Multispecific fusion proteins that target angiogenic and inflammatory factors

Abstract

An antibody fusion protein or its antigen-binding fragment or domain is provided, comprising a binding construct capable of targeting and binding to at least one of Ang-2, the IL-6 receptor, and a member of the VEGF family. The antibody fusion protein comprises, as a binding component, an antibody or binding polypeptide against Ang-2, an antibody against IL-6R, and multiple extracellular domains of the VEGF receptor. This antibody fusion protein is useful for the treatment or control of ocular or systemic diseases, conditions, or disorders pathogenic to abnormal angiogenesis, increased vascular leakage, or inflammation.

Description

[Technical Field]

[0001] The present invention relates to multispecific fusion proteins that target angiogenic and inflammatory factors. Specifically, the present invention relates to multispecific fusion proteins that target angiopoietin-2 ("Ang-2"), specific members of the vascular endothelial growth factor family ("VEGF"), and the interleukin-6 receptor ("IL-6R"). More specifically, the present invention relates to multispecific antibody fusion proteins that target Ang-2, VEGF-A, VEGF-B, PlGF, and IL-6R. The present invention also relates to such fusion proteins, their uses, and production processes. [Background technology]

[0002] The major cellular components of the mammalian vascular system are endothelium, smooth muscle cells, and pericytes. Endothelial cells form the inner lining of all mammalian blood vessels and constitute the non-thrombus-forming interface between blood and tissue. Therefore, the proliferation of endothelial cells is a crucial component of the development of new capillaries and blood vessels, and the development of new capillaries and blood vessels is a necessary process for the growth and / or regeneration of mammalian tissues.

[0003] In recent years, various signaling molecules have been identified as playing crucial roles in angiogenesis and increased vascular permeability (vascular leakage). These signaling molecules include members of the VEGF family ("VEGF family members"), angiopoietin, ephrin, delta-like 4 ligands, and specific members of the interleukin family (such as IL-3, IL-6, IL-8, and IL-17). Secreted polypeptides belonging to the VEGF family have been shown to play a critical role in promoting endothelial cell proliferation and angiogenesis. The pathological feature of uncontrolled angiogenesis resulting from VEGF overexpression is increased vascular permeability, leading to fluid leakage and swelling into surrounding tissues. In mammals, this family consists of five related growth factors with highly conserved receptor-binding structures: vascular endothelial growth factors A-D ("VEGF-A", "VEGF-B", "VEGF-C", and "VEGF-D") and placental growth factor ("PlGF"). In this disclosure, this family of growth factors is also referred to as the VEGF family.

[0004] The cytokine interleukin-6 ("IL-6") plays a crucial role in host defense against environmental stressors such as infection and injury. Under physiological conditions, IL-6 is barely detectable, but its levels can increase more than 100,000-fold in the early stages of inflammation. However, not all IL-6 stimulation is beneficial. Dysregulated and persistent production of IL-6 is associated with the development of various autoimmune and chronic inflammatory diseases. There is evidence that unsuppressed production of IL-6 in inflammatory tissues induces overproduction of VEGF-A and VEGF-C.

[0005] The angiopoietin / Tie ligand / receptor system plays a crucial regulatory role in regulating vascular integrity and quiescence. In addition to its role in angiogenesis, it is an important regulator in many diseases, including inflammation. Key members of the angiopoietin family are Ang-1 and Ang-2. Ang-1-mediated activation of Tie-2 is necessary to maintain endothelial quiescence. The function of the agonist Ang-1 is antagonized by Ang-2, which is thought to inhibit Ang-1 / Tie-2 signaling. Ang-2 destabilizes quiescent endothelium and primes it to respond to exogenous stimuli, thereby promoting the activity of inflammatory and angiogenic cytokines. Ang-2 has been shown to enhance the angiogenic effects of VEGF, and VEGF has been shown to upregulate Ang-2 expression in endothelial cells.

[0006] VEGF family growth factors stimulate angiogenesis by acting via a family of homogeneous receptor tyrosine kinases that are present only on the surface of vascular endothelial cells (VEGF receptor 1 (VEGFR-1), also known as "flt-1," VEGF receptor 2 (VEGFR-2, also known as "KDR" in humans and "flk-1" in mice), and VEGF receptor 3 (VEGFR-3, also known as "flt-4").

[0007] VEGF-A (sometimes simply called VEGF) appears as the most important member of this family of growth factors. Human VEGF-A is expressed in various tissues as multiple homodimeric forms (121, 145, 165, 183, 189, and 206 amino acids per monomer), each form resulting from alternative splicing of a single RNA transcript.

[0008] Because VEGF promotes the proliferation and angiogenesis of vascular endothelial cells, its growth-promoting activity on vascular endothelial cells may be beneficial in treating many conditions where this activity is important, such as ulcers, vascular injuries, and myocardial infarction.

[0009] In contrast, while vascular endothelial cell proliferation is desirable under certain circumstances, endothelial cell proliferation and angiogenesis are also undesirable components of a variety of diseases and disorders, including tumor growth and metastasis, rheumatoid arthritis, psoriasis, atherosclerosis, diabetic retinopathy, postlentic fibroplasia, neovascular glaucoma, neovascular age-related macular degeneration, hemangiomas, immune rejection of transplanted corneal tissue and other tissues, and chronic inflammation. In individuals suffering from any of these disorders, it is desirable to inhibit, or at least significantly reduce, the endothelial cell proliferation activity of the aforementioned angiogenic factors.

[0010] Each of the flt-1, KDR, and flt-4 tyrosine kinase receptors possesses seven extracellular immunoglobulin-like ("Ig-like") domains available for ligand binding, a transmembrane domain that helps fix the receptor to the cell surface on which it is expressed, and an intracellular catalytic tyrosine kinase domain. Flt-1 binds to VEGF-A, VEGF-B, and PlGF. KDR binds to VEGF-A, VEGF-C, and VEGF-D. Flt-4 binds to VEGF-C and VEGF-D.

[0011] Given the roles of VEGF family growth factors in vascular endothelial cell proliferation and angiogenesis, and the roles these processes play in many different diseases and disorders, therapies targeting the regulation of these growth factors have been devised. However, anti-VEGF therapy alone has not been able to completely inhibit the progression of angiogenic diseases.

[0012] Therefore, in patients with conditions resulting from abnormal angiogenesis, pharmacological means to more completely reduce or inhibit one or more of the biological activities of these growth factors are desirable. Furthermore, pharmacological means to improve the treatment or control of conditions resulting from abnormal angiogenesis are also desirable. [Overview of the Initiative]

[0013] As used herein, the term “control” also includes mitigation, mitigation, improvement, or prevention.

[0014] Generally, the present invention provides multispecific fusion proteins or chimeric proteins, methods for producing the same, compositions comprising the same, and methods for treating or controlling at least pathological conditions in a subject (conditions characterized by abnormal angiogenesis, increased vascular permeability (vascular leakage), and inflammation). In this disclosure, “fusion proteins” may be referred to as “chimeric proteins” because they contain components of different origins.

[0015] Specifically, the multispecific fusion protein of the present invention comprises an Ang-2 binding unit, an IL-6R binding unit, and a VEGF binding unit. The Ang-2 binding unit is a polypeptide or protein capable of binding to or substantially binding to Ang-2. The IL-6R binding unit is a polypeptide or protein capable of binding to or substantially binding to IL-6R. The VEGF binding unit is a polypeptide or protein capable of binding to or substantially binding to one or more VEGF family members.

[0016] In one embodiment, such a multispecific fusion protein is a multispecific antibody fusion protein.

[0017] In another embodiment, such a multispecific antibody fusion protein is a triplespecific fusion protein.

[0018] In yet another embodiment, the present invention provides a triply specific antibody fusion protein or chimeric protein, or an antigen-binding fragment or antigen-binding domain thereof, that can substantially bind to Ang-2, IL-6R (including membrane-bound or soluble IL-6R), and one or more VEGF family members, thereby simultaneously reducing or inhibiting the signaling of Ang-2, IL-6, and VEGF family members.

[0019] In a further embodiment, the tripspecific antibody fusion protein or chimeric protein of the present invention comprises linked Ang-2, IL-6R, and VEGF binding units. Such binding units include, consist of, or essentially consist of antigen-binding domains or moieties that target Ang-2, IL-6R, and one or more VEGF family members.

[0020] In yet another embodiment, the triplicate antibody fusion protein or chimeric protein of the present invention comprises a VEGF-binding unit linked to an IL-6R-binding unit and a VEGF / IL-6R-binding unit including an Ang-2-binding unit linked to the VEGF / IL-6R-binding unit.

[0021] The present invention also provides antigen-binding fragments or domains of such fusion proteins.

[0022] In one embodiment, the IL-6R binding unit contains an IL-6R antibody.

[0023] In another embodiment, the triplicate antibody fusion protein of the present invention comprises an antibody against IL-6R linked to an Ang-2 binding unit and a VEGF binding unit. In a further embodiment, the Ang-2 binding unit comprises an antibody or bioactive polypeptide capable of binding to or substantially binding to Ang-2. In this disclosure, the term “antibody” includes full-length antibodies, single-chain F2 antibodies, and others. V Antibody (scF V This includes, but is not limited to, Fab antibodies, Fab' antibodies, (Fab')2 antibodies, single-domain antibodies (sdAbs, also known as nanobodies), minibodies, maxibodies, diabodies, and peptidebodies.

[0024] In yet another embodiment, the VEGF-binding unit contained in the triplicate antibody fusion protein comprises an Ig-like domain selected from the group consisting of Ig-like domains of one or more VEGF receptors. In one embodiment, the VEGF family members that bind to such a VEGF-binding unit include VEGF-A, VEGF-B, and PlGF. In another embodiment, such family members are VEGF-A, VEGF-B, and PlGF. Thus, in one embodiment, the antibody fusion protein of the present invention may be considered as a triplicate construct capable of binding to at least three different types of ligands involved in pathological angiogenesis and vascular leakage. In one embodiment, the VEGF-binding unit comprises multiple Ig-like domains of one or more VEGF receptors. In some embodiments, the VEGF-binding unit comprises multiple Ig-like domains of VEGF receptors 1 and 2 ("VEGFR-1" and "VEGFR-2"). In some other embodiments, the VEGF-binding unit comprises the Ig-like domain (extracellular domain) 2, or substantially the Ig-like domain 2, of VGFR-1 ("VEGFR-1-D2"), and the Ig-like domain 3, or substantially the domain 3, of VEGFR-2 ("VEGFR-2-D3"). In some other embodiments, the VEGF-binding unit comprises VEGFR-1-D2 and VEGFR-2-D3 ligated to the Fc domain of IgG1.

[0025] In yet another embodiment, IL-6R is human IL-6R, Ang-2 is human Ang-2, and VEGFR-1 and VEGFR-2 are human VEGFR-1 and VEGFR-2.

[0026] In yet another embodiment, the antibody fusion protein or chimeric protein of the present invention, or its antigen-binding fragment or domain, comprises an antibody against IL-6R linked to an Ang-2 binding unit and a VEGF binding unit, wherein the antibody against IL-6R comprises a full-length antibody or sdAb against IL-6R.

[0027] In one embodiment, the Ang-2 binding unit comprises an sdAb or a bioactive polypeptide for Ang-2.

[0028] In a further embodiment, the triplicate antibody fusion protein of the present invention comprises the Fc domain of human IgG1.

[0029] In yet another embodiment, the VEGF-binding unit includes human VEGFR-1-D2 linked to human VEGFR-2-D3.

[0030] In further embodiments, the VEGF-binding unit comprises (a) human VEGFR-1-D2, (b) human VEGFR-2-D3, and (c) the Fc domain of IgG1, with VEGFR-1-D2 and VEGFR-2-D3 linked in series. In some embodiments, the C-terminus of VEGFR-2-D3 is linked to the N-terminus of the Fc domain. In other embodiments, the C-terminus of VEGFR-1-D2 is linked to the N-terminus of the Fc domain.

[0031] In a further embodiment, the sdAb against Ang-2 or IL-6R, or its antigen-binding fragment or domain, includes the heavy chain variable region of a heavy chain antibody against Ang-2 or IL-6R. Such an sdAb can specifically bind to Ang-2 or IL-6R without requiring a complementary variable region, as is the case with conventional quadrilateral immunoglobulin molecules.

[0032] In yet another aspect, the present invention provides isolated nucleic acid molecules encoding antibody fusion proteins or chimeric proteins.

[0033] In yet another aspect, the present invention provides a vector comprising the nucleic acid molecule, the vector comprising an expression vector comprising the nucleic acid molecule operably ligated to an expression regulatory sequence. As used herein, the term “operably ligated” means that the components of a construct are arranged in a functional relationship with one another, and each component retains its function. A nucleic acid is “operably ligated” if it is arranged in a functional relationship with another nucleic acid sequence. For example, a pre-sequence or secretion leader DNA is “operably ligated” to the DNA encoding a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide. A promoter or enhancer is operably ligated to a coding sequence if it affects the transcription of the sequence. A ribosome binding site is also operably ligated to a coding sequence if it is positioned to facilitate translation.

[0034] In yet another aspect, the present invention provides a host vector system for producing the antibody fusion protein or chimeric protein, comprising an expression vector within a suitable host cell.

[0035] In another embodiment, the present invention provides a method for producing an antibody fusion protein or chimeric protein, the method comprising (a) growing cells of a host vector system under conditions that enable the production of an antibody fusion protein or chimeric protein, and (b) recovering the antibody fusion protein or chimeric protein thus produced. Such a method may further comprise the step of purifying the antibody fusion protein or chimeric protein.

[0036] In yet another aspect, the present invention provides a method for treating or controlling at least one disease, condition, or disorder in a subject whose etiology is selected from the group consisting of abnormal angiogenesis, vascular leakage, inflammation, and combinations thereof, or a composition for use in treating or controlling such a condition.

[0037] In certain embodiments, such disease, condition, or disorder is a disease, condition, or disorder of the eye. In some other embodiments, such disease, condition, or disorder is related to tumor growth and metastasis. In other embodiments, such disease, condition, or disorder is rheumatoid arthritis, psoriasis, or atherosclerosis.

[0038] Other features and advantages of the present invention will become apparent from the following detailed description and claims. [Brief explanation of the drawing]

[0039] [Figure 1] This is a schematic diagram of the first embodiment of the present invention. [Figure 2] This is a schematic diagram of a second embodiment of the present invention. [Figure 3A] This is a schematic diagram of a third embodiment of the present invention. [Figure 3B] This is a schematic diagram of a third embodiment of the present invention. [Figure 4A] This is a schematic diagram of a fourth embodiment of the present invention. [Figure 4B] This is a schematic diagram of a fourth embodiment of the present invention. [Figure 5A] This shows the purity of the antibody fusion proteins EB-105BI-7CA and EB-105BIc1 as indicated by SEC-HPLC chromatograms. [Figure 5B] This shows the purity of the antibody fusion proteins EB-105BI-7CA and EB-105BIc1 as indicated by SEC-HPLC chromatograms. [Figure 6A] This document demonstrates the ELISA binding affinity of the antibody fusion protein of the present invention to human VEGF-A165. [Figure 6B] This document demonstrates the ELISA binding affinity of the antibody fusion protein of the present invention to human VEGF-A165. [Figure 6C] This document demonstrates the ELISA binding affinity of the antibody fusion protein of the present invention to human VEGF-A165. [Figure 7]This document demonstrates the ELISA binding affinity of several antibody fusion proteins of the present invention to human VEGF-B167. [Figure 8A] This document demonstrates the ELISA binding affinity of several antibody fusion proteins of the present invention to human P1GF. [Figure 8B] This document demonstrates the ELISA binding affinity of several antibody fusion proteins of the present invention to human P1GF. [Figure 9A] This document demonstrates the ELISA binding affinity of several antibody fusion proteins of the present invention to human IL-6R. [Figure 9B] This document demonstrates the ELISA binding affinity of several antibody fusion proteins of the present invention to human IL-6R. [Figure 9C] This document demonstrates the ELISA binding affinity of several antibody fusion proteins of the present invention to human IL-6R. [Figure 10A] This document demonstrates the ELISA binding affinity of several antibody fusion proteins of the present invention to human Ang-2. [Figure 10B] This document demonstrates the ELISA binding affinity of several antibody fusion proteins of the present invention to human Ang-2. [Figure 10C] This document demonstrates the ELISA binding affinity of several antibody fusion proteins of the present invention to human Ang-2. [Figure 11] This document demonstrates the ELISA binding affinity of the antibody fusion protein, nesbakumab, and falisimab of the present invention to human Ang-1. [Figure 12A] This demonstrates the inhibition of VEGF-A165-mediated VEGFR-2 signaling by the antibody fusion protein of the present invention. [Figure 12B] This demonstrates the inhibition of VEGF-A165-mediated VEGFR-2 signaling by the antibody fusion protein of the present invention. [Figure 12C] This demonstrates the inhibition of VEGF-A165-mediated VEGFR-2 signaling by the antibody fusion protein of the present invention. [Figure 13A] This demonstrates the inhibition of the Ang-2 / Tie-2 interaction by the antibody fusion protein of the present invention, nesbakumab, and falisimab. [Figure 13B] This demonstrates the inhibition of the Ang-2 / Tie-2 interaction by the antibody fusion protein of the present invention, nesbakumab, and falisimab. [Figure 14A] This demonstrates the inhibition of the Ang-1 / Tie-2 interaction by the antibody fusion protein of the present invention and nesbacumab. [Figure 14B] This demonstrates the inhibition of the Ang-1 / Tie-2 interaction by the antibody fusion protein of the present invention and nesbacumab. [Figure 15A] This invention demonstrates the inhibition of IL-6 binding to IL-6R by the antibody fusion protein, tocilizumab, and bovalilizumab. [Figure 15B] This invention demonstrates the inhibition of IL-6 binding to IL-6R by the antibody fusion protein, tocilizumab, and bovalilizumab. [Figure 16] This study demonstrates the effect of fusion protein B21138002 on inhibiting vascular leakage from preretinal neovascularization (PRN) induced by DL-α-aminoadipic acid (DL-AAA) in Dutch belt rabbits. [Figure 17] This shows quantitative analysis of vascular leakage in DL-AAA-induced PRN in Dutch belt rabbits after intravitreal (IVT) injection of B21138002, aflibercept (Eylea), and falisimab. [Figure 18] This study demonstrates the effects of B21138002, aflibercept, and falisimab on inhibiting vascular leakage from laser-induced choroidal neovascularization (CNV) in monkeys. [Figure 19] This shows quantitative analysis of changes in grade IV CNV lesions after IVT injections of B21138002, aflibercept, and falisimab. [Figure 20] This paper presents quantitative analysis of vascular leakage in laser-induced CNV in monkeys after IVT injection of B21138002, aflibercept, and falisimab. [Modes for carrying out the invention]

[0040] In this specification, the terms “protein,” “polypeptide,” and “peptide” are used interchangeably to refer to polymers of amino acid residues.

[0041] Generally, the present invention provides a multispecific fusion protein or chimeric protein, or an antigen-binding fragment or domain thereof, that can reduce or inhibit the signaling of Ang-2, IL-6R, and one or more VEGF family members by binding to or substantially binding to Ang-2, IL-6R, and one or more VEGF family members.

[0042] In one embodiment, the multispecific fusion protein or chimeric protein is an antibody fusion protein or a chimeric protein.

[0043] In this disclosure, the term “antibody fusion protein” may be used in place of “antibody fusion protein or chimeric protein.”

[0044] In one embodiment, the antibody fusion protein or its antigen-binding fragment is a trispecific antibody fusion protein capable of reducing or inhibiting the signaling of Ang-2, IL-6, and VEGF family members by binding or substantially binding to Ang-2, IL-6R (both soluble and membrane-bound), and one or more VEGF family members.

[0045] In another embodiment, the multispecific fusion protein or chimeric protein of the present invention comprises binding units linked together, targeting Ang-2, IL-6R, and one or more VEGF family members. Such binding units include, consist of, or essentially consist of antigen-binding domains or moieties that target Ang-2, IL-6R, and one or more VEGF family members. In a further embodiment, such a multispecific fusion protein or chimeric protein is a triplespecific antibody fusion protein or chimeric protein.

[0046] In yet another embodiment, the triplicate antibody fusion protein of the present invention does not bind to Ang-1. In yet another embodiment, the triplicate antibody fusion protein of the present invention has an affinity of approximately 10 to Ang-2. -3 It has an affinity for Ang-1 that is less than twice its original value.

[0047] In another embodiment, the triplicate antibody fusion protein of the present invention comprises an antibody against IL-6R, or an antigen-binding fragment or domain thereof, linked to an Ang-2 binding unit and a VEGF binding unit.

[0048] In some embodiments, at least one of the VEGF-binding unit and the Ang-2-binding unit is directly linked to an antibody against IL-6R, or its antigen-binding fragment or domain.

[0049] In some other embodiments, one of the VEGF-binding unit and the Ang-2-binding unit is linked to an antibody against IL-6R, or its antigen-binding fragment or domain, via an intervening polypeptide. Such an intervening polypeptide may include an IgG1 Fc domain.

[0050] In a further embodiment, the Ang-2 binding unit comprises an antigen-binding fragment of an antibody against Ang-2, or a bioactive peptide that binds to or substantially binds to Ang-2.

[0051] In yet another embodiment, the VEGF-binding unit contained in the triplicate antibody fusion protein comprises an Ig-like domain selected from the group consisting of Ig-like domains of one or more VEGF receptors. The VEGF-binding unit binds or substantially binds to at least one of the VEGF family members. In one embodiment, such VEGF family members include VEGF-A, VEGF-B, and PlGF. In another embodiment, such family members are VEGF-A, VEGF-B, and PlGF. Thus, in one embodiment, the antibody fusion protein of the present invention may be considered as at least a triplicate construct capable of binding to three different types of ligands involved in pathological angiogenesis and vascular leakage. In one embodiment, the VEGF-binding unit comprises multiple Ig-like domains of one or more VEGF receptors. In some embodiments, the VEGF-binding unit comprises multiple Ig-like domains of VEGFR-1 and VEGFR-2. In some other embodiments, the VEGF-binding unit comprises VEGFR-1-D2 and VEGFR-2-D3.

[0052] In yet another embodiment, IL-6R is human IL-6R, Ang-2 is human Ang-2, and VEGFR-1 and VEGFR-2 are human VEGFR-1 and VEGFR-2.

[0053] In yet another embodiment, the antibody fusion protein or chimeric protein of the present invention, or its antigen-binding fragment or domain, comprises an antibody against IL-6R linked to an Ang-2 binding unit and a VEGF binding unit, wherein the antibody against IL-6R comprises a full-length antibody against IL-6R, its IL-6R-binding fragment, or an sdAb. In some embodiments, the IL-6R-binding antibody fragment comprises complementarity-determining regions ("CDRs") of the heavy and light chains of the IL-6R antibody. In some other embodiments, the IL-6R-binding antibody fragment comprises fewer CDRs than all of the CDRs of the heavy and light chains of the IL-6R antibody combined.

[0054] In one embodiment, the binding unit targeting Ang-2 comprises sdAb or a biologically active polypeptide against Ang-2.

[0055] In a further embodiment, the triplicate antibody fusion protein of the present invention comprises the Fc domain of human IgG1.

[0056] In yet another embodiment, the VEGF-binding unit comprises human VEGFR-1-D2 linked directly to human VEGFR-2-D3 or via a peptide linker. Such a peptide linker, if used, is preferably a short peptide linker having, for example, fewer than 20 amino acid residues. Peptide linkers are known in the art, such as peptides containing glycine, serine, and / or threonine residues. Typical peptide linkers consist of short sequences of glycine and serine residues. The amino acid sequences of human VEGFR-1-D2 and VEGFR-2-D3 are shown below as Sequence ID No. 1 and Sequence ID No. 2.

[0057] In further embodiments, the VEGF-binding unit comprises (a) human VEGFR-1-D2, (b) human VEGFR-2-D3, and (c) the Fc domain of IgG1, with VEGFR-1-D2 and VEGFR-2-D3 linked in series. In some embodiments, the C-terminus of VEGFR-2-D3 is linked to the N-terminus of the Fc domain. In other embodiments, the C-terminus of VEGFR-1-D2 is linked to the N-terminus of the Fc domain. The amino acid sequence of the Fc domain of human IgG1 is shown below as Sequence ID No. 3.

[0058] In further embodiments, an sdAb against Ang-2 or IL-6R, or its antigen-binding fragment or domain, comprises a heavy-chain variable region of a heavy-chain antibody (VHH) against Ang-2 or IL-6R. Such an sdAb can specifically bind to Ang-2 or IL-6R without requiring a complementary variable region, as is the case with conventional four-chain immunoglobulin molecules. An sdAb against Ang-2 may be referred to herein as "Ang-2 sdAb." An sdAb against IL-6R may be referred to herein as "IL-6R sdAb."

[0059] When disclosed herein, “antigen-binding fragment or domain” of an antibody means a fragment or portion of such antibody that is conjugable to or substantially conjugable to an antigen. In one embodiment, such antigen-binding fragment or domain of an antibody comprises, essentially consists of, or consists of a variable domain of the heavy chain (VH) of such antibody.

[0060] sdAbs against Ang-2 or IL-6R, as included in some embodiments of the present invention, consist of three CDRs in the antibody heavy chain, each CDR flanked by a framework domain. Such sdAbs lack the CH1 domain in the antibody heavy chain. Despite having only three CDRs, the sdAbs exhibit comparable antigen-binding affinity and other effector functions compared to conventional antibodies containing six CDRs (three in the heavy chain and three in the light chain). Single-domain antibodies are described, for example, in Bathula et al., Cancer Biotherapy and Radiopharmaceuticals, Vol.36, No.2, 109-122 (2021).

[0061] The sdAb for Ang-2 or IL-6R included in embodiments of the present invention provides approximately 1 × 10⁻¹⁶ sdAbs to their corresponding ligand (Ang-2 or IL-6R). -6 M to 1x10 -12It is capable of binding with an equilibrium dissociation constant (KD) within the range up to M. In some embodiments, the sdAb against Ang-2 or IL-6R is capable of binding to its corresponding ligand with a K -7 D ranging from about 1×10 -12 M to about 1×10 D M. In some other embodiments, the sdAb against Ang-2 or IL-6R is capable of binding to its corresponding ligand with a K -8 D ranging from about 1×10 -12 M to about 1×10 D M. In still some embodiments, the sdAb against Ang-2 or IL-6R is capable of binding to its corresponding ligand with a K -9 D ranging from about 1×10 -12 M to about 1×10 D M. First embodiment of the trispecific fusion protein of the present invention

[0062] In one aspect, the first embodiment of the trispecific fusion protein of the present invention comprises (a) an IL-6R binding unit comprising an antibody against IL-6R comprising a heavy chain and a light chain ("IL-6R antibody"), (b) a VEGF binding unit comprising VEGFR-1-D2 and VEGFR-2-D3 linked to each other in series, and (c) a polypeptide capable of binding Ang-2 ("Ang-2 binding polypeptide"), wherein the C-terminus of the VEGF binding unit is linked to the N-terminus of the light chain of the IL-6R antibody, and the C-terminus of the heavy chain of the IL-6R antibody is linked to the N-terminus of the Ang-2 binding polypeptide. See Figure 1. Alternatively, the C-terminus of the VEGF binding unit is linked to the N-terminus of the heavy chain of the IL-6R antibody. In one aspect, the C-terminus of the heavy chain of the IL-6R antibody is the C-terminus of the Fc domain of IgG1.

[0063] In another aspect, the IL-6R binding unit of the first embodiment of the trispecific fusion protein of the present invention comprises (1) CDR1, CDR2, and CDR3 of the heavy chain of the IL-6R antibody linked to at least one of CH2 and CH3 of the Fc domain of IgG1, and (2) CDR1, CDR2, and CDR3 of the light chain of the IL-6R antibody.

[0064] Movable linkers of varying lengths may be used to ligate the Ang-2 binding polypeptide to the C-terminus of the heavy chain Fc domain of the IL-6R antibody. A non-limiting example of a peptide linker is (GGGGS). x Examples include those that contain or consist of the motif (x=1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). Specific examples of such possibility linkers include GGGGSGGGGSGGGGS, GGGGSGGGGS, and GGGGGSGGGS.

[0065] The amino acid sequences disclosed or claimed herein also include conserved amino acid substitutions in these sequences, but these substitutions generally do not alter the biological activity of the protein or peptide. The most commonly occurring substitutions, in both directions, are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0066] Non-limiting examples of IL-6R antibodies include tocilizumab (e.g., U.S. Patents 10,323,095, 7,479,543, and 5,795,965), sarilumab (e.g., U.S. Patent 7,582,298), and satralizumab (e.g., U.S. Patent 8,562,991). The amino acid sequences of the heavy and light chains of tocilizumab are shown in SEQ ID NOs. 4 and 5. Further non-limiting examples of IL-6R antibodies are disclosed in U.S. Patents 8,753,634 (Apexigen) and 8,748,581 (Ablynx NV). The aforementioned patents are incorporated herein by reference to the extent of the disclosure of IL-6R antibodies.

[0067] Non-limiting examples of Ang-2-binding peptides are shown in SEQ ID NOs. 6 to 11. Other Ang-2-binding polypeptides that may be used to construct the fusion protein of the first embodiment are disclosed, for example, in U.S. Patents 7,138,370 and 7,205,275, which are incorporated herein by reference to the extent of the disclosure of Ang-2-binding polypeptides.

[0068] Nine triple-specific fusion proteins were constructed (denoted as EB-105BI-7CA, EB-105BI-7CB, EB-105BI-10C, EB-105BI-15CA, EB-105BI-15CB, EB-105BI-21C, EB-105BI-Con4C, EB-105BI-Con4-40CA, and EB-105BI-Con4-40CB). Each fusion protein comprises (a) tocilizumab (IL-6R antibody) including a heavy chain and a light chain, (b) a VEGF-binding unit containing VEGFR-1-D2 and VEGFR-2-D3 linked in series in that order, and (c) an Ang-2 binding polypeptide selected from the group consisting of SEQ ID NOs. 6 to SEQ ID NOs. 11, wherein the C-terminus of the VEGF-binding unit is ligated to the N-terminus of the tocilizumab light chain, and the C-terminus of the tocilizumab heavy chain is ligated to the N-terminus of the Ang-2 binding polypeptide.

[0069] The complete sequences of the heavy and light chains of these fusion proteins are disclosed in SEQ ID NOs: 12 to 29.

[0070] Other triplicate fusion proteins having the same binding unit may be constructed, with the C-terminus of the VEGF binding unit ligated to the N-terminus of the tocilizumab heavy chain.

[0071] Further trispecific fusion proteins of the first embodiment may be constructed, and the IL-6R antibody may include sarilumab or satralizumab.

[0072] Further trispecific fusion proteins of the first embodiment may be constructed, the IL-6R binding unit comprising (1) CDR1, CDR2, and CDR3 ("HCCDR1", "HCCDR2", and "HCCDR3") of the tocilizumab heavy chain ligated to at least one of the CH2 and CH3 of the Fc domain of IgG1 (represented as SEQ ID NOs. 63, 64, and 65, respectively), and (2) CDR1, CDR2, and CDR3 ("LCCDR1", "LCCDR2", and "LCCDR3") of the tocilizumab light chain (represented as SEQ ID NOs. 66, 67, and 68, respectively).

[0073] Further trispecific fusion proteins of the first embodiment may be constructed, the IL-6R binding unit comprising (1) HCCDR1, HCCDR2, and HCCDR3 of sarilumab or satralizumab ligated to at least one of CH2 and CH3 of the Fc domain of IgG1, and (2) LCCDR1, LCCDR2, and LCCDR3 of sarilumab or satralizumab.

[0074] Second embodiment of the triplicate fusion protein of the present invention In one embodiment, a second embodiment of the triplicate fusion protein of the present invention comprises (a) an IL-6R binding unit containing an IL-6R antibody including a heavy chain and a light chain, (b) a VEGF binding unit containing VEGFR-1-D2 and VEGFR-2-D3 linked in series, and (c) sdAb(VHH) for Ang-2 ("Ang-2 sdAb"), wherein the C-terminus of the VEGF binding unit is ligated to the N-terminus of the light chain of the IL-6R antibody, and the C-terminus of the heavy chain of the IL-6R antibody is ligated to the N-terminus of the Ang-2 sdAb. See Figure 2. Alternatively, the C-terminus of the VEGF binding unit is ligated to the N-terminus of the heavy chain of the IL-6R antibody.

[0075] In another embodiment, the IL-6R binding unit comprises (1) CDR1, CDR2, and CDR3 of the heavy chain of the IL-6R antibody ligated to at least one of the CH2 and CH3 of the Fc domain of IgG1, and (2) CDR1, CDR2, and CDR3 of the light chain of the IL-6R antibody.

[0076] One of the mobile linkers disclosed herein may be used to ligate the N-terminus of Ang-2 sdAb to the C-terminus of the Fc domain of the IL-6R antibody.

[0077] Non-limiting examples of IL-6R antibodies are disclosed herein above.

[0078] The inventors generated Ang-2 sdAbs in a search program, and the amino acid sequences of eight of them are shown in SEQ ID NOs. 30 to 37. Other Ang-2 sdAbs that may be used to construct the fusion protein of the second embodiment are disclosed, for example, in U.S. Patent No. 9,527,925 (Boehringer Ingelheim GmbH), which is incorporated herein by reference to the extent of the disclosure of Ang-2 sdAbs.

[0079] A triple-specific fusion protein of the second embodiment was constructed. Each fusion protein comprises (a) tocilizumab (IL-6R antibody) including a heavy chain and a light chain, (b) a VEGF-binding unit including VEGFR-1-D2 and VEGFR-2-D3 linked in series in that order, and (c) Ang-2 sdAb having an amino acid sequence selected from the group consisting of SEQ ID NOs. 30 to 37, wherein the C-terminus of the VEGF-binding unit is linked to the N-terminus of the light chain of tocilizumab, and the C-terminus of the heavy chain of tocilizumab is linked to the N-terminus of Ang-2 sdAb.

[0080] Two such fusion proteins (denoted as EB-105BI-c1 and EB-105BI-c2) containing Ang-2 sdAb having sequence numbers 31 and 32, respectively, were constructed.

[0081] The complete amino acid sequences of the heavy and light chains of EB-105BI-c1 and EB-105BI-c2 are shown in SEQ ID NOs. 38 to 41.

[0082] Other trispecific fusion proteins may be constructed according to the second embodiment, and the IL-6R antibody may include sarilumab or satralizumab.

[0083] Further other triple-specific fusion proteins of the second embodiment may be constructed, the IL-6R binding unit comprising (1) HCCDR1, HCCDR2, and HCCDR3 of tocilizumab ligated to at least one of CH2 and CH3 of the Fc domain of IgG1, and (2) LCCDR1, LCCDR2, and LCCDR3 of tocilizumab.

[0084] Further other triple-specific fusion proteins of the second embodiment may be constructed, the IL-6R binding unit comprising (1) HCCDR1, HCCDR2, and HCCDR3 of sarilumab or satralizumab ligated to at least one of CH2 and CH3 of the Fc domain of IgG1, and (2) LCCDR1, LCCDR2, and LCCDR3 of sarilumab or satralizumab. Third embodiment of the fusion protein of the present invention

[0085] In one embodiment, a third embodiment of the tripspecific fusion protein of the present invention comprises two fusion polypeptides, each comprising (a) an sdAb for IL-6R ("IL-6R sdAb"), (b) a VEGF-binding unit comprising VEGFR-1-D2 and VEGFR-2-D3 linked in series, (c) an Ang-2-binding polypeptide, and (d) an Fc domain of IgG1, wherein the C-terminus of the VEGF-binding unit is linked to the N-terminus of the Fc domain, the N-terminus of the VEGF-binding unit is linked to the C-terminus of IL-6R sdAb, and the N-terminus of the Ang-2-binding polypeptide is linked to the C-terminus of the Fc domain. See Figure 3A. The N-terminus of the VEGF-binding unit is linked to the C-terminus of IL-6R sdAb directly or via a mobile linker. The N-terminus of the Ang-2-binding polypeptide is linked to the C-terminus of the Fc domain directly or via a mobile linker. Possible linkers disclosed above in this specification, or other movable linkers known in the art, may be used.

[0086] In another embodiment, a third embodiment of the triplicate fusion protein of the present invention comprises two fusion polypeptides, each comprising (a) IL-6R sdAb, (b) a VEGF-binding unit comprising VEGFR-1-D2 and VEGFR-2-D3 linked in series, (c) an Ang-2-binding polypeptide, and (d) an Fc domain of IgG1, wherein the C-terminus of the VEGF-binding unit is linked to the N-terminus of the Fc domain, the N-terminus of the VEGF-binding unit is linked to the C-terminus of the Ang-2 polypeptide, and the N-terminus of IL-6R sdAb is linked to the C-terminus of the Fc domain. See Figure 3B. The N-terminus of the VEGF-binding unit is linked directly to the C-terminus of the Ang-2-binding polypeptide or via a mobile linker. The N-terminus of IL-6R sdAb is linked directly to the C-terminus of the Fc domain or via a mobile linker. A mobile linker disclosed herein may be used.

[0087] The IL-6R sdAb contained in the two fusion polypeptides may be the same or different. The Ang-2 binding polypeptides contained in the two fusion polypeptides may be the same or different.

[0088] The Ang-2 polypeptides disclosed herein (including SEQ ID NOs: 6 to 11) may be used to construct the fusion protein of the third embodiment.

[0089] The inventors generated IL-6R sdAbs in a search program, and the amino acid sequences of 15 of them are shown in SEQ ID NOs. 42 to 56. Other IL-6R sdAbs that may be used to construct the fusion protein of the third embodiment are disclosed, for example, in U.S. Patent No. 10,618,964 (assigned to Ablynx NV), No. 8,753,634 (assigned to Apexigen), and No. 8,748,581 (assigned to Ablynx NV), which are incorporated herein by reference to the extent of the disclosure of IL-6R sdAbs.

[0090] Two triplicate fusion proteins (denoted EB-105BId1 and EB-105BId3) of the third embodiment were constructed. The fusion protein comprises two fusion polypeptides, each comprising (a) an IL-6R sdAb having SEQ ID NO: 42 (contained in EB-105BId1) or SEQ ID NO: 44 (contained in EB-105BId3), (b) a VEGF-binding unit comprising VEGFR-1-D2 (SEQ ID NO: 1) and VEGFR-2-D3 (SEQ ID NO: 2) linked in series, (c) an Ang-2-binding polypeptide having SEQ ID NO: 6, and (d) an Fc domain of IgG1 (SEQ ID NO: 3), wherein the C-terminus of the VEGF-binding unit is ligated to the N-terminus of the Fc domain, the N-terminus of the VEGF-binding unit is ligated to the C-terminus of the IL-6R sdAb, and the N-terminus of the Ang-2-binding polypeptide is ligated to the C-terminus of the Fc domain. The N-terminus of the VEGF-binding unit is linked to the C-terminus of IL-6R sdAb via a mobile linker. The N-terminus of the Ang-2-binding polypeptide is linked to the C-terminus of the Fc domain via a mobile linker.

[0091] The complete amino acid sequences of the antibody fusion proteins EB-105BId1 and EB-105BId3 are shown in SEQ ID NO: 57 and SEQ ID NO: 58, respectively.

[0092] Two triplicate fusion proteins (denoted as EB-105BId2 and EB-105BId4) of the present invention were constructed. The fusion protein comprises two fusion polypeptides, each comprising (a) an IL-6R sdAb having SEQ ID NO: 42 (contained in EB-105BId2) or SEQ ID NO: 44 (contained in EB-105BId4), (b) a VEGF-binding unit comprising VEGFR-1-D2 (SEQ ID NO: 1) and VEGFR-2-D3 (SEQ ID NO: 2) linked in series, (c) an Ang-2-binding polypeptide having SEQ ID NO: 6, and (d) an Fc domain of IgG1 (SEQ ID NO: 3), wherein the C-terminus of the VEGF-binding unit is ligated to the N-terminus of the Fc domain, the N-terminus of the VEGF-binding unit is ligated to the C-terminus of the Ang-2 polypeptide, and the N-terminus of the IL-6R sdAb is ligated to the C-terminus of the Fc domain. The N-terminus of the VEGF-binding unit is linked to the C-terminus of the Ang-2-binding polypeptide via a mobile linker. The N-terminus of IL-6R sdAb is linked to the C-terminus of the Fc domain via a mobile linker.

[0093] The complete amino acid sequences of the antibody fusion proteins EB-105BId2 and EB-105BId4 are shown in SEQ ID NO: 59 and SEQ ID NO: 60, respectively.

[0094] Fourth embodiment of the fusion protein of the present invention In one embodiment, a fourth embodiment of the triplicate fusion protein of the present invention comprises two fusion polypeptides, each comprising (a) IL-6R sdAb, (b) a VEGF-binding unit comprising VEGFR-1-D2 and VEGFR-2-D3 linked in series, (c) Ang-2 sdAb, and (d) an Fc domain of IgG1, wherein the C-terminus of the VEGF-binding unit is linked to the N-terminus of the Fc domain, the N-terminus of the VEGF-binding unit is linked to the C-terminus of Ang-2 sdAb, and the N-terminus of IL-6R sdAb is linked to the C-terminus of the Fc domain. See Figure 4A. The N-terminus of the VEGF-binding unit is linked to the C-terminus of Ang-2 sdAb directly or via a mobile linker. The N-terminus of IL-6R sdAb is linked to the C-terminus of the Fc domain directly or via a mobile linker. Possible linkers disclosed above in this specification, or other movable linkers known in the art, may be used.

[0095] In another embodiment, a fourth embodiment of the triplicate fusion protein of the present invention comprises two fusion polypeptides, each comprising (a) IL-6R sdAb, (b) a VEGF-binding unit comprising VEGFR-1-D2 and VEGFR-2-D3 linked in series, (c) Ang-2 sdAb, and (d) an Fc domain of IgG1, wherein the C-terminus of the VEGF-binding unit is linked to the N-terminus of the Fc domain, the N-terminus of the VEGF-binding unit is linked to the C-terminus of IL-6R sdAb, and the N-terminus of Ang-2 sdAb is linked to the C-terminus of the Fc domain. See Figure 4B. The N-terminus of the VEGF-binding unit is linked directly to the C-terminus of IL-6R sdAb or via a mobile linker. The N-terminus of Ang-2 sdAb is linked directly to the C-terminus of the Fc domain or via a mobile linker. A mobile linker disclosed herein may be used.

[0096] The IL-6R sdAbs in the two fusion polypeptides may be the same or different. The Ang-2 sdAbs in the two fusion polypeptides may be the same or different.

[0097] The Ang-2 sdAb (including SEQ ID NOs. 30 to 37) disclosed herein may be used to construct the fusion protein of the fourth embodiment.

[0098] The IL-6R sdAbs disclosed herein (including SEQ ID NOs. 42 to 56) may be used to construct the fusion protein of the fourth embodiment.

[0099] A triple-specific fusion protein (denoted as EB-105BIe1) of the fourth embodiment was constructed. The fusion protein comprises two fusion polypeptides, each comprising (a) an IL-6R sdAb having SEQ ID NO: 42, (b) a VEGF-binding unit containing VEGFR-1-D2 (SEQ ID NO: 1) and VEGFR-2-D3 (SEQ ID NO: 2) linked in series, (c) an Ang-2 sdAb having SEQ ID NO: 31, and (d) an Fc domain of IgG1 (SEQ ID NO: 3), wherein the C-terminus of the VEGF-binding unit is linked to the N-terminus of the Fc domain, the N-terminus of the VEGF-binding unit is linked to the C-terminus of the Ang-2 sdAb, and the N-terminus of the IL-6R sdAb is linked to the C-terminus of the Fc domain. The N-terminus of the VEGF-binding unit is linked to the C-terminus of the Ang-2 sdAb via a movable linker. The N-terminus of IL-6R sdAb is linked to the C-terminus of the Fc domain via a movable linker.

[0100] The complete amino acid sequence of the antibody fusion protein EB-105BIe1 is shown in SEQ ID NO: 61.

[0101] Another triple-specific fusion protein (denoted EB-105BIe2) of the fourth embodiment was constructed. The fusion protein comprises two fusion polypeptides, each comprising (a) an IL-6R sdAb having SEQ ID NO: 42, (b) a VEGF-binding unit containing VEGFR-1-D2 (SEQ ID NO: 1) and VEGFR-2-D3 (SEQ ID NO: 2) linked in series, (c) an Ang-2 sdAb having SEQ ID NO: 31, and (d) an Fc domain of IgG1 (SEQ ID NO: 3), wherein the C-terminus of the VEGF-binding unit is linked to the N-terminus of the Fc domain, the N-terminus of the VEGF-binding unit is linked to the C-terminus of the IL-6R sdAb, and the N-terminus of the Ang-2 sdAb is linked to the C-terminus of the Fc domain. The N-terminus of the VEGF-binding unit is linked to the C-terminus of the IL-6R sdAb via a movable linker. The N-terminus of Ang-2 sdAb is linked to the C-terminus of the Fc domain via a movable linker.

[0102] The complete amino acid sequence of the antibody fusion protein EB-105BIe2 is shown in SEQ ID NO: 62.

[0103] The IL-6R binding unit contained in the triplicate antibody fusion protein of the present invention is approximately 1 × 10⁶ -6 From M to approximately 1 x 10 -12 K within the range up to M D It can be coupled to IL-6R. In some embodiments, the above K D Approximately 1 x 10 -7 From M to approximately 1 x 10 -12 In some other embodiments, the above K D Approximately 1 x 10 -8 From M to approximately 1 x 10 -12 It is within the range up to M. Furthermore, in some embodiments, the above K D Approximately 1 x 10 -9 From M to approximately 1 x 10 -12 It falls within the range of M. As a result, the antibody fusion protein controls pathological conditions caused by abnormal angiogenesis and inflammation by substantially inhibiting the biological activity of IL-6R in promoting angiogenesis and inflammation.

[0104] The Ang-2 binding unit contained in the triplicate antibody fusion protein of the present invention is approximately 1 × 10⁶ -6 From M to approximately 1 x 10 -12 K within the range up to M D It can be coupled to Ang-2. In some embodiments, the above K D Approximately 1 x 10 -7 From M to approximately 1 x 10 -12 In some other embodiments, the above K D Approximately 1 x 10 -8 From M to approximately 1 x 10 -12 It is within the range up to M. Furthermore, in some embodiments, the above K D Approximately 1 x 10 -9 From M to approximately 1 x 10 -12 It falls within the range of M. As a result, the antibody fusion protein substantially inhibits the biological activity of Ang-2 in promoting angiogenesis and increasing vascular permeability, thereby controlling pathological conditions caused by abnormal angiogenesis and vascular leakage.

[0105] The VEGF-binding unit contained in the triplicate antibody fusion protein of the present invention binds at least one of VEGF-A, VEGF-B, and PlGF to approximately 1 × 10⁻¹⁶ VEGF-A, VEGF-B, and PlGF. -6 From M to approximately 1 x 10 -12 K within the range up to M D It can be coupled with the above K. In some embodiments, the above K D Approximately 1 x 10 -7 From M to approximately 1 x 10 -12 The range is up to M. In some other embodiments, the above K D Approximately 1 x 10 -8 From M to approximately 1 x 10 -12 In some other embodiments, the above K D Approximately 1 x 10 -9 From M to approximately 1 x 10 -12 It falls within the range of M. As a result, the antibody fusion protein controls pathological conditions involving abnormal angiogenesis and vascular leakage by substantially inhibiting the biological activity of at least one of the VEGF family members in promoting angiogenesis.

[0106] In some embodiments, the present invention also provides a binding construct comprising, consisting of, or essentially consisting of, a plurality of triplicate antibody fusion proteins or chimeric proteins described herein, linked or associated with one another by covalent bonds or other forms of attachment, wherein the triplicate antibody fusion proteins of such a binding construct may be identical or different. Such binding constructs of the present invention are capable of binding with high affinity to Ang-2, IL-6R, and at least one of VEGF-A, VEGF-B, and PlGF. If the triplicate antibody fusion proteins are different, each triplicate antibody fusion protein may comprise different binding units to Ang-2, IL-6R, or VEGF family members, selected from the binding units disclosed herein.

[0107] The triplicate antibody fusion protein or conjugation construct may further contain heterologous peptides or other chemical moieties. Such additions may modify properties such as stability, solubility, toxicity, serum half-life, immunogenicity, detectability, or other characteristics.

[0108] The term "high affinity" is used in a physiological context relating to the relative affinity of a tripspecific antibody fusion protein to Ang-2, IL-6R, and the above-mentioned VEGF family members in vivo in mammals such as experimental animals, livestock, pets, or humans. In this invention, the tripspecific antibody fusion protein binding to Ang-2, IL-6R, and the above-mentioned VEGF family members has a specific affinity (typically, the equilibrium dissociation constant (K)) for their ligands in vivo. D It may have a sub-nanomolecular value of ) measured as such. For the purposes of the present invention, the triplicate antibody fusion protein of the present invention has the K of the native ligand / receptor pair D K that is approximately 1x or less, or approximately 5x or less, or approximately 10x or less, or approximately 50x or less, or approximately 100x or less, or approximately 500x or less, or approximately 1000x or less D It can then bind to its target ligand.

[0109] The triple-specific antibody fusion protein of the present invention is approximately 1 × 10⁶ -6 M to 1x10 -12 Equilibrium dissociation constant (K) within the range up to M D ) can be coupled to Ang-2, IL-6R, and at least VEGF family members. In some embodiments, K D The value is approximately 1 × 10 -7 From M to approximately 1 × 10⁻¹² M, or approximately 1 × 10⁻¹² M. -8 From M to approximately 1 x 10 -12 Up to M, or approximately 1 x 10 -9 It is within the range of M to approximately 1 × 10⁻¹² M.

[0110] In another embodiment, the triplicate antibody fusion protein may contain two or more Ang-2 binding units, VEGF binding units, and IL-6R binding units.

[0111] In one embodiment, the amino acid sequences of various non-limiting parts or embodiments of the triplicate antibody fusion protein of the present invention are listed in Table 1.

[0112] [Table 1-1] [Table 1-2]

[0113] In another embodiment, the nucleic acid sequences encoding the amino acid sequences in Table 1 are listed in Table 2.

[0114] [Table 2-1] [Table 2-2]

[0115] In a further embodiment, the triplicate antibody fusion protein or chimeric protein of the present invention comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 1 to SEQ ID NOs: 72.

[0116] In yet another embodiment, the triplicate antibody fusion protein or chimeric protein of the present invention comprises an amino acid sequence that is at least 95% identical to any one of SEQ ID NOs: 1 to SEQ ID NOs: 72.

[0117] In yet another embodiment, the present invention provides a triplicate fusion protein comprising a polypeptide having a pair of amino acid sequences selected from the group consisting of SEQ ID NOs: 12 and 13, 14 and 15, 16 and 17, 18 and 19, 20 and 21, 22 and 23, 24 and 25, 26 and 27, 28 and 29, 38 and 39, and 40 and 41.

[0118] In yet another aspect, the present invention provides a triplicate fusion protein comprising a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs. 57 to 62.

[0119] In yet another embodiment, one or more amino acid substitutions may be made in any one of the above-described amino acid sequences. Preferably, such substitutions are conservative substitutions, where one amino acid from the following groups: (1) A, G, (2) D, E, (3) N, Q, (4) R, K, (5) I, L, M, V, (6) F, Y, W, (7) S, T, and (8) C, M is substituted with another amino acid from the same group. Such substitutions are selected to substantially preserve the binding activity of the fusion protein. In one embodiment, the triplicate antibody fusion protein of the present invention having a conservative substitution is K of Ang-2, IL-6R, VEGF-A, VEGF-B, or PlGF ligand. D The value is K before such substitution. D It is less than approximately 120% of the value. Preferably, K D The value is K before such substitution. D It is less than approximately 110% of the value. More preferably, K D The value is K before such substitution.D It is less than approximately 105% of the value. More preferably, K D The value is K before such substitution. D It is less than approximately 100% of the value.

[0120] Furthermore, the amino acid sequences disclosed or claimed herein also include “conservatively modified variants” which are the result of substitutions, deletions, or additions of a single amino acid or a small number of amino acids (such as ≤5, ≤4, ≤3, ≤2, or ≤1%) in the sequence of the original peptide, polypeptide, or protein, without substantially altering the biological activity of the original peptide, polypeptide, or protein. For example, such a conservatively modified variant may retain about ≥95, ≥96, ≥97, ≥98, ≥99, or 100% of the biological activity of the original peptide, polypeptide, or protein.

[0121] Most conservative substitutions are not expected to result in fundamental changes to the properties of the Ig-like domain or other domains of the fusion polypeptide. However, when it is difficult to predict the exact effect of a substitution in advance, those skilled in the art will understand that the effect can be evaluated by routine screening assays. For example, variants of the Ig-like domain or other domains are typically prepared by site-directed mutagenesis of the nucleic acid encoding the complete fusion polypeptide, expression of the variant nucleic acid in recombinant cell culture, purification of the variant fusion polypeptide from cell culture, and detection of the ability of the variant fusion polypeptide to specifically bind to Ang-2, IL-6R, or the aforementioned VEGF ligands. Exemplary binding assays that can be employed to determine whether a particular substitution in the Ig-like domain or other domain affects the ability of the fusion polypeptide to bind to Ang-2, IL-6R, or the aforementioned VEGF family members and inhibit their activity are described in Park et al., J. Biol. Chem., 269:25646-25654 (1994).

[0122] The VEGFR-1-D2 binding unit of the fusion protein can bind to free VEGF-A, VEGF-B, and PlGF with high affinity (Davis-Smyth et al., EMBO J., 15(18):4919 (1996)). The VEGFR-2-D3 binding unit of the fusion protein can bind to free VEGF-A, VEGF-C, and VEGF-D with high affinity (Stuttfeld et al., Life, 61(9):915 (2009)). The Ang-2 and IL-6R binding units can inhibit the activation of Tie-2 by Ang-2 and the activation of IL-6R by IL-6, respectively. Therefore, the fusion protein of the present invention can substantially inhibit the angiogenesis of these growth factors to endothelial cells at the site of disease.

[0123] In yet another embodiment, the present invention provides an isolated nucleic acid molecule encoding the above-mentioned triplicate antibody fusion protein.

[0124] In a further embodiment, the present invention provides an isolated nucleic acid molecule encoding a triply specific antibody fusion protein, the isolated nucleic acid molecule comprising (a) a nucleic acid sequence encoding an IL-6R binding unit containing an IL-6R antibody, (b) a nucleic acid sequence encoding a VEGF binding unit containing VEGFR-1-D2-VEGFR-2-D3 operably linked to the nucleic acid sequence encoding the IL-6R binding unit, and (c) a nucleic acid sequence encoding an Ang-2 binding polypeptide operably linked to the nucleic acid sequence encoding the IL-6R binding unit.

[0125] In a further embodiment, the present invention provides an isolated nucleic acid molecule encoding a triplicate antibody fusion protein of the first embodiment disclosed herein.

[0126] In yet another aspect, the present invention provides an isolated nucleic acid molecule encoding the triple-specific antibody fusion protein, the isolated nucleic acid molecule comprising (a) nucleic acid sequences encoding the heavy and light chains of tocilizumab having sequences listed in SEQ ID NOs. 76 and 77; (b) a nucleic acid sequence encoding a VEGF-binding unit including VEGFR-1-D2-VEGFR-2-D3 having sequences listed in SEQ ID NOs. 73 and 74, operably ligated to the 5' end of the nucleic acid sequence encoding the light chain of tocilizumab; and (c) a nucleic acid sequence encoding an Ang-2 binding peptide having a sequence selected from the group consisting of SEQ ID NOs. 78 to 83, operably ligated to the 3' end of the nucleic acid sequence encoding the heavy chain of tocilizumab.

[0127] In a further embodiment, the present invention provides an isolated nucleic acid molecule encoding a tripspecific antibody fusion protein, the isolated nucleic acid molecule comprising (a) a nucleic acid sequence encoding an IL-6R binding unit containing an IL-6R antibody, (b) a nucleic acid sequence encoding a VEGF binding unit containing VEGFR-1-D2-VEGFR-2-D3 operably linked to the nucleic acid sequence encoding the IL-6R binding unit, and (c) a nucleic acid sequence encoding Ang-2 sdAb operably linked to the nucleic acid sequence encoding the IL-6R binding unit. In one embodiment, the 3' end of the nucleic acid sequence encoding the VEGF binding unit is linked to the 5' end of the nucleic acid sequence encoding the light chain of the IL-6R antibody, and the 5' end of the nucleic acid sequence encoding Ang-2 sdAb is linked to the 3' end of the nucleic acid sequence encoding the heavy chain of the IL-6R antibody.

[0128] In a further embodiment, the present invention provides an isolated nucleic acid molecule encoding a triplicate antibody fusion protein of the second embodiment disclosed herein.

[0129] In yet another aspect, the present invention provides an isolated nucleic acid molecule encoding the triple-specific antibody fusion protein, the isolated nucleic acid molecule comprising (a) nucleic acid sequences encoding the heavy and light chains of tocilizumab having sequences listed in SEQ ID NOs. 76 and 77, (b) a nucleic acid sequence encoding a VEGF-binding unit including VEGFR-1-D2-VEGFR-2-D3 having sequences listed in SEQ ID NOs. 73 and 74, operably ligated to the 5' end of the nucleic acid sequence encoding the light chain of tocilizumab, and (c) a nucleic acid sequence encoding Ang-2 sdAb having a sequence selected from the group consisting of SEQ ID NOs. 102 to 109, operably ligated to the 3' end of the nucleic acid sequence encoding the heavy chain of tocilizumab.

[0130] In yet another embodiment, the present invention provides an isolated nucleic acid molecule encoding a triply specific antibody fusion protein of a third embodiment, the isolated nucleic acid molecule comprising: (a) a nucleic acid sequence encoding IL-6R sdAb having a sequence selected from the group consisting of SEQ ID NOs: 114-128; (b) a nucleic acid sequence encoding a VEGF-binding unit including VEGFR-1-D2 and VEGFR-2-D3 linked in series, having sequences listed in SEQ ID NOs: 73 and 74; (c) a nucleic acid sequence encoding an Ang-2-binding polypeptide having a sequence selected from the group consisting of SEQ ID NOs: 78-83; and (d) a nucleic acid sequence encoding the Fc domain of IgG1. The 3' end of the nucleic acid sequence encoding the VEGF-binding unit is operably ligated to the N-terminus of the nucleic acid of the Fc domain; the 5' end of the nucleic acid sequence encoding the VEGF-binding unit is operably ligated to the 3' end of the nucleic acid sequence encoding IL-6R sdAb; and the 5' end of the nucleic acid sequence encoding the Ang-2-binding polypeptide is operably ligated to the 3' end of the nucleic acid sequence encoding the Fc domain.

[0131] In yet another embodiment, the present invention provides an isolated nucleic acid molecule encoding another trispecific antibody fusion protein of a third embodiment, the isolated nucleic acid molecule comprising (a) a nucleic acid sequence encoding IL-6R sdAb selected from the group consisting of SEQ ID NOs: 114-128, (b) a nucleic acid sequence encoding a VEGF-binding unit comprising VEGFR-1-D2 and VEGFR-2-D3 linked in series, having sequences listed in SEQ ID NOs: 73 and 74, (c) a nucleic acid sequence encoding an Ang-2-binding polypeptide selected from the group consisting of SEQ ID NOs: 78-83, and (d) a nucleic acid sequence encoding the Fc domain of IgG1 listed in SEQ ID NO: 75. The 3' end of the nucleic acid sequence encoding the VEGF-binding unit is operably ligated to the 5' end of the nucleic acid of the Fc domain, the 5' end of the nucleic acid sequence encoding the VEGF-binding unit is operably ligated to the 3' end of the nucleic acid sequence encoding the Ang-2-binding polypeptide, and the 5' end of the nucleic acid sequence encoding IL-6R sdAb is operably ligated to the 3' end of the nucleic acid sequence encoding the Fc domain.

[0132] In another embodiment, the present invention provides an isolated nucleic acid molecule encoding one of the two chains of a triple-specific antibody fusion protein of a fourth embodiment, the isolated nucleic acid molecule comprising (a) a nucleic acid sequence encoding IL-6R sdAb selected from the group consisting of SEQ ID NOs: 114-128, (b) a nucleic acid sequence encoding a VEGF-binding unit including VEGFR-1-D2 and VEGFR-2-D3 linked in series, having sequences listed in SEQ ID NOs: 73 and 74, (c) a nucleic acid sequence encoding Ang-2 sdAb selected from the group consisting of SEQ ID NOs: 102-109, and (d) a nucleic acid sequence encoding the Fc domain of IgG1 listed in SEQ ID NO: 75. The 3' end of the nucleic acid sequence encoding the VEGF-binding unit is operably ligated to the 5' end of the nucleic acid sequence encoding the Fc domain, the 5' end of the nucleic acid sequence encoding the VEGF-binding unit is operably ligated to the 3' end of the nucleic acid sequence encoding Ang-2 sdAb, and the 5' end of the nucleic acid sequence encoding IL-6R sdAb is operably ligated to the 3' end of the nucleic acid sequence encoding the Fc domain.

[0133] In yet another embodiment, the present invention provides an isolated nucleic acid molecule encoding one of two chains of another trispecific antibody fusion protein of a fourth embodiment, the isolated nucleic acid molecule comprising (a) a nucleic acid sequence encoding IL-6R sdAb selected from the group consisting of SEQ ID NOs: 114-128, (b) a nucleic acid sequence encoding a VEGF binding unit including VEGFR-1-D2 and VEGFR-2-D3 linked in series, having sequences listed in SEQ ID NOs: 73 and 74, (c) a nucleic acid sequence encoding Ang-2 sdAb selected from the group consisting of SEQ ID NOs: 102-109, and (d) a nucleic acid sequence encoding the Fc domain of IgG1 listed in SEQ ID NO: 75. The 3' end of the nucleic acid sequence encoding the VEGF binding unit is operably ligated to the 5' end of the nucleic acid sequence encoding the Fc domain, the 5' end of the nucleic acid sequence encoding the VEGF binding unit is operably ligated to the 3' end of the nucleic acid sequence encoding IL-6R sdAb, and the 5' end of the nucleic acid sequence encoding Ang-2 sdAb is operably ligated to the 3' end of the nucleic acid sequence encoding the Fc domain.

[0134] In another aspect, the present invention provides an isolated nucleic acid molecule encoding a tripspecific antibody fusion protein or chimeric protein of the present invention, wherein the isolated nucleic acid molecule contains a nucleic acid sequence that differs in one or more codons from the nucleic acid sequences enumerated in this disclosure as a result of degeneracy of the genetic code. Such different nucleic acid sequences are within the scope of the present invention.

[0135] In yet another aspect, the present invention provides a vector comprising any of the nucleic acid molecules disclosed herein, the vector comprising an expression vector comprising any of the nucleic acid molecules operably ligated to an expression control sequence.

[0136] In a further embodiment, the vector comprises nucleic acid sequences encoding tripspecific antibody fusion proteins listed in SEQ ID NOs. 84-101, 110-113, and 129-134.

[0137] In yet another aspect, the present invention provides a host vector system for the production of either the triplicate antibody fusion protein or the chimeric protein, the host vector system comprising an expression vector in a suitable host cell.

[0138] In one embodiment, the present invention provides the construction of a nucleic acid molecule encoding a tripspecific antibody fusion protein disclosed herein, which, upon introduction into a suitable host cell, is inserted into a vector capable of expressing the antibody fusion protein. Suitable host cells include, but are not limited to, bacterial cells, yeast cells, insect cells, and mammalian cells. Any method known to those skilled in the art for inserting DNA fragments into vectors may be used to construct an expression vector encoding a chimeric polypeptide molecule under the control of transcriptional / translational regulatory signals. These methods may include in vitro recombinant DNA and synthetic techniques, as well as in vivo recombination (genetic recombination) (see, for example, Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory; Current Protocols in Molecular Biology, Eds. Ausubel et al., Greene Publ. Assoc., Wiley-Interscience, NY).

[0139] The expression of the nucleic acid molecule encoding the antibody fusion protein of the present invention may be regulated by a second nucleic acid sequence (promoter), so that the antibody fusion protein is expressed in a host transformed with that nucleic acid molecule. For example, the expression of the antibody fusion protein described herein may be controlled by any promoter / enhancer element known in the art.

[0140] Generally, plasmid vectors containing species-derived replicons and regulatory sequences compatible with these host cells are used for connection to these hosts. The vectors typically carry replication sites as well as marking sequences that can provide phenotypic selection in transformed cells. For example, *Escherichia coli* is transformed using pBR322, a plasmid typically derived from the *Escherichia coli* species (e.g., Bolivar et al., Gene, 2:95 (1977)). Because the pBR322 plasmid contains ampicillin and tetracycline resistance genes, it provides a convenient means of identifying transformed cells. The pBR322 plasmid, or other microbial plasmids or phages, also need to contain, or be modified to contain, a promoter that can be used by the microorganism for protein expression.

[0141] The promoters most commonly used for recombinant DNA construction include β-lactamase (penicillinase) and lactose promoter systems, or tryptophan (trp) promoter systems (Goeddel et al., Nucleic Acids Res., 8:4057 (1980)). While these are the most commonly used, other microbial promoters have also been discovered and utilized. For example, the tac promoter is a synthetically constructed DNA promoter made from a combination of promoters from the trp operon and the lac operon (de Boer et al., PNAS, (1983-01-80(1):21-25 (1983)). This is commonly used for protein production in E. coli (Amann et al., Gene, 25:167 (1983)). Any of these promoters may be used in connection with the method for producing antibody fusion proteins of the present invention.

[0142] In addition to prokaryotes, eukaryotic microorganisms such as yeast cultures may also be used. Saccharomyces cerevisiae or common baker's yeast are the most commonly used eukaryotic microorganisms, but numerous other strains are generally available. Plasmid YRp7, e.g. (Stinchcomb et al., Nature, 282:39 (1979)), is commonly used for expression in Saccharomyces. Other exemplary plasmids are disclosed in U.S. Patent No. 4,615,974 and Struhl et al., PNAS, 76(3):1035 (1979). Plasmid YRp7 contains the trp1 gene, which provides a selection marker for yeast mutants lacking the ability to grow without tryptophan (e.g., ATCC No. 44,076 or RH218) (Jones, Genetics, 85:23 (1977)). The presence of trp1 lesions as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation due to growth in the absence of tryptophan.

[0143] Appropriate promoter sequences in yeast vectors include promoters for 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem., 255:2073 (1980)) or other glycolytic enzymes such as glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, and glucokinase (Romanos et al., Yeast, 8:423 (1992), Weinhandl et al., Microb. Cell Factories, 13:5 (2014)). When constructing an appropriate expression plasmid, termination sequences associated with these genes are also ligated into the expression vector at the 3' end of the sequence to be expressed to provide polyadenylation and termination of mRNA. Other promoters, such as the promoter region of alcohol dehydrogenase 2 or enzymes responsible for the utilization of maltose and galactose (Romanos et al., Weinhandl et al., cited above), which have the added advantage of transcription being regulated by growth conditions, may also be used in vector construction. Any plasmid vector containing a yeast-compatible promoter, origin of replication, and termination sequence is suitable.

[0144] In addition to microorganisms, cell cultures derived from multicellular organisms may also be used as hosts. In principle, any such cell culture can be used, whether it is derived from a vertebrate or an invertebrate. However, there is considerable interest in vertebrate cells, and in recent years, propagation of vertebrate cells in tissue culture has become commonplace. Examples of such useful host cell lines include VERO cells, HeLa cells, Chinese hamster ovary (CHO) cell lines, as well as W138, BHK, COS-7, HEK293, and MDCK cell lines. Expression vectors for such cells typically contain (if necessary) an origin of replication, a promoter located before the gene to be expressed, along with any necessary ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription termination sequences.

[0145] For use in mammalian cells, regulatory functions for expression vectors are often provided by viral material. For example, commonly used promoters are derived from polyomas, adenovirus 2, and most frequently, Simianvirus 40 (SV40). Both the early and late promoters of the SV40 virus are particularly useful because they can be readily obtained from the virus as fragments that also contain the SV40 virus origin of replication (Fiers et al., Nature, 273:113 (1978)). Smaller or larger SV40 fragments may also be used, as long as they contain a sequence of approximately 250 bp extending from the HindIII site to the BglI site located at the viral origin of replication. Furthermore, it is possible, and often desirable, to utilize promoters or regulatory sequences that are usually associated with the desired gene sequence. However, such regulatory sequences must be compatible with the host cell system.

[0146] Accordingly, according to the present invention, an expression vector reproducible in a bacterial, yeast, insect, or mammalian cell host, comprising a nucleic acid encoding an antibody fusion protein as described herein, is used to transfect the host and thereby induce the expression of such nucleic acid to produce a fusion polypeptide, which may then be recovered in a bioactive form. When used herein, the bioactive form includes at least a form capable of binding to a VEGF family member.

[0147] In some embodiments, the host cell may be Escherichia coli, COS cells, HEK293 cells (simply known as 293 cells), or Chinese hamster ovary ("CHO") cells. Preferably, the host cell is HEK293 or CHO cells.

[0148] A non-limiting example is plasmid pcDNA3.4, which is suitable for use in mammalian host cells such as CHO cells. Plasmid pcDNA3.4 contains the ampicillin resistance gene and the SV40 and CMV promoter genes. Vector construction

[0149] Standard ligation techniques are employed to construct suitable vectors containing the desired coding and control sequences. Isolated plasmids or DNA fragments are cut, processed, and ligated into the desired form to form the required plasmid. The method employed is independent of the DNA source or the target host. Cutting is performed by treatment with restriction enzymes (or multiple restriction enzymes) in a suitable buffer.

[0150] Nucleic acid sequences substantially encoding one or more Ig-like domains of VEGFR-1 or VEGFR-2 can be generated according to the method disclosed in U.S. Patent No. 6,897,294.

[0151] In the first embodiment, nucleic acid sequences substantially encoding the Ig-like domain 2 of VEGFR-1 and the Ig-like domain 3 of VEGFR-2 are tandem-linked in a desired order. This construct is then linked to the 5' end of the nucleic acid sequence encoding the light chain of the IL-6R antibody (e.g., tocilizumab). The nucleic acid sequence encoding the Ang-2 binding is linked to the 3' end of the nucleic acid sequence encoding the heavy chain of the IL-6R antibody. Such a whole set of nucleic acid sequences is called a chimeric nucleic acid sequence.

[0152] The entire chimeric nucleic acid sequence is then positioned in a vector containing a promoter within a gene reading frame and compatible with the intended host cell. In the production of the antibody fusion protein of the present invention, numerous plasmids may be used, such as those described in U.S. Patents 4,456,748, 5,460,811, 5,888,808, and 6,333,147.

[0153] In one embodiment, the vector system pcDNA3.4 is suitable for expressing the antibody fusion protein of the present invention in mammalian cells.

[0154] In one embodiment, the antibody fusion protein of the present invention may be produced according to the method described in U.S. Patent No. 7,070,959. For example, the chimeric nucleic acid sequences of SEQ ID NOs. 84 and 85 are inserted into an expression vector pcDNA3.4 having a CMV promoter.

[0155] In one embodiment, CHO cells are transfected with pcDNA3.4 / SEQ ID NOs. 84 and 85. The antibody fusion protein obtained from the CHO cells may be purified and characterized by a binding assay, as described in U.S. Patent No. 7,070,959.

[0156] Similarly, nucleic acid molecules encoding other triplicate antibody fusion proteins described above may be produced by ligating nucleic acid sequences encoding various desired ligand-binding units in a desired order and then inserting them into an expression vector pcDNA3.4. CHO cells are transfected with such vectors and grown. The antibody fusion proteins obtained from these CHO cells may also be purified and characterized.

[0157] In one embodiment, the antibody fusion protein of the present invention is derived from human Ang-2 ("hAng-2"), human IL-6R ("hIL-6R"), and a VEGF family member K D ≤10 -9 It can be bound with M. In another embodiment, the antibody fusion protein of the present invention is bound to hAng-2, hIL-6R, and VEGF family members with K D ≤ 5 × 10 -10 It can be bound with M. In yet another embodiment, the antibody fusion protein of the present invention is K to hAng-2, hIL-6R, and VEGF family members. D ≤10 -10 They can be joined using M.

[0158] In one embodiment, the present invention provides compounds, compositions, and methods for treating or controlling diseases, conditions, or disorders caused by abnormal angiogenesis and inflammation.

[0159] In another embodiment, the present invention provides a method for treating or controlling at least ocular or systemic diseases, conditions, or disorders in a subject that are pathogenic to abnormal angiogenesis and inflammation. The method comprises the step of administering a composition comprising a trispecific antibody fusion protein disclosed herein to a subject requiring such treatment or control. Non-limiting embodiments of such antibody fusion proteins have amino acid sequences listed in SEQ ID NOs: 12-29, 38-41, and 57-62.

[0160] In yet another aspect, the present invention provides a composition for use in treating or controlling at least ocular or systemic diseases, conditions, or disorders in a subject that are pathogenic to abnormal angiogenesis and inflammation, the composition comprising a trispecific antibody fusion protein disclosed herein. Non-limiting embodiments of such antibody fusion proteins have amino acid sequences listed in SEQ ID NOs: 12-29, 38-41, and 57-62.

[0161] In yet another aspect, the present invention provides the use of the tripspecific antibody fusion proteins disclosed herein in the preparation of pharmaceutical compositions or medicinal products for the treatment or control of at least ocular or systemic diseases, conditions, or disorders in a subject, which are pathogenic to abnormal angiogenesis and inflammation. Non-limiting embodiments of such antibody fusion proteins have amino acid sequences listed in SEQ ID NOs: 12-29, 38-41, and 57-62.

[0162] In yet another embodiment, the above-mentioned eye diseases, conditions, or disorders are selected from the group consisting of macular edema, uveitis, central retinal vein occlusion and branch retinal vein occlusion, choroidal neovascularization, neovascular age-related macular degeneration (exudative age-related macular degeneration), polypoid choroidal vasculopathy ("PCV"), myopic choroidal neovascularization, vascular leakage, non-proliferative and proliferative diabetic retinopathy, retinopathy of prematurity, corneal neovascularization, corneal inflammation, and neovascular glaucoma.

[0163] In one embodiment, the subject is administered a dose of approximately 25 to 4000 micrograms of the fusion protein. In another embodiment, the subject is administered a dose of approximately 50 to 8000, approximately 100 to 8000, approximately 500 to 8000, approximately 1000 to 8000, approximately 2000 to 8000, approximately 50 to 6000, approximately 50 to 5000, approximately 50 to 4000, approximately 50 to 3000, approximately 50 to 2000, or approximately 50 to 1000 micrograms of the fusion protein.

[0164] In yet another embodiment, the composition comprising the fusion protein is in the form of eye drops or intraocular injection (e.g., intravitreous, anterior chamber, periorbital, sub-Tenon's capsule, subretinal, or suprachoroidal injection). Such compositions include ophthalmic compositions. The antibody fusion protein of the present invention may also be incorporated into medical devices that can be implanted in or near lesional tissue.

[0165] In one embodiment, the present invention provides a method or composition for treating or controlling anterior segment diseases, conditions, or disorders such as corneal neovascularization, corneal inflammation, or neovascular glaucoma. The composition comprising the fusion protein may be in the form of eye drops, intrachorporeal injection, or subconjunctival injection. In another embodiment, the present invention provides a method or composition for treating or controlling posterior segment diseases, conditions, or disorders (e.g., choroidal neovascularization, neovascular age-related macular degeneration (exudative age-related macular degeneration), polypoid choroidal vasculopathy ("PCV"), myopic choroidal neovascularization, vascular leakage, macular edema due to diabetes, uveitis, central retinal vein occlusion and branch retinal vein occlusion, non-proliferative and proliferative diabetic retinopathy, retinopathy of prematurity, etc.). In this case, the composition comprising the fusion protein may be administered in the form of intravitreal injection.

[0166] In a further embodiment, the eye drops are administered to the subject at least once daily, at least once weekly, or at least once monthly until the disease, condition, or disorder is substantially treated or controlled.

[0167] In a further embodiment, the composition is administered to the subject via sustained drug release for at least one month, at least two months, at least three months, or at least six months.

[0168] In yet another embodiment, intravitreal injections, or injections into or near lesional tissue, are administered to the subject according to a regimen recommended by a physician for the particular patient. For example, injections may be administered at least once a month, at least once every two months, at least once every three months, at least once every four months, or at least once every six months until the disease, condition, or disorder is substantially treated or controlled. In one embodiment, treatment may be administered at a relatively high frequency at the outset and at a relatively low frequency after a certain period. Such periods may be determined by a physician.

[0169] The concentration of the antibody fusion protein of the present invention in such an ophthalmic composition may be in the range of about 0.1 to about 200 mg / ml (or alternatively, about 0.25 to about 200 mg / ml, or about 0.25 to about 160 mg / ml, or about 0.5 to about 100 mg / ml, or about 0.25 to about 80 mg / ml, or about 0.5 to about 200 mg / ml, or about 0.5 to about 160 mg / ml, or about 0.5 to about 100 mg / ml, or about 0.5 to about 80 mg / ml, or about 1 to about 200 mg / ml, or 1 to about 160 mg / ml, or about 0.5 to about 100 mg / ml, or about 1 to about 80 mg / ml).

[0170] In yet another embodiment, a method for preparing a composition of the present invention comprises the step of combining (a) a certain amount of the tripspecific antibody fusion protein of the present invention and (b) a physiologically acceptable carrier.

[0171] In one embodiment, such a physiologically acceptable carrier may be a sterile saline solution or a physiologically acceptable buffer. In another embodiment, such a carrier comprises a hydrophobic medium such as a pharmaceutically acceptable oil. In yet another embodiment, such a carrier comprises an emulsion of a hydrophobic material and water. In yet another embodiment, the antibody fusion protein of the present invention may associate with or ligate with a high molecular weight material to provide a long circulation time.

[0172] Physiologically acceptable buffers include, but are not limited to, phosphate buffers or Tris-HCl buffers (containing tris(hydroxymethyl)aminomethane and HCl). For example, a Tris-HCl buffer with pH 7.4 contains 3 g / l of tris(hydroxymethyl)aminomethane and 0.76 g / l of HCl. In further embodiments, the buffer is 10-fold phosphate-buffered saline ("PBS") or a 5-fold PBS solution. Non-limiting examples of buffers used in injectable compositions containing biological products include phosphoric acid, citrate, acetate, tromethamine, histidine, arginine, gluconic acid, lactic acid, tartaric acid, aspartic acid, and glutamic acid.

[0173] Depending on the situation, other buffers (pK at 25°C) a A buffer solution based on HEPES (N-{2-hydroxyethyl}peperazine-N'-{2-ethanesulfonic acid}) with a pH of 7.5 and a pH in the range of 6.8 to 8.2, at 25°C. a A buffer solution based on BES (N,N-bis{2-hydroxyethyl}2-aminoethanesulfonic acid) with a pH of 7.1 and a pH in the range of 6.4 to 7.8, pK at 25°C a MOPS (3-{N-morpholino}propanesulfonic acid) with a pH of 7.2 and a pH in the range of 6.5-7.9, pK at 25°C a TES (N-tris{hydroxymethyl}-methyl-2-aminoethanesulfonic acid) with a pH of 7.4 and a pH in the range of 6.8-8.2, pK at 25°C a MOBS (4-{N-morpholino}butanesulfonic acid) with a pH of 7.6 and a range of 6.9-8.3, pK at 25°C aDIPSO(3-(N,N-bis{2-hydroxyethyl}amino)-2-hydroxypropane) has a pH of 7.52 and is in the range of 7-8.2, at 25°C. a TAPSO (such as 2-hydroxy-3{tris(hydroxymethyl)methylamino}-1-propanesulfonic acid) with a pH of 7.61 and a pH in the range of 7 to 8.2 may also be considered suitable or desirable.

[0174] In certain embodiments, the compositions of the present invention are formulated in a buffer having an acidic pH (from about 4 to about 6.8, or alternatively, from about 5 to about 6.8, etc.). In such embodiments, the buffering capacity of the composition preferably allows the composition to rapidly reach a physiological pH after administration to a patient.

[0175] In addition to the buffer solution, the composition of the present invention may contain materials selected from the group consisting of surfactants, stabilizers, preservatives, cosolvents, humectants, emollients, chelating agents, tonicity modifiers, and antioxidants.

[0176] In one embodiment, any of these materials that may be used in the composition of the present invention are physiologically acceptable materials. In a particular embodiment, any of these substances that may be used in the composition of the present invention are ophthalmologically or systemically acceptable materials.

[0177] Suitable water-soluble preservatives include quaternary ammonium compounds such as benzalkonium chloride and various polyquaternium compounds. Each of these agents may be present in an amount of about 0.001 to about 2% by weight (preferably about 0.01 to about 0.05% by weight).

[0178] Non-exclusive examples of surfactants include, but are not limited to, nonionic surfactants such as polysorbates (e.g., polysorbate 20, polysorbate 80), 4-(1,1,3,3-tetramethylbutyl)phenol / poly(oxyethylene) polymers (such as polymers sold under the registered trademark Tyloxapol), poly(oxyethylene)-poly(oxypropylene) block copolymers, glycolic acid esters of fatty acids, and mixtures thereof.

[0179] In one embodiment, the pH of the composition is in the range of about 4 to about 8. Alternatively, the pH of the composition is in the range of about 6 to about 8, or about 6.5 to about 8, or about 6.5 to about 7.5.

[0180] In another embodiment, the composition has a pH of about 7. Alternatively, the composition has a pH in the range of about 7 to about 7.5.

[0181] In yet another embodiment, the composition has a pH of approximately 7.4.

[0182] In further embodiments, the composition may also include viscosity-modifying compounds designed to facilitate the administration of the composition to a subject or to enhance its bioavailability within the subject. In yet another embodiment, the viscosity-modifying compounds may be selected to prevent the composition from dispersing easily after it has been administered to the ocular environment. Such compounds may enhance the viscosity of the composition and may include, but are not limited to, monomeric polyols (such as glycerol, propylene glycol, and ethylene glycol), polymeric polyols (such as polyethylene glycol), various polymers of the cellulose family (such as hydroxypropyl methylcellulose ("HPMC"), carboxymethylcellulose ("CMC") sodium, hydroxypropylcellulose ("HPC")), polysaccharides (such as hyaluronic acid and its salts, chondroitin sulfate and its salts, and dextrans such as dextran 70), water-soluble proteins (such as gelatin), vinyl polymers (such as polyvinyl alcohol, polyvinylpyrrolidone, and povidone), carbomers (such as carbomer 934P, carbomer 941, carbomer 940, or carbomer 974P), and acrylic acid polymers. Generally, the desired viscosity can range from approximately 1 to approximately 400 centipoise ("cps") or mPa·s.

[0183] Non-exclusive examples of chelating agents include ethylenediaminetetraacetic acid ("EDTA"), diethylenetriaminepentakis (methylphosphonic acid), etidronic acid, and tetrasodium etidronate (also known as "HAP").

[0184] While the buffer itself is a "tonicity adjuster" and "pH adjuster" that broadly maintains ophthalmic solutions at specific ionic concentrations and pH levels, the final tonicity of the solution can be adjusted by adding additional "tonicity adjusters." Such tonicity adjusters are well known to those skilled in the art and include, but are not limited to, mannitol, sorbitol, dextrose, sucrose, urea, propylene glycol, and glycerin. Various salts containing monovalent cation halide salts (e.g., NaCl or KCl) can also be used. Typically, the tonicity of the formulations of the present invention is in the range of about 200–400 mOsm / kg. Alternatively, the tonicity of the formulations of the present invention is in the range of about 220–400 mOsm / kg, or about 220–350 mOsm / kg, or about 220–300 mOsm / kg, or about 250–350 mOsm / kg.

[0185] Non-exclusive examples of antioxidants include ascorbic acid (vitamin C) and its salts and esters, tocopherols (such as α-tocopherol) and tocotrienols (vitamin E) and their salts and esters (such as vitamin E TGPS (D-α-tocopheryl polyethylene glycol 1000 succinate)), glutathione, lipoic acid, uric acid, butylated hydroxyanisole ("BHA"), butylated hydroxytoluene ("BHT"), tertiary butylhydroquinone ("TBHQ"), and polyphenolic antioxidants (such as gallic acid, cinnamic acid, flavonoids, and their salts, esters, and derivatives).

[0186] Non-specific examples of stabilizers include sucrose, mannitol, sorbitol, and trehalose.

[0187] Please understand that the proportions of various ingredients or mixtures may be adjusted as appropriate for the situation.

[0188] In another embodiment, an appropriate amount of the antibody fusion protein of the present invention and one or more desired excipients is incorporated into a formulation for topical administration or injection into a portion of the eye (anterior or posterior segment, or vitreous humor). The injection formulation may preferably include a carrier that provides sustained release of the active ingredient (e.g., for about one week or more (or about one, two, three, four, five, or six months or more)). In certain embodiments, the antibody fusion protein of the present invention is contained in a delivery device for sustained release of the active ingredient over a long period (e.g., four, five, or six months or more). Examples of such delivery devices are described in U.S. Patents 8,399,006 and 9,417,238.

[0189] In yet another embodiment, a composition comprising the antibody fusion protein and the desired excipient of the present invention is lyophilized and substantially reconstituted with a physiologically acceptable liquid carrier immediately before administration to a subject.

[0190] In one embodiment, the compounds or compositions of the present invention may be injected with a fine-gauge needle, such as a 25-35 gauge needle. Typically, about 25 μl to about 100 μl of a composition containing about 25-4000 μg of the antibody fusion protein of the present invention is administered to a subject. In one embodiment, the antibody fusion protein has an amino acid sequence selected from the group consisting of SEQ ID NOs: 12-29, 38-41, and 57-62, as well as conservatively modified variants thereof. The concentration of such an antibody fusion protein is selected from the range disclosed above. Other antibody fusion proteins containing various Ang-2, VEGF, and IL-6R binding units disclosed herein may also be incorporated into the compositions disclosed herein.

[0191] In yet another embodiment, the antibody fusion protein of the present invention is incorporated into an ophthalmic device comprising a biodegradable material, and this device is implanted in the posterior ocular tissue of a subject to provide long-term treatment or control (e.g., for about one week or more, or for about one, two, three, four, five, or six months or more) of angiogenic disease, condition, or disorder. Such a device may be implanted in the eyeball or periocular tissue of a subject by a skilled physician. Non-limiting examples of ophthalmic implantation systems or devices for sustained release of active ingredients are disclosed in U.S. Patents 5,378,475, 5,773,019, 5,902,598, 6,001,386, 6,051,576, and 6,726,918.

[0192] In yet another embodiment, a method for treating or controlling an ophthalmic neovascular disease, condition, or disorder comprises the step of administering a composition comprising the antibody fusion protein of the present invention to a subject of interest.

[0193] In yet another embodiment, a method for treating or controlling an ophthalmic neovascular disease, condition, or disorder comprises the step of administering a composition comprising an antibody fusion protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 12-29, 38-41, and 57-62, and a conservatively modified variant thereof, to a subject requiring such treatment or control. Various other antibody fusion proteins containing Ang-2, VEGF, and IL-6R binding units disclosed herein may also be used in such methods.

[0194] In yet another embodiment, a method for treating or controlling an ophthalmic neovascular disease, condition, or disorder caused by abnormal neovascularization in the posterior part of the eye comprises the step of intravitreal injection of a composition comprising an antibody fusion protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 12-29, 38-41, and 57-62, and conservatively modified variants thereof.

[0195] In another embodiment, such diseases, conditions, or disorders are selected from the group consisting of macular edema, uveitis, central retinal vein occlusion and branch retinal vein occlusion, choroidal neovascularization (including neovascular age-related macular degeneration (exudative age-related macular degeneration), polypoid choroidal vasculopathy (PCV) and myopic choroidal neovascularization), vascular leakage, non-proliferative and proliferative diabetic retinopathy, retinopathy of prematurity, corneal neovascularization, corneal inflammation, and neovascular glaucoma.

[0196] In further embodiments, the compositions of the present invention are administered once a week, once a month, once a year, twice a year, three times a year, four times a year, or at a frequency deemed appropriate to treat or control an anterior segment inflammatory disease, condition, or disorder.

[0197] In yet another embodiment, the antibody fusion protein of the present invention may also be used to treat or control tumors, systemic inflammatory diseases or conditions, or autoimmune diseases such as arthritis. Such treatment or control may be carried out, for example, by systemic administration. Dosages and regimens for treating such diseases or conditions may be determined or recommended by a physician depending on the specific disease or condition. Example 1: Expression and purification of the fusion protein of the present invention

[0198] The fusion protein of the present invention was successfully expressed in Chinese hamster ovary ("CHO") cells. A single purification by affinity chromatography achieved a purity of over 95% for most of the produced proteins.

[0199] cDNAs encoding the amino acid sequences of the fusion proteins identified in Table 3 were synthesized, and expression vectors based on the circular pcDNA3.4 vector system were constructed for each cDNA. CHO cells were transiently transfected using the expression vectors in chemically defined culture media. The produced proteins were purified by ultrafiltration using a protein A affinity column, and then subjected to 0.2 μm sterile filtration to obtain high-purity bulk. After a single purification by affinity chromatography and analysis by size exclusion chromatography (SEC-HPLC), the purity ranged from 82.9% to 100%.

[0200] [Table 3]

[0201] Recombinant human VEGF-A 165 An ELISA assay was performed at +4°C for 16 hours using a 96-well plate coated with (4 μg / ml, 50 μl / well). After nonspecific blockage with 1% BSA at 25°C for 1 hour, serial dilutions of the test antibody ("Abs") were added to the coated wells and incubated at 25°C for 1 hour. Binding antibodies were detected using a secondary Ab (goat anti-human IgG1-Fc) conjugated with HRP, followed by OD (Oral Dissociation). 450 Submitted for reading. A series of EB-105 molecules of human VEGF-A 165 The binding affinity for these proteins is in the sub-nanomolecal scale, comparable to aflibercept, falisimab, and convercept. See Figures 6A-C. Aflibercept and convercept are two human Fc fusion proteins that bind to VEGF-A, B, and PlGF, respectively. Falicimab is a bispecific antibody that binds to VEGF-A and Ang-2. Example 3: Human VEGF-B21138001-B21138009 167 ELISA binding affinity

[0202] The same ELISA procedure was modified to analyze human VEGF-B 167This was carried out using a series of EB-105 molecules in human VEGF-B 167 The binding affinity to this substance is in the sub-nanomolecal scale, comparable to that of aflibercept and convercept. See Figure 7. Example 4: ELISA binding affinity of B21138001-B21138009 to human PlGF

[0203] A similar ELISA procedure was performed using recombinant human PlGF. The binding affinity of the EB-105 series molecules to human PlGF was in the sub-nanomolecal scale, comparable to that of aflibercept and convercept. See Figures 8A and 8B. Example 5: ELISA binding affinity to human IL-6R

[0204] The same ELISA procedure was performed with recombinant human IL-6R. The binding affinity of the series of EB-105 molecules to human IL-6R was in the sub-nanomolecal scale, comparable to tocilizumab and bovalilizumab. Tocilizumab is a clinical-stage mAb against IL-6R. Bovalilizumab is a single-domain antibody against IL-6R. B781402 and B781405 are bispecific fusion antibodies that inhibit VEGF-A, B, PlGF, and IL-6R. See Figures 9A-C. Example 6: ELISA binding affinity to human Ang-2

[0205] The same ELISA procedure was performed with recombinant human Ang-2. The binding affinity of the series of EB-105 molecules to human Ang-2 was in the sub-nanomolar scale, comparable to that of nesbukumab and falisimab. Nevasukumab is an mAb for human Ang-2. Falicimab is a clinical-stage bispecific antibody for inhibiting VEGF-A and Ang-2. See Figures 10A-C. Example 7: ELISA binding affinity of B21138001, B21138003, B21138004, B21138006, B21138008, and B21138009 to human Ang-1

[0206] The same ELISA procedure was performed using recombinant human Ang-1. B21138001 showed very low binding affinity for human Ang-1. The affinities of B21138003 and B21138006 for human Ang-1 are equivalent to that of nesvacumab, but slightly higher than that of faricimab. See Figure 11. Example 8: SPR Biacore Binding Affinity Assay

[0207] The SPR Biacore assay was performed at 25°C in HBS-EP+ as running buffer. This assay was carried out by immobilizing an anti-human IgG (Fc) antibody onto the surface of a CM5 sensor chip, and the level of ligand immobilization was determined. The amount of anti-Fc antibody bound to the CM5 sensor chip was approximately 7,000 to 14,000 response units (RU). The test antibody was injected over the surface of a Series S sensor chip CM5 for capture. The analyte (target protein) was diluted to different concentrations in running buffer and injected over the sensor surface for the association phase to determine affinity and kinetics. Binding affinity and kinetics were measured using a 1:1 binding model.

[0208] The SPR Biacore assay shows that the EB-105 molecules bind to the target protein on the picomolar or subnanomolar scale (Tables 4 and 5). B21138001 and B21138002 bind human VEGF-A 165 , VEGF-B 167 , and PlGF with high affinity, and their binding affinities are equivalent to that of aflibercept. They also bind human IL-6R and Ang-2, and their binding affinities are equivalent to those of tocilizumab and faricimab (Tables 4 and 5). Tocilizumab is a clinical-stage mAb targeting human IL-6R. Faricimab is the first bispecific antibody approved by the FDA that binds VEGF-A and Ang-2 for the treatment of DME.

[0209] [Table 4]

[0210]

Table 5

[0211] Example 9: VEGF-A 165 Inhibition of Mediated VEGFR-2 Signaling

[0212] An engineered HEK-293 cell line, in which the firefly luciferase gene is expressed under the control of nuclear factor of activated T-cell response element (VEGFR-2-NF-AT), was used to investigate the effect of EB-105 molecules on VEGF-A 165 mediated inhibition of VEGFR-2 signaling. Aflibercept and faricimab were used as two comparators. The results show that the EB-105 molecule is comparable to aflibercept and faricimab in dose-dependent inhibition of VEGF-A 165 stimulated VEGFR-2 signaling. See FIGS. 12A to 12C.

[0213] Serial dilutions of EB-105 molecule, aflibercept and faricimab were mixed with 60 ng / ml of human VEGF-A 165 and incubated at room temperature for 30 minutes, after which VEGFR-2 luciferase reporter cells were added to each well to study the effect of the test substance on the inhibition of VEGF-A 165 mediated VEGFR-2 signaling. The EB-105 molecule exhibits an IC 50 50 value as indicated, and is comparable to aflibercept and faricimab in a dose-response manner in the inhibition of VEGF-A 165 stimulated VEGFR-2 signaling. Aflibercept is a human Fc fusion protein comprising VEGFR-1-D2 and VEGFR-2-D3. Faricimab is a bispecific antibody that binds to VEGF-A and Ang-2. Example 10: Inhibition of Ang-2 / Tie-2 Interaction

[0214] The effect of the EB-105 molecule on inhibiting Ang-2 / Tie-2 interaction was studied using fluorescence-activated cell sorting (FACS) analysis with engineered HEK-293 cells overexpressing the human Tie-2 receptor, comparing it to falisimab and nesvaskumab. Nesvaskumab is an mAb for Ang-2. Serial dilutions of EB-105, falisimab, and nesvaskumab were incubated with 100 ng / ml human Ang-2 at 4°C for 60 minutes. Tie-2-expressing cells were then added to each well, and the effect of the test antibodies on inhibiting Ang-2 binding to the Tie-2 receptor was studied by FACS analysis. The results show that the EB-105 molecule exhibited a dose-response similar effect to nesvaskumab and was more potent than falisimab in inhibiting Ang-2 binding to the Tie-2 receptor. See Figures 13A and 13B. Example 11: Inhibition of Ang-1 / Tie-2 interaction

[0215] The effect of the EB-105 molecule on Ang-1 / Tie-2 interactions was studied using FACS analysis with engineered HEK-293 cells overexpressing the human Tie-2 (hTie-2) receptor. Falicimab and nesvasukumab were used as two control groups. Serial dilutions of EB-105, falisimab, nesvasukumab, and hTie-2 were incubated with 2 μg / ml human Ang-1-Fc at 4°C for 60 minutes. Subsequently, Tie-2-expressing cells were added to each well to study the effect of each test substance on inhibiting Ang-1 binding to the Tie-2 receptor. Recombinant hTie-2 was used as a positive control. Similar to falisimab and nesvasukumb, most EB-105 molecules, including B21138001, B21138002, B21138003, B21138006, and B21138007, do not affect the binding of Ang-1 to the Tie-2 receptor. B21138004, B21138008, and B21138009 showed some effect on inhibiting Ang-1 binding to the Tie-2 receptor. See Figures 14A and 14B. Example 12: Inhibition of IL-6 binding to IL-6R

[0216] Using engineered CHO-S cells overexpressing human IL-6R, the effect of the EB-105 molecule on inhibiting IL-6 binding to IL-6R was studied by FACS analysis, with tocilizumab and bovalilizumab used as comparison targets. Bovalilizumab is a single-domain antibody (also called a nanobody) against human IL-6R. Serial dilutions of EB-105, tocilizumab, and bovalilizumab were incubated with biotinylated human IL-6 at 4°C for 60 minutes. Subsequently, IL-6R-expressing CHO-S cells were added to each well to study the effect of each test substance on inhibiting biotinylated IL-6 binding to IL-6R. The results demonstrate that the EB-105 molecule is equivalent to tocilizumab and bovalilizumab in inhibiting IL-6 binding to IL-6R in a dose-response manner. See Figures 15A and 15B. Example 13: Interspecies activity of the lead molecule of EB-105 (B21138002) against target proteins in humans, monkeys, rabbits, and rats.

[0217] Enzyme-linked immunosorbent assay (ELISA) was performed to evaluate the interspecies binding affinity of lead molecule B21138002 to VEGF-A, IL-6R, and Ang-2 in humans, rhesus monkeys, rabbits, and rats. B21138002 was found to be the lead molecule of VEGF-A in humans, rhesus monkeys, and rabbits. 165 It strongly binds to Ang-2 and IL-6R at the sub-nanomole level. B21138002 is rat VEGF-A 165 It binds to Ang-2 but not to IL-6R. See Table 6.

[0218] [Table 6]

[0219] As described below, the lead antibody fusion protein B21138002 of the present invention was formulated in a phosphate-buffered solution for intravitreous (IVT) injection, and its efficacy and exploratory safety were studied in two animal models: preretinal neovascularization (PRN) in Dutch belt rabbits and laser-induced choroidal neovascularization (CNV) in cynomolgus monkeys. Example 14: Effect of a single IVT injection of B21138002 on vascular leakage from PRN in rabbits

[0220] PRN was induced in Dutch belt rabbits by IVT injection of DL-α-aminoadipic acid (DL-AAA, 80 mM, 50 μl / eye). After confirming the development of PRN 8 weeks after DL-AAA injection (indicated by the arrows in the top row / first row of the synthetic fundus fluorescence angiography image in Figure 15), each eye was treated with 50 μl of vehicle and equimolar amounts of aflibercept (EYLEA). (R) , 0.28mg / eye), faricimab (VABYSMO (R) Patients received a single IVT injection of either aflibercept (0.36 mg / eye) or B21138002 (0.5 mg / eye) to study their effects on inhibiting vascular leakage from pre-existing PRNs. Fundus fluorescein angiography (FFA) was performed to monitor changes in vascular leakage over time. As demonstrated, B21138002 effectively inhibited vascular leakage, and its apparent efficacy was comparable to that of aflibercept and falisimab (Figures 16 and 17). Aflibercept and falisimab are FDA-approved treatments for DME.

[0221] Sustained vascular leakage in the vehicle treatment group at 7 and 14 days after IVT injection (n=12). Eyes treated with aflibercept (n=12), faricimab (n=6), or B21138002 (n=6) showed highly effective inhibition of vascular leakage at 7 and 14 days after treatment. The large arrow in FIG. 16 indicates extensive leakage from a well-developed PRN, while the small arrow indicates residual fluorescein leakage from one region of an eye treated with faricimab. FIG. 17 shows the results of quantitative analysis of vascular leakage area at 7 and 14 days after IVT injection of vehicle and test substances. Aflibercept, faricimab, and B21138002 effectively inhibited vascular leakage at 7 and 14 days after IVT injection with a p-value < 0.01 compared with pre-dose leakage.

[0222] Example 15: Effect of a single IVT injection of B21138002 on vascular leakage in laser-induced choroidal neovascularization (CNV) in monkeys

[0223] The effect of B21138002 on inhibiting vascular leakage in laser-induced CNVs in cynomolgus monkeys was studied. Aflibercept and falisimab were used as two clinical comparison groups. After 12 days of intensive laser photocoagulation, CNV development was confirmed by FFA. Two days later (14 days post-laser photocoagulation), each eye received a single IVT injection of 50 μl vehicle, 0.5 mg / eye of aflibercept, falisimab, or B21138002. Color fundus photography and FFA were performed at 1, 2, and 4 weeks post-treatment to monitor changes in fundus and vascular leakage. Similar to findings in the DL-AAA-induced PRN rabbit study, B21138002 effectively inhibited leakage from CNV lesions, and its apparent efficacy was comparable to that of aflibercept and falisimab (Figures 18-20). Persistent vascular leakage from CNV lesions in the vehicle-treated group before administration and at 1, 2, and 4 weeks post-treatment (n=6). Eyes treated with aflibercept, falisimab, or B21138002 (n=3 / group) showed near-complete inhibition of vascular leakage at 1, 2, and 4 weeks post-treatment. Fundus photographs showed no apparent signs of retinal inflammation up to 4 weeks after IVT injection of the test substance (including vehicle, aflibercept, falisimab, and B21138002) (Figure 18, last row of fundus images).

[0224] Figure 19 shows that the proportion of grade IV CNV lesions in the vehicle treatment group showed a slight decreasing trend over time, but this change was not statistically significant. (R) ) dramatically reduced the number of grade IV CNV lesions. Falicimab and B21138002 completely inhibited the development of grade IV CNV lesions at 1, 2, and 4 weeks post-treatment. (CNV lesion grades: Grade I: no hyperfluorescence, Grade II: hyperfluorescence staining with no fluorescein leakage, Grade III: early hyperfluorescence with mild fluorescein leakage localized to the laser burn boundary in late FFA, Grade IV: early hyperfluorescence with severe fluorescein leakage beyond the laser burn boundary in late. Grade IV lesions are considered to be of extremely high clinical relevance.)

[0225] Figure 20 shows the results of quantitative analysis of vascular leakage in laser-induced CNV in monkeys. The vehicle-treated group showed relatively sustained leakage from CNV lesions before administration, and at 1, 2, and 4 weeks after treatment. B21138002 dramatically inhibited CNV leakage, and its efficacy was comparable to aflibercept (Eylea (R) ) and faricimab at 1, 2, and 4 weeks after treatment.

[0226] Nucleic acid and amino acid sequence listing

[0227] SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTII(SEQ ID NO: 1)

[0228] DVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK(SEQ ID NO: 2)

[0229] VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP(SEQ ID NO: 3)

[0230] QVQLQESGPGLVRPSQTLSLTCTVSGYSITSDHAWSWVRQPPGRGLEWIGYISYSGITTYNPSLKSRVTMLRDTSKNQFSLRLSSVTAADTAVYYCARSLARTTAMDYWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP(SEQ ID NO:4)

[0231] DIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:5)

[0232] AQTNFMPMDDLEQRLYEQFILQQGLE(SEQ ID NO:6)

[0233] MGAQQKFQPLDELEQTLYEQFMLQQALE(SEQ ID NO:7)

[0234] MGAQQKYQPLDELDKTLYDQFMLQQGLE(SEQ ID NO:8)

[0235] MGAQHTFQPLDELEETLYYQWLYDQLLE(SEQ ID NO:9)

[0236] AQQEECEWDPWTCEHMLE(SEQ ID NO:10)

[0237] AQTNIQEECEWDPWTCDHMPGKLE(SEQ ID NO:11)

[0238] QVQLQESGPGLVRPSQTLSLTCTVSGYSITSDHAWSWVRQPPGRGLEWIGYISYSGITTYNPSLKSRVTMLRDTSKNQFSLRLSSVTAADTAVYYCARSLARTTAMDYWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGGSGGGGSAQTNFMPMDDLEQRLYEQFILQQGLE(SEQ ID NO:12)

[0239] SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:13)

[0240] QVQLQESGPGLVRPSQTLSLTCTVSGYSITSDHAWSWVRQPPGRGLEWIGYISYSGITTYNPSLKSRVTMLRDTSKNQFSLRLSSVTAADTAVYYCARSLARTTAMDYWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGGSAQTNFMPMDDLEQRLYEQFILQQGLE(SEQ ID NO:14)

[0241] SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:15)

[0242] QVQLQESGPGLVRPSQTLSLTCTVSGYSITSDHAWSWVRQPPGRGLEWIGYISYSGITTYNPSLKSRVTMLRDTSKNQFSLRLSSVTAADTAVYYCARSLARTTAMDYWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGGSGGGGSMGAQQKFQPLDELEQTLYEQFMLQQALE(SEQ ID NO:16)

[0243] SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:17)

[0244] QVQLQESGPGLVRPSQTLSLTCTVSGYSITSDHAWSWVRQPPGRGLEWIGYISYSGITTYNPSLKSRVTMLRDTSKNQFSLRLSSVTAADTAVYYCARSLARTTAMDYWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGGSGGGGSMGAQQKYQPLDELDKTLYDQFMLQQGLE(SEQ ID NO:18)

[0245] SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:19)

[0246] QVQLQESGPGLVRPSQTLSLTCTVSGYSITSDHAWSWVRQPPGRGLEWIGYISYSGITTYNPSLKSRVTMLRDTSKNQFSLRLSSVTAADTAVYYCARSLARTTAMDYWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGGSMGAQQKYQPLDELDKTLYDQFMLQQGLE(SEQ ID NO:20)

[0247] SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:21)

[0248] QVQLQESGPGLVRPSQTLSLTCTVSGYSITSDHAWSWVRQPPGRGLEWIGYISYSGITTYNPSLKSRVTMLRDTSKNQFSLRLSSVTAADTAVYYCARSLARTTAMDYWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGGSGGGGSMGAQHTFQPLDELEETLYYQWLYDQLLE(SEQ ID NO:22)

[0249] SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:23)

[0250] QVQLQESGPGLVRPSQTLSLTCTVSGYSITSDHAWSWVRQPPGRGLEWIGYISYSGITTYNPSLKSRVTMLRDTSKNQFSLRLSSVTAADTAVYYCARSLARTTAMDYWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGGSGGGGSAQQEECEWDPWTCEHMLE(SEQ ID NO:24)

[0251] SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:25)

[0252] QVQLQESGPGLVRPSQTLSLTCTVSGYSITSDHAWSWVRQPPGRGLEWIGYISYSGITTYNPSLKSRVTMLRDTSKNQFSLRLSSVTAADTAVYYCARSLARTTAMDYWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGGSGGGGSAQTNIQEECEWDPWTCDHMPGKLE(SEQ ID NO:26)

[0253] SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:27)

[0254] QVQLQESGPGLVRPSQTLSLTCTVSGYSITSDHAWSWVRQPPGRGLEWIGYISYSGITTYNPSLKSRVTMLRDTSKNQFSLRLSSVTAADTAVYYCARSLARTTAMDYWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGGSAQTNIQEECEWDPWTCDHMPGKLE(SEQ ID NO:28)

[0255] SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:29)

[0256] EVQLVESGGGLVQPGGSLRLSCAASGRTFSSYDFVWVRQAPGKGLEFVSAISVEGEHTYYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCNAVKWSDYGPDNDYWGQGTLVTVSS(SEQ ID NO:30)

[0257] EVQLVESGGGLVQPGGSLRLSCAASGLYFSSYDIAWVRQAPGKGLEFVSAIDVSGEHTYYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCNAVKWSDYGPDNDYWGQGTLVTVSS(SEQ ID NO:31)

[0258] EVQLQESGGGLVQPGGSLRLSCAASGFTLDAYAIGWFRQAPGKEREGVSTIGKSDGSTCYADSVKGRFTISRDNAKNTVWLQMNSLRAEDTAVYYCAARPWWVGDAPSIAAEYEYDLWGQGTQVTVSS(SEQ ID NO:32)

[0259] EVQLQESGGGLVQPGGSLRLSCAASGFTLDEYAIGWFRQAPGKEREGVSCIGKADGSTCYADSVKGRFTISRDNAKNTVWLQMNSLRAEDTAVYYCAARPWWVGDPPSIGAEYEYDLWGQGTQVTVSS(SEQ ID NO:33)

[0260] QVQLVESGGGLVQPGGSLRLSCAASGFRFSSYAMSWVRQAPGKGLEWVSKINSGGGITYYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCRDEGPFGSWGQGTQVTVSS(SEQ ID NO:34)

[0261] QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSDINSGGSETYYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCRDKGPFGSWGQGTQVTVSS(SEQ ID NO:35)

[0262] EVQLVESGGGLVQPGGSLRLSCAASGRTFSSYDIIWVRQAPGKGLEFVSAISTSGEHTYYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCNAVKWSDYGPQNDYWGQGTLVTVSS(SEQ ID NO:36)

[0263] EVQLQESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSVIGKSDGSTCYADSVKGRFTISRDNAKNTVWLQMNSLRAEDTAVYYCAARPWWVGDRPSIAAEYEYDLWGQGTQVTVSS(SEQ ID NO:37)

[0264] MHSSALLCCLVLLTGVRAQVQLQESGPGLVRPSQTLSLTCTVSGYSITSDHAWSWVRQPPGRGLEWIGYISYSGITTYNPSLKSRVTMLRDTSKNQFSLRLSSVTAADTAVYYCARSLARTTAMDYWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGLYFSSYDIAWVRQAPGKGLEFVSAIDVSGEHTYYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCNAVKWSDYGPDNDYWGQGTLVTVSS(SEQ ID NO:38)

[0265] MHSSALLCCLVLLTGVRASDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:39)

[0266] MHSSALLCCLVLLTGVRAQVQLQESGPGLVRPSQTLSLTCTVSGYSITSDHAWSWVRQPPGRGLEWIGYISYSGITTYNPSLKSRVTMLRDTSKNQFSLRLSSVTAADTAVYYCARSLARTTAMDYWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGGSEVQLQESGGGLVQPGGSLRLSCAASGFTLDAYAIGWFRQAPGKEREGVSTIGKSDGSTCYADSVKGRFTISRDNAKNTVWLQMNSLRAEDTAVYYCAARPWWVGDAPSIAAEYEYDLWGQGTQVTVSS(SEQ ID NO:40)

[0267] MHSSALLCCLVLLTGVRASDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:41)

[0268] EVQLVESGGGLVQAGGSLRLSCAASGDDLSSVDMGWYRQAPGKGLEFVGVISRGGGSTYYVDSVQGRFTISRDNAKNTVYLQMNSLRAEDTAVYYCKTEIPYFDDYWGQGTLVTVSS(SEQ ID NO:42)

[0269] EVQLVESGGGLVQAGGSLRLSCAASGESLSSVDMGWYRQAPGKGLEFVGVISRGGESTYYVDSVQGRFTISRDNAKNTVYLQMNSLRAEDTAVYYCKTEIPFGDDYWGQGTLVTVSS(SEQ ID NO:43)

[0270] EVQLVESGGGLVQAGGSLRLSCAASGGDLSSVDMGWYRQAPGKGLEFVGVISRTGGSTYYVDSVQGRFTISRDNAKNTVYLQMNSLRAEDTAVYYCKTEIPGGDDYWGQGTLVTVSS(SEQ ID NO:44)

[0271] EVQLVESGGGLVQAGGSLRLSCAASGESLSSVDMGWYRQAPGKGLEFVGVISRTGGSTYYVDSVQGRFTISRDNAKNTVYLQMNSLRAEDTAVYYCKTEIPGGDDVWGQGTLVTVSS(SEQ ID NO:45)

[0272] EVQLVESGGGLVQPGGSLRLSCAASGGTFIDLDMGWYRQAPGNERQFVGVISRTGGSTYYADSVQGRFTISRDNAKNTLYLQMNSLRAEDTATYYCKTEIPGGDDYWGQGTLVTVSS(SEQ ID NO:46)

[0273] EVQLVESGGGLVQPGGSLRLSCAASGGTFHLYDMGWYRQAPGNERQFVGVISRGGGSTYYADSVQGRFTISRDNAKNTLYLQMNSLRAEDTATYYCKTEIPGFQDYWGQGTLVTVSS(SEQ ID NO:47)

[0274] EVQLVESGGGLVQPGGSLRLSCAASGPEFIDYDMGWYRQAPGNERQFVGVISRGGGSTYYADSVQGRFTISRDNAKNTLYLQMNSLRAEDTATYYCKTEIPFFDDYWGQGTLVTVSS(SEQ ID NO:48)

[0275] EVQLVESGGGLVQPGGSLRLSCAASGGTFIDLDMGWYRQAPGNERQFVGVISRGGGSVYYADSVQGRFTISRDNAKNTLYLQMNSLRAEDTATYYCKTEIPGGPDYWGQGTLVTVSS(SEQ ID NO:49)

[0276] EVQLVESGGGLVQPGGSLRLSCAASGGTFIDYDMGWYRQAPGNERQFVGVISRGGGAVYYADSVQGRFTISRDNAKNTLYLQMNSLRAEDTATYYCKTEIPFFDDYWGQGTLVTVSS(SEQ ID NO:50)

[0277] QVQLVESGGGLVQPGGSLRLSCAASGPTFSNLDMGWYRQAPGKGLELVGVISRTGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCKTEVPFSDDYWGQGTLVTVSS(SEQ ID NO:51)

[0278] QVQLVESGGGLVQPGGSLRLSCAASVPTFGDLDMGWYRQAPGKGLELVGVISRTGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCKTEVPFGDDYWGQGTLVTVSS(SEQ ID NO:52)

[0279] QVQLVESGGGLVQPGGSLRLSCAASGDTFSNLDMGWYRQAPGKGLELVGVISRTGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCKTEVPWADDYWGQGTLVTVSS(SEQ ID NO:53)

[0280] QVQLVESGGGLVQPGGSLRLSCAASVPTFSDVDMGWYRQAPGKGLELVGVISRTGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCKTEVPFADDYWGQGTLVTVSS(SEQ ID NO:54)

[0281] QVQLVESGGGLVQPGGSLRLSCAASVPWFSNLDMGWYRQAPGKGLELVGVISRTGGMTYYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCKTEVPGSDDYWGQGTLVTVSS(SEQ ID NO:55)

[0282] QVQLVESGGGLVQPGGSLRLSCAASGATFSNLDMGWYRQAPGKGLELVGVISRTGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCKTEVPFDDDYWGQGTLVTVSS(SEQ ID NO:56)

[0283] MHSSALLCCLVLLTGVRAEVQLVESGGGLVQAGGSLRLSCAASGDDLSSVDMGWYRQAPGKGLEFVGVISRGGGSTYYVDSVQGRFTISRDNAKNTVYLQMNSLRAEDTAVYYCKTEIPYFDDYWGQGTLVTVSSGGSGGGSGGGGSGGGGGSGGGGSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSAQTNFMPMDDLEQRLYEQFILQQGLE(SEQ ID NO:57)

[0284] MHSSALLCCLVLLTGVRAEVQLVESGGGLVQAGGSLRLSCAASGGDLSSVDMGWYRQAPGKGLEFVGVISRTGGSTYYVDSVQGRFTISRDNAKNTVYLQMNSLRAEDTAVYYCKTEIPGGDDYWGQGTLVTVSSGGSGGGSGGGGSGGGGGSGGGGSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSAQTNFMPMDDLEQRLYEQFILQQGLE(SEQ ID NO:58)

[0285] MHSSALLCCLVLLTGVRAAQTNFMPMDDLEQRLYEQFILQQGLEGGGGSGGGGSGGGGSSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGDDLSSVDMGWYRQAPGKGLEFVGVISRGGGSTYYVDSVQGRFTISRDNAKNTVYLQMNSLRAEDTAVYYCKTEIPYFDDYWGQGTLVTVSS(SEQ ID NO:59)

[0286] MHSSALLCCLVLLTGVRAAQTNFMPMDDLEQRLYEQFILQQGLEGGGGSGGGGSGGGGSSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGGDLSSVDMGWYRQAPGKGLEFVGVISRTGGSTYYVDSVQGRFTISRDNAKNTVYLQMNSLRAEDTAVYYCKTEIPGGDDYWGQGTLVTVSS(SEQ ID NO:60)

[0287] (SEQ ID NO:61)

[0288] MHSSALLCCLVLLTGVRAEVQLVESGGGLVQAGGSLRLSCAASGDDLSSVDMGWYRQAPGKGLEFVGVISRGGGSTYYVDSVQGRFTISRDNAKNTVYLQMNSLRAEDTAVYYCKTEIPYFDDYWGQGTLVTVSSGGGGSGGGGSGGGGSSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGLYFSSYDIAWVRQAPGKGLEFVSAIDVSGEHTYYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCNAVKWSDYGPDNDYWGQGTLVTVSS(SEQ ID NO:62)

[0289] · SDHAWS(SEQ ID NO:63)

[0290] YISYSGITTYNPSLKS(SEQ ID NO:64)

[0291] ARTTAMDY(SEQ ID NO:65)

[0292] RASQDISSYLN(SEQ ID NO:66)

[0293] YTSRLHS(SEQ ID NO:67)

[0294] QQGNTLPYT(SEQ ID NO:68)

[0295] EVQLVESGGGLVQPGRSLRLSCAASRFTFDDYAMHWVRQAPGKGLEWVSGISWNSGRIGYADSVKGRFTISRDNAENSLFLQMNGLRAEDTALYYCAKGRDSFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVTYLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:69)

[0296] DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYGASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFASYYCQQANSFPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:70)

[0297] QVQLQESGPGLVKPSETLSLTCAVSGHSISHDHAWSWVRQPPGEGLEWIGFISYSGITNYNPSLQGRVTISRDNSKNTLYLQMNSLRAEDTAVYYCARSLARTTAMDYWGEGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHAHYTQKSLS LSP(SEQ ID NO:71)

[0298] DIQMTQSPSSLSASVGDSVTITCQASTDISSHLNWYQQKPGKAPELLIYYGSHLLSGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCGQGNRLPYTFGQGTKVEIERTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:72)

[0299] AGCGACACCGGCAGACCCTTCGTGGAGATGTACAGCGAGATCCCCGAGATCATCCACATGACCGAGGGCAGAGAGCTGGTGATCCCCTGCAGAGTGACCAGCCCCAACATCACCGTGACCCTGAAGAAGTTCCCCCTGGACACCCTGATCCCCGACGGCAAGAGAATCATCTGGGACAGCAGAAAGGGCTTCATCATCAGCAACGCCACCTACAAGGAGATCGGCCTGCTGACCTGCGAGGCCACCGTGAACGGCCACCTGTACAAGACCAACTACCTGACCCACAGACAGACCAACACCATCATC(SEQ ID NO:73)

[0300] GACGTGGTGCTGAGCCCCAGCCACGGCATCGAGCTGAGCGTGGGCGAGAAGCTGGTGCTGAACTGCACCGCCAGAACCGAGCTGAACGTGGGCATCGACTTCAACTGGGAGTACCCCAGCAGCAAGCACCAGCACAAGAAGCTGGTGAACAGAGACCTGAAGACCCAGAGCGGCAGCGAGATGAAGAAGTTCCTGAGCACCCTGACCATCGACGGCGTGACCAGAAGCGACCAGGGCCTGTACACCTGCGCCGCCAGCAGCGGCCTGATGACCAAGAAGAACAGCACCTTCGTGAGAGTGCACGAGAAG(SEQ ID NO:74)

[0301] GTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCG(SEQ ID 75)

[0302]

[0303] GACATTCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGAGACAGAGTGACCATCACCTGCAGAGCCAGCCAGGACATCTCCAGCTACCTGAACTGGTATCAGCAGAAACCCGGCAAAGCCCCAAAACTGCTGATCTACTACACCAGTAGACTGCACAGCGGCGTGCCCAGCAGATTCTCAGGAAGCGGCTCCGGAACCGACTTCACCTTCACTATCAGCAGCCTGCAGCCCGAAGATATTGCTACTTACTACTGCCAGCAGGGGAACACCCTGCCCTATACCTTCGGCCAGGGCACCAAGGTGGAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGTTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTGATTCTAGA(SEQ ID NO:77)

[0304] AGTGCTCAGACCAATTTTATGCCTATGGATGATCTGGAACAGAGACTGTACGAACAGTTCATCCTCCAACAGGGACTG(SEQ ID NO:78)

[0305] ATGGGCGCTCAGCAGAAGTTCCAGCCCCTGGACGAGCTGGAGCAGACCCTGTACGAGCAGTTCATGCTCCAGCAGGCCCTGGAG(SEQ ID NO:79)

[0306] ATGGGCGCTCAGCAGAAGTACCAGCCCCTGGACGAGCTGGACAAGACCCTGTACGACCAGTTCATGCTCCAGCAGGGGCTGGAA(SEQ ID NO:80)

[0307] ATGGGCGCTCAGCACACCTTCCAGCCCCTGGACGAGCTGGAGGAGACACTGTACTACCAGTGGCTCTACGACCAGCTGCTGGAG(SEQ ID NO:81)

[0308] GCTCAGCAAGAAGAGTGTGAATGGGACCCTTGGACCTGTGAACACATGCTGGAA(SEQ ID NO:82)

[0309] GCTCAGACCAATATTCAAGAAGAATGTGAATGGGACCCTTGGACCTGTGATCACATGCCCGGCAAACTGGAA(SEQ ID NO:83)

[0310]

[0311]

[0312]

[0313]

[0314]

[0315]

[0316]

[0317]

[0318]

[0319]

[0320]

[0321]

[0322]

[0323]

[0324]

[0325]

[0326]

[0327]

[0328] GAGGTGCAGCTGGTGGAGAGCGGCGGCGGACTGGTGCAGCCTGGAGGATCTCTGAGGCTGTCCTGCGCCGCTTCTGGCCGGACCTTCTCCAGCTACGACTTTGTTTGGGTGCGGCAGGCCCCTGGCAAGGGACTGGAGTTCGTGTCCGCCATCTCCGTTGAGGGCGAGCACACCTACTATGCTGATAGCGTGAAGGGCCGGTTCACCATCAGCAGGGATAACGCCAAGAATACCCTGTATCTGCAGATGAATAGCCTGCGGGCTGAGGACACCGCCGTGTATTACTGTAACGCCGTGAAGTGGAGCGACTACGGCCCCGACAACGATTACTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCC(SEQ ID NO:102)

[0329] GAGGTGCAGCTGGTGGAGAGCGGCGGCGGACTGGTGCAGCCTGGAGGATCTCTGAGGCTGTCCTGCGCCGCTTCTGGCCTTTATTTCTCCAGCTACGACATCGCTTGGGTGCGGCAGGCCCCTGGCAAGGGACTGGAGTTCGTGTCCGCCATCGATGTGTCCGGCGAGCACACCTACTATGCTGATAGCGTGAAGGGCCGGTTCACCATCAGCAGGGATAACGCCAAGAATACCCTGTATCTGCAGATGAATAGCCTGCGGGCTGAGGACACCGCCGTGTATTACTGTAACGCCGTGAAGTGGAGCGACTACGGCCCCGACAACGATTACTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCC(SEQ ID NO:103)

[0330] GAGGTGCAGCTGCAGGAGAGCGGCGGCGGACTGGTGCAGCCAGGAGGAAGCCTGCGGCTGTCCTGTGCCGCTTCCGGCTTTACCCTGGATGCGTATGCTATCGGCTGGTTTAGGCAGGCCCCTGGCAAGGAGAGGGAGGGCGTGTCCACGATCGGCAAGTCCGACGGCAGCACCTGTTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCTAAGAATACCGTGTGGCTGCAGATGAACTCCCTGCGGGCTGAGGACACCGCCGTGTACTATTGCGCCGCTCGGCCCTGGTGGGTGGGCGACGCGCCTTCTATCGCTGCCGAGTACGAGTACGATCTGTGGGGCCAGGGCACCCAGGTGACCGTGAGCTCC(SEQ ID NO:104)

[0331] GAGGTGCAGCTGCAGGAGAGCGGCGGCGGACTGGTGCAGCCAGGAGGAAGCCTGCGGCTGTCCTGTGCCGCTTCTGGCTTTACCCTGGATGAGTATGCTATCGGCTGGTTTAGGCAGGCCCCTGGCAAGGAGAGGGAGGGCGTGTCCTGTATCGGCAAGGCGGACGGCAGCACCTGTTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCTAAGAATACCGTGTGGCTGCAGATGAACTCCCTGCGGGCTGAGGACACCGCCGTGTACTATTGCGCCGCTCGGCCCTGGTGGGTGGGCGACCCTCCTTCTATCGGTGCCGAGTACGAGTACGATCTGTGGGGCCAGGGCACCCAGGTGACCGTGAGCTCC(SEQ ID NO:105)

[0332] CAGGTGCAGCTGGTGGAGAGCGGCGGCGGACTGGTGCAGCCTGGAGGAAGCCTGCGGCTGTCCTGTGCCGCCAGCGGATTCAGGTTTAGCAGCTACGCCATGTCCTGGGTGAGGCAGGCTCCTGGCAAGGGCCTGGAGTGGGTGAGCAAGATCAATAGCGGCGGCGGCATCACCTACTACGCTGACTCCGTGAAGGGCAGGTTTACCATCTCCAGGGATAACGCCAAGAATACCCTGTACCTGCAGATGAATAGCCTGCGGGCTGAGGACACCGCCGTGTATTACTGTCGGGACGAGGGCCCTTTTGGCAGCTGGGGCCAGGGCACCCAGGTGACAGTGAGCTCC(SEQ ID NO:106)

[0333] CAGGTGCAGCTGGTGGAGAGCGGCGGCGGACTGGTGCAGCCTGGAGGAAGCCTGCGGCTGTCCTGTGCCGCCAGCGGATTCACCTTTAGCAGCTACGCCATGTCCTGGGTGAGGCAGGCTCCTGGCAAGGGCCTGGAGTGGGTGAGCGACATCAATAGCGGCGGCAGTGAGACCTACTACGCTGACTCCGTGAAGGGCAGGTTTACCATCTCCAGGGATAACGCCAAGAATACCCTGTACCTGCAGATGAATAGCCTGCGGGCTGAGGACACCGCCGTGTATTACTGTCGGGACAAGGGCCCTTTTGGCAGCTGGGGCCAGGGCACCCAGGTGACAGTGAGCTCC(SEQ ID NO:107)

[0334] GAGGTGCAGCTGGTGGAGAGCGGCGGCGGACTGGTGCAGCCTGGAGGATCTCTGAGGCTGTCCTGCGCCGCTTCTGGCCGGACCTTCTCCAGCTACGACATCATTTGGGTGCGGCAGGCCCCTGGCAAGGGACTGGAGTTCGTGTCCGCCATCTCCACGTCCGGCGAGCACACCTACTATGCTGATAGCGTGAAGGGCCGGTTCACCATCAGCAGGGATAACGCCAAGAATACCCTGTATCTGCAGATGAATAGCCTGCGGGCTGAGGACACCGCCGTGTATTACTGTAACGCCGTGAAGTGGAGCGACTACGGCCCCCAGAACGATTACTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCC(SEQ ID NO:108)

[0335] GAGGTGCAGCTGCAGGAGAGCGGCGGCGGACTGGTGCAGCCAGGAGGAAGCCTGCGGCTGTCCTGTGCCGCTTCCGGCTTTACCCTGGATTACTATGCTATCGGCTGGTTTAGGCAGGCCCCTGGCAAGGAGAGGGAGGGCGTGTCCGTGATCGGCAAGTCCGACGGCAGCACCTGTTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCTAAGAATACCGTGTGGCTGCAGATGAACTCCCTGCGGGCTGAGGACACCGCCGTGTACTATTGCGCCGCTCGGCCCTGGTGGGTGGGCGACAGGCCTTCTATCGCTGCCGAGTACGAGTACGATCTGTGGGGCCAGGGCACCCAGGTGACCGTGAGCTCC(SEQ ID NO:109)

[0336]

[0337]

[0338]

[0339]

[0340] GAGGTGCAGTTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCACTGAGACTCTCCTGTGCAGCCTCTGGAGATGATTTAAGTAGCGTAGACATGGGCTGGTACCGCCAGGCTCCAGGGAAGGGCCTGGAGTTTGTCGGCGTTATTAGCCGTGGTGGTGGTAGCACATACTATGTAGACTCCGTGCAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGCGGGCCGAGGACACGGCCGTGTATTACTGTAAGACAGAAATTCCATATTTTGATGACTACTGGGGCCAGGGGACCCTGGTCACCGTCTCCTCA(SEQ ID NO:114)

[0341] GAGGTGCAGTTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCACTGAGACTCTCCTGTGCAGCCTCTGGAGAGTCCTTAAGTAGCGTAGACATGGGCTGGTACCGCCAGGCTCCAGGGAAGGGCCTGGAGTTTGTCGGCGTTATTAGCCGTGGTGGTGAGAGCACATACTATGTAGACTCCGTGCAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGCGGGCCGAGGACACGGCCGTGTATTACTGTAAGACAGAAATTCCATTTGGGGATGACTACTGGGGCCAGGGGACCCTGGTCACCGTCTCCTCA(SEQ ID NO:115)

[0342] GAGGTGCAGTTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCACTGAGACTCTCCTGTGCAGCCTCTGGAGGTGATTTAAGTAGCGTAGACATGGGCTGGTACCGCCAGGCTCCAGGGAAGGGCCTGGAGTTTGTCGGCGTTATTAGCAGGACGGGTGGTAGCACATACTATGTAGACTCCGTGCAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGCGGGCCGAGGACACGGCCGTGTATTACTGTAAGACAGAAATTCCAGGGGGGGATGACTACTGGGGCCAGGGGACCCTGGTCACCGTCTCCTCA(SEQ ID NO:116)

[0343] GAGGTGCAGTTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCACTGAGACTCTCCTGTGCAGCCTCTGGAGAGTCCTTAAGTAGCGTAGACATGGGCTGGTACCGCCAGGCTCCAGGGAAGGGCCTGGAGTTTGTCGGCGTTATTAGCCGTACGGGTGGTAGCACATACTATGTAGACTCCGTGCAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGCGGGCCGAGGACACGGCCGTGTATTACTGTAAGACAGAAATTCCAGGGGGGGATGACGTTTGGGGCCAGGGGACCCTGGTCACCGTCTCCTCA(SEQ ID NO:117)

[0344] GAGGTGCAGTTGGTGGAGAGCGGGGGGGGGCTGGTGCAGCCTGGAGGAAGTTTGAGGTTGAGCTGTGCCGCAAGCGGGGGGACATTCATTGATTTGGACATGGGATGGTACAGGCAGGCACCTGGAAACGAGAGGCAGTTTGTGGGGGTGATTTCCCGGACTGGAGGATCCACATACTACGCTGACAGCGTGCAGGGGAGGTTCACCATCTCCCGGGACAACGCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGGGCTGAGGACACTGCTACCTATTACTGCAAGACCGAGATTCCCGGAGGAGACGACTACTGGGGCCAGGGCACACTGGTGACCGTGAGCTCC(SEQ ID NO:118)

[0345] GAGGTGCAGTTGGTGGAGAGCGGGGGGGGGCTGGTGCAGCCTGGAGGAAGTTTGAGGTTGAGCTGTGCCGCAAGCGGGGGGACATTCCATCTGTACGACATGGGATGGTACAGGCAGGCACCTGGAAACGAGAGGCAGTTTGTGGGGGTGATTTCCCGCGGCGGAGGATCCACATACTACGCTGACAGCGTGCAGGGGAGGTTCACCATCTCCCGGGACAACGCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGGGCTGAGGACACTGCTACCTATTACTGCAAGACCGAGATTCCCGGATTTCAGGACTACTGGGGCCAGGGCACACTGGTGACCGTGAGCTCC(SEQ ID NO:119)

[0346] GAGGTGCAGTTGGTGGAGAGCGGGGGGGGGCTGGTGCAGCCTGGAGGAAGTTTGAGGTTGAGCTGTGCCGCAAGCGGGCCGGAGTTCATTGATTACGACATGGGATGGTACAGGCAGGCACCTGGAAACGAGAGGCAGTTTGTGGGGGTGATTTCCCGCGGCGGAGGATCCACATACTACGCTGACAGCGTGCAGGGGAGGTTCACCATCTCCCGGGACAACGCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGGGCTGAGGACACTGCTACCTATTACTGCAAGACCGAGATTCCCTTTTTTGACGACTACTGGGGCCAGGGCACACTGGTGACCGTGAGCTCC(SEQ ID NO:120)

[0347] GAGGTGCAGTTGGTGGAGAGCGGGGGGGGGCTGGTGCAGCCTGGAGGAAGTTTGAGGTTGAGCTGTGCCGCAAGCGGGGGGACATTCATTGATCTGGACATGGGATGGTACAGGCAGGCACCTGGAAACGAGAGGCAGTTTGTGGGGGTGATTTCCCGCGGCGGAGGATCCGTTTACTACGCTGACAGCGTGCAGGGGAGGTTCACCATCTCCCGGGACAACGCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGGGCTGAGGACACTGCTACCTATTACTGCAAGACCGAGATTCCCGGAGGACCTGACTACTGGGGCCAGGGCACACTGGTGACCGTGAGCTCC(SEQ ID NO:121)

[0348] GAGGTGCAGTTGGTGGAGAGCGGGGGGGGGCTGGTGCAGCCTGGAGGAAGTTTGAGGTTGAGCTGTGCCGCAAGCGGGGGGACATTCATTGATTACGACATGGGATGGTACAGGCAGGCACCTGGAAACGAGAGGCAGTTTGTGGGGGTGATTTCCCGCGGCGGAGGAGCTGTTTACTACGCTGACAGCGTGCAGGGGAGGTTCACCATCTCCCGGGACAACGCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGGGCTGAGGACACTGCTACCTATTACTGCAAGACCGAGATTCCCTTTTTTGACGACTACTGGGGCCAGGGCACACTGGTGACCGTGAGCTCC(SEQ ID NO:122)

[0349] CAGGTCCAGCTCGTCGAGTCAGGCGGCGGCCTCGTGCAGCCTGGAGGAAGTTTGAGGTTGAGTTGTGCAGCCAGCGGTCCTACATTTTCCAACCTCGACATGGGCTGGTACAGGCAGGCACCCGGCAAAGGGCTGGAGCTGGTGGGCGTTATTTCTCGGACAGGAGGGTCCACATACTACGCCGATTCCGTGAAGGGCAGATTCACCATCAGCAGGGACAATGCTAAGAACACCCTGTACCTGCAGATGAACTCCTTGAGGGCTGAGGACACCGCAGTGTACTACTGCAAGACCGAGGTGCCCTTTTCTGACGATTACTGGGGACAGGGCACACTGGTGACCGTGAGCTCC(SEQ ID NO:123)

[0350] CAGGTCCAGCTCGTCGAGTCAGGCGGCGGCCTCGTGCAGCCTGGAGGAAGTTTGAGGTTGAGTTGTGCAGCCAGCGTGCCGACATTTGGGGATCTCGACATGGGCTGGTACAGGCAGGCACCCGGCAAAGGGCTGGAGCTGGTGGGCGTTATTTCTCGGACAGGAGGGTCCACATACTACGCCGATTCCGTGAAGGGCAGATTCACCATCAGCAGGGACAATGCTAAGAACACCCTGTACCTGCAGATGAACTCCTTGAGGGCTGAGGACACCGCAGTGTACTACTGCAAGACCGAGGTGCCCTTTGGGGACGATTACTGGGGACAGGGCACACTGGTGACCGTGAGCTCC(SEQ ID NO:124)

[0351] CAGGTCCAGCTCGTCGAGTCAGGCGGCGGCCTCGTGCAGCCTGGAGGAAGTTTGAGGTTGAGTTGTGCAGCCAGCGGGGATACATTTTCCAACCTCGACATGGGCTGGTACAGGCAGGCACCCGGCAAAGGGCTGGAGCTGGTGGGCGTTATTTCTCGGACAGGAGGGTCCACATACTACGCCGATTCCGTGAAGGGCAGATTCACCATCAGCAGGGACAATGCTAAGAACACCCTGTACCTGCAGATGAACTCCTTGAGGGCTGAGGACACCGCAGTGTACTACTGCAAGACCGAGGTGCCCTGGGCGGACGATTACTGGGGACAGGGCACACTGGTGACCGTGAGCTCC(SEQ ID NO:125)

[0352] CAGGTCCAGCTCGTCGAGTCAGGCGGCGGCCTCGTGCAGCCTGGAGGAAGTTTGAGGTTGAGTTGTGCAGCCAGCGTGCCGACATTTTCCGATGTGGACATGGGCTGGTACAGGCAGGCACCCGGCAAAGGGCTGGAGCTGGTGGGCGTTATTTCTCGGACAGGAGGGTCCACATACTACGCCGATTCCGTGAAGGGCAGATTCACCATCAGCAGGGACAATGCTAAGAACACCCTGTACCTGCAGATGAACTCCTTGAGGGCTGAGGACACCGCAGTGTACTACTGCAAGACCGAGGTGCCCTTTGCTGACGATTACTGGGGACAGGGCACACTGGTGACCGTGAGCTCC(SEQ ID NO:126)

[0353] CAGGTCCAGCTCGTCGAGTCAGGCGGCGGCCTCGTGCAGCCTGGAGGAAGTTTGAGGTTGAGTTGTGCAGCCAGCGTGCCGTGGTTTTCCAACCTCGACATGGGCTGGTACAGGCAGGCACCCGGCAAAGGGCTGGAGCTGGTGGGCGTTATTTCTCGGACAGGAGGGATGACATACTACGCCGATTCCGTGAAGGGCAGATTCACCATCAGCAGGGACAATGCTAAGAACACCCTGTACCTGCAGATGAACTCCTTGAGGGCTGAGGACACCGCAGTGTACTACTGCAAGACCGAGGTGCCCGGCTCGGACGATTACTGGGGACAGGGCACACTGGTGACCGTGAGCTCC(SEQ ID NO:127)

[0354] CAGGTCCAGCTCGTCGAGTCAGGCGGCGGCCTCGTGCAGCCTGGAGGAAGTTTGAGGTTGAGTTGTGCAGCCAGCGGGGCGACATTTTCCAACCTCGACATGGGCTGGTACAGGCAGGCACCCGGCAAAGGGCTGGAGCTGGTGGGCGTTATTTCTCGGACAGGAGGGTCCACATACTACGCCGATTCCGTGAAGGGCAGATTCACCATCAGCAGGGACAATGCTAAGAACACCCTGTACCTGCAGATGAACTCCTTGAGGGCTGAGGACACCGCAGTGTACTACTGCAAGACCGAGGTGCCCTTTGATGACGATTACTGGGGACAGGGCACACTGGTGACCGTGAGCTCC(SEQ ID NO:128)

[0355]

[0356]

[0357]

[0358]

[0359]

[0360]

[0361] AGCGACCACGCCTGGAGC(SEQ ID NO: 135)

[0362] TACATCAGCTACAGCGGCATCACCACCTACAACCCCAGCCTGAAGAGC(SEQ ID NO: 136)

[0363] GCCAGGACCACCGCCATGGACTAC(SEQ ID NO: 137)

[0364] TACACCAGCAGGCTGCACAGC(SEQ ID NO: 138)

[0365] AGGGCCAGCCAGGACATCAGCAGCTACCTGAAC(SEQ ID NO: 139)

[0366] CAGCAGGGCAACACCCTGCCCTACACC(SEQ ID NO: 140)

[0367]

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

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[0368] Reference to electronic sequence listings The contents of the electronic sequence listing (076908-8004WO01.xml, size: 242KB, and creation date: September 7, 2023) are incorporated herein by reference in their entirety.

[0369] While specific embodiments of the present invention have been described above, it will be understood by those skilled in the art that many equivalents, modifications, substitutions, and variations may be made thereto without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

1. An antibody fusion protein or its antigen-binding fragment or domain comprising an Ang-2 binding unit, an IL-6R binding unit, and a VEGF binding unit, wherein the fusion protein is substantially capable of binding to Ang-2, IL-6R, and one or more VEGF family members.

2. The antibody fusion protein or antigen-binding fragment or domain according to claim 1, wherein the Ang-2 binding unit, the IL-6R binding unit, and the VEGF binding unit are linked to each other.

3. The antibody fusion protein or antigen-binding fragment or domain thereof according to claim 2, wherein the Ang-2 binding unit comprises an antibody or a biologically active polypeptide substantially capable of binding to Ang-2.

4. The antibody fusion protein or its antigen-binding fragment or domain according to claim 2, wherein the IL-6R binding unit comprises an antibody against IL-6R.

5. The antibody fusion protein or antigen-binding fragment or domain according to claim 2, wherein the VEGF-binding unit comprises multiple Ig-like domains of one or more VEGF receptors.

6. The antibody fusion protein or its antigen-binding fragment or domain according to claim 3, wherein the antibody substantially capable of binding to Ang-2 comprises Ang-2 sdAb.

7. The antibody fusion protein or its antigen-binding fragment or domain according to claim 4, wherein the antibody against IL-6R comprises IL-6R sdAb.

8. The antibody fusion protein or antigen-binding fragment or domain according to claim 5, wherein the VEGF-binding unit comprises VEGFR-1-D2 and VEGFR-2-D3 linked together.

9. The antibody fusion protein or antigen-binding fragment or domain according to claim 2, comprising: (a) an IL-6R antibody comprising a heavy chain and a light chain; (b) a VEGF-binding unit comprising VEGFR-1-D2 and VEGFR-2-D3 linked in series with respect to each other; and (c) an Ang-2-binding polypeptide, wherein the C-terminus of the VEGF-binding unit is linked to the N-terminus of the light chain or the heavy chain of the IL-6R antibody, and the C-terminus of the heavy chain of the IL-6R antibody is linked to the N-terminus of the Ang-2-binding polypeptide.

10. An antibody fusion protein or antigen-binding fragment or domain according to claim 2, comprising (a) an IL-6R antibody comprising a heavy chain and a light chain, (b) a VEGF-binding unit comprising VEGFR-1-D2 and VEGFR-2-D3 linked in series, and (c) Ang-2 sdAb, wherein the C-terminus of the VEGF-binding unit is linked to the N-terminus of the light chain or heavy chain of the IL-6R antibody, and the C-terminus of the heavy chain of the IL-6R antibody is linked to the N-terminus of Ang-2 sdAb.

11. The antibody fusion protein or antigen-binding fragment or domain according to claim 2, comprising two fusion polypeptides, each of which comprises (a) IL-6R sdAb, (b) a VEGF-binding unit comprising VEGFR-1-D2 and VEGFR-2-D3 linked in series, (c) an Ang-2-binding polypeptide, and (d) an IgG1 Fc domain, wherein the C-terminus of the VEGF-binding unit is linked to the N-terminus of the Fc domain, the N-terminus of the VEGF-binding unit is linked to the C-terminus of the IL-6R sdAb, and the N-terminus of the Ang-2-binding polypeptide is linked to the C-terminus of the Fc domain.

12. The antibody fusion protein or antigen-binding fragment or domain according to claim 2, comprising two fusion polypeptides, each of which comprises (a) IL-6R sdAb, (b) a VEGF-binding unit comprising VEGFR-1-D2 and VEGFR-2-D3 linked in series, (c) an Ang-2-binding polypeptide, and (d) an IgG1 Fc domain, wherein the C-terminus of the VEGF-binding unit is linked to the N-terminus of the Fc domain, the N-terminus of the VEGF-binding unit is linked to the C-terminus of the Ang-2 polypeptide, and the N-terminus of the IL-6R sdAb is linked to the C-terminus of the Fc domain.

13. The antibody fusion protein or antigen-binding fragment or domain according to claim 2, comprising two fusion polypeptides, each of which comprises (a) IL-6R sdAb, (b) a VEGF-binding unit comprising VEGFR-1-D2 and VEGFR-2-D3 linked in series, (c) Ang-2 sdAb, and (d) an Fc domain of IgG1, wherein the C-terminus of the VEGF-binding unit is ligated to the N-terminus of the Fc domain, the N-terminus of the VEGF-binding unit is ligated to the C-terminus of the Ang-2 sdAb, and the N-terminus of the IL-6R sdAb is ligated to the C-terminus of the Fc domain.

14. The antibody fusion protein or antigen-binding fragment or domain according to claim 2, comprising two fusion polypeptides, each of which comprises (a) IL-6R sdAb, (b) a VEGF-binding unit comprising VEGFR-1-D2 and VEGFR-2-D3 linked in series, (c) Ang-2 sdAb, and (d) an Fc domain of IgG1, wherein the C-terminus of the VEGF-binding unit is linked to the N-terminus of the Fc domain, the N-terminus of the VEGF-binding unit is linked to the C-terminus of the IL-6R sdAb, and the N-terminus of the Ang-2 sdAb is linked to the C-terminus of the Fc domain.

15. The antibody fusion protein or antigen-binding fragment or domain according to claim 2, comprising two fusion polypeptides, each of which comprises (a) IL-6R sdAb, (b) a VEGF-binding unit comprising VEGFR-1-D2 and VEGFR-2-D3 linked in series, (c) Ang-2 sdAb, and (d) an Fc domain of IgG1, wherein the C-terminus of the VEGF-binding unit is linked to the N-terminus of the Fc domain, the N-terminus of the VEGF-binding unit is linked to the C-terminus of the IL-6R sdAb, and the N-terminus of the Ang-2 sdAb is linked to the C-terminus of the Fc domain.

16. The antibody fusion protein or antigen-binding fragment or domain according to claim 8, comprising a polypeptide having a pair of amino acid sequences selected from the group consisting of SEQ ID NOs: 12 and 13, 14 and 15, 16 and 17, 18 and 19, 20 and 21, 22 and 23, 24 and 25, 26 and 27, 28 and 29, 38 and 39, and 40 and 41.

17. The antibody fusion protein according to claim 8, comprising a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs. 57 to 62, or an antigen-binding fragment or domain thereof.

18. The antibody fusion protein or its antigen-binding fragment or domain is approximately 1 × 10 －６ From M, approximately 1 x 10 －１２ Up to M, or approximately 1 x 10 －８ From M, approximately 1 x 10 －１２ Equilibrium dissociation constant (K) within the range up to M Ｄ The antibody fusion protein or its antigen-binding fragment or domain according to claim 2, which is capable of binding to IL-6R, Ang-2, and at least a VEGF family member.

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

20. The isolated nucleic acid molecule according to claim 19, wherein the nucleic acid molecule includes a sequence selected from the group consisting of SEQ ID NOs: 78 to 134.

21. An expression vector comprising a nucleic acid molecule according to any one of claims 19 to 20.

22. The expression vector according to claim 21, comprising the nucleic acid molecule operably linked to an expression control sequence.

23. A host vector system comprising the expression vector described in claim 22 in a host cell.

24. A method for producing a substantially purified antibody fusion protein, comprising: (a) growing cells of the host vector system described in claim 23 under conditions that enable the production of the antibody fusion protein; (b) recovering the antibody fusion protein to produce a recovered antibody fusion protein; and (c) purifying the recovered antibody fusion protein to produce the substantially purified antibody fusion protein.

25. A method for treating or controlling at least one disease, condition, or disorder in a subject of interest, which is pathogenic to abnormal angiogenesis or inflammation, the method comprising the step of administering to the subject a certain amount of a composition of an antibody fusion protein or antigen-binding fragment or domain according to any one of claims 1 to 18.

26. The method according to claim 25, wherein the disease, condition, or disorder is selected from the group consisting of macular edema, uveitis, central retinal vein occlusion and branch retinal vein occlusion, choroidal neovascularization, neovascular age-related macular degeneration, polypoid choroidal vasculopathy, myopic choroidal neovascularization, vascular leakage, non-proliferative and proliferative diabetic retinopathy, corneal neovascularization, corneal inflammation, myopic neovascularization, and neovascular glaucoma.

27. The method according to claim 26, wherein the subject is administered a dose of approximately 25 to 4000 micrograms of the antibody fusion protein or its antigen-binding fragment or domain.

28. The method according to claim 27, wherein the composition is administered to the subject as eye drops, a punctal plug, intrachorally, retroocularly, subconjunctivally, periocularly, subtenon's capsule, near the sclera, transsclerally, intravitreously, subretinally, or suprachoroidally.

29. The method according to claim 27, wherein, when administered intraocularly, the composition is administered to the subject over a period of at least one month.

30. The method according to claim 27, wherein the composition is administered to the subject at a frequency of at least once a month when administered intraocularly.

31. A method for treating or controlling at least one systemic disease, condition, or disorder in a subject of interest, which is pathogenic to abnormal angiogenesis or inflammation, comprising the step of administering to the subject a certain amount of a composition of an antibody fusion protein or antigen-binding fragment or domain according to any one of claims 1 to 18.

32. The method according to claim 32, wherein the systemic disease, condition, or disorder involves tumor growth, tumor metastasis, a combination of tumor growth and metastasis, atherosclerosis, and psoriasis.

33. A pharmaceutical composition for use in the treatment or control of at least one disease, condition, or disorder caused by abnormal angiogenesis or inflammation, comprising an antibody fusion protein or its antigen-binding fragment or domain as described in any one of claims 1 to 18.

34. The pharmaceutical composition according to claim 33, wherein the disease, condition, or disorder is selected from the group consisting of macular edema, uveitis, central retinal vein occlusion and branch retinal vein occlusion, choroidal neovascularization, neovascular age-related macular degeneration, polypoid choroidal vasculopathy, myopic choroidal neovascularization, vascular leakage, non-proliferative and proliferative diabetic retinopathy, corneal neovascularization, corneal inflammation, myopic neovascularization, neovascular glaucoma, tumor growth, tumor metastasis, a combination of tumor growth and metastasis, atherosclerosis, and psoriasis.

35. A pharmaceutical composition comprising an antibody fusion protein or an antigen-binding fragment or domain thereof according to any one of claims 1 to 18, and a pharmaceutically acceptable carrier.

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