variable region domain (VH) of the heavy (H) chain and the first constant domain (CH1) of one heavy chain. Pepsin treatment of antibodies produces a single large F(ab')2 fragment, which roughly corresponds to two disulfide-linked Fab fragments with divalent antigen-binding activity and is still capable of cross-linking antigen. Fab' fragments differ from Fab fragments in having an additional small number of residues at the carboxyl terminus of the CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residues of the constant domains carry free thiol groups. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical coupling reactions for antibody fragments are also known.

[0073] The term "Fc region" herein is used to define the C-terminal region of at least a portion of the constant region of an immunoglobulin heavy chain. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) in the Fc region may or may not exist. Unless otherwise specified herein, the amino acid residue numbering in the Fc region or constant region is according to the EU numbering system (also referred to as the EU index), as described in Kabat et al., Sequences of Proteins of Immunological Interest [immunological purpose protein sequence], 5th edition, Public Health Service [U.S. Public Health Service Center], National Institutes of Health [U.S. National Institutes of Health], Bethesda, Maryland (1991).

[0074] "Fv" consists of a dimer of one heavy chain variable region domain and one light chain variable region domain in tight non-covalent association. Six hypervariable loops (3 loops each for the H chain and L chain) are generated from the folding of these two domains, which provide amino acid residues for antigen binding and give the antibody antigen binding specificity. However, even a single variable domain (or half of an Fv containing only three CDRs specific for an antigen) has the ability to recognize and bind to an antigen, although the affinity is usually lower than that of the entire binding site.

[0075] "Single-chain Fv" (also abbreviated as "sFv" or "scFv") is an antibody fragment comprising VH and VL antibody domains linked into a single polypeptide chain. The sFv polypeptide may further comprise a polypeptide linker between the VH and VL domains that enables the sFv to form the structure required for antigen binding. For a review of sFv, see Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0076] The term "diabodies" refers to small antibody fragments prepared by constructing sFv fragments (see the preceding paragraph) with a short linker (about 5-10 residues) between the VH and VL domains, so that interchain rather than intrachain pairing of the V domains is achieved, thereby generating a bivalent fragment, i.e., a fragment with two antigen-binding sites. Bispecific diabodies are heterodimers of two "crossover" sFv fragments, in which the VH and VL domains of the two antibodies are present on different polypeptide chains. Diabodies are more fully described in, for example, EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America], 90:6444-6448, 1993.

[0077] A "blocking" antibody or "antagonist" antibody is an antibody that inhibits or reduces the biological activity of the antigen to which it binds. Certain blocking or antagonist antibodies substantially or completely inhibit the biological activity of the antigen.

[0078] "Agonistic" or "activating" antibodies are antibodies that activate, stimulate, or increase the biological activity or signaling activity of the antigen to which they bind. In some cases, it is contemplated that agonistic antibodies can act in a manner similar to how a ligand engages and activates its cognate receptor. In other cases, it is contemplated that if the bispecific antibodies of the present invention induce Tie2 signaling (as determined by an increase in the level of one or more of intracellular phosphorylated Tie2 (pTie2) and / or phosphorylated Akt (pAkt) and / or phosphorylated ERK (pERK)), the bispecific antibodies of the present invention are considered to be agonistic antibodies. In addition, it is further contemplated that the agonistic Tie2 antibodies of the present invention can also activate downstream signaling of their target antigen in the presence or absence of endogenous activating (i.e., Ang1) or inhibitory (i.e., Ang2) ligands. See Damiano J., et al., WO / 2021 / 102173, for examples of assays for determining this.

[0079] As used herein, the term "bicomplementarity" refers to a bispecific antibody wherein the first antigen binding moiety and the second antigen binding moiety bind to different epitopes on the same antigen.

[0080] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0081] The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0082] "Complement factor" means the various proteins and glycoproteins that make up the complement cascade, which is part of the immune system that enhances (supplements) the ability of antibodies and phagocytes to remove microorganisms and damaged cells from an organism, promote inflammation, and attack pathogen cell membranes. It is part of the innate immune system. Complement factors contemplated herein include, for example, C1, C2, C2a, C2b, C3, C3a, C3b, C4, C4a, C4b, C5, C5a, C5b, C6, C7, C8, and C9.

[0083] "Effector functions" refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0084] "Framework" or "framework region" or "FR" refers to the variable domain residues other than the hypervariable region (CDR) residues. The FR of the variable domain is generally composed of four FR domains: FR1, FR2, FR3 and FR4.

[0085] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.

[0086] The term "fusion polypeptide" encompasses bispecific antibodies of the present invention fused to, for example, an immunoglobulin Fc region. The Fc region may comprise, for example, a CH3 domain of an immunoglobulin, which may be naturally occurring or modified in some manner. Such an Fc fusion polypeptide may exhibit a longer in vivo half-life than an unfused polypeptide. In addition, fusion with the Fc region allows dimerization / multimerization of the fusion polypeptide. As contemplated, the Fc region may be a naturally occurring Fc region, or may be altered to improve certain properties, such as therapeutic properties, circulation time, and reduced aggregation problems. In another embodiment, the fusion polypeptide contemplates a bispecific antibody fragment fused to a ligand (such as Ang1) to enhance the agonistic activity of the fusion polypeptide. In another embodiment, the fusion polypeptide contemplates a bispecific antibody fragment fused to, for example, a cytokine to induce other desired biological properties.

[0087] A "human antibody" is an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human or human cell or derived from an antibody of a non-human source utilizing a human antibody library or other human antibody encoding sequences. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen binding residues.

[0088] "Human consensus framework" is a framework representing the most common amino acid residues in a plurality of human immunoglobulin VL or VH framework sequences that are available for selection. Typically, the plurality of human immunoglobulin VL or VH sequences that are available for selection are from a subset of variable domain sequences. Typically, a subset of sequences is a subset such as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, NIH publication 91-3242, Bethesda, Maryland (1991), volumes 1-3. In one embodiment, for VL, the subgroup is subgroup κI such as in Kabat et al., supra. In one embodiment, for VH, the subgroup is subgroup III such as in Kabat et al., supra.

[0089] The non-human (e.g., rodent) antibody of "humanization" form is a chimeric antibody containing the minimum sequence derived from a non-human antibody. In most cases, a humanized antibody is a human immunoglobulin (receptor antibody), wherein the residues from the receptor hypervariable region are replaced by the residues from the hypervariable region (donor antibody) with the desired antibody specificity, affinity and ability from a non-human species (e.g., mouse, rat, rabbit or non-human primate). In some cases, the FR residues of the human immunoglobulin are replaced by corresponding non-human residues. In addition, the humanized antibody may be included in residues not found in the receptor antibody or the donor antibody. These modifications are made to further improve antibody performance. Generally, a humanized antibody will include substantially all of at least one and typically two variable domains, all or substantially all of its hypervariable loops corresponding to those of non-human immunoglobulins, and all or substantially all of FRs are those of human immunoglobulin sequences. A humanized antibody will also optionally include at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr Op. Struct. Biol. 2:593-596 (1992).

[0090] The term "variable" refers to the fact that certain segments of the variable domains differ greatly in sequence between antibodies. The variable domain or "V" domain mediates antigen binding and defines the specificity of a particular antibody to its specific antigen. However, variability is not evenly distributed across the span of the variable domain. On the contrary, the V region is composed of relatively constant segments called framework regions (FR) of 15-30 amino acids, which are separated by shorter regions with extreme variability called "hypervariable regions", each of which is 9-12 amino acids in length. When used herein, the term "hypervariable region" or "CDR" refers to the amino acid residues responsible for antigen binding of an antibody. The hypervariable regions generally include amino acid residues from about, for example, residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in VL and about residues 26-35 (H1), 49-65 (H2), and 95-102 (H3) in VH (in one embodiment, H1 is about residues 31-35); Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service, National Institutes of Health. Health [National Institutes of Health], Bethesda, Maryland (1991) and / or those residues from a "hypervariable loop" (e.g., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in VL, and residues 26-32 (H1), 53-55 (H2), and 96-101 (H3) in VH; Chothia and Lesk, J. Mol. Biol. [Journal of Molecular Biology] 196:901-917 (1987). The variable domains of native heavy and light chains each contain four FRs, largely adopting a β-sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the β-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, together with the hypervariable region from the other chain, contribute to the formation of the antibody antigen-binding site (see Kabat et al., Sequences of Proteins of Immunological Biology). [Protein Sequences of Immunological Interest], 5th Edition Public Health Service, National Institutes of Health, Bethesda, Maryland (1991). Thus, CDR and FR sequences typically appear in the following sequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.The constant domains are not involved directly in binding the antibody to an antigen, but exhibit various effector functions, such as adding antibody-dependent cellular cytotoxicity (ADCC) to the antibody.

[0091] The terms "residue numbering as in Kabat," "Kabat amino acid residues," or "amino acid position numbering as in Kabat," and variations thereof, refer to the numbering system used for heavy chain variable domains or light chain variable domains in the compilation of antibodies in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to shortening or insertion of a FR or CDR of the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and an inserted residue after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c, etc. according to Kabat). The Kabat numbering of residues for a given antibody can be determined by aligning the antibody sequence with a region of homology to the "standard" Kabat numbering sequence.

[0092] When referring to the residues in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain), the Kabat numbering system is generally used (e.g., Kabat et al., Sequences of Immunological Interest, 5th Edition Public Health Service, National Institutes of Health, Bethesda, Maryland (1991)). When referring to the residues in the constant region of the immunoglobulin heavy chain, the "EU numbering system" or "EU index" is generally used (e.g., the EU index reported by Kabat et al. above). The "EU index as in Kabat" refers to the residue numbering of human IgG1EU antibodies. Unless otherwise specified herein, reference to the residue numbering in the antibody variable domain means the residue numbering by the Kabat numbering system. Unless otherwise specified herein, reference to the residue numbering in the antibody constant domain means the residue numbering by the EU numbering system. Unless otherwise indicated herein, the CDR residues and other residues (e.g., FR residues) in the variable domain are numbered herein according to Kabat et al., supra.

[0093] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules that deliver cell-killing or cell-altering activity, including but not limited to small molecule drugs (inhibitors or activators) or cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof), or radioactive isotopes.

[0094] When used to describe the various bispecific antibodies disclosed herein, the term "isolated bispecific antibody" means a bispecific antibody that has been identified and separated and / or recovered from a cell or cell culture in which it is expressed. Contaminating components of its natural environment are substances that typically interfere with the diagnostic or therapeutic use of the polypeptide, and may include enzymes, hormones, and other proteins or non-protein solutes. In some embodiments, the bispecific antibodies of the present invention are purified to a purity greater than 95% or 99%, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC) methods. For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B [Journal of Chromatography B] 848: 79-87 (2007). In certain embodiments, the bispecific antibodies of the invention are purified (1) to a sufficient extent to obtain at least 15 N-terminal or internal amino acid sequence residues by using a spinning cup sequenator, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or silver stain. The isolated bispecific antibodies of the invention include bispecific antibodies in situ within recombinant cells, since at least one component of the bispecific antibodies of the invention's natural environment will not be present. Typically, the isolated bispecific antibodies of the invention will be prepared by at least one purification step.

[0095] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, that is, the single antibody comprising the population is identical and / or binds to the same epitope, except for example containing naturally occurring mutations or possible variant antibodies occurring during the production of monoclonal antibody preparations, such variants are generally present in small amounts. Compared with polyclonal antibody preparations typically including different antibodies for different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed to a single determinant on an antigen. Therefore, the modifier "monoclonal" represents the characteristics of the antibody obtained from a substantially homogeneous antibody population, and should not be interpreted as requiring antibody production by any method. For example, the monoclonal antibody used according to the present invention can be prepared by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of human immunoglobulin loci, such methods and other exemplary methods for preparing monoclonal antibodies as described herein.

[0096] The term "bispecific antibody" is used in the broadest sense and specifically encompasses antibodies or antibody fragments with polyepitopic specificity (i.e., capable of binding to two different epitopes on one biomolecule or each epitope on different biomolecules). Such bispecific antibodies include, but are not limited to, full-length antibodies, antibodies with two or more VL and VH domains, antibody fragments (e.g., Fab, Fv, dsFv, scFv), diabodies, bispecific diabodies and triabodies, covalently or non-covalently linked antibody fragments. "Polyepitopic specificity" refers to the ability to specifically bind to two or more different epitopes on the same or different targets. According to one embodiment, the bispecific antibody of the present invention in the form of IgG1 binds to each epitope with an affinity of 5 μM to 0.001 pM, 3 μM to 0.001 pM, 1 μM to 0.001 pM, 0.5 μM to 0.001 pM, or 0.1 μM to 0.001 pM.

[0097] As used herein, the term "linker" refers to a fusion protein linker, which may be, for example, a flexible linker well known in the art. (See, e.g., Hollinger P, et al., PNAS USA [Proceedings of the National Academy of Sciences of the United States of America]. 90: 6444-6448 (1993); Poljak RJ. Structure [Structure] 2: 1121-1123 (1994)). Commonly used flexible linkers have sequences consisting of segments of Gly and Ser residues ("GS" or "G4S" linkers). An example of such a GS flexible linker has a sequence of (Gly-Gly-Gly-Gly-Ser)-n. By adjusting the copy number "n", the length of the GS linker can be optimized to achieve appropriate separation of functional domains, or to maintain necessary domain interactions, such as in the construct of the bispecific antibody of the present invention (VH and VL domains, two scFv antibody fragments or variable domains and extracellular trap proteins or scFv antibody fragments and extracellular trap proteins). Other examples of suitable non-immunogenic linker peptides are: (SG4)n or G4(SG4)n or GQSSRSS(G4S)n flexible peptide linkers, or rigid / non-flexible linkers (EAAAK)n or (XP)n (in both cases, where "n" is a number between 1 and 10 or between 1 and 4), and oligomers of such linkers.

[0098] With respect to the binding of the bispecific antibodies of the present invention to a target molecule, the term "specifically binds" to a specific polypeptide or an epitope on a specific polypeptide target or "specifically binds" to a specific polypeptide or an epitope on a specific polypeptide target or "has specificity" for a specific polypeptide or an epitope on a specific polypeptide target means binding that is measurably different from non-specific interactions. Specific binding can be measured, for example, by determining the binding of a molecule compared to the binding of a control molecule. For example, specific binding can be determined by competition with a control molecule similar to the target (e.g., an excess of unlabeled target). In this case, if the binding of the labeled target to the probe is competitively inhibited by an excess of unlabeled target, specific binding is indicated. As used herein, the term "specifically binds" to a specific polypeptide or an epitope on a specific polypeptide target or "specifically binds" to a specific polypeptide or an epitope on a specific polypeptide target or "has specificity" for a specific polypeptide or an epitope on a specific polypeptide target can be exhibited, for example, by a bispecific antibody of the present invention that has 10 -4 M or lower, alternatively 10 -5 M or lower, alternatively 10 -6 M or lower, alternatively 10 -7 M or lower, alternatively 10 -8 M or lower, alternatively 10 -9 M or lower, alternatively 10 -10 M or lower, alternatively 10 -11 M or lower, alternatively 10 -12 KD of M or less, or range of 10 -4 M to 10 -6 M or 10 -6 M to 10 -10 M or 10 -7 M to 10 -9 As will be appreciated by the skilled artisan, affinity and KD values ​​are negatively correlated. High affinity for an antigen is measured by a low KD value. In one embodiment, the term "specific binding" refers to a binding in which a molecule (e.g., a bispecific antibody of the invention) binds to a specific polypeptide or an epitope on a specific polypeptide without substantially binding to any other polypeptide or polypeptide epitope.

[0099] The term "Tie2 non-ligand competitive binder" refers to a bispecific antibody of the present invention that does not compete with Ang 1 or Ang 2 for the active site of Tie2 while still allowing Ang 1 or Ang 2 to bind at the active site.

[0100] "Nucleic acid encoding a bispecific antibody of the invention" refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such nucleic acid molecules in a single vector or separate vectors, and such nucleic acid molecules present at one or more locations in a host cell.

[0101] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures, as well as vectors that are incorporated into the genome of a host cell into which the vector has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0102] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably to refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," including the primary transformed cell and progeny derived therefrom, without regard to the number of passages. The nucleic acid content of the progeny may not be exactly the same as that of the parent cell, but may contain mutations. Mutant progeny having the same function or biological activity screened or selected for in the originally transformed cell are included herein.

[0103] "Percentage (%) of amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, after alignment of the sequences and introduction of gaps (if necessary) to achieve maximum percentage sequence identity and not considering any conservative substitutions as part of the sequence identity. Alignment for determining percentage amino acid sequence identity can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. One skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required to achieve maximum alignment over the full length of the compared sequences.

[0104] As used herein, "administering" means a method of administering a dose of a bispecific antibody or composition of the invention (e.g., a pharmaceutical composition comprising a bispecific antibody of the invention) to a subject in need thereof. Such compositions used in the methods of treatment described herein can be, for example, topically administered to a tissue or organ affected by a disease. In one embodiment, such administration is the injection of a composition of the invention directly into the affected eye, for example, via intravitreal, ocular, intraocular, subretinal, or suprachoroidal routes. In another embodiment, the composition used in the methods of treatment described herein can be administered subcutaneously, for example, near a diseased tissue or organ.

[0105] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, an individual or subject is a human. A "subject" may be a "patient."

[0106] As used herein, "treatment" (and "treat" or "treating") refers to clinical intervention that attempts to alter the natural course of the individual being treated, and can be used for prevention or performed during the course of clinical pathology. Desired effects of treatment include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviating the symptoms of such a disease, reducing any direct or indirect pathological consequences of the disease, reducing the rate of disease progression, ameliorating or alleviating the disease state, and alleviating or improving prognosis. In some embodiments, the bispecific antibodies of the invention are used to delay the development of a disease or slow the progression of a disease.

[0107] As used herein, the expressions "cell", "cell line" and "cell culture" are used interchangeably, and all such designations include progeny. Thus, the words "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom, regardless of the number of transfers. It is also understood that the DNA content of all progeny may not be identical due to intentional or unintentional mutations. Mutant progeny are included that have the same function or biological activity as screened for in the originally transformed cell. Where different designations are contemplated, this will be clear from the context.

[0108] A "mutation" is a deletion, insertion or substitution of nucleotides relative to a reference nucleotide sequence (eg, a wild-type sequence).

[0109] A "variant" or "mutant" of a starting or reference bispecific antibody (e.g., a reference antibody or its variable domain / CDR) is a polypeptide that (1) has an amino acid sequence that is different from the amino acid sequence of the starting or reference bispecific antibody, and (2) is derived from the starting or reference bispecific antibody by natural or artificial (man-made) mutagenesis. Such variants include, for example, deletions and / or insertions and / or substitutions of residues within the amino acid sequence of the bispecific antibody of interest, referred to herein as "amino acid residue changes." Thus, a variant CDR refers to a CDR comprising a variant sequence relative to a starting or reference bispecific antibody sequence (e.g., a starting or reference bispecific antibody sequence of a source antibody or antigen-binding fragment). In this context, an amino acid residue change refers to an amino acid that is different from the amino acid at the corresponding position in the starting or reference bispecific antibody sequence (e.g., a starting or reference bispecific antibody sequence of a reference antibody or its fragment). Any combination of deletions, insertions, and substitutions may be performed to obtain a final variant or mutant construct, provided that the final construct has the desired functional characteristics. Amino acid changes may also alter post-translational processes of the bispecific antibody, such as changing the number or position of glycosylation sites.

[0110] A "wild-type (WT)" or "reference" sequence or the sequence of a "wild-type" or "reference" antibody (e.g., a CDR or variable domain of a reference antibody) can be a reference sequence from which a variant antibody is derived by introducing mutations. Typically, the "wild-type" sequence of a given antibody is the most common sequence found in nature. Similarly, a "wild-type" gene sequence is the sequence of the most common gene found in nature. Mutations can be introduced into a "wild-type" gene (and therefore the protein it encodes) by natural processes or by artificial induction. The product of such a process is a "variant" or "mutant" form of the original "wild-type" protein or gene.

[0111] As used herein, "reference antibody" refers to an antibody or fragment thereof whose antigen binding sequence is used as a template sequence on which diversification is performed according to the criteria described herein. The antigen binding sequence generally includes an antibody variable region, at least one CDR, including framework regions.

[0112] Compositions and methods

[0113] The present invention provides novel bispecific antibodies that bind to Tie2 and VEGF, and methods of making and using the same, for example, for therapeutic and diagnostic applications. The bispecific antibodies of the invention can be used, for example, to diagnose or treat various disorders described herein.

[0114] Suitable Tie2 binders for generating the bispecific antibodies of the present invention are those disclosed in WO / 2021 / 102173 filed by Damiano, J., et al. on November 19, 2020, which are agonistic anti-Tie2 monoclonal antibodies. In one embodiment, the Tie2 binders for generating the bispecific antibodies of the present invention are the variable heavy and light chain domains of clone 2, which are disclosed as SEQ ID NOs: 244 and 245, respectively. In another embodiment, the Tie2 binders for generating the bispecific antibodies of the present invention are the variable heavy and light chain domains of clone 3, which are disclosed as SEQ ID Nos: 246 and 247, respectively. WO / 2021 / 102173 and the sequences therein are hereby incorporated by reference in their entirety. See Figure 1 and Table 1.

[0115] In some embodiments, suitable VEGF binders for generating bispecific antibodies of the invention utilize VEGF from aflibercept or (Regeneron-Bayer HealthCare, Tarrytown, NY, USA) or the ligand binding sequence from ranibizumab or (Genentech Inc., a member of the Roche Group, California, USA). In other embodiments, antigen binding sequences from the B20 anti-VEGF monoclonal antibody are used (see Liang, WC, et al., JBC [Journal of Biological Chemistry], 2006: 281, 951-961). See Figure 1 and Table 1.

[0116] The techniques and procedures described or referenced herein are generally well understood by those skilled in the art and are commonly employed using conventional methods, such as, for example, the widely used methods described in Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd ed. (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (FM Ausubel, et al., eds., (2003)); Methods in Enzymology series (Academic Press, Inc.): PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR Taylor, eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Animal Husbandry, Inc. Cell Culture [Antibodies, Laboratory Manual, and Animal Cell Culture] (RI Freshney, ed. (1987)); Oligonucleotide Synthesis [Oligonucleotide Synthesis] (MJ Gait, ed., 1984); Methods in Molecular Biology [Molecular Biology Methods], Humana Press [Humana Press]; Cell Biology: A Laboratory Notebook [Cell Biology: Laboratory Notebook] (JE Cellis, ed., 1998) Academic Press [Academic Press]; Animal Cell Culture [Animal Cell Culture] (RI Freshney, ed., 1987); Introduction to Cell and Tissue Culture [Cell and Tissue Culture Introduction] (JP Thayer and PE Roberts, 1998) Plenum Press [Plenum Press]; Cell and Tissue Culture: Laboratory Procedures [Cell and Tissue Culture: Laboratory Procedures] (A. Doyle, JB Griffiths, and DG Newell, eds., 1993-8) J.Wiley and Sons; Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); Current Protocols in Immunology (JE Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (CA Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach [Antibodies: A Practical Approach] (D. Catty., ed., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach [Monoclonal Antibodies: A Practical Approach] (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual [Using Antibodies: A Laboratory Manual] (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies [Antibodies] (M. Zanetti and JD Capra, eds., Harwood Academic Publishers, 1995).

[0117] The bispecific antibodies of the invention described herein, as well as any antibodies used in the methods described herein, may have any of the features described herein, alone or in combination.

[0118] In one embodiment, KD is measured by radiolabeled antigen binding assay (RIA). In one embodiment, RIA is performed using a Fab version of the antibody of interest and its antigen. For example, by titrating the antibody with a minimum concentration of ( 125 I) equilibrate Fab with labeled antigen, and then capture bound antigen with an anti-Fab antibody-coated plate to measure solution binding affinity of Fab for antigen (see, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). To establish the conditions for the assay, MICROTITER TM Multiwell plates (Thermo Scientific) were coated overnight with 5 μg / ml 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 two to five hours at room temperature (approximately 23° C.). In non-adsorbent plates (Nunc No. 269620), 100 pM or 26 pM [ 125 I]-antigen is mixed with serial dilutions of the Fab of interest (see Presta et al., Cancer Res. [Cancer Research] 57:4593-4599 (1997)). The Fab of interest is then incubated overnight; however, the incubation can be continued for a longer period of time (e.g., about 65 hours) to ensure that equilibrium is reached. Thereafter, the mixture is transferred to a capture plate for incubation at room temperature (e.g., for one hour). The solution is then removed and the plate is washed with 0.1% polysorbate 20 (TWEEN-20 in PBS). TM ) were washed eight times. When the plate had dried, 150 μl / well of scintillating material (MICROSCINT-20 TM ; Hewlett-Packard Company (Packard)), and the board is placed on TOPCOUNT TM The counts were performed on a gamma counter (Packard) for tens of minutes. The concentration of each Fab that gave less than or equal to 20% of maximal binding was chosen for competitive binding assays.

[0119] According to another embodiment, KD uses BIACORE TM Surface plasmon resonance (SPR) is used to measure the surface plasmon resonance (SPR) of the sample. TM -2000 or BIACORE TM-3000 (BIAcore, Piscataway, New Jersey) assays were performed at 25°C with an immobilized antigen CM5 chip of approximately 10 response units (RU). In one embodiment, carboxymethylated dextran biosensor chips (CM5, BIAcore) were activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Antigen was diluted to 5 μg / ml (approximately 0.2 μM) with 10 mM sodium acetate (pH 4.8) and then injected at a flow rate of 5 μl / min to obtain approximately 10 response units (RU) of coupled protein. After the injection of the antigen, 1 M ethanolamine was injected to block unreacted groups. For kinetic measurements, at 25°C, the cells were plated on a plate with 0.05% polysorbate 20 (TWEEN TM Two-fold serial dilutions of Fab (0.78 nM to 500 nM) in PBS containing a surfactant (PBST) were injected at a flow rate of approximately 25 μl / min. on ) and dissociation rate (k off ) is a simple one-to-one Langmuir binding model (BIACORE TM Evaluation software version 3.2) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (KD) was calculated as the ratio k off / k on See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). If the on-rate measured by the above surface plasmon resonance method exceeds 10 6 M -1 s -1 , the association rate can be determined by using a fluorescence quenching technique at increasing antigen concentrations (e.g., a spectrophotometer in a flow-through configuration (Aviv Instruments) or a 8000 Series SLM-AMINCO TM The increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) of 20 nM anti-antigen antibody (Fab form) in PBS (pH 7.2) is measured at 25°C in the presence of 5% paraformaldehyde (pH 7.2) and 1% paraformaldehyde (pH 7.2). KD can also be measured using BIACORE® known in the art. TM SPR assay was used to measure the

[0120] The amino acid sequences of the bispecific antibodies of the present invention are disclosed in Table 1 and the sequence listing herein:

[0121] Table 1

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[0141]

[0142]

[0143]

[0144]

[0145]

[0146] In certain embodiments, the bispecific antibodies provided herein are human antibodies. Human antibodies can be produced using different techniques known in the art. Human antibodies are generally described in van Dijk et al., Curr. Opin. Pharmacol. [Current Views in Pharmacology] 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. [Current Views in Immunology] 20: 450-459 (2008).

[0147] Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce complete human antibodies or complete antibodies with human variable regions in response to an antigenic attack. Such animals typically contain all or part of a human immunoglobulin locus that replaces an endogenous immunoglobulin locus, or it is present outside the chromosome or randomly integrated into the animal chromosome. In such transgenic animals, the endogenous immunoglobulin locus is usually inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. [Natural Biotechnology] 23: 1117-1125 (2005). See also, for example, description of XENOMOUSE TM US Patent Nos. 6,075,181 and 6,150,584 for technology; describing HUANTIBODIES TM US Patent No. 5,770,429 for technology; describes KM MOUSE TM US Patent No. 7,041,870, and describes the VELOCIMOUSE TM The U.S. patent application publication number US2007 / 0061900 describes OmniChicken TM Nos. 9,809,642 and 9,380,769 of the US Patent Application Publication No. 200400618). Human variable regions from intact antibodies produced by such animals can be further modified, for example by combining with a different human constant region.

[0148] Human antibodies can also be prepared by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for producing human monoclonal antibodies have been described. (See, e.g., Kozbor J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147:86 (1991)). Human antibodies generated via human B cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional methods include those described in, for example, U.S. Pat. No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue [Modern Immunology], 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology [Histology and Histopathology], 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology [Experimental and Clinical Pharmacology Methods and Discovery], 27(3):185-91 (2005).

[0149] It is also possible to generate human antibodies by separating the Fv clone variable domain sequence selected from a phage display library derived from humans. Then, such variable domain sequence can be combined with required human constant domains. The technology for selecting human antibodies from an antibody library is described below.

[0150] The bispecific antibodies of the invention can be isolated by screening combinatorial libraries for antibodies having the desired one or more activities. For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies having the desired binding characteristics. Such methods are reviewed, for example, in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., eds., Human Press, Totowa, NJ, 2001), and further described, for example, in 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, Methods in Molecular Biology 248:161-175 (Lo, eds., Human Press, Totowa, NJ, 2001). Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004).

[0151] In certain phage display methods, repertoires of VH and VL genes are cloned individually by polymerase chain reaction (PCR) and randomly recombined in phage libraries, which can then be screened for antigen binding phage, as described in Winter et al., Ann. Rev. Immunol. [Annual Review of Immunology], 12: 433-455 (1994). Phage typically display antibody fragments, as single-chain Fv (scFv) fragments or Fab fragments. Libraries from immune sources can provide high-affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, an initial library (e.g., obtained from humans) can be cloned without any immunization to provide a single source of antibodies to a wide range of non-self and self antigens, as described in Griffiths et al., EMBO J [Journal of the European Molecular Biology Association], 12: 725-734 (1993). Finally, the initial library can also be prepared synthetically by cloning unrearranged V-gene segments from stem cells and using PCR primers containing random sequences to encode the highly variable CDR3 regions and complete rearrangement in vitro, as described by Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example, U.S. Pat. No. 5,750,373, and U.S. Pat. Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0152] Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments herein.

[0153] Techniques for preparing the bispecific antibodies of the invention include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537 (1983), WO 93 / 08829, and Traunecker et al., EMBO J. [Journal of the European Molecular Biology Association] 10:3655 (1991)) and "knob-in-hole" engineering (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can also be prepared by engineering electrostatic steering effects for preparing antibody Fc-heterodimers (WO 2009 / 089004A1); cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science, 229:81 (1985)); using leucine zippers to generate bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); using "diabody" technology for preparing bispecific antibody fragments (see, e.g., USA, 90:6444-6448 (1993); and the use of single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. immunol., 152:5368 (1994)); and the preparation of trispecific antibodies as described, e.g., in Tutt et al., J. Immunol. 147:60 (1991).

[0154] The antibodies or fragments herein also include "dual-acting FAbs" or "DAFs" that comprise antigen binding sites that bind to both Tie2 and VEGF (see, e.g., US 2008 / 0069820).

[0155] In certain embodiments, amino acid sequence variants of the bispecific antibodies provided herein are contemplated (e.g., antibody variants comprising one or more amino acid residue changes). For example, it may be desirable to improve the binding affinity and / or other biological properties of the bispecific antibody. Amino acid sequence variants of the bispecific antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the bispecific antibody or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues within the amino acid sequence of the bispecific antibody. Any combination of deletions, insertions, and substitutions may be performed to obtain the final construct, provided that the final construct has the desired characteristics, such as antigen binding to Tie2 or VEGF.

[0156] In certain embodiments, bispecific antibody variants with one or more amino acid substitutions are provided. The target sites for substitution mutagenesis include CDRs and FRs. Conservative substitutions are contemplated and are well known in the art.

[0157] Other amino acid substitutions are described below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into the bispecific antibody of interest, and the products screened for desired activity (e.g., retained / improved antigen binding, reduced immunogenicity, or improved or reduced ADCC or CDC). Amino acids can be grouped according to common side chain properties:

[0158] (1) Hydrophobicity: norleucine, Met, Ala, Val, Leu, Lie;

[0159] (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gin;

[0160] (3) Acidic: Asp, Glu;

[0161] (4) Basic: His, Lys, Arg;

[0162] (5) Residues that affect chain orientation: Gly, Pro;

[0163] (6) Aromatic: Trp, Tyr, Phe.

[0164] Non-conservative substitutions will entail exchanging a member of one of these classes for another.

[0165] A type of substitution variant relates to one or more hypervariable region residues and / or FR residues that replace a parent antibody (e.g., humanized or human antibody). Typically, the resulting variant selected for further study will have modification (e.g., improvement) relative to some biological properties of the parent antibody (e.g., increased affinity, increased stability, increased expression, changed pI and / or reduced immunogenicity) and / or will substantially retain some biological properties of the parent antibody. Exemplary substitution variants are affinity-matured antibodies, which can be easily generated, for example, using affinity maturation techniques (e.g., those described herein) based on phage display. In short, one or more CDR residues are mutated, and the variant antibody is displayed on phage and screened for specific biological activity (e.g., binding affinity).

[0166] Changes (e.g., substitutions) can be made in the CDR, for example, to improve antibody affinity. Such changes can be made in CDR "hot spots", i.e., residues encoded by codons that are mutated at high frequency during somatic maturation (see, e.g., Chowdhury, Methods Mol Biol. [Molecular Biology Methods] 207: 179-196 (2008)) and / or residues that contact antigens, wherein the binding affinity of the resulting variant VH or VL is tested. Affinity maturation by construction and reselection from a secondary library is described in, e.g., Hoogenboom et al., Methods in Molecular Biology [Molecular Biology Methods] 178: 1-37 (O'Brien et al., ed., Human Press [Humana Press], Totowa, New Jersey, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A second library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method of introducing diversity involves a CDR-guided approach in which several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues that participate in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 are often targeted in particular.

[0167] In certain embodiments, substitutions, insertions or deletions may occur within one or more CDRs, as long as such changes do not substantially reduce the ability of the antibody to bind to antigens. For example, conservative changes (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in the CDRs. Such changes may, for example, be outside the antigen contact residues in the CDRs. In certain embodiments of the variant VH and VL sequences provided above, each CDR is unchanged, or contains no more than one, two or three amino acid substitutions.

[0168] In certain embodiments, substitution, insertion or deletion may occur in one or more FRs, as long as such changes do not substantially reduce the ability of the antibody to bind antigen. Such changes may, for example, improve antibody affinity and / or stability (e.g., as assessed by increased melting temperature).

[0169] In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of the bispecific antibody provided herein, thereby generating Fc region variants. Fc region variants can be included in a human Fc region sequence (e.g., human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid residue change (e.g., substitution) at one or more amino acid positions. In certain embodiments, the present invention contemplates variants with some but not all effector functions, which make it an ideal candidate for applications in which the half-life of bispecific antibodies in vivo is important but some effector functions (e.g., complement and ADCC) are unnecessary or harmful. In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / depletion of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that bispecific antibodies lack FcγR binding (therefore, ADCC activity may be lacking) but retain FcRn binding ability. Primary cell NK cells used to mediate ADCC express only FcγRIII, while monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991).

[0170] Non-limiting examples of in vitro assays for evaluating ADCC activity of bispecific antibodies of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986) and Hellstrom et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); U.S. Pat. No. 5,821,337; and Bruggemann et al., J. Exp. Med.. [Journal of Experimental Medicine] 166:1351-1361 (1987)). Alternatively, non-radioactive assays can be employed (see, e.g., ACTI for flow cytometry). TM Nonradioactive cytotoxicity assay (Cell Technology, Inc., Mountain View, CA; and CYTOTOX 96 TMNon-radioactive cytotoxicity assay (Promega, Madison, Wisconsin). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or in addition, ADCC activity of the bispecific antibody of interest can be assessed in vivo, for example, in an animal model such as that disclosed in Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay can also be performed to confirm that the bispecific antibody cannot bind to C1q and therefore lacks CDC activity. See, for example, C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996); Cragg et al., Blood 101: 1045-1052 (2003); and Cragg et al., Blood 103: 2738-2743 (2004)). FcRn binding and in vivo clearance / half-life assays can also be performed using methods known in the art (see, e.g., Petkova et al., Int'l. Immunol. 18(12): 1759-1769 (2006)).

[0171] The bispecific antibodies of the present invention can be engineered to have reduced effector functions, such as reduced complement dependent cytotoxicity (CDC), antibody dependent cellular cytotoxicity (ADCC) and antibody dependent cellular phagocytosis (ADCP) by reducing the affinity for binding to human FcγRIIIA and / or FcγRIIA and / or FcγRI and / or CIq. In some cases, such reduced effector functions are achieved by amino acid substitution of one or more of the following Fc region residues: N297, L234, L235, D265 and P329, according to EU numbering in Kabat. See US6,737,056, US7,332,581 and WO / 2012 / 130831. In some embodiments, the substitution mutation is one or more of the following: N297G, N297A, L234A, L235A, D265A and / or P329G. In some embodiments, the substitution mutation is N297A or N297G substitution mutation. In some embodiments, substitution mutations include so-called "DANA" Fc mutants in which residues D265 and N297 are substituted with alanine (U.S. Pat. No. 7,332,581). In some embodiments, substitution mutations include "LALA" Fc mutants in which residues L234 and L235 are substituted with alanine (see Lund, J., et al., (1992) Mol. Immunol., 29, 53-59; and Tamm, A. and Schmidt, RE (1997) Int. Rev. Immunol., 16, 57-85). In other embodiments, substitution mutations include "LALA-PG" Fc mutants in which residues L234 and L235 are substituted with alanine and P329 is substituted with glycine (see Brünker, P., et al. (2016) Mol. Cancer Ther.

[0172] Certain antibody variants with improved or reduced binding to FcRs are described (see, e.g., U.S. Pat. No. 6,737,056; WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001)).

[0173] In some embodiments, alterations are made in the Fc region that result in altered (i.e., improved or reduced) C1q binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol. 164:4178-4184 (2000).

[0174] US2005 / 0014934A1 (Hinton et al.) describes bispecific antibodies with increased half-life and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. [Journal of Immunology] 117:587 (1976) and Kim et al., J. Immunol. [Journal of Immunology] 24:249 (1994)). Those antibodies comprise an Fc region having one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more of the following Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424 or 434, e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826). See also Duncan and Winter, Nature 322:738-40 (1988); U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; and WO 94 / 29351, for other examples of Fc region variants.

[0175] The invention also provides immunoconjugates comprising a bispecific antibody herein conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof), or radioactive isotopes.

[0176] In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC) in which the bispecific antibody is conjugated to one or more drugs, including but not limited to maytansinoids (see U.S. Pat. Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235B1); an auristatin, such as the monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483 and 5,780,588, and 7,498,298); a dolastatin; a calicheamicin or a derivative thereof (see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode et al., Cancer Res. 53:3336-3342 (1993); Res. [Cancer Research] 58:2925-2928 (1998)); anthracyclines such as daunorubicin or doxorubicin (see Kratz et al., Current Med. Chem. [Current Medicinal Chemistry] 13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters [Biological Organic and Medicinal Chemistry Letters] 16:358-362 (2006); Torgov et al., Bioconj. Chem. [Bioconjugate Chemistry] 16:717-721 (2005); Nagy et al., Proc. Na tl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 97:829-834 (2000); Dubowchik et al., Bioorg. & Med. Chem. Letters [Bioorg. & Med. Chem. Letters] 12:1529-1532 (2002); King et al., J. Med. Chem. [Journal of Medicinal Chemistry] 45:4336-4343 (2002); and U.S. Patent No. 6,630,579); methotrexate; vindesine; taxanes, such as docetaxel, paclitaxel, larotaxel, tesetaxel and ortataxel; trichothecenes; and CC1065.

[0177] In another embodiment, the immunoconjugate comprises a bispecific antibody as described herein conjugated to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria A chain, a non-binding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthus toxin proteins, pokeweed proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curculin, crotonin, saponin inhibitor, gelonin, mitomycin, restrictocin, phenomycin, enomycin, and trichothecenes.

[0178] In another embodiment, the immunoconjugate comprises a bispecific antibody as described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioisotopes can be used to produce radioconjugates. Examples include At 211 ,I 131 ,I 125 , Y 90 、Re 186 、Re 188 、Sm 153 、Bi 212 , P 32 , Pb 212 , and radioisotopes of Lu. When the radioconjugate is used for detection, it can contain a radioactive atom (e.g., TC99M or 1123) for scintigraphic studies, or a spin label (e.g., iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron) for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI).

[0179] In another embodiment, the immunoconjugate comprises a bispecific antibody as described herein conjugated to a non-cytotoxic agent such as, for example, an artemisinin (e.g., artesunate) or a cannabinoid, or naltrexone, or aspirin, or a statin, or a metabolic agent (e.g., metformin, doxycycline, and anthelmintics).

[0180] Conjugates of the bispecific antibodies of the invention and cytotoxic or non-cytotoxic agents can be prepared using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science 238: 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies. See WO 94 / 11026. The linker can be a "cleavable linker" that promotes the release of the cytotoxic drug in the cell. For example, an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker can be used (Chari et al., Cancer Res. 52: 127-131 (1992); U.S. Pat. No. 5,208,020).

[0181] The immunoconjugates or ADCs herein specifically contemplate, but are not limited to, such conjugates prepared with cross-linkers including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, STAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC and sulfo-SMPB, and SVSB (succinimidyl-(4-vinyl sulfone) benzoate), which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL, USA).

[0182] Any bispecific antibody described herein can be produced using recombinant methods and compositions, for example, as described in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid encoding a bispecific antibody described herein is provided. Such a nucleic acid can encode an amino acid sequence of a VL comprising each binding end of a bispecific antibody (e.g., a light chain and / or a heavy chain of an antibody) and / or an amino acid sequence of a VH comprising each binding end of a bispecific antibody (e.g., a light chain and / or a heavy chain of an antibody). In another embodiment, one or more vectors (e.g., an expression vector) comprising such a nucleic acid are provided. In another embodiment, a host cell comprising such a nucleic acid is provided. In one such embodiment, the host cell comprises (e.g., has been transformed with): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising a VL of a bispecific antibody and an amino acid sequence comprising a VH of a bispecific antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising a VL of a bispecific antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising a VH of a bispecific antibody. In one embodiment, the host cell is a eukaryotic cell, e.g., a Chinese hamster ovary (CHO) cell or a lymphocyte (e.g., a YO, NSO, Sp20 cell). In one embodiment, a method for preparing a bispecific antibody is provided, wherein the method includes culturing a host cell comprising a nucleic acid encoding a bispecific antibody as provided above under conditions suitable for expressing the antibody, and optionally recovering the bispecific antibody from the host cell (or host cell culture medium).

[0183] In order to recombinantly produce bispecific antibodies, nucleic acid encoding the antibody (e.g., as described above) is separated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acid can be easily separated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding antibody heavy and light chains).

[0184] Suitable host cells for cloning or expressing antibody encoding vectors include prokaryotes or eukaryotic cells as described herein. For example, bispecific antibodies can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199 and 5,840,523. Also see Charlton, Methods in Molecular Biology [Molecular Biology Methods], Vol. 248 (BKCLo, ed., Humana Press [Humana Press], Totowa, New Jersey, 2003), pp. 245-254, describing the expression of antibody fragments in Escherichia coli (E. coli). After expression, bispecific antibodies can be separated from bacterial cell paste with soluble fractions and can be further purified.

[0185] In addition to prokaryotes, eukaryotic microorganisms (e.g., filamentous fungi or yeast) are suitable cloning or expression hosts for antibody encoding vectors, including fungi and yeast strains whose glycosylation pathways have been "humanized," resulting in the production of bispecific antibodies with partially or fully human glycosylation patterns. See Gerngross, Nat. Biotech. [Natural Biotechnology] 22: 1409-1414 (2004), and Li et al., Nat. Biotech. [Natural Biotechnology] 24: 210-215 (2006).

[0186] Suitable host cells for expressing glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Many baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfecting Spodoptera frugiperda cells.

[0187] Plant cell cultures can also be used as hosts. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES producing antibodies in transgenic plants). TM technology).

[0188] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in suspension can be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line (COS-7) transformed by SV40; human embryonic kidney line (293 or 293 cells, as described, for example, in Graham et al., J. Gen Virol. [Journal of General Virology] 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (T M4 cells, as described, e.g., in Mather, Biol. Reprod. [Biology of Reproduction] 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; Buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., in Mather et al., Annals NY Acad. Sci. [Annals of the New York Academy of Sciences] 383:44-68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines, such as YO, NSO and Sp2 / 0. For a review of certain mammalian host cell lines suitable for producing antibodies, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0189] The bispecific antibodies provided herein can be identified, screened or characterized for their physical / chemical properties and / or biological activities by various assays known in the art and described herein in the Examples and throughout the specification.

[0190] In one aspect, the antigen binding activity of the bispecific antibodies of the present invention to Tie2 and VEGF is determined, for example, by known methods (e.g., ELISA, Western blot, surface plasmon resonance assay (e.g., BIACORE TM ) etc.) to test.

[0191] In one aspect, antigen binding activity (e.g., as indicated by KD) is measured using BIACORE TMSurface plasmon resonance (SPR) is used to measure the surface plasmon resonance (SPR) of the sample. TM -2000 or BIACORE TM -3000 (BIAcore, Piscataway, NJ) assays were performed at 25°C with an immobilized antigen CM5 chip of approximately 10 response units (RU). In one embodiment, carboxymethylated dextran biosensor chips (CM5, BIAcore) were activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Tie2 or VEGF antigens were diluted to 5 μg / ml (approximately 0.2 μM) with 10 mM sodium acetate (pH 4.8) and then injected at a flow rate of 5 μl / min to obtain approximately 10 response units (RU) of coupled protein. After injection of the antigen, 1 M ethanolamine was injected to block unreacted groups. For kinetic measurements, at 25°C, the cells were plated on a plate with 0.05% polysorbate 20 (TWEEN TM Two-fold serial dilutions of Fab (0.78 nM to 500 nM) in PBS containing a -20) surfactant (PBST) were injected at a flow rate of approximately 25 μl / min. on ) and dissociation rate (k off ) is a simple one-to-one Langmuir binding model (BIACORE TM Evaluation software version 3.2) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (KD) was calculated as the ratio k off / k on See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). If the on-rate measured by the above surface plasmon resonance method exceeds 10 6 M -1 s -1 , the association rate can be determined by using a fluorescence quenching technique at increasing antigen concentrations (e.g., a spectrophotometer in a flow-through configuration (Aviv Instruments) or a 8000 Series SLM-AMINCO TM The increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) of 20 nM antibody (Fab form) in PBS (pH 7.2) was measured at 25°C in the presence of 5% paraformaldehyde (BPA) and 4% paraformaldehyde (BPA-1). KD can also be measured using the BIACORE TM SPR assay was used to measure the

[0192] In another aspect, competition assays can be used to identify bispecific antibodies that compete with another antibody for binding to Tie2 and / or VEGF. In certain embodiments, such a competing antibody binds to the same epitope (e.g., a linear or conformational epitope) as that bound by an antibody as described herein. Detailed exemplary methods for locating epitopes to which antibodies bind are provided in Morris (1996) "Epitope Mapping Protocols," Methods in Molecular Biology, Vol. 66 (Humana Press, Totowa, NJ).

[0193] In an exemplary competition assay, immobilized Tie2 or VEGF is incubated in a solution comprising a first labeled antibody that binds Tie2 or VEGF and a second unlabeled antibody that is being tested for its ability to compete with the first antibody for binding to Tie2 or VEGF. The second antibody may be present in the hybridoma supernatant. As a control, immobilized Tie2 or VEGF is incubated in a solution comprising the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow the first antibody to bind to Tie2 or VEGF, excess unbound antibody is removed and the amount of label associated with immobilized Tie2 or VEGF is measured. If the amount of label associated with immobilized Tie2 or VEGF in the test sample is significantly reduced relative to the control sample, it indicates that the second antibody competes with the first antibody for binding to Tie2 or VEGF. See Harlow and Lane (1988) Antibodies: A Laboratory Manual, Chapter 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York).

[0194] In one aspect, assays are provided for identifying bispecific antibodies that have biological activity. See the Examples section. Biological activity can include, for example, activation, excitement, increase, enhancement of one or more biological activities of Tie2 or VEGF. Also provided herein are bispecific antibodies that have such biological activity in vivo and / or in vitro, see Table 1.

[0195] In certain embodiments, labeled bispecific antibodies are provided. Labels include, but are not limited to, directly detected labels or moieties (e.g., fluorescent labels, chromogenic labels, electron-dense labels, chemiluminescent labels, and radioactive labels), as well as indirectly detected moieties (e.g., enzymes or ligands) (e.g., via enzymatic reactions or molecular interactions). Exemplary labels include, but are not limited to, radioisotopes. 32 P. 14 C. 125 I.3 H and 131 I, fluorophores (e.g., rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone), luciferases (e.g., firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456)), luciferin, 2,3-dihydrophthalazinedione, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, carbohydrate oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (e.g., uricase and xanthine oxidase, coupled to enzymes that use hydrogen peroxide to oxidize dye precursors (e.g., HRP, lactoperoxidase, or microperoxidase)), biotin / avidin, spin labels, phage labels, stable free radicals, etc. In another embodiment of the present invention, the bispecific antibody does not need to be labeled, and its presence can be detected using a labeled bispecific antibody bound to the bispecific antibody, such as, for example, an anti-horseradish peroxidase antibody as is well known in the art.

[0196] The bispecific antibodies of the invention can be used in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. Zola et al., Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987).

[0197] Competitive binding assays rely on the ability of a labeled standard to compete with the test sample analyte for binding to a limited amount of antibody. The amount of antigen in the test sample is inversely proportional to the amount of standard that will bind to the antibody. To facilitate determination of the amount of standard that will bind, the antibody is usually insolubilized before or after competition so that the standard and analyte bound to the antibody can be conveniently separated from the standard and analyte that remain unbound.

[0198] Sandwich assays involve the use of two antibodies, each capable of binding to a different immunogenic portion or epitope of the protein to be detected. In a sandwich assay, the test sample analyte is bound by a first antibody immobilized on a solid support, and then a second antibody binds to the analyte, thereby forming an insoluble three-part complex. See, e.g., U.S. Patent No. 4,376,110. The second antibody itself can be labeled with a detectable moiety (direct sandwich assay) or can be measured using an anti-immunoglobulin antibody labeled with a detectable moiety (indirect sandwich assay). For example, one type of sandwich assay is an ELISA assay, in which case the detectable moiety is an enzyme.

[0199] Pharmaceutical preparations

[0200] Therapeutic formulations of the bispecific antibodies of the invention or the immunoconjugates of the invention or the fusion polypeptides of the invention provided herein can be prepared as lyophilized formulations or aqueous solutions for storage by mixing the polypeptides having the desired purity with optional "pharmaceutically acceptable" carriers, excipients or stabilizers (all of which are referred to as "excipients") typically used in the art. For example, buffers, stabilizers, preservatives, isotonic agents, nonionic detergents, antioxidants and other various additives. See, for example, Remington's Pharmaceutical Sciences, 16th edition, A. Osol, ed. (1980). Such additives must be nontoxic to the receptor at the dosages and concentrations used.

[0201] Treatment

[0202] The bispecific antibodies of the invention provided herein can be used in therapeutic methods for treating, preventing, alleviating, diagnosing various diseases, including but not limited to eye diseases, ischemic diseases or injuries, and infectious diseases as defined herein.

[0203] In one embodiment, the bispecific antibodies of the invention provided herein can be used to treat, for example, diabetic retinopathy (DR), diabetic macular edema (DME), proliferative diabetic retinopathy (PDR) (non-proliferative or proliferative), age-related macular degeneration (AMD) (wet or dry), geographic atrophy (GA), retinopathy of prematurity (ROP), glaucoma, retinal vein occlusion (RVO), central RVO or branched RVO, etc.

[0204] In another embodiment, the bispecific antibodies of the invention provided herein can be used to treat, for example, sepsis, dengue virus infection, tuberculosis, coronavirus infection, or influenza.

[0205] In yet another embodiment, the bispecific antibodies of the present invention provided herein can be used to treat, for example, diabetic nephropathy, acute kidney injury, chronic kidney disease, kidney or other organ transplantation, and the like.

[0206] Examples

[0207] The following are examples of methods and bispecific antibody constructs of the present invention.It should be understood that various other embodiments can be practiced, given the general description provided above.

[0208] Example 1: Tie2 / VEGF bispecific antibody activates Akt pathway in vitro

[0209] This is a cell-based assay for determining the levels of phosphorylated Akt protein in HUVEC cells. This determines whether the bispecific antibody constructs of the invention are able to activate the correct pathway in vitro.

[0210] HUVEC cell treatment in 96-well plates: HUVEC cells from a single donor were cultured in endothelial cell growth medium with supplements in a 37°C incubator at 5% CO2 and 3% oxygen. The day before the assay, cells were plated in 96-well tissue culture plates at 80,000 cells / well. The next day, the cells were starved for 4h in unsupplemented endothelial basal medium. After cell starvation, unsupplemented endothelial basal medium containing bispecific antibody constructs or angiopoietin-2 (Tie2 ligand) at concentrations ranging from 100nM to 0.14nM was added to the cells. The cells were then incubated for 20min in a 37°C incubator at 5% CO2 and 3% oxygen. After incubation, the culture medium was removed from the 96-well plate and stored in a -80°C refrigerator.

[0211] MSD ELISA Assay: MSD-TRIS 5% (w / v) blocking buffer was added to a streptavidin-coated small spot 96-well MSD plate at a volume of 150 uL / well and incubated at 600 rpm on an orbital shaker for 1 h at room temperature (RT). After the blocking step, the plate was immediately washed once with 150 uL of MSD-TRIS wash buffer. Biotinylated anti-pAkt (s473) capture antibody at a concentration of 100 ng / well was added to the plate and incubated for 2 h at RT. The previously treated HUVEC 96-well cell plate was taken out of the -80°C freezer and 65 uL of MSD-TRIS lysis buffer with protease and phosphatase inhibitors was added to each well. After adding the lysis buffer, the plate was incubated at RT on a shaker at 600 rpm for 1 h. After incubation with the capture antibody for 2 h, the plate was washed 3 times with 150 ul of MSD-TRIS wash buffer. Cell lysate was added at 50ul / well and the plate was incubated overnight at 4°C on a shaker at 600rpm. After incubation, the plate was washed 3 times with 150ul of MSD-TRIS wash buffer, sulfo-tagged anti-Akt antibody was added to each well at a concentration of 200ng / well and incubated at room temperature on a shaker at 600rpm for 2h. After incubation, the plate was washed 3 times with 150ul of MSD-TRIS wash buffer, and 150uL of 2-fold diluted concentrated MSD read buffer was added to each well (the original stock solution concentration was 4 times). The plate was read on an MSD S-600 instrument.

[0212] Electrochemiluminescent signals were determined. Signals were normalized by subtracting background signals from cells treated with unsupplemented endothelial basal medium. Normalized signals from cells treated with different concentrations of drug or angiogenin were fitted to the "log (agonist) vs. normalized response - variable slope" equation in GraphPad Prism. Efficacy was reported as EC50. See Figure 4 (panels AB) and Figure 11 (panels AB).

[0213] The results showed that construct No. 1 activated downstream AKT signaling similar to the anti-Tie2 clone No. 3. Construct No. 1 was approximately 5-fold more potent than construct No. 5. See also Figure 7 In contrast, the Ang2-mVEGF bispecific antibody, which is a faricitumab-like bispecific antibody that acts as an Ang2-dependent Tie2 activator, did not activate the AKT downstream pathway. Similarly, in a separate experiment, both constructs No. 1 and No. 3 activated the downstream AKT pathway, in contrast to the Ang1 control ( FIG. 11 , panels AB).

[0214] Example 2: In vitro determination of Tie2 potency

[0215] A pTie2 cell-based assay was developed to determine the levels of phosphorylated Tie2 protein in human umbilical vein endothelial cells (HUVECs). Using this assay, the potency of various bispecific antibody constructs was tested and quantified to enable ranking of the bispecific constructs of the invention.

[0216] HUVEC cell treatment in 96-well plates: HUVEC cells from a single donor have been used in all assays. The cells were cultured in endothelial cell growth medium with supplements in a 37°C incubator at 5% CO2 and 3% oxygen. The day before the assay, the cells were plated in 96-well tissue culture plates at 80,000 cells / well. The next day, the cells were starved for 4h in unsupplemented endothelial basal medium. After cell starvation, unsupplemented endothelial basal medium containing bispecific antibody constructs or angiopoietin-2 (Tie2 ligand) at concentrations ranging from 100nM to 0.14nM was added to the cells. The cells were then incubated for 20min in a 37°C incubator at 5% CO2 and 3% oxygen. After incubation, the culture medium was removed from the 96-well plate and the plate was stored in a -80°C refrigerator.

[0217] Mesoscale Diagnostics (MSD) ELISA Assay: The MSD ELISA assay was employed to determine the levels of phosphorylated Tie2 present in treated cells. MSD-TRIS 5% (w / v) blocking buffer was added to a streptavidin-coated small spot 96-well MSD plate at a volume of 150 uL / well and incubated for 1 h at RT on an orbital shaker at 600 rpm. After the blocking step, the plate was immediately washed once with 150 μL of MSD-TRIS wash buffer. Biotinylated anti-Tie2 capture antibody at a concentration of 50 ng / well was added to the plate and incubated for 2 h at room temperature. The previously treated HUVEC 96-well cell plate was removed from the -80°C freezer and 65 μL of MSD-TRIS lysis buffer with protease and phosphatase inhibitors was added to each well. After the addition of lysis buffer, the plate was incubated for 1 h at RT on a shaker at 600 rpm. 3-fold serial dilutions of standard recombinant human pTie2 protein ranging from 25 ng / mL to 0.39 ng / mL were prepared in MSD-TRIS lysis buffer with protease and phosphatase inhibitors. After incubation with capture antibody for 2 h, the plate was washed 3 times with 150 μl of MSD-TRIS wash buffer. Standards or cell lysates were added at 50 ul / well and the plate was incubated overnight at 4 °C on a shaker at 600 rpm. After incubation, the plate was washed 3 times with 150 ul of MSD-TRIS wash buffer, and sulfo-tagged anti-phosphotyrosine detection antibody was added to each well at a concentration of 100 ng / well, and the plate was then incubated on a shaker at room temperature at 600 rpm for 2 h. After incubation, the plate was washed 3 times with 150 ul of MSD-TRIS wash buffer, and 150 μL of 2x concentrated MSD read buffer was added to each well (the original stock concentration was 4x). The plate was read on an MSD S-600 instrument.

[0218] Using Discovery Workbench TM Software analyzes electrochemiluminescent signals. The concentration of pTie2 in HUVEC cells treated with different concentrations of the tested bispecific constructs (constructs No. 1, No. 2, No. 3, No. 4, or No. 5) or controls (anti-Tie2 clone No. 3 or Ang1) was determined by four-parameter fitting logistic regression analysis based on the standard curve. The normalized signal was plotted against the concentration and fitted to the "log (agonist) versus normalized response-variable slope" equation in GraphPad Prism. The efficacy is reported as EC50nM. See Figure 5 (panel B), Figure 7 , Figure 8A-B , Figure 9 (Panel B) and Figure 9 (Panel C).

[0219] Different bispecific antibody constructs exhibited different p-Tie2 EC50 values ​​measured in vitro, which emphasizes the unpredictability of a specific construct with regard to its p-Tie2 in vitro potency.

[0220] Example 3: In vitro determination of VEGF potency

[0221] The VEGF bioassay from Promega is a luminescent cell-based assay that measures VEGF inhibition via KDR / VEGFR2. KDR / NFAT-RE HEK293 cells have been engineered to express NFAT response elements upstream of Luc2P and exogenous KDR. Bio-GloTM luciferase assay system is used to detect and quantify bioluminescent signals. This assay has been used to determine the in vitro VEGF efficacy of various bispecific antibody constructs.

[0222] Treatment of KDR / NFAT-RE HEK293 cells in 96-well plates: Frozen cells were thawed and resuspended in pre-warmed assay buffer (DMEM medium with 10% fetal bovine serum). Cells were plated at 5000 cells / well in 96-well tissue culture plates. Serial dilutions of the tested bispecific antibody constructs (Constructs No. 1, 2, 3, 4, or 5) or controls (aflibercept or aflibercept-LALA-PG or anti-Tie2 clone No. 3) were added to the plates in duplicate at final concentrations ranging from 20 nM to 1 pM, or serial dilutions of bevacizumab were added to the plates in duplicate at final concentrations ranging from 40 nM to 2 pM. In the case of Figure 5, this step was followed by the addition of 0.5 nM human recombinant VEGF-165 protein. The negative control included in each assay was cells treated with 0.5 nM VEGF-165 alone. The plate was incubated in a humidified incubator at 37°C with 5% CO2 for 6 hours. After incubation, 75 μl of Bio-Glo TM The reagent was added to all wells and the plate was incubated at room temperature for 10 min. TM Luminescence was measured by a luminescence plate reader. Percent inhibition was calculated based on cells treated with VEGF-165 as 0% inhibition. Percent inhibition was plotted against concentration and fitted to the "log (inhibitor) versus normalized response - variable slope" equation in GraphPadPrism, and potency was reported as EC50 nM. See Figure 5 (panel A), Figure 7 , Figure 8A-B , Figure 9 (Panel A) and Figure 9 (Panel C).

[0223] The results indicate that different bispecific constructs can exhibit different VEGF inhibitory potencies.

[0224] Example 4: Determination of in vitro phosphorylated Tie2 potency in HUVECs in the presence of recombinant human VEGF-165

[0225] A pTie2 cell-based assay was developed to determine the levels of phosphorylated Tie2 protein in HUVECs to investigate the effect of VEGF on the bispecific antibody constructs in activating the Tie2 pathway. Potency was determined in the presence and absence of VEGF.

[0226] HUVEC cell treatment in 96-well plates: HUVECs were cultured in endothelial cell growth medium with supplements in a 37°C incubator at 5% CO2 and 3% oxygen. The day before the assay, cells were plated in 96-well tissue culture plates at 80,000 cells / well. The next day, cells were starved for 4 h in unsupplemented endothelial basal medium. After cell starvation, cells were treated with or without 20 nM (final concentration) VEGF-165 recombinant protein in unsupplemented endothelial basal medium for 5 min. After VEGF treatment, bispecific antibody constructs or angiopoietin-2 were added to cells at concentrations ranging from 100 nM to 0.14 nM. The cells were then incubated in a 37°C incubator at 5% CO2 and 3% oxygen for 20 min, and after incubation, the medium was removed from the 96-well plate and the plate was stored in an ultra-low temperature freezer at -80°C.

[0227] MSD ELISA assay: The assay was performed in streptavidin-coated small-spot 96-well MSD plates. MSD-TRIS 5% (w / v) blocking buffer was added to the plate at a volume of 150 μL / well and incubated on an orbital shaker at 600 rpm for 1 h at RT. After the blocking step, the plate was immediately washed once with 150 μL of MSD-TRIS wash buffer. Biotinylated anti-Tie2 capture antibody at a concentration of 50 ng / well was added to the plate and incubated for 2 h at RT. The previously treated HUVEC 96-well cell plate was taken out of the -80°C ultra-low temperature freezer, and 65 μL of MSD-TRIS lysis buffer with protease and phosphatase inhibitors was added to each well. After the addition of the lysis buffer, the plate was incubated on a shaker at 600 rpm for 1 h at RT. 3-fold serial dilutions of standard recombinant human pTie2 protein ranging from 25 ng / mL to 0.39 ng / mL were prepared in MSD-TRIS lysis buffer with protease and phosphatase inhibitors. After incubation with capture antibody for 2 h, the plate was washed 3 times with 150 ul of MSD-TRIS wash buffer. Standards or cell lysates were added at 50 ul / well and the plate was incubated overnight at 4 °C on a shaker at 600 rpm. After incubation, the plate was washed 3 times with 150 μl of MSD-TRIS wash buffer, and sulfo-tagged anti-pan phosphotyrosine detection antibody was added to each well at a concentration of 100 ng / well, and the plate was incubated on a shaker at room temperature at 600 rpm for 2 h. After incubation, the plate was washed 3 times with 150 ul of MSD-TRIS wash buffer, and 150 μL of 2-fold diluted concentrated MSD read buffer was added to each well (the original stock concentration was 4 times). Plates were read on an MSD S-600 instrument.

[0228] The reading from the plate is an electrochemiluminescent signal, which is analyzed using discovery work bench software. The concentration of pTie2 in HUVEC cells treated with different concentrations of bispecific antibody constructs (constructs No. 1, No. 3) or Ang1 or anti-Tie2 clone No. 3 was determined by four-parameter fitting logistic regression analysis based on the standard curve. The normalized signal was plotted against the concentration and fitted to the "logarithm (agonist) versus normalized response-variable slope" equation in GraphPad Prism. The efficacy is reported as EC50nM. See Figure 6 (panel AE) and Figure 10 (panel AD).

[0229] The results show that the tested bispecific antibody constructs have improved in vitro Tie2 potency in the presence of VEGF (Figure 6 (Panel E) and Figure 10 (Panel D)), while the tested Ang1 and anti-Tie2 clone No. 3 are not significantly affected by VEGF (Figure 6 (Panel A) and Figure 6 (Panel E) and Figure 10 (Panel A) and Figure 10 (Panel D)). This is a surprising result and suggests that in vivo, the endogenous presence of VEGF in the microenvironment of diseased tissues can enhance the Tie2 activity of certain bispecific antibody constructs of the present invention.

[0230] Example 5: Measuring Tie2 / VEGF bispecific antibody yield, purity and stability

[0231] Important to the successful generation of a viable biopharmaceutical product is the manufacturability of the product. Given the unpredictability of bispecific antibodies in this regard, certain CMC criteria are used to determine which bispecific antibodies of the invention will show the most promise in terms of each molecule: (a) titer; (b) yield; (c) purity; and (d) stability.

[0232] Plasmid preparation: The gene sequence of each specific bispecific construct is amplified by PCR and inserted into the corresponding expression vector. The sequence of the constructed vector is verified by sequencing. The correct plasmid for each bispecific antibody construct is amplified and delivered downstream.

[0233] Transient transfection and cell culture: For transient transfection, mix the plasmid with the transfection reagent at an optimal ratio and then add to the flask using transient CHO-K1 or HEK293 cell lines. The cells are cultured in serum-free medium and maintained in Erlenmeyer flasks on an orbital shaker or bioreactor at 37°C with appropriate agitation speed for 6 days.

[0234] Purification and Analysis: A standard purification procedure was used: (a) cell culture fluid of each bispecific antibody construct was centrifuged; (b) cell culture supernatant was purified using protein A affinity chromatography and then affinity purification using gel filtration; and (c) purified bispecific antibodies were analyzed for endotoxin load by SDS-PAGE and SEC-HPLC.

[0235] Measurement of Titer or Yield: In biologics manufacturing, titer is the concentration of total product in the production medium, as measured by some analytical method (ELISA or HPLC) using a standard curve of reference materials. Yield refers to the total mass of the final, purified and processed material or drug substance. Intermediate yield is the mass of total product recovered at various stages of product harvest and processing. Yield is calculated using the formula [conc]*volume, and these numbers are specific to each step / intermediate / product. Intermediate yields will be higher than final yields because inherent product losses are unavoidable while removing impurities. Concentration [conc] is the strength of the DS protein in its final formulation buffer. It can be determined experimentally, or calculated using known extinction coefficients and A280 measurements. See Figure 7 and Figure 8A-B .

[0236] Purity Assessment by SEC-HPLC: Size Exclusion HPLC (SEC-HPLC) separates proteins based on hydrodynamic volume, which is generally correlated to molecular weight. SEC resins have pores into which proteins can diffuse as they pass through the column. The time it takes for proteins to pass through the pores (which delays their elution from the column) is inversely proportional to their hydrodynamic volume. Therefore, the elution time in SEC is (to a first approximation) inversely proportional to the molecular weight. When SEC is employed to assess the quality / purity of a protein sample, typically, in addition to the main product peak, there may be higher order species (or high molecular weight, HMW) eluting at shorter retention times due to aggregation or multimerization of the product, and / or degradation / truncation products (LMW) eluting later than the main product peak. HMW and LMW species are generally considered product-related impurities. Impurities from the process or raw materials may also be present. For biologics, typically, >98% main peak and <2% HMW in DS would be acceptable as an initial SEC purity standard. See Figure 7 and Figure 8A-B .

[0237] Stress stability testing: Each batch of bispecific antibody constructs produced and purified as described above was aliquoted into 8 or more equal fractions. Half of the aliquots were stored at 37°C for two weeks, while the other half were stored or frozen at 5°C as a "control time zero" or (T0). At the end of the stress test, the 37°C samples were compared to the T0 samples using SEC-HPLC and p-Tie2 and VEGF potency assays, as described above in Examples 2 and 3. See Figure 7 and Figure 8A-B .

[0238] The bispecific antibody constructs tested and Figure 7 and Figure 8A-BThe results shown in showed different CMC properties, suggesting that not all generated Tie2 / VEGF bispecific antibody constructs were suitable from a CMC perspective for further development as possible drug candidates.

[0239] Example 6: Evaluation of the Tie2 / VEGF bispecific antibody in the oxygen-induced retinopathy (OIR) mouse model Standard bonding (TE)

[0240] The OIR model is based on the exposure of mouse pups to hyperoxia at a stage when their retinal vasculature is still developing. This leads to capillary depletion and, upon return to room air, retinal ischemia and proliferative vascular disease in the retinal vasculature, or oxygen-induced retinopathy. The objectives of the experiment were: first, to evaluate the ability of bispecific molecule constructs No. 1 and No. 3 to bind to their target protein Tie2 receptor (TE: target engagement) after intravitreal (IVT) delivery to hyperoxic pups compared to faricimab mouse surrogate or "FMS" (which is a bispecific construct containing the known anti-mouse VEGF clone B20 (see Liang, WC, et al., JBC [Journal of Biological Chemistry], 2006: 281, 951-961) and the known anti-Ang2 clone LC10 (Roche RO5485202); second, to evaluate the Ang2 neutralization effect of bispecific molecule constructs No. 1 and No. 3 compared to FMS after IVT delivery in hyperoxic pups.

[0241] Postnatal day 7 (P7) C57BL / 6J pups were placed in a hyperoxic chamber (75% O2) for 5 days (n=10 / cage), resulting in vascular regression in the central retina. CD-1 foster mothers were rotated 2-3 days before and after entering the chamber. At P12, pups were returned to room air, which triggers aberrant neovascularization, and then given bispecific constructs of the invention or negative controls intravitreally as described below.

[0242] All groups (including naive OIR mice) were euthanized at P17. Eyes were enucleated and fixed in 4% paraformaldehyde for 1 hour.

[0243] Retinas were dissected and incubated overnight with rhodamine-labeled lectin from Bandeiraea simplicifolia (Griffonia simplicifolia) (1:100) in 1 mM CaCl2 in PBS to visualize areas of vascular occlusion (VO) or neovascularization (NV). Stained retinas were mounted flat on slides and Imaging on AxioScan. Images were analyzed to determine %VO or %NV of the total retina. All data are presented as mean ± SD, where each data point represents the mean of two retinas from one pup. Standard one-way ANOVA was used, followed by analysis of variance from The statistical significance of the tested bispecific constructs was determined using Prism 8 software (San Diego, CA, USA) at the corresponding time points using Dunnett's multiple comparison test against the NSIgG group, where statistical significance was determined as p<0.05.

[0244] Alternatively, the harvested retina can be evaluated by a mesoscale diagnostic (MSD) ELISA assay as described above in Example 2 to determine the level of phosphorylated Tie2 present in the retinal tissue of treated OIR mice. MSD-TRIS 5% (w / v) blocking buffer was added to a streptavidin-coated small spot 96-well MSD plate at a volume of 150uL / well and incubated at 600rpm for 1h at RT on an orbital shaker. After the blocking step, the plate was immediately washed once with 150μL of MSD-TRIS wash buffer. A biotinylated anti-Tie2 capture antibody at a concentration of 50ng / well was added to the plate and incubated at room temperature for 2h. The previously treated HUVEC 96-well cell plate was removed from a -80°C freezer and 65μL of MSD-TRIS lysis buffer with protease and phosphatase inhibitors was added to each well. After the addition of the lysis buffer, the plate was incubated at 600rpm on a shaker at RT for 1h. 3-fold serial dilutions of standard recombinant human pTie2 protein ranging from 25 ng / mL to 0.39 ng / mL were prepared in MSD-TRIS lysis buffer with protease and phosphatase inhibitors. After incubation with capture antibody for 2 h, the plate was washed 3 times with 150 μl of MSD-TRIS wash buffer. Standards or cell lysates were added at 50 ul / well and the plate was incubated overnight at 4 °C on a shaker at 600 rpm. After incubation, the plate was washed 3 times with 150 ul of MSD-TRIS wash buffer, and sulfo-tagged anti-phosphotyrosine detection antibody was added to each well at a concentration of 100 ng / well, and the plate was then incubated on a shaker at room temperature at 600 rpm for 2 h. After incubation, the plate was washed 3 times with 150 ul of MSD-TRIS wash buffer, and 150 μL of 2x concentrated MSD read buffer was added to each well (the original stock concentration was 4x). The plate was read on an MSD S-600 instrument.

[0245] This experiment was performed to test the following: 10ug / eye of each of the bispecific constructs #1, #3, FMS control as comparator, and an irrelevant negative control NSIgG. OIR mouse pups were dosed at P13 and retinas harvested at P15 (48 hour collection), or dosed at P15 and retinas harvested at P16 (24 hour collection). Comparisons were made relative to a time-matched NSIgG control group 10ug / eye dose. One-way ANOVA and Dunnet post hoc test. **p<0.01; ****p<0.0001.

[0246] When dosed at P13 and harvested at 48h, Construct No. 3 (10ug), Construct No. 1 (10ug) and FMS (10ug) did not show significant upregulation of pTie2 in hyperoxic retina compared to NSIgG control (10ug). See Figure 16 (Panel A). However, all three molecules showed significant reduction in Ang2. See Figure 16 (Panel B). When dosed at P15 and harvested at 24h, both Construct No. 3 (10ug) and Construct No. 1 (10ug) showed significant upregulation of pTie2 compared to NSIgG control (10ug). See Figure 17 (Panel A). However, this was not observed with FMS (10ug). See Figure 17 (Panel A). All three molecules significantly downregulated Ang2. See Figure 17 (Panel B).

[0247] Both constructs #1 and #3 exhibited engagement with their target Tie2 receptor, significantly upregulated pTie2 and modulated Ang2 levels in hyperoxic eyes at P15. In contrast, FMS appeared to neutralize Ang2 but did not show any changes in pTie2 levels, which may be due to the ineffectiveness of the Ang1 protein at P15. At P13, all three molecules showed significant Ang2 reduction, while the pTie2 signal was probably lost before 48h. This study shows the ability of the tested bispecific constructs of the present invention to trigger pTie2 induction in disease states by modulating the biomarker Ang2, regardless of Ang1 and Ang2 levels.

[0248] Example 7: Tie2 / VEGF bispecific antibody in laser-induced choroidal neovascularization (CNV) rat model Effects of

[0249] This study will determine the effects of certain bispecific antibody constructs on vascular leakage and choroidal neovascularization in a rat laser CNV model. The laser-induced CNV rat model involves rupture of Bruch's membrane, resulting in an inflammatory / wound healing response and concomitant CNV, thereby mimicking wet or neovascular AMD. The choriocapillaris is clearly involved in the neovascular response, and this model produces an angiographic appearance similar to wet or neovascular AMD. Animal experiments will be performed by EyeCRO TMLimited Liability Company (Oklahoma City, Oklahoma).

[0250] Animal Housing and Welfare: Female brown Norway rats, 9-12 weeks old and weighing 126-150 g, were obtained for the study. Rats were housed in groups of 3-5 in large cages on ventilated racks under the supervision of the IUCUC and the attending veterinarian and maintained under standard animal care conditions (30%-70% humidity, 68°F-79°F, and 12h light / dark cycle). Rats were monitored daily for gross behavioral changes, such as lack of activity, lack of grooming, lack of food / drinking, and intolerance as humane endpoints. Any rats exhibiting these behaviors were removed from the study on the advice of the attending veterinarian.

[0251] Study Design: The study design is depicted in Table 2 below. The rats were pupillary dilated and protected from light. They were then sedated with ketamine / xylazine. The fundus of the rats' eyes was photographed with a Micron IV Small Animal Ophthalmoscope (Phoenix Research). A 540 nm wavelength thermal laser connected to a Micron IV custom laser attachment was used to create 3 lesions per eye. The fundus was then recorded to ensure that bubbles were formed in Bruch's membrane, confirming successful laser injury. The laser injury was bilateral with 3 lesions per eye or 6 lesions per rat.

[0252] On the 3rd and 10th days, after sedation, 5 μL bispecific antibody constructs or vehicles were administered intravitreally to the pars plana region using a 33G Hamilton syringe. For the fluorescein angiography on the 22nd day, rats were sedated with ketamine / xylazine and injected with 10% sodium fluorescein at a dose of 1 μL / g. Fundus images under 488nm excitation wavelength were captured. Fundus images were quantified using image analysis software Photoshop CS Adobe Inc. Lesions were measured to calculate neovascularization, while the integral density of fluorescein intensity was measured to indicate vascular leakage in the neovascular region. Overlapping or hemorrhagic lesions will be excluded from analysis. After euthanasia, the right eye was removed and fixed overnight in Davidson at room temperature, then transferred to 70% EtOH at 4°C until ready for analysis. The left eye was removed, dissected into retina and RPE / choroid, and snap frozen until ready for analysis.

[0253] Quantification of neovascularization in the lesion area and vascular density in the retina can be performed by ImageJ (National Institutes of Health Health). P values ​​(significant change, p < 0.05) can be evaluated by Student's t test.

[0254] Table 2: Study design

[0255]

[0256] Example 8: Pharmacokinetic (PK) study of Tie2 / VEGF bispecific antibody in adult mice

[0257] This study will compare the bispecific antibody constructs with clinical controls at different time points. PK profiles of the IgG or non-related IgG in whole eye or retinal or choroidal tissues and serum to assess persistence.

[0258] Intravitreal Administration and Terminal Harvest: All animals will be handled in accordance with the Association for the Research in Vision and Ophthalmology statement for the use of animals in ophthalmic and vision research and will be approved by the Institutional Animal Care and Use Committee of UNITY Biotechnology.

[0259] Male C57BL / 6J mice, 10-12 weeks old, were obtained from the Jackson Laboratory and acclimated for 48 hours prior to the start of the study. Intravitreal injections will be performed using a Hamilton syringe connected to a Harvard apparatus pump at a flow rate of 12 μL / min. After a small incision approximately 1-2 mm posterior to the ora serrata, a 45° bevel needle (34G) will be inserted into the vitreous cavity. After sedation by isoflurane exposure, mice will be injected unilaterally (right eye only) with 2 μL of IVT at 10 μg / eye. (aflibercept) or farximab murine surrogate ("FMS") or non-related IgG (NSigG) or bispecific antibody constructs. During the experiment, mice will be sacrificed at 2h, 6h, 1d, 2d, 3d, 5d, 7d and 14d after IVT administration. At these specific time points, deeply anesthetized mice will be sacrificed by cervical dislocation. The chest cavity will be exposed and blood will be collected from the lower ventricle by cardiac puncture using a 25G needle into serum separator tubes and incubated at 4°C for 30min (BD Vacutainer TM Venous blood collection tubes, Fisher Scientific). The right eye was enucleated, the retina and choroid were dissected, snap frozen, and then stored at -80°C. Once incubated, the blood was centrifuged at 3000G for 10 min. The resulting serum was aliquoted into microtubes and then stored at -80°C.

[0260] Meso Scale Diagnostics MSD IgG Assay: PK analysis will be performed using the Human / NHP IgG Kit (Meso Scale Discovery). Frozen eyes or eye tissues will be homogenized with MSD Tris lysis buffer (MSD) in a 0.5 mL Precellys tube with ceramic beads (Bertin instruments) using a Precellys homogenizer. Samples will be centrifuged at 13,000G for 10 min. The supernatant will be collected and incubated at 4°C until the plate is run.

[0261] Thaw the serum at 4°C. Add Diluent 100 TM Buffer was used to dilute the whole eye lysate 1:10. Serum was also diluted 1:10,000 with Diluent 100 TM Preparation. To prepare the standard, 200,000 pg / mL of Stock solutions of the 1:1 or 2:1 bispecific antibody constructs will be serially diluted 4-fold to generate an 8-point standard curve. Standards will be prepared in undiluted or diluted whole eye lysate or eye tissue lysate or diluted serum lysate.

[0262] MSD Tris 5% (w / v) blocking buffer was added to a streptavidin-coated 96-well 4-spot plate and incubated at 600 rpm for 30 min on an orbital shaker. Upon completion, the plate was washed 3 times with Tris-MSD wash buffer and then 25 μL of standards, undiluted whole eye lysate, diluted whole eye lysate, and diluted serum were added. The samples were incubated at 600 rpm on an orbital shaker for 2 hours. After washing 3 times, the plate was incubated with 25 μL / well of Diluent 100 TM The plates were incubated with 1x SULPHO-TAG anti-Hu / NHP IgG antibody prepared in . The plates were incubated at 600 rpm on an orbital shaker away from direct sunlight for 2 hours. The plates were washed 3 times for the last time. 150 μL of 2x read buffer T was added to each well and then read on the MSD S-600 instrument.

[0263] You will use Discovery Workbench TM The software analyzes the electrochemiluminescent signal generated from each well. The sample concentration will be determined by a four-parameter fit logistic regression analysis based on the standard curve. Human IgG levels in the whole eye, retina, choroid, and serum were plotted and compared to PK profiles of the bispecific antibody constructs with or without relevant IgG control.

[0264] The experiment was performed with 2.5ug / eye, 5ug / eye or 10ug / eye of bispecific construct No. 3 or non-related IgG (NSigG) as a control, wherein target engagement was assessed on retinal tissue or choroidal tissue at time points 2h, 6h, 24h, 72h, 120h and 168h or 7 days for construct No. 3 and at time points 2h and 168h or 7 days for NSigG control. For choroidal tissue, the results are shown in Figure 13 (panels AC), and for retinal tissue, the results are shown in Figure 14 (panels AC). Construct No. 3 was able to engage the target by activating the Tie2 pathway in the choroid in a dose-dependent manner at 2h and 6h after IVT administration. See Figure 13 (panels A and B). Similarly, construct No. 3 was also able to engage the target by activating the Tie2 pathway in the retinal tissue in a dose-dependent manner at 2h after IVT administration. See Figure 14 (panels A and B).

[0265] Example 9: ERK pathway activation by Western blot

[0266] This study measured ERK pathway activation in HUVEC primary endothelial cells treated with the bispecific constructs of the invention compared to the natural ligand Ang1 by Western blotting.

[0267] HUVEC cells from a single donor were used in the assay. The cells were cultured in endothelial cell growth medium with supplements in a 37°C incubator at 5% CO2 and 3% oxygen. The day before the assay, the cells were plated in 96-well tissue culture plates at 80,000 cells / well. The next day, the cells were starved for 4h in unsupplemented endothelial basal medium. After cell starvation, unsupplemented endothelial basal medium containing bispecific construct No. 3 or Ang1 control was added to the cells at three different concentrations - 100, 25 and 6.25nM. The cells were then incubated for 20min in a 37°C incubator at 5% CO2 and 3% oxygen. After incubation, the medium was removed from the 96-well plate and the plate was stored in a -80°C ultra-low temperature refrigerator.

[0268] Treated HUVEC cells were lysed in 96-well plates using MSD TRIS lysis buffer. The lysis step was accomplished by adding 50 μL of lysis buffer and incubating the plate on a shaker at RT for 1 h. After cell lysis, the lysates were collected and run on SDS PAGE gels for western blot analysis. Total ERK / phospho-ERK was assessed for all samples. Lysates were run on NuPAGE TMThe gel was run on a 4%-12% gel (Cat. No. NP0321BOX). After electrophoresis, the gel was blotted onto a nitrocellulose membrane and blocked with 5% NFDM in TRIS-buffered saline for 30 min at RT. The blot was then mounted on a 5% NFDM membrane against ERK (Cat. No. Rabbit mAb 4695, Cell Signaling Technologies, Inc. and pERK (Cat. No. Rabbit mAb 3192, ) at 1:1000 at 4°C for 16 hours. After incubation, the blot was washed and then incubated with the secondary antibody - anti-rabbit antibody (Catalog No. 7074, ) at RT for 2 h. The blot was exposed to substrate solution (Catalog No. 34095, Thermo Fisher Scientific The results are Fig.12 Displayed in.

[0269] This experiment showed that for bispecific construct No. 3, activation of the downstream ERK pathway was measured in a dose-dependent manner. Fig.12 .

[0270] Example 10: In vivo efficacy of neovascularization in the OIR model

[0271] The OIR model was used to test the efficacy of the bispecific constructs of the present invention on neovascularization. Specifically, 78 mice were exposed to hyperoxia for 5 days at 7 days of age (P7). Afterwards, the mice were returned to normoxic conditions and treatment was administered via intraperitoneal (IP) injection at P12. The treatment consisted of: 20 mg / kg of (a) anti-Tie2 clone 3 (n=11); (b) aflibercept-LALA-PG (n=11); (c) construct 3 (n=10); (d) aflibercept and (e) 5 mg / kg of construct No. 3 (n=10) and (f) non-specific IgG negative control (NSIgG) (n=11). Five control mice were maintained under normoxic conditions.

[0272] Animals were killed and tissues were collected at P17, and the whole eye was processed into retinal flat mounts and stained with isolectin B4 to observe blood vessels and new blood vessel formation. In brief, the flat mounts were washed with Tris buffered saline (TBS) and blocked for 1 hour with 10% normal goat serum (NGS), 0.5% Triton X-100 (TBST) in TBS (pH 7.4) at room temperature (RT). The samples were washed with TBS and incubated with fluorescein-labeled isolectin GS-IB4 (1:100, Invitrogen) Incubate overnight at +4°C in 1% NGS diluted in 0.1% TBST. Thereafter, wash samples 3x 10 min with 1% NGS diluted in 0.1% TBST and rinse in TBS at room temperature. (1:10 000, Sigma Andrrich Counterstain for 30 minutes and wash with TBS. TM Mounting medium Flat coverslips were mounted on microscope slides. Fluorescence microscopy (Leica Thunder 3D Tissue Leica Microsystems Retinal flat mounts were imaged. Avascular area (AVA) and neovascularization (NV) were quantified from flat mounts by computational analysis using (Kuopio, Finland) using a proprietary algorithm that uses a combination of convolutional neural networks (CNNs) designed for semantic segmentation and traditional computer vision algorithms. Quantitative data were plotted, analyzed, and presented as mean ± standard deviation (SD). Statistical analysis was performed using Prism software (v9.2, GraphPad Software, Inc., USA). The ROUT method was performed to detect outliers in the data set. Animal weight data were subjected to two-way RM analysis of variance, followed by Multiple comparison test. One-way ANOVA followed by Dunnett's multiple comparison test was performed for NV and AVA regions. Differences were considered statistically significant at the p < 0.05 level.

[0273] Results: Regarding the reduction in avascular area, the following was observed.

[0274] Table 3: Comparison of reduction in avascular area

[0275]

[0276] Results: Regarding neovascularization, the following was observed:

[0277] Table 4: Comparison of reduction in neovascularization

[0278]

[0279] Compared with the control group (NSIgG), the OIR model behaved as expected, and the positive control (20 mg / kg) resulted in a significant reduction in both avascular and neovascular areas. Both avascular (Fig. 15 (panel A) and Table 3) and neovascular (Fig. 15 (panel B) and Table 4) areas were significantly reduced by construct No. 3 (20 mg / kg). In addition to these, anti-Tie2 clone No. 3 (20 mg / kg), Aflibercept-LALA-PG (20 mg / kg) and construct No. 3 (5 mg / kg) reduced the size of the avascular area (Fig. 15 (panel A) and Table 3).

[0280] Although the foregoing invention has been described in detail by way of illustration and example for purposes of clarity of understanding, the description and example should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety.

Claims

1. An isolated bispecific antibody that specifically binds to human Tie2 and human VEGF, the isolated bispecific antibody being selected from the group consisting of: (a) an IgG heavy chain comprising the amino acid sequence of SEQ ID NO: 4; and a light chain κ comprising the amino acid sequence of SEQ ID NO: 2; (b) an IgG heavy chain comprising the amino acid sequence of SEQ ID NO: 3; and a light chain κ comprising the amino acid sequence of SEQ ID NO: 2; (c) an IgG heavy chain comprising the amino acid sequence of SEQ ID NO: 1; and a light chain κ comprising the amino acid sequence of SEQ ID NO: 2; (d) an IgG heavy chain comprising the amino acid sequence of SEQ ID NO: 5; and a light chain κ comprising the amino acid sequence of SEQ ID NO: 2; (e) an IgG heavy chain comprising the amino acid sequence of SEQ ID NO: 6 and a light chain κ comprising the amino acid sequence of SEQ ID NO: 2; (f) the amino acid sequence of SEQ ID NO:7; (g) the amino acid sequence of SEQ ID NO: 8; (h) the amino acid sequence of SEQ ID NO: 9; (i) the amino acid sequence of SEQ ID NO: 10; (j) an IgG heavy chain comprising the amino acid sequence of SEQ ID NO: 19; and a light chain κ comprising the amino acid sequence of SEQ ID NO: 2; (k) an IgG heavy chain comprising the amino acid sequence of SEQ ID NO: 20; and a light chain κ comprising the amino acid sequence of SEQ ID NO: 2; (l) an IgG heavy chain comprising the amino acid sequence of SEQ ID NO: 21; and a light chain κ comprising the amino acid sequence of SEQ ID NO: 2; (m) an IgG heavy chain comprising the amino acid sequence of SEQ ID NO: 22; and a light chain κ comprising the amino acid sequence of SEQ ID NO: 2; (n) an IgG heavy chain comprising the amino acid sequence of SEQ ID NO: 23; and a light chain κ comprising the amino acid sequence of SEQ ID NO: 2; (o) an IgG heavy chain comprising the amino acid sequence of SEQ ID NO: 24; and a light chain κ comprising the amino acid sequence of SEQ ID NO: 2; (p) an IgG heavy chain comprising the amino acid sequence of SEQ ID NO: 25; and a light chain κ comprising the amino acid sequence of SEQ ID NO: 2; (q) an IgG heavy chain comprising the amino acid sequence of SEQ ID NO: 26; and a light chain κ comprising the amino acid sequence of SEQ ID NO: 2; (r) an IgG heavy chain comprising the amino acid sequence of SEQ ID NO: 27; and a light chain kappa comprising the amino acid sequence of SEQ ID NO: 2; and (s) an IgG heavy chain comprising the amino acid sequence of SEQ ID NO:28; and a light chain κ comprising the amino acid sequence of SEQ ID NO:

2.

2. An isolated nucleic acid encoding the isolated bispecific antibody of claim 1.

3. A vector comprising the isolated nucleic acid according to claim 2. A host cell comprising the vector according to claim 3 .

5. A method for producing an isolated bispecific antibody, the method comprising culturing the host cell according to claim 4 in a culture medium and isolating the obtained bispecific antibody. An immunoconjugate comprising the isolated bispecific antibody of claim 1 .

7. A fusion polypeptide comprising the isolated bispecific antibody according to claim 1.

8. A pharmaceutical composition comprising the isolated bispecific antibody of claim 1, the immunoconjugate of claim 6, or the fusion polypeptide of claim 7.

9. A method of treating a disease in a subject in need thereof, the method comprising administering to the subject the pharmaceutical composition of claim 8.

10. The method of claim 9, wherein the disease comprises an eye disease, an infectious disease, or an ischemic disease or injury.

11. The method of claim 10, wherein the infectious diseases include sepsis, dengue virus infection, tuberculosis, coronavirus infection or influenza.

12. The method of claim 10, wherein the ischemic diseases include diabetic nephropathy, acute kidney injury, chronic kidney disease, organ transplantation, or critical limb ischemia.

13. The method of claim 10, wherein the eye diseases include diabetic retinopathy (DR), diabetic macular edema (DME), non-proliferative diabetic retinopathy (nPDR), proliferative diabetic retinopathy (PDR), wet age-related macular degeneration (AMD), dry AMD, geographic atrophy (GA), retinopathy of prematurity (ROP), glaucoma, retinal vein occlusion (RVO), central RVO or branched RVO.

14. The isolated bispecific antibody of claim 1, or the immunoconjugate of claim 6, or the fusion polypeptide of claim 7, for use in treating a disease according to claims 10-13.

15. Use of the isolated bispecific antibody of claim 1, or the immunoconjugate of claim 6, or the fusion polypeptide of claim 7 for the manufacture of a medicament for treating a disease according to claims 10-13.

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