Multispecific fusion proteins targeting angiogenic and inflammatory factors

Abstract

This invention provides an antibody fusion protein comprising a binding construct that targets and is capable of binding to Ang-2, IL-6 receptors, and at least one VEGF family member; or an antigen-binding fragment or domain of the antibody fusion protein. The antibody fusion protein comprises an antibody or binding peptide against Ang-2, an antibody against IL-6R, and multiple extracellular domains of the VEGF receptor as binding components. The antibody fusion protein is suitable for treating or controlling ocular or systemic diseases, symptoms, or conditions caused by abnormal angiogenesis, increased vascular leakage, or inflammation.

Description

Background Technology

[0001] This invention relates to multispecific fusion proteins that target angiogenic factors and inflammatory factors. Specifically, this invention relates to multispecific fusion proteins that target angiogenesis factor-2 (“Ang-2”), certain members of the vascular endothelial growth factor (“VEGF”) family, and interleukin-6 receptor (“IL-6R”). More specifically, this invention relates to multispecific antibody fusion proteins that target Ang-2, VEGF-A, VEGF-B, P1GF, and IL-6R. This invention also relates to these fusion proteins, their uses, and methods of production.

[0002] The main cellular components of the mammalian vascular system are endothelial cells, smooth muscle cells, and pericytes. Endothelial cells form the inner lining of all blood vessels in mammals and constitute the non-thrombotic interface between blood and tissues. Therefore, endothelial cell proliferation is an important component of new capillary and vascular development, which in turn is a necessary process for mammalian tissue growth and / or regeneration.

[0003] In recent decades, a variety of signaling molecules have been identified as playing important 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 certain members of the interleukin family (e.g., IL-3, IL-6, IL-8, and IL-17). VEGF family members that secrete peptides have been shown to play a crucial role in promoting endothelial cell proliferation and angiogenesis. A pathological feature of uncontrolled angiogenesis caused by VEGF overexpression is increased vascular permeability, which leads to fluid leakage into surrounding tissues and swelling of those tissues. In mammals, this family consists of five related growth factors with highly conserved receptor-binding structures: vascular endothelial growth factor AD (“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 stresses such as infection and injury. Under physiological conditions, IL-6 is virtually undetectable, but its levels can increase by more than 100,000-fold during the early stages of inflammation. However, not all IL-6 stimulation is beneficial. Dysregulation and persistent production of IL-6 have been involved in the development of various autoimmune and chronic inflammatory diseases. Evidence suggests that uncontrolled IL-6 production in inflamed tissues can induce excessive production of VEGF-A and VEGF-C.

[0005] The angiopoietin / Tie ligand / receptor system plays a crucial regulatory role in controlling vascular integrity and quiescence. Besides its role in angiogenesis, it is also an important regulator in various diseases, including inflammation. Key members of the angiopoietin family are Ang-1 and Ang-2. Ang-1-mediated Tie-2 activation is essential for maintaining endothelial quiescence. Prokinetic Ang-1 function is antagonized by Ang-2, which is thought to inhibit Ang-1 / Tie-2 signaling. Ang-2 destabilizes quiescent endothelium and induces a response to exogenous stimuli, thereby promoting the activity of inflammatory and angiogenic cytokines. It has been demonstrated that Ang-2 promotes the pro-angiogenic effects of VEGF, and VEGF upregulates Ang-2 expression in endothelial cells.

[0006] VEGF family growth factors act by stimulating angiogenesis through a series of homologous 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 referred to as VEGF) has become the most important member of this family of growth factors. Human VEGF-A is expressed in multiple homodimeric forms in various tissues (each monomer contains 121, 145, 165, 183, 189, and 206 amino acids), each of which is produced by alternative splicing of a single RNA transcript.

[0008] Because VEGF promotes vascular endothelial cell proliferation and angiogenesis, it can be used to treat a variety of conditions that have significant benefits from the growth-promoting activity of vascular endothelial cells; for example, the treatment of ulcers, vascular injuries, and myocardial infarction.

[0009] However, in contrast, while vascular endothelial proliferation is desirable in some cases, it and angiogenesis are also undesirable components of a variety of diseases and conditions, including tumor growth and metastasis, rheumatoid arthritis, psoriasis, atherosclerosis, diabetic retinopathy, posterior lens fibrosis, neovascular glaucoma, neovascular age-related macular degeneration, hemangioma, immune rejection of transplanted corneal tissue and other tissues, and chronic inflammation. In subjects suffering from any of these conditions, it is desirable to inhibit or at least sufficiently reduce the endothelial proliferation activity of the aforementioned angiogenic factors.

[0010] The flt-1, KDR, and flt-4 tyrosine kinase receptors each possess seven extracellular immunoglobulin-like (“Ig-like”) domains for ligand binding, a transmembrane domain for anchoring the receptor to the cell surface expressing the receptor, and an intracellular catalytic tyrosine kinase domain. Flt-1 binds to VEGF-A, VEGF-B, and P1GF. KDR binds to VEGF-A, VEGF-C, and VEGF-D. Flt-4 binds to VEGF-C and VEGF-D.

[0011] Given the role of VEGF family growth factors in vascular endothelial proliferation and angiogenesis, and the function of these processes in many different diseases and conditions, therapies targeting these growth factors have been designed. However, anti-VEGF therapy alone cannot completely halt the progression of angiogenic diseases.

[0012] Therefore, a pharmacological approach is desired for more completely reducing or inhibiting the biological activity of one or more of these growth factors in a patient whose pathological symptoms originate from abnormal angiogenesis. A pharmacological approach is also desired for improving the treatment or control of pathological symptoms originating from abnormal angiogenesis. Summary of the Invention

[0013] As used in this article, the term “control” also includes mitigation, relief, improvement, or prevention.

[0014] Generally, the present invention provides multispecific fusion proteins or chimeric proteins, methods for their production, and compositions comprising them, as well as methods for treating or controlling at least one pathological condition in a subject, said condition being caused by abnormal angiogenesis, increased vascular permeability (vascular leakage), and inflammation. In this disclosure, a "fusion protein" may be referred to as a "chimeric protein" because it comprises components from different sources.

[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 members of the VEGF family.

[0016] In one respect, this multispecific fusion protein is a multispecific antibody fusion protein.

[0017] On the other hand, this multispecific antibody fusion protein is a trispecific fusion protein.

[0018] In another aspect, the present invention provides a trispecific antibody fusion protein or chimeric protein, or its antigen-binding fragment or antigen-binding domain, which is capable of substantially binding to Ang-2, IL-6R (including membrane-bound or soluble forms of IL-6R) and one or more VEGF family members; thereby simultaneously reducing or inhibiting the signal transduction of Ang-2, IL-6 and VEGF family members.

[0019] In another aspect, the trispecific antibody fusion protein or chimeric protein of the present invention comprises an Ang-2 binding unit, an IL-6R binding unit, and a VEGF binding unit linked together. These binding units comprise, or are substantially comprise, antigen-binding domains or portions targeting Ang-2, IL-6R, and one or more VEGF family members.

[0020] In another aspect, the trispecific antibody fusion protein or chimeric protein of the present invention comprises: a VEGF / IL-6R binding unit comprising a VEGF binding unit linked to an IL-6R binding unit; and 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 these fusion proteins.

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

[0023] In another aspect, the trispecific 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 yet another aspect, 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" encompasses, but is not limited to, full-length antibodies, single-chain antibodies, and VEGF-linked antibodies. V Antibody (scF) V ), Fab antibodies, Fab' antibodies, (Fab')2 antibodies, single-domain antibodies (sdAb, also known as nanobodies), micro antibodies, large antibodies, bifunctional antibodies and peptides.

[0024] In another aspect, the VEGF binding unit included in the trispecific antibody fusion protein comprises Ig-like domains selected from the group consisting of Ig-like domains of one or more VEGF receptors. In one aspect, VEGF family members binding to this VEGF binding unit include VEGF-A, VEGF-B, and PlGF. In another aspect, these family members are VEGF-A, VEGF-B, and PlGF. Therefore, in one aspect, the antibody fusion protein of the present invention can be considered at least as a trispecific construct that can bind to three different types of ligands involved in pathological angiogenesis and vascular leakage. In one aspect, 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 an Ig-like domain (extracellular domain) 2 or substantially Ig-like domain 2 of VEGFR-1 (“VEGFR-1-D2”) and an Ig-like domain 3 or substantially Ig-like domain 3 of VEGFR-2 (“VEGFR-2-D3”). In some other embodiments, the VEGF binding unit comprises VEGFR-1-D2 and VEGFR-2-D3 linked to the Fc domain of IgG1.

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

[0026] In another aspect, 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 aspect, the Ang-2 binding unit comprises an sdAb or a bioactive peptide targeting Ang-2.

[0028] In another aspect, the trispecific antibody fusion protein of the present invention comprises the Fc domain of human IgG1.

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

[0030] In another embodiment, the VEGF binding unit comprises: (a) human VEGFR-1-D2; (b) human VEGFR-2-D3; and (c) an Fc domain of IgG1, wherein VEGFR-1-D2 and VEGFR-2-D3 are connected in tandem. In some embodiments, the C-terminus of VEGFR-2-D3 is connected to the N-terminus of the Fc domain. In other embodiments, the C-terminus of VEGFR-1-D2 is connected to the N-terminus of the Fc domain.

[0031] On the other hand, sdAbs targeting Ang-2 or IL-6R, or their antigen-binding fragments or domains, contain the heavy chain variable region of heavy chain antibodies targeting Ang-2 or IL-6R. These sdAbs can specifically bind to Ang-2 or IL-6R without requiring the complementary variable region found in conventional four-stranded immunoglobulin molecules.

[0032] In another aspect, the present invention provides an isolated nucleic acid molecule that encodes an antibody fusion protein or a chimeric protein.

[0033] In another aspect, the present invention provides a vector comprising the nucleic acid molecule, including an expression vector comprising the nucleic acid molecule operably linked to an expression control sequence. As used herein, the phrase “operably linked” means that the components of a construct are positioned in a functional relationship with each other and each component retains its function. The nucleic acids are “operably linked” when one nucleic acid is positioned in a functional relationship with another nucleic acid sequence. For example, if a pre-sequence or secretory leader sequence of DNA is expressed as a pre-protein form involved in polypeptide secretion, then the DNA is operably linked to the DNA encoding the polypeptide; if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is operably linked to the coding sequence; or if a ribosome binding site is localized to promote translation, then the ribosome binding site is operably linked to the coding sequence.

[0034] In another aspect, the present invention provides a host-vector system for generating said antibody fusion protein or chimeric protein, which comprises an expression vector suitable for host cells.

[0035] In another aspect, the present invention provides a method for producing an antibody fusion protein or chimeric protein, the method comprising: (a) growing cells of the host-vector system under conditions that allow for the production of the antibody fusion protein or chimeric protein; and (b) recovering the resulting antibody fusion protein or chimeric protein. This method may further comprise purifying the antibody fusion protein or chimeric protein.

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

[0037] In some embodiments, such diseases, symptoms, or conditions are eye diseases, symptoms, or conditions. In some other embodiments, such diseases, symptoms, or conditions involve tumor growth and metastasis. In other embodiments, such diseases, symptoms, or conditions are rheumatoid arthritis, psoriasis, or atherosclerosis.

[0038] Other features and advantages of the present invention will become apparent from the following description and claims. Attached Figure Description

[0039] Figure 1 A schematic diagram of the first embodiment of the present invention is shown.

[0040] Figure 2 A schematic diagram of the second embodiment of the present invention is shown.

[0041] Figure 3 shows a schematic diagram of the third embodiment of the present invention.

[0042] Figure 4 shows a schematic diagram of the fourth embodiment of the present invention.

[0043] Figure 5A Figure B shows the purity of the antibody fusion proteins EB-105BI-7CA and EB-105BIc1 as presented by SEC-HPLC chromatograms.

[0044] Figure 6A -C shows the antibody fusion protein of the present invention as determined by ELISA for human VEGF-A. 165 The combination of affinity.

[0045] Figure 7 This invention demonstrates the effectiveness of some antibody fusion proteins of the present invention against human VEGF-B, as determined by ELISA. 167 The combination of affinity.

[0046] Figure 8A The results show that some of the antibody fusion proteins of the present invention, as determined by ELISA, have binding affinity for human PlGF.

[0047] Figure 9A -C indicates the binding affinity of some antibody fusion proteins of the present invention to human IL-6R as determined by ELISA.

[0048] Figure 10A-C indicates the binding affinity of some antibody fusion proteins of the present invention to human Ang-2, as determined by ELISA.

[0049] Figure 11 The binding affinity of some of the antibody fusion proteins of the present invention, nesvacumab, and faricimab to human Ang-1 was shown by ELISA.

[0050] Figure 12A -C shows the antibody fusion protein of the present invention against VEGF-A. 165 Inhibition of VEGFR-2 signaling mediated by VEGFR-2.

[0051] Figure 13A Figures B and B demonstrate the inhibitory effect of the antibody fusion protein, nevasulamab, and fareximab of the present invention on the Ang-2 / Tie-2 interaction.

[0052] Figure 14A Figures B and B demonstrate the inhibitory effect of the antibody fusion protein and nevasuzumab of the present invention on the Ang-1 / Tie-2 interaction.

[0053] Figure 15A Figures B and B show that the antibody fusion protein, tocilizumab, and vobarilizumab of the present invention block the binding of IL-6 to IL-6R.

[0054] Figure 16 The fusion protein B21138002 was shown to inhibit vascular leakage caused by DL-α-aminoadipic acid (DL-AAA)-induced preretinal neovascularization (PRN) in Dutch belted rabbits.

[0055] Figure 17 This presents a quantitative analysis of vascular leakage in the PRN induced by DL-AAA in Dutch Black Belt rabbits following intravitreal (IVT) injections of B21138002, aflibercept (Eylea), and fareximab.

[0056] Figure 18 This study demonstrates the effects of B21138002, aflibercept, and faraximab on inhibiting vascular leakage caused by laser-induced choroidal neovascularization (CNV) in monkeys.

[0057] Figure 19 This presents a quantitative analysis of changes in grade IV CNV lesions following IVT administration of B21138002, aflibercept, and fareximab.

[0058] Figure 20This presents a quantitative analysis of vascular leakage in laser-induced CNV in monkeys following IVT injection of B21138002, aflibercept, and fareximab. Detailed Implementation

[0059] The terms “protein,” “polypeptide,” and “peptide” are used interchangeably in this document to refer to polymers of amino acid residues.

[0060] Generally, the present invention provides a multispecific fusion protein or chimeric protein or its antigen-binding fragment or domain that can bind to or substantially bind to Ang-2, IL-6R and one or more VEGF family members; thereby reducing or inhibiting the signal transduction of Ang-2, IL-6 and VEGF family members.

[0061] In one aspect, the multispecific fusion protein or chimeric protein is an antibody fusion protein or chimeric protein.

[0062] In this disclosure, the term "antibody fusion protein" may be used in place of "antibody fusion protein or chimeric protein".

[0063] In one aspect, the antibody fusion protein or its antigen-binding fragment is a trispecific antibody fusion protein capable of binding to or substantially binding to Ang-2, IL-6R (in soluble and membrane-bound forms) and one or more VEGF family members; thereby reducing or inhibiting the signaling of Ang-2, IL-6 and VEGF family members.

[0064] In another aspect, the multispecific fusion protein or chimeric protein of the present invention comprises binding units that target Ang-2, IL-6R, and one or more VEGF family members and are linked together. These binding units comprise, or are substantially comprise, antigen-binding domains or portions thereof targeting Ang-2, IL-6R, and one or more VEGF family members. In another aspect, such multispecific fusion proteins or chimeric proteins are trispecific antibody fusion proteins or chimeric proteins.

[0065] In another aspect, the trispecific antibody fusion protein of the present invention does not bind to Ang-1. In another aspect, the trispecific antibody fusion protein of the present invention has an affinity for Ang-1 that is about 10% lower than its affinity for Ang-2. -3 times.

[0066] In another aspect, the trispecific 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.

[0067] In some implementations, at least one of the VEGF binding unit and the Ang-2 binding unit is directly linked to the antibody against IL-6R or its antigen-binding fragment or domain.

[0068] In some other embodiments, one of the VEGF binding unit and the Ang-2 binding unit is linked to the antibody against IL-6R or its antigen-binding fragment or domain via an insert polypeptide. Such insert polypeptides may contain an IgG1 Fc domain.

[0069] In another aspect, the Ang-2 binding unit comprises an antigen-binding fragment of an antibody against Ang-2, or a bioactive peptide that is bound to or substantially bound to Ang-2.

[0070] In another aspect, the VEGF binding unit included in the trispecific antibody fusion protein comprises Ig-like domains selected from the group consisting of Ig-like domains of one or more VEGF receptors. The VEGF binding unit binds to or substantially binds to at least one of the VEGF family members. In one aspect, these VEGF family members include VEGF-A, VEGF-B, and PlGF. In another aspect, these family members are VEGF-A, VEGF-B, and PlGF. Thus, in one aspect, the antibody fusion protein of the present invention can be considered at least as a trispecific construct that can bind to three different types of ligands involved in pathological angiogenesis and vascular leakage. In one aspect, 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.

[0071] In another aspect, the IL-6R is human IL-6R, the Ang-2 is human Ang-2, and the VEGFR-1 and VEGFR-2 are human VEGFR-1 and VEGFR-2.

[0072] In another aspect, 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 than all CDRs of the heavy and light chains of the IL-6R antibody.

[0073] In one aspect, the binding unit targeting Ang-2 comprises an sdAb or a bioactive peptide targeting Ang-2.

[0074] In another aspect, the trispecific antibody fusion protein of the present invention comprises the Fc domain of human IgG1.

[0075] In another aspect, the VEGF binding unit comprises human VEGFR-1-D2 directly or via a peptide linker linked to human VEGFR-2-D3. Such peptide linkers, when used, are preferably short peptide linkers, for example, having fewer than 20 amino acid residues. Peptide linkers are known in the art, for example, peptides containing glycine, serine, and / or threonine residues. Common peptide linkers comprise short sequences containing glycine and serine residues. The amino acid sequences of human VEGFR-1-D2 and VEGFR-2-D3 are shown below as SEQ ID NO:1 and SEQ ID NO:2.

[0076] In another embodiment, the VEGF binding unit comprises: (a) human VEGFR-1-D2; (b) human VEGFR-2-D3; and (c) an Fc domain of IgG1, wherein VEGFR-1-D2 is tandemly linked with VEGFR-2-D3. In some embodiments, the C-terminus of VEGFR-2-D3 is attached to the N-terminus of the Fc domain. In other embodiments, the C-terminus of VEGFR-1-D2 is attached to the N-terminus of the Fc domain. The amino acid sequence of the Fc domain of human IgG1 is shown below as SEQ ID NO:3.

[0077] On the other hand, sdAbs targeting Ang-2 or IL-6R, or their antigen-binding fragments or domains, contain the heavy chain variable region of a heavy chain antibody (VHH) targeting Ang-2 or IL-6R. These sdAbs can specifically bind to Ang-2 or IL-6R without requiring the complementary variable region found in conventional four-chain immunoglobulin molecules. sdAbs targeting Ang-2 are sometimes referred to herein as "Ang-2 sdAbs." sdAbs targeting IL-6R are sometimes referred to herein as "IL-6R sdAbs."

[0078] As disclosed herein, an "antigen-binding fragment or domain" of an antibody refers to a fragment or portion of the antibody that is capable of binding to or substantially binding to an antigen. In one embodiment, the antigen-binding fragment or domain of the antibody comprises, is substantially composed of, or is composed of a variable domain of the heavy chain (VH) of the antibody.

[0079] In some embodiments of the invention, the sdAb targeting Ang-2 or IL-6R comprises three CDRs of the antibody heavy chain, each CDR side-mounted with a framework domain. This sdAb lacks the CH1 domain of the antibody heavy chain. Despite having only three CDRs, the sdAb exhibits similar antigen-binding affinity and other effector functions compared to conventional antibodies containing six CDRs (three CDRs of the heavy chain and three CDRs of the light chain). Single-domain antibodies are described, for example, by Bathula et al. Cancer Biotherapy and Radiopharmaceuticals Volume 36, Issue 2, pp. 109-122 (2021).

[0080] The sdAbs for Ang-2 or IL-6R included in embodiments of the present invention are capable of reaching approximately 1 × 10⁻⁶ ppm. -6 M to 1×10 -12 The equilibrium dissociation constant (K) within the M range D It binds to its corresponding ligand (Ang-2 or IL-6R). In some embodiments, the sdAb targeting Ang-2 or IL-6R can bind at approximately 1 × 10⁻⁶ ppm. -7 M to approximately 1×10 -12 K within the range of M D It binds to its corresponding ligand. In some other embodiments, the sdAb targeting Ang-2 or IL-6R can bind at approximately 1 × 10⁻⁶. -8 M to approximately 1×10 -12 K within the range of M D It binds to its corresponding ligand. In some embodiments, sdAb targeting Ang-2 or IL-6R can bind at approximately 1 × 10⁻⁶ mmol / L. -9 M to approximately 1×10 -12 K within the range of M D It binds to its corresponding ligand.

[0081] First Embodiment of the Trispecific Fusion Protein of the Present Invention In one aspect, a first embodiment of the trispecific fusion protein of the present invention comprises: (a) an IL-6R binding unit comprising an antibody against IL-6-R (“IL-6R antibody”), said antibody comprising a heavy chain and a light chain; (b) a VEGF binding unit comprising VEGFR-1-D2 and VEGFR-2-D3 linked together in tandem; and (c) a polypeptide capable of binding Ang-2 (“Ang-2 binding polypeptide”), wherein the C-terminus of the VEGF binding unit is attached 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 attached to the N-terminus of the Ang-2 binding polypeptide. See Fig. 1. Alternatively, the C-terminus of the VEGF binding unit is attached 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.

[0082] 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 an 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.

[0083] Flexible linkers of various lengths can be used to link Ang-2 binding peptides to the C-terminus of the Fc domain of the heavy chain of an IL-6R antibody. Non-limiting examples of peptide linkers may include or consist of the following motifs: (GGGGS) x (x = 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). Specific examples of these flexible connectors are GGGGSGGGGSGGGGS, GGGGSGGGGS, and GGGGSGGGS.

[0084] The amino acid sequences disclosed or claimed herein also encompass conserved amino acid substitutions in these sequences that generally do not alter the biological activity of the protein or peptide. The most common substitutions 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, and these are bidirectional substitutions.

[0085] Non-limiting examples of IL-6R antibodies are tocilizumab (described, for example, in U.S. Patents 10,323,095; 7,479,543; and 5,795,965), sarilumab (described, for example, in U.S. Patent 7,582,298), and satralizumab (described, for example, in U.S. Patent 8,562,991). The amino acid sequences of the heavy and light chains of tocilizumab are shown in SEQ ID NO:4 and SEQ ID NO:5. Additional non-limiting examples of IL-6R antibodies are disclosed in U.S. Patents 8,753,634 (Apexigen, Inc.) and 8,748,581 (Ablynx NV). The foregoing patents are incorporated herein by reference in respect of their disclosures regarding IL-6R antibodies.

[0086] Non-limiting examples of Ang-2 binding peptides are shown in SEQ ID NO: 6 to SEQ ID NO: 11. Other Ang-2 binding peptides that can 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, the disclosures of which are incorporated herein by reference in respect of the Ang-2 binding peptides.

[0087] Nine trispecific 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 (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 tandem in sequence; and (c) an Ang-2 binding polypeptide selected from the group consisting of SEQ ID NO: 6 to SEQ ID NO: 11, wherein the C-terminus of the VEGF binding unit is attached to the N-terminus of the light chain of the tocilizumab; and the C-terminus of the heavy chain of the tocilizumab is attached to the N-terminus of the Ang-2 binding polypeptide.

[0088] The complete sequences of the heavy and light chains of these fusion proteins are disclosed in SEQ ID NO: 12 to SEQ ID NO: 29.

[0089] Other trispecific fusion proteins with the same binding unit can be constructed, wherein the C-terminus of the VEGF binding unit is linked to the N-terminus of the heavy chain of tocilizumab.

[0090] Other trispecific fusion proteins of the first embodiment can be constructed, wherein the IL-6R antibody comprises thalidomide or salidomide.

[0091] Other trispecific fusion proteins of the first embodiment can be constructed, wherein the IL-6R binding unit comprises: (1) CDR1, CDR2 and CDR3 (“HCCDR1”, “HCCDR2” and “HCCDR3”) of the heavy chain of tocilizumab (as shown in SEQ ID NO: 63, 64 and 65, respectively), which are linked to at least one of CH2 and CH3 of the Fc domain of IgG1; and (2) CDR1, CDR2 and CDR3 (“LCCDR1”, “LCCDR2” and “LCCDR3”) of the light chain of tocilizumab (as shown in SEQ ID NO: 66, 67 and 68, respectively).

[0092] Other trispecific fusion proteins of the first embodiment can be constructed, wherein the IL-6R binding unit comprises: (1) HCCDR1, HCCDR2 and HCCDR3 of thalidomide or salidomide, which are linked to at least one of CH2 and CH3 of the Fc domain of IgG1; and (2) LCCDR1, LCCDR2 and LCCDR3 of thalidomide or salidomide.

[0093] Second embodiment of the trispecific fusion protein of the present invention In one aspect, a second embodiment of the trispecific fusion protein of the present invention comprises: (a) an IL-6R binding unit comprising an IL-6R antibody, said antibody comprising a heavy chain and a light chain; (b) a VEGF binding unit comprising VEGFR-1-D2 and VEGFR-2-D3 linked in tandem; and (c) an Ang-2 sdAb (VHH) (“Ang-2 sdAb”), wherein the C-terminus of the VEGF binding unit is attached 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 attached to the N-terminus of the Ang-2 sdAb. See also Figure 2 Alternatively, the C-terminus of the VEGF binding unit is linked to the N-terminus of the heavy chain of the IL-6R antibody.

[0094] In another aspect, the IL-6R binding unit comprises: (1) CDR1, CDR2 and CDR3 of the heavy chain of the IL-6R antibody, which are 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.

[0095] The N-terminus of Ang-2 sdAb can be linked to the C-terminus of the Fc domain of IL-6R antibody using a flexible linker disclosed above.

[0096] Non-limiting examples of IL-6R antibodies are disclosed above.

[0097] The inventors of this case generated an Ang-2 sdAb in a research and development project, wherein the amino acid sequences of eight of them are shown in SEQ ID NO: 30 to SEQ ID NO: 37. Other Ang-2 sdAbs that can be used to construct the fusion protein of the second embodiment are disclosed, for example, in U.S. Patent 9,527,925 (Boehringer Ingelheim GmbH), the disclosure of which is incorporated herein by reference in respect of the Ang-2 sdAb disclosure.

[0098] Construct a trispecific fusion protein according to the second embodiment. Each fusion protein comprises: (a) a tocilizumab (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 tandem in sequence; and (c) an Ang-2 sdAb having an amino acid sequence selected from the group consisting of SEQ ID NO: 30 to SEQ ID NO: 37, wherein the C-terminus of the VEGF binding unit is attached to the N-terminus of the light chain of the tocilizumab; and the C-terminus of the heavy chain of the tocilizumab is attached to the N-terminus of the Ang-2 sdAb.

[0099] Two such fusion proteins (denoted as EB-105BI-c1 and EB-105BI-c2) were constructed, each containing Ang-2 sdAbs having SEQ ID NO: 31 and SEQ ID NO: 32, respectively.

[0100] The complete amino acid sequences of the heavy and light chains of EB-105BI-c1 and EB-105BI-c2 are shown in SEQ ID NO: 38 to SEQ ID NO: 41.

[0101] Other trispecific fusion proteins of the second embodiment can be constructed, wherein the IL-6R antibody comprises thalidomide or salidomide.

[0102] Other trispecific fusion proteins of the second embodiment can be constructed, wherein the IL-6R binding unit comprises: (1) HCCDR1, HCCDR2 and HCCDR3 of tocilizumab, which are linked to at least one of CH2 and CH3 of the Fc domain of IgG1; and (2) LCCDR1, LCCDR2 and LCCDR3 of tocilizumab.

[0103] Other trispecific fusion proteins of the second embodiment can be constructed, wherein the IL-6R binding unit comprises: (1) HCCDR1, HCCDR2 and HCCDR3 of thalidomide or salidomide, which are linked to at least one of CH2 and CH3 of the Fc domain of IgG1; and (2) LCCDR1, LCCDR2 and LCCDR3 of thalidomide or salidomide.

[0104] Third embodiment of the fusion protein of the present invention In one aspect, a third embodiment of the trispecific fusion protein of the present invention comprises two fusion polypeptides, each comprising: (a) an sdAb targeting IL-6R (“IL-6R sdAb”); (b) a VEGF binding unit comprising tandemly linked VEGFR-1-D2 and VEGFR-2-D3; (c) an Ang-2 binding polypeptide; and (d) an Fc domain of IgG1, wherein the C-terminus of the VEGF binding unit is attached to the N-terminus of the Fc domain, the N-terminus of the VEGF binding unit is attached to the C-terminus of the IL-6R sdAb, and the N-terminus of the Ang-2 binding polypeptide is attached to the C-terminus of the Fc domain. See also Figure 3A The N-terminus of the VEGF binding unit is directly or via a flexible linker to the C-terminus of the IL-6R sdAb. The N-terminus of the Ang-2 binding peptide is directly or via a flexible linker to the C-terminus of the Fc domain. The flexible linkers disclosed above or other flexible linkers known in the art can be used.

[0105] In another aspect, a third embodiment of the trispecific fusion protein of the present invention comprises two fusion polypeptides, each comprising: (a) an IL-6R sdAb; (b) a VEGF binding unit comprising tandemly linked VEGFR-1-D2 and VEGFR-2-D3; (c) an Ang-2 binding polypeptide; and (d) an Fc domain of IgG1, wherein the C-terminus of the VEGF binding unit is attached to the N-terminus of the Fc domain, the N-terminus of the VEGF binding unit is attached to the C-terminus of the Ang-2 binding polypeptide, and the N-terminus of the IL-6R sdAb is attached to the C-terminus of the Fc domain. See also Figure 3B The N-terminus of the VEGF binding unit is directly or via a flexible linker to the C-terminus of the Ang-2 binding peptide. The N-terminus of the IL-6R sdAb is directly or via a flexible linker to the C-terminus of the Fc domain. Flexible linkers disclosed herein can be used.

[0106] The IL-6R sdAb included in the two fusion peptides may be the same or different. The Ang-2 binding peptide included in the two fusion peptides may be the same or different.

[0107] The Ang-2 peptides disclosed above, including SEQ ID NO: 6 to SEQ ID NO: 11, can be used to construct the fusion protein of the third embodiment.

[0108] The inventors of this case generated an IL-6R sdAb in a research and development project, wherein the amino acid sequences of fifteen of them are shown in SEQ ID NO:42 to SEQ ID NO:56. Other IL-6R sdAbs that can be used to construct the fusion protein of the third embodiment are disclosed, for example, in U.S. Patents 10,618,964 (Ablynx NV); 8,753,634 (Apexigen, Inc.); and 8,748,581 (Ablynx NV), each of which is incorporated herein by reference in connection with the disclosure of the IL-6R sdAb.

[0109] Two trispecific fusion proteins (represented as EB-105BId1 and EB-105BId3) according to the third embodiment were constructed. The fusion proteins comprise two fusion polypeptides, each comprising: (a) an IL-6R sdAb having SEQ ID NO:42 (included in EB-105BId1) or SEQ ID NO:44 (included in EB-105BId3); (b) a VEGF binding unit comprising tandemly linked VEGFR-1-D2 (SEQ ID NO: 1) and VEGFR-2-D3 (SEQ ID NO: 2); (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 attached to the N-terminus of the Fc domain, the N-terminus of the VEGF binding unit is attached to the C-terminus of the IL-6R sdAb, and the N-terminus of the Ang-2 binding polypeptide is attached to the C-terminus of the Fc domain. The N-terminus of the VEGF binding unit is connected to the C-terminus of the IL-6R sdAb via a flexible linker. The N-terminus of the Ang-2 binding peptide is connected to the C-terminus of the Fc domain via a flexible linker.

[0110] 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.

[0111] Two trispecific fusion proteins of the present invention (denoted as EB-105BId2 and EB-105BId4) are constructed. The fusion proteins comprise two fusion polypeptides, each comprising: (a) an IL-6R sdAb having SEQ ID NO:42 (included in EB-105BId2) or SEQ ID NO:44 (included in EB-105BId4); (b) a VEGF binding unit comprising tandemly linked VEGFR-1-D2 (SEQ ID NO: 1) and VEGFR-2-D3 (SEQ ID NO: 2); (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 attached to the N-terminus of the Fc domain, the N-terminus of the VEGF binding unit is attached to the C-terminus of the Ang-2 binding polypeptide, and the N-terminus of the IL-6R sdAb is attached to the C-terminus of the Fc domain. The N-terminus of the VEGF binding unit is connected to the C-terminus of the Ang-2 binding peptide via a flexible linker. The N-terminus of the IL-6R sdAb is connected to the C-terminus of the Fc domain via a flexible linker.

[0112] 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.

[0113] Fourth embodiment of the fusion protein of the present invention In one aspect, a fourth embodiment of the trispecific fusion protein of the present invention comprises two fusion polypeptides, each comprising: (a) an IL-6R sdAb; (b) a VEGF binding unit comprising tandemly linked VEGFR-1-D2 and VEGFR-2-D3; (c) an Ang-2 sdAb; and (d) an Fc domain of IgG1, wherein the C-terminus of the VEGF binding unit is attached to the N-terminus of the Fc domain, the N-terminus of the VEGF binding unit is attached to the C-terminus of the Ang-2 sdAb, and the N-terminus of the IL-6R sdAb is attached to the C-terminus of the Fc domain. See also Figure 4A The N-terminus of the VEGF binding unit is connected directly or via a flexible connector to the C-terminus of the Ang-2 sdAb. The N-terminus of the IL-6R sdAb is connected directly or via a flexible connector to the C-terminus of the Fc domain. The flexible connectors disclosed above or other flexible connectors known in the art can be used.

[0114] In another aspect, a fourth embodiment of the trispecific fusion protein of the present invention comprises two fusion polypeptides, each comprising: (a) an IL-6R sdAb; (b) a VEGF binding unit comprising VEGFR-1-D2 and VEGFR-2-D3 linked in tandem; (c) an Ang-2 sdAb; and (d) an Fc domain of IgG1, wherein the C-terminus of the VEGF binding unit is attached to the N-terminus of the Fc domain, the N-terminus of the VEGF binding unit is attached to the C-terminus of the IL-6R sdAb, and the N-terminus of the Ang-2 sdAb is attached to the C-terminus of the Fc domain. See also Figure 4B The N-terminus of the VEGF binding unit is connected directly or via a flexible connector to the C-terminus of the IL-6R sdAb. The N-terminus of the Ang-2 sdAb is connected directly or via a flexible connector to the C-terminus of the Fc domain. The flexible connectors disclosed above can be used.

[0115] The IL-6R sdAb included in the two fusion peptides may be the same or different. The Ang-2 sdAb included in the two fusion peptides may be the same or different.

[0116] The Ang-2 sdAbs disclosed above, including SEQ ID NO: 30 to SEQ ID NO: 37, can be used to construct the fusion protein of the fourth embodiment.

[0117] The IL-6R sdAb disclosed above, including SEQ ID NO:42 to SEQ ID NO:56, can be used to construct the fusion protein of the fourth embodiment.

[0118] A fourth embodiment of a trispecific fusion protein (denoted as EB-105BIe1) 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 comprising tandemly linked VEGFR-1-D2 (SEQ ID NO:1) and VEGFR-2-D3 (SEQ ID NO:2); (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 connected to the N-terminus of the Fc domain, the N-terminus of the VEGF binding unit is connected to the C-terminus of the Ang-2 sdAb, and the N-terminus of the IL-6R sdAb is connected to the C-terminus of the Fc domain. The N-terminus of the VEGF binding unit is connected to the C-terminus of the Ang-2 sdAb via a flexible linker. The N-terminus of the IL-6R sdAb is connected to the C-terminus of the Fc structural domain via a flexible connector.

[0119] The complete amino acid sequence of the antibody fusion protein EB-105BIe1 is shown in SEQ ID NO: 61.

[0120] Another trispecific fusion protein (denoted as EB-105BIe2) according to 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 comprising VEGFR-1-D2 (SEQ ID NO:1) and VEGFR-2-D3 (SEQ ID NO:2) linked in tandem; (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 connected to the N-terminus of the Fc domain, the N-terminus of the VEGF binding unit is connected to the C-terminus of the IL-6R sdAb, and the N-terminus of the Ang-2 sdAb is connected to the C-terminus of the Fc domain. The N-terminus of the VEGF binding unit is connected to the C-terminus of the IL-6R sdAb via a flexible linker. The N-terminus of the Ang-2 sdAb is connected to the C-terminus of the Fc structural domain via a flexible connector.

[0121] The complete amino acid sequence of the antibody fusion protein EB-105BIe2 is shown in SEQ ID NO: 62.

[0122] The IL-6R binding unit included in the trispecific antibody fusion protein of the present invention can achieve a binding rate of approximately 1 × 10⁻⁶. -6 M to approximately 1×10 -12 K within the range of M D Combined with IL-6R. In some embodiments, the K D In approximately 1×10 -7 M to approximately 1×10 -12 Within the range of M. In some other embodiments, K... D In approximately 1×10 -8 M to approximately 1×10 -12 Within the range of M. In some implementations, the K... D In approximately 1×10 -9 M to approximately 1×10 -12 Within the M range. Therefore, the antibody fusion protein essentially inhibits the bioactivity of IL-6R in promoting angiogenesis and inflammation; thus, the pathogenesis lies in the abnormal angiogenesis and inflammatory symptoms.

[0123] The Ang-2 binding unit included in the trispecific antibody fusion protein of the present invention is capable of binding at approximately 1 × 10⁻⁶. -6 M to approximately 1×10 -12 K within the range of M D Combined with Ang-2. In some embodiments, the K D In approximately 1×10 -7 M to approximately 1×10 -12 Within the range of M. In some other embodiments, K... D In approximately 1×10 -8 M to approximately 1×10 -12 Within the range of M. In some implementations, the K... D In approximately 1×10 -9 M to approximately 1×10 -12 Within the M range. Therefore, the antibody fusion protein essentially inhibits the biological activity of Ang-2 in promoting angiogenesis and increasing vascular permeability; thereby controlling the pathological symptoms caused by abnormal angiogenesis and vascular leakage.

[0124] The VEGF binding unit included in the trispecific antibody fusion protein of the present invention is capable of binding at approximately 1 × 10⁻⁶ ppm. -6 M to approximately 1×10 -12 K within the range of M D It combines at least one of VEGF-A, VEGF-B, and PlGF. In some embodiments, the K... D In approximately 1×10 -7 M to approximately 1×10 -12 Within the range of M. In some other embodiments, K... DIn approximately 1×10 -8 M to approximately 1×10 -12 Within the range of M. In some other embodiments, K... D In approximately 1×10 -9 M to approximately 1×10 -12 Within the M range. Therefore, the antibody fusion protein substantially inhibits the angiogenesis-promoting biological activity of at least one of the VEGF family members; thereby controlling the pathological symptoms caused by abnormal angiogenesis and vascular leakage.

[0125] In some embodiments, the present invention also provides a binding construct comprising, or substantially comprising, a plurality of trispecific antibody fusion proteins or chimeric proteins described herein, which are linked or associated with each other by covalent bonds or other linkages, wherein the trispecific antibody fusion proteins of such binding constructs may be the same or different. Such binding constructs of the present invention are capable of binding with high affinity to at least one of Ang-2, IL-6R, and VEGF-A, VEGF-B, and PlGF. In cases where the trispecific antibody fusion proteins are different, each trispecific antibody fusion protein may comprise different binding units selected from the binding units disclosed herein targeting members of the Ang-2, IL-6R, or VEGF family.

[0126] Trispecific antibody fusion proteins or binding constructs may also include heteropeptides or other chemical motifs. These additions can alter their properties, such as stability, solubility, toxicity, serum half-life, immunogenicity, detectability, or other characteristics.

[0127] The term "high affinity" is used in the physiological context of the relative affinity of trispecific antibody fusion proteins for Ang-2, IL-6R, and members of the VEGF family in vivo in mammals, including laboratory test animals, domestic farm animals or pets, or humans. The trispecific antibody fusion proteins of this invention that bind to Ang-2, IL-6R, and members of the VEGF family can exhibit specific affinity for their ligands in vivo, typically expressed as an equilibrium dissociation constant (K0). D The sub-nanomolar concentration value of the protein was measured. For the purposes of this invention, the trispecific antibody fusion protein of this invention can be less than or equal to the K0 of the natural ligand / receptor pair. D Approximately 1, or approximately 5, or approximately 10, or approximately 50, or approximately 100, or approximately 500, or approximately 1000 times K D Combined with its target ligand.

[0128] The trispecific antibody fusion protein of the present invention can achieve a yield of approximately 1 × 10⁻⁶. -6 M to 1×10-12 The equilibrium dissociation constant (K) within the M range D ) combines with Ang-2, IL-6R, and at least one VEGF family member. In some implementations, K D The value is approximately 1×10 -7 M to approximately 1×10 -12 M, or approximately 1×10 -8 M to approximately 1×10 -12 M, or approximately 1×10 -9 M to approximately 1×10 -12 Within the range of M.

[0129] On the other hand, a trispecific antibody fusion protein may contain more than one of each of the Ang-2 binding unit, VEGF binding unit, and IL-6R binding unit.

[0130] In one aspect, the amino acid sequences of various non-limiting portions or embodiments of the trispecific antibody fusion protein of the present invention are listed in Table 1.

[0131] Table 1 amino acid sequence On the other hand, the nucleic acid sequences encoding the amino acid sequences in Table 1 are listed in Table 2.

[0132] Table 2 Nucleic acid sequence In another aspect, the trispecific 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 NO:1 to SEQ ID NO:72.

[0133] In another aspect, the trispecific 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 NO:1 to SEQ ID NO:72.

[0134] In another aspect, the present invention provides a trispecific fusion protein comprising a polypeptide having a pair of amino acid sequences selected from the group consisting of: SEQ ID NO: 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.

[0135] In another aspect, the present invention provides a trispecific fusion protein comprising a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 57-62.

[0136] In another aspect, one or more amino acid substitutions may be made in any of the amino acid sequences described above. Preferably, this substitution is a conservative substitution, wherein one amino acid from the following group is replaced by another amino acid from the same group: (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; and the choice of this substitution results in substantially maintaining the binding activity of the fusion protein. In one embodiment, the trispecific antibody fusion protein of the present invention having a conservative substitution is effective against K of Ang-2, IL-6R, VEGF-A, VEGF-B or PlGF ligands. D The value is less than the K that was replaced here. D The value is approximately 120%. Preferably, K D The value is less than the K that was replaced here. D The value is approximately 110%. More preferably, K D The value is less than the K that was replaced here. D The value is approximately 105%. More preferably, K D The value is less than the K that was replaced here. D The value is approximately 100%.

[0137] Furthermore, the amino acid sequences disclosed or claimed herein also encompass their “conserved variants,” which are the result of substitution, deletion, or addition of a single amino acid or a low percentage of amino acids (e.g., ≤5%, ≤4%, ≤3%, ≤2%, or ≤1%) in the original sequence of a peptide, polypeptide, or protein, wherein the substitution, deletion, or addition substantially does not alter the biological activity of the original peptide, polypeptide, or protein. For example, these conserved variants may retain approximately ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% of the biological activity of the original peptide, polypeptide, or protein.

[0138] Most conserved substitutions are not expected to cause fundamental changes in the characterization of the Ig-like domain or other domains in the fusion peptide. However, when the exact effect of the substitution is difficult to predict before substitution, those skilled in the art will understand that the effect can be evaluated by routine screening assays. For example, Ig-like domain or other domain variants are typically prepared by inducing site-specific mutations in the nucleic acid encoding the intact fusion peptide, expressing the variant nucleic acid in a recombinant cell culture, purifying the variant fusion peptide from the cell culture, and detecting the ability of the variant fusion peptide to specifically bind to Ang-2 IL-6R or the aforementioned VEGF ligands. Exemplary binding assays that can be used to determine whether one or more specific substitutions in the Ig-like domain or other domain affect the binding of the fusion peptide and inhibit the activity of Ang-2, IL-6R, or the aforementioned VEGF family members are described in Park et al. J. Biol. Chem. In the article , 269:25646-25654 (1994).

[0139] The VEGFR-1-D2 binding unit of the fusion protein can bind 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 free VEGF-A, VEGF-C and VEGF-D with high affinity (Stuttfeld et al., 15(18):4919(1996)). Life , 61(9):915(2009)). The Ang-2 binding unit and the IL-6R binding unit can respectively inhibit the activation of Tie-2 by Ang-2 and the activation of IL-6R by IL-6. Therefore, the fusion protein of the present invention can substantially inhibit the angiogenic activity of these growth factors on endothelial cells at disease sites.

[0140] In another aspect, the present invention provides isolated nucleic acid molecules that encode the trispecific antibody fusion protein.

[0141] In another aspect, the present invention provides an isolated nucleic acid molecule encoding a trispecific antibody fusion protein, wherein the isolated nucleic acid molecule comprises: (a) a nucleic acid sequence encoding an IL-6R binding unit comprising an IL-6R antibody; (b) a nucleic acid sequence encoding a VEGF binding unit comprising VEGFR-1-D2-VEGFR-2-D3, the nucleic acid sequence being operatively linked to the nucleic acid sequence encoding the IL-6R binding unit; and (c) a nucleic acid sequence encoding an Ang-2 binding polypeptide, the nucleic acid sequence being operatively linked to the nucleic acid sequence encoding the IL-6R binding unit.

[0142] In another aspect, the present invention provides isolated nucleic acid molecules that encode the trispecific antibody fusion protein of the first embodiment disclosed above.

[0143] In another aspect, the present invention provides an isolated nucleic acid molecule encoding the trispecific antibody fusion protein, wherein the isolated nucleic acid molecule comprises: (a) a nucleic acid sequence encoding the heavy and light chains of tocilizumab, having the sequences listed in SEQ ID NO:76 and SEQ ID NO:77; (b) a nucleic acid sequence encoding a VEGF binding unit comprising VEGFR-1-D2-VEGFR-2-D3, having the sequences listed in SEQ ID NO:73 and SEQ ID NO:74 and operatively linked 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 NO:78-83 and operatively linked to the 3' end of the nucleic acid sequence encoding the heavy chain of tocilizumab.

[0144] In another aspect, the present invention provides an isolated nucleic acid molecule encoding a trispecific antibody fusion protein, wherein the isolated nucleic acid molecule comprises: (a) a nucleic acid sequence encoding an IL-6R binding unit comprising an IL-6R antibody; (b) a nucleic acid sequence encoding a VEGF binding unit comprising VEGFR-1-D2-VEGFR-2-D3, the nucleic acid sequence being operatively linked to the nucleic acid sequence encoding the IL-6R binding unit; and (c) a nucleic acid sequence encoding an Ang-2 sdAb, the nucleic acid sequence being operatively 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 the Ang-2 sdAb is linked to the 3' end of the nucleic acid sequence encoding the heavy chain of the IL-6R antibody.

[0145] In another aspect, the present invention provides isolated nucleic acid molecules that encode the trispecific antibody fusion protein of the second embodiment disclosed above.

[0146] In another aspect, the present invention provides an isolated nucleic acid molecule encoding the trispecific antibody fusion protein, wherein the isolated nucleic acid molecule comprises: (a) a nucleic acid sequence encoding the heavy and light chains of tocilizumab, having the sequences listed in SEQ ID NO:76 and SEQ ID NO:77; (b) a nucleic acid sequence encoding a VEGF binding unit comprising VEGFR-1-D2-VEGFR-2-D3, having the sequences listed in SEQ ID NO:73 and SEQ ID NO:74 and operatively linked 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 NO:102-109 and operatively linked to the 3' end of the nucleic acid sequence encoding the heavy chain of tocilizumab.

[0147] In another aspect, the present invention provides an isolated nucleic acid molecule encoding a trispecific antibody fusion protein of a third embodiment, wherein the isolated nucleic acid molecule comprises: (a) a nucleic acid sequence encoding IL-6R sdAb having a sequence selected from the group consisting of SEQ ID NO:114-128; (b) a nucleic acid sequence encoding a VEGF binding unit comprising tandemly linked VEGFR-1-D2 and VEGFR-2-D3 having the sequences listed in SEQ ID NO: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 NO:78-83; and (d) a nucleic acid sequence encoding the Fc domain of IgG1, wherein the 3' end of the nucleic acid sequence encoding the VEGF binding unit is operatively linked to the N-terminus of the nucleic acid encoding the Fc domain, and the 5' end of the nucleic acid sequence encoding the VEGF binding unit is operatively linked to the nucleic acid encoding IL-6R. The 3' end of the sdAb nucleic acid sequence and the 5' end of the nucleic acid sequence encoding the Ang-2 binding polypeptide are operatively linked to the 3' end of the nucleic acid sequence encoding the Fc domain.

[0148] In another aspect, the present invention provides an isolated nucleic acid molecule encoding another trispecific antibody fusion protein according to a third embodiment, wherein the isolated nucleic acid molecule comprises: (a) a nucleic acid sequence encoding IL-6R sdAb selected from the group consisting of SEQ ID NO:114-128; (b) a nucleic acid sequence encoding VEGF binding units comprising tandemly linked VEGFR-1-D2 and VEGFR-2-D3, having the sequences listed in SEQ ID NO:73 and 74; (c) a nucleic acid sequence encoding an Ang-2 binding polypeptide selected from the group consisting of SEQ ID NO:78-83; and (d) a nucleic acid sequence encoding the Fc domain of IgG1 listed in SEQ ID NO:75, wherein the 3' end of the nucleic acid sequence encoding the VEGF binding unit is operatively linked 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 operatively linked to the 3' end of the nucleic acid sequence encoding the Ang-2 binding polypeptide, and encoding IL-6R The 5' end of the sdAb nucleic acid sequence is operatively linked to the 3' end of the nucleic acid sequence encoding the Fc domain.

[0149] In another aspect, the present invention provides an isolated nucleic acid molecule encoding one of the two chains of a trispecific antibody fusion protein according to a fourth embodiment, wherein the isolated nucleic acid molecule comprises: (a) a nucleic acid sequence encoding IL-6RsdAb, selected from the group consisting of SEQ ID NO:114-128; (b) a nucleic acid sequence encoding a VEGF binding unit comprising tandemly linked VEGFR-1-D2 and VEGFR-2-D3, having the sequences listed in SEQ ID NO:73 and 74; (c) a nucleic acid sequence encoding Ang-2 sdAb, selected from the group consisting of SEQ ID NO:102-109; and (d) a nucleic acid sequence encoding the Fc domain of IgG1, as listed in SEQ ID NO:75, wherein the 3' end of the nucleic acid sequence encoding the VEGF binding unit is operatively linked 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 operatively linked to the 3' end of the nucleic acid encoding Ang-2 sdAb, and encoding IL-6R The 5' end of the sdAb nucleic acid sequence is operatively linked to the 3' end of the nucleic acid encoding the Fc domain.

[0150] In another aspect, the present invention provides an isolated nucleic acid molecule encoding one of the two chains of another trispecific antibody fusion protein according to a fourth embodiment, wherein the isolated nucleic acid molecule comprises: (a) a nucleic acid sequence encoding IL-6R sdAb selected from the group consisting of SEQ ID NO:114-128; (b) a nucleic acid sequence encoding VEGF binding units comprising tandemly linked VEGFR-1-D2 and VEGFR-2-D3, having the sequences listed in SEQ ID NO:73 and 74; (c) a nucleic acid sequence encoding Ang-2 sdAb selected from the group consisting of SEQ ID NO:102-109; and (d) a nucleic acid sequence encoding the Fc domain of IgG1 listed in SEQ ID NO:75, wherein the 3' end of the nucleic acid sequence encoding the VEGF binding unit is operatively linked 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 operatively linked to the 3' end of the nucleic acid encoding IL-6R sdAb, and encoding Ang-2 The 5' end of the sdAb nucleic acid sequence is operatively linked to the 3' end of the nucleic acid encoding the Fc domain.

[0151] In another aspect, the present invention provides isolated nucleic acid molecules encoding the trispecific antibody fusion protein or chimeric protein of the present invention, wherein the isolated nucleic acid molecule comprises a nucleic acid sequence in which one or more codons differ from the nucleic acid sequences listed in this disclosure due to the degeneracy of the genetic code. Such different nucleic acid sequences are within the scope of the present invention.

[0152] In another aspect, the present invention provides a vector comprising any of the nucleic acid molecules disclosed herein, including an expression vector comprising any of the nucleic acid molecules operatively linked to an expression control sequence.

[0153] In another embodiment, the vector contains the nucleic acid sequences encoding trispecific antibody fusion proteins listed in SEQ ID NO:84-101, 110-113 and 129-134.

[0154] In another aspect, the present invention provides a host-vector system for generating any of the three-specific antibody fusion proteins or chimeric proteins, the host-vector system comprising an expression vector suitable for host cells.

[0155] In one aspect, the present invention provides the construction of a nucleic acid molecule encoding a trispecific antibody fusion protein disclosed herein, said nucleic acid molecule being inserted into a vector that, when introduced into a suitable host cell, is 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 a vector can be used to construct an expression vector encoding a chimeric polypeptide molecule under the control of transcription / translation control 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).

[0156] The expression of the nucleic acid molecule encoding the antibody fusion protein of the present invention can be regulated by a second nucleic acid sequence (promoter) to enable the expression of the antibody fusion protein in a host transformed by the nucleic acid molecule. For example, the expression of the antibody fusion protein described herein can be controlled by any promoter / enhancer element known in the art.

[0157] Generally, plasmid vectors containing replicons and control sequences derived from a species compatible with the host cell are used in conjunction with these hosts. The vectors typically carry replication sites as well as marker sequences that provide phenotypic selection in the transformed cells. For example, *E. coli* is often transformed using pBR322 (a plasmid derived from the *E. coli* species) (see, for example, Bolivar et al., ...). Gene , 2:95 (1977). The plasmid pBR322 contains ampicillin and tetracycline resistance genes and thus provides a simple method for identifying transformed cells. The pBR322 plasmid, or other microbial plasmids or phages, must also contain or be modified to contain promoters that can be used by microorganisms to express proteins.

[0158] 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)). Although these promoters are the most commonly used, other microbial promoters have also been discovered and utilized. For example, the TAC promoter is a synthetic DNA promoter produced by combining promoters from the TRP and LC operons (de Boer et al., 8:4057 (1980)). PNAS , (1983-01-80 (1):21-25 (1983)). It is commonly used for protein production in Escherichia coli. (Amann et al., Gene , 25:167 (1983). All of these promoters can be used in conjunction with the methods for producing the antibody fusion proteins of the present invention.

[0159] Besides prokaryotes, eukaryotic microorganisms, such as yeast cultures, can also be used. Among eukaryotic microorganisms, *Saccharomyces cerevisiae* or common baking yeast are the most commonly used, but many other strains can also be used. For expression in yeast, plasmids such as YRp7 (Stinchcomb et al.) are commonly used. Nature , 282:39(1979)). Other exemplary plasmids are disclosed in U.S. Patent 4,615,974; Struhl et al., PNAS ,76(3):1035(1979). Plasmid YRp7 contains the trp1 gene, which provides a selection marker for mutant yeast strains (e.g., ATCC No. 44,076 or RH218) lacking the ability to grow in tryptophan-free conditions (Jones, 76(3):1035(1979). Genetics , 85:23(1977)). The presence of trp1 lesions, a characteristic of the yeast host cell genome, then provides an efficient environment for detecting transformation by growing the yeast in the absence of tryptophan.

[0160] Suitable promoter sequences for yeast vectors include 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, 255:2073 (1980)), Yeast , 8:423 (1992); Weinhandl et al., Microb. Cell FactoriesThe promoters of these genes (13:5 (2014)) are used. When constructing suitable expression plasmids, the termination sequences associated with these genes are also ligated to the 3' end of the sequence to be expressed in the expression vector to provide polyadenylation and termination of the mRNA. Other promoters with the added advantage of transcription being controlled by growth conditions, such as the promoter regions of alcohol dehydrogenase 2 and enzymes responsible for maltose and galactose utilization (Romanos et al., Weinhandl et al., see above), can also be used for vector construction. Any plasmid vector containing a yeast-compatible promoter, origin of replication, and termination sequence is suitable.

[0161] Besides microorganisms, cell cultures derived from multicellular organisms can also be used as hosts. In principle, any such cell culture can be used, whether from vertebrates or invertebrates. However, vertebrate cells are of greatest interest, and in recent years, the propagation of vertebrate cells in cultures (tissue cultures) has become a routine procedure. Examples of these useful host cell lines include VERO and HeLa cells, the Chinese hamster ovary (CHO) cell line, and the W138, BHK, COS-7, HEK293, and MDCK cell lines. Expression vectors used for these cells typically include (if necessary) an origin of replication, a promoter preceding the gene to be expressed, and any necessary ribosome binding sites, RNA splicing sites, polyadenylation sites, and transcription terminator sequences.

[0162] For use in mammalian cells, the control over the expression vector is typically provided by viral material. Common promoters, for example, are derived from polyomaviruses, adenovirus 2, and most commonly, simian virus 40 (SV40). Early and late promoters of SV40 are particularly useful because both are readily available from the virus in fragment form, which also contains the SV40 viral origin of replication (Fiers et al.). Nature , 273:113 (1978). Smaller or larger SV40 fragments can also be used, as long as they include a sequence of about 250 bp extending from the HindIII site toward the BglI site located at the origin of viral replication. Alternatively, it is possible to utilize promoters or control sequences that are usually associated with the desired gene sequence, and are generally desirable, provided that these control sequences are compatible with the host cell system.

[0163] Therefore, according to the present invention, a host is transfected with an expression vector capable of replicating in bacterial, yeast, insect, or mammalian cell hosts and containing nucleic acids encoding the antibody fusion protein described herein, and thereby these nucleic acids are directly expressed to produce a fusion polypeptide, which can then be recovered in a biologically active form. As used herein, the biologically active form includes a form capable of binding at least one member of the VEGF family.

[0164] In some embodiments, the host cell may be *Escherichia coli*, COS cells, HEK 293 cells (also referred to as 293 cells), or Chinese hamster ovary (“CHO”) cells. Preferably, the host cell is HEK 293 or CHO cells.

[0165] A non-restrictive example is plasmid pcDNA3.4, which is suitable for mammalian host cells, such as CHO cells. Plasmid pcDNA3.4 contains the ampicillin resistance gene as well as genes for the SV40 and CMV promoters.

[0166] Carrier construction Suitable vectors containing the desired coding and control sequences are constructed using standard ligation techniques. Isolated plasmids or DNA fragments are lysed, customized, and ligated in the desired form to form the desired plasmid. The methods used are independent of the DNA source or intended host. Lysis is performed by treatment with one or more restriction enzymes in a suitable buffer.

[0167] The nucleic acid sequence 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 6,897,294.

[0168] In the first embodiment, the nucleic acid sequences encoding the Ig-like domain 2 of VEGFR-1 and the Ig-like domain 3 of VEGFR-2 are tandemly linked in the desired order. This construct is then linked to the 5' end of the nucleic acid sequence encoding the light chain of an IL-6R antibody (e.g., tocilizumab). The nucleic acid sequence encoding Ang-2 binding is linked to the 3' end of the nucleic acid sequence encoding the heavy chain of the IL-6R antibody. These entire nucleic acid sequences are referred to as a chimeric nucleic acid sequence.

[0169] Next, the entire chimeric nucleic acid sequence is placed in a reading frame containing the gene and compatible with the proposed host cell within a vector containing a promoter. Various plasmids, such as those described in U.S. Patents 4,456,748, 5,460,811, 5,888,808, and 6,333,147, can be used to generate the antibody fusion protein of the present invention.

[0170] In one aspect, the vector system pcDNA3.4 is adapted to express the antibody fusion protein of the present invention in mammalian cells.

[0171] In one embodiment, the antibody fusion protein of the present invention can be produced according to the method described in U.S. Patent 7,070,959. For example, the chimeric nucleic acid sequences of SEQ ID NO: 84 and 85 are inserted into the expression vector pcDNA3.4 having a CMV promoter.

[0172] In one embodiment, CHO cells are transfected with pcDNA3.4 / SEQ ID NO:84 and 85. The antibody fusion protein obtained from CHO cells can be purified and characterized by binding assays, as described in U.S. Patent 7,070,959.

[0173] Similarly, a nucleic acid molecule encoding another trispecific antibody fusion protein described above can be generated by linking nucleic acid sequences encoding various desired ligand-binding units in the desired order and then inserting them into the expression vector pcDNA3.4. CHO cells were transfected with this vector and allowed to grow. The antibody fusion proteins obtained from these CHO cells can be purified and characterized in a similar manner.

[0174] In one embodiment, the antibody fusion protein of the present invention can K D ≤10 -9 M binds to human Ang-2 (“hAng-2”), human IL-6R (“hIL-6R”), and VEGF family members. In another embodiment, the antibody fusion protein of the present invention can K D ≤5×10 -10 M binds to hAng-2, hIL-6R, and VEGF family members. In another embodiment, the antibody fusion protein of the present invention can K D ≤10 -10 M binds to hAng-2, hIL-6R, and VEGF family members.

[0175] In one aspect, the present invention provides compounds, compositions, and methods for treating or controlling diseases, symptoms, or conditions caused by abnormal angiogenesis and inflammation.

[0176] In another aspect, the present invention provides a method for treating or controlling at least one ocular or systemic disease, symptom, or condition in a subject whose cause is abnormal angiogenesis and inflammation. The method comprises administering to the subject requiring this treatment or control a composition comprising a trispecific antibody fusion protein disclosed herein. Non-limiting embodiments of such antibody fusion proteins have the amino acid sequences set forth in SEQ ID NO: 12-29, 38-41, and 57-62.

[0177] In another aspect, the present invention provides a composition for treating or controlling at least one ocular or systemic disease, symptom, or condition in a subject whose etiology is abnormal angiogenesis and inflammation, wherein the composition comprises the trispecific antibody fusion protein disclosed herein. Non-limiting embodiments of such antibody fusion proteins have the amino acid sequences listed in SEQ ID NO:12-29, 38-41, and 57-62.

[0178] In another aspect, the present invention provides the use of the trispecific antibody fusion protein disclosed herein for the preparation of pharmaceutical compositions or agents for the treatment or control of at least one ocular or systemic disease, symptom, or condition in subjects caused by abnormal angiogenesis and inflammation. Non-limiting embodiments of such antibody fusion proteins have the amino acid sequences listed in SEQ ID NO:12-29, 38-41, and 57-62.

[0179] In another aspect, the eye disease, symptom, or condition is selected from the group consisting of: macular edema caused by diabetes, uveitis, central and branch retinal vein occlusion, choroidal neovascularization, neovascular age-related macular degeneration (wet age-related macular degeneration), polypoid choroidal angiopathy (“PCV”), myopic choroidal neovascularization, vascular leakage, non-proliferative and proliferative diabetic retinopathy, retinopathy of prematurity, corneal neovascularization, corneal inflammation, and neovascular glaucoma.

[0180] In one embodiment, a dose of approximately 25-4000 micrograms of the fusion protein is administered to the subject. In another embodiment, a dose of approximately 50-8000 micrograms, approximately 100-8000 micrograms, approximately 500-8000 micrograms, approximately 1000-8000 micrograms, approximately 2000-8000 micrograms, approximately 50-6000 micrograms, approximately 50-5000 micrograms, approximately 50-4000 micrograms, approximately 50-3000 micrograms, approximately 50-2000 micrograms, or approximately 50-1000 micrograms of the fusion protein is administered to the subject.

[0181] In another aspect, the composition comprising the fusion protein is in the form of eye drops or ophthalmic injections (e.g., intravitreal, intra-anterior chamber, periorbital, subfascial, subretinal, or suprachoroidal injections). Such compositions comprise ophthalmic compositions. The antibody fusion protein of the present invention can also be incorporated into a medical device that can be implanted in or near diseased tissue.

[0182] In one embodiment, the present invention provides a method or composition for treating or controlling anterior segment diseases, symptoms, or conditions, such as corneal neovascularization, keratitis, or neovascular glaucoma. The composition containing the fusion protein may be in the form of eye drops or intra-anterior chamber or subconjunctival injections. In another embodiment, the present invention provides a method or composition for treating or controlling posterior segment diseases, symptoms, or conditions, such as choroidal neovascularization, neovascular age-related macular degeneration (wet age-related macular degeneration), polypoid choroidal angiopathy (“PCV”), myopic choroidal neovascularization, vascular leakage, macular edema due to diabetes, uveitis, central and branch retinal vein occlusion, non-proliferative and proliferative diabetic retinopathy, and retinopathy of prematurity. In this case, the composition containing the fusion protein may be administered as an intravitreal injection.

[0183] In another case, the eye drops are administered to the subject at least once a day, at least once a week, or at least once a month until the disease, symptom, or condition is substantially treated or controlled.

[0184] In another embodiment, the composition is administered to the subject via sustained drug release for a period of at least one month, at least two months, at least three months, or at least six months.

[0185] In another embodiment, intravitreal injections, or injections in or near diseased tissue, are administered to the subject according to a protocol recommended by a medical professional for the specific patient. For example, the injection 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, symptom, or condition is substantially treated or controlled. In one implementation, treatment may be administered at a higher frequency initially, followed by a lower frequency after a period of time. This period of time may be determined by the medical professional.

[0186] The concentration of the antibody fusion protein of the present invention in such ophthalmic compositions 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 about 1 to about 160 mg / ml, or about 0.5 to about 100 mg / ml, or about 1 to about 80 mg / ml).

[0187] In another aspect, a method for preparing the composition of the present invention comprises combining: (a) an amount of the antibody fusion protein of the present invention; and (b) a physiologically acceptable carrier.

[0188] In one embodiment, such physiologically acceptable carriers may be sterile saline solutions or physiologically acceptable buffer solutions. In another embodiment, such carriers comprise hydrophobic media, such as pharmaceutically acceptable oils. In yet another embodiment, such carriers comprise emulsions of hydrophobic materials and water. In yet another embodiment, the antibody fusion protein of the present invention may be conjugated or linked to high molecular weight materials to provide long cycling times.

[0189] 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 at pH 7.4 contains 3 g / L tris(hydroxymethyl)aminomethane and 0.76 g / L HCl. In another case, the buffer is 10× phosphate-buffered saline (“PBS”) or a 5× PBS solution. Non-limiting examples of buffers for injectable compositions containing biological agents include phosphate, citric acid, acetic acid, thiamethoxam, histidine, arginine, gluconic acid, lactate, tartaric acid, aspartic acid, and glutamate buffers.

[0190] Other buffers may also be suitable or appropriate in certain situations, such as those based on pK at 25°C. a HEPES (N-{2-hydroxyethyl}piperazine-N'-{2-ethanesulfonic acid}) with a pH of 7.5 and a pH range of 6.8-8.2; pK at 25°C a BES (N,N-bis{2-hydroxyethyl}-2-aminoethanesulfonic acid) with a pH of 7.1 and a pH range of 6.4-7.8; pK at 25°C a MOPS (3-{N-morpholino}propanesulfonic acid) with a pH of 7.2 and a pH range of 6.5-7.9; pK at 25°C a TES (N-tris(hydroxymethyl)-2-aminoethanesulfonic acid) with a pH of 7.4 and a pH 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 pH range of 6.9-8.3; pK at 25°C a DIPSO (3-(N,N-bis{2-hydroxyethyl}amino)-2-hydroxypropane) with a pH of 7.52 and a pH range of 7-8.2; pK at 25°C aTAPSO (2-hydroxy-3{tris(hydroxymethyl)methylamino}-1-propanesulfonic acid) with a pH of 7.61 and a pH range of 7-8.2.

[0191] In some embodiments, the compositions of the present invention are formulated in a buffer solution having an acidic pH value, for example, from about 4 to about 6.8, or alternatively from about 5 to about 6.8. In these embodiments, the buffering capacity of the composition desirably allows the composition to rapidly reach a physiological pH value after administration to a patient.

[0192] In addition to buffer solutions, the compositions of the present invention may contain materials selected from the group consisting of surfactants, stabilizers, preservatives, cosolvents, humectants, emollients, chelating agents, tension modifiers, and antioxidants.

[0193] In one aspect, all of these materials that can be used in the compositions of the present invention are physiologically acceptable materials. In some embodiments, all of these materials that can be used in the compositions of the present invention are ocularly or systemically acceptable materials.

[0194] Water-soluble preservatives that can be used include quaternary ammonium compounds, such as benzalkonium chloride and various polyquaternary ammonium compounds. These agents may be present individually in amounts of about 0.001% by weight to about 2% by weight (preferably about 0.01% by weight to about 0.05% by weight).

[0195] Non-limiting 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 (e.g., polymers sold under the trademark Tyloxapol), poly(oxyethylene)-poly(oxypropylene) block copolymers, glycolates of fatty acids, and mixtures thereof.

[0196] In one aspect, the pH value of the composition is in the range of about 4 to about 8. Alternatively, the pH value 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.

[0197] In another aspect, the pH value of the composition is about 7. Alternatively, the pH value of the composition is in the range of about 7 to about 7.5.

[0198] In another aspect, the pH value of the composition is approximately 7.4.

[0199] In another aspect, the composition may also comprise a viscosity-modifying compound designed to facilitate the application of the composition to a subject or to enhance bioavailability in a subject. In yet another aspect, the choice of viscosity-modifying compound may make the composition less easily dispersed upon application to the ocular environment. These compounds can enhance the viscosity of the composition and 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 sodium hydroxypropyl methylcellulose (“HPMC”), sodium carboxymethyl cellulose (“CMC”), and hydroxypropyl cellulose (“HPC”); polysaccharides, such as hyaluronic acid and its salts, chondroitin sulfate and its salts, and polydextrose, such as polydextrose 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 polymers. Generally, the required viscosity can be in the range of about 1 to about 400 centipoise (“cp”) or mPa.s.

[0200] Non-limiting examples of chelating agents include ethylenediaminetetraacetic acid (“EDTA”), diethylenetriaminepenta (methylphosphonic acid), etidronic acid, and tetrasodium etidronic acid (also known as “HAP”).

[0201] Although the buffer solution itself is a "tonolator" and "pH adjuster" that substantially maintains the ophthalmic solution at a specific ion concentration and pH value, additional "tonolators" may be added to adjust the final tension of the solution. These tonicators are well known to those skilled in the art and include, but are not limited to, mannitol, sorbitol, dextrose, sucrose, urea, propylene glycol, and glycerol. Additionally, various salts, including halide salts of monovalent cations (e.g., NaCl or KCl), may be used. Typically, the formulations of the present invention have a tension in the range of about 200 to 400 mOsm / kg. Alternatively, the formulations of the present invention have a tension in the range of about 220 to 400 mOsm / kg, or about 220 to 350 mOsm / kg, or about 220 to 300 mOsm / kg, or about 250 to 350 mOsm / kg.

[0202] Non-limiting examples of antioxidants include ascorbic acid (vitamin C) and its salts and esters; tocopherols (e.g., α-tocopherol) and tocotrienols (vitamin E) and their salts and esters (e.g., vitamin E TGPS (D-α-tocopherol polyethylene glycol 1000 succinate)); glutathione; lipoic acid; uric acid; butylated hydroxyanisole (“BHA”); butylated hydroxytoluene (“BHT”); tert-butylhydroquinone (“TBHQ”); and polyphenolic antioxidants (e.g., gallic acid, cinnamic acid, flavonoids and their salts, esters and derivatives).

[0203] Non-limiting examples of stabilizers include sucrose, mannitol, sorbitol, and trehalose.

[0204] It should be understood that the proportions of various components or mixtures can be adjusted as appropriate.

[0205] In another aspect, the antibody fusion protein of the present invention and appropriate amounts of one or more desired excipients are incorporated into a formulation for surface application or injection into a portion of the eye, such as the anterior or posterior segment, or a vitreous solution. The injectable formulation may desirablely include a carrier that provides sustained release of the active ingredient, for example, for a period longer than about one week (or longer than about one, two, three, four, five, or six months). In some embodiments, the antibody fusion protein of the present invention is included in a delivery device to sustain release of the active ingredient over a longer period, such as four, five, six months, or longer. Examples of such delivery devices are described in U.S. Patents 8,399,006 and 9,417,238.

[0206] In another aspect, a composition comprising the antibody fusion protein of the present invention and the desired excipient is lyophilized and substantially reconstituted with a physiologically acceptable liquid carrier just before administration to a subject.

[0207] In one embodiment, the compounds or compositions of the present invention can be injected using, for example, a 25-35 gauge fine 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 patient. In one aspect, the antibody fusion protein has an amino acid sequence selected from the group consisting of SEQ ID NO: 12-29, 38-41, and 57-62, and their conserved variants. The concentration of this antibody fusion protein is selected from the range disclosed above. Other antibody fusion proteins comprising the various Ang-2 binding units, VEGF binding units, and IL-6R binding units disclosed herein may also be incorporated into the compositions disclosed herein.

[0208] In another aspect, the antibody fusion protein of the present invention is incorporated into an ophthalmic device comprising a biodegradable material, and said device is implanted in the posterior segment of a subject's eye to provide long-term (e.g., longer than about 1 week or longer than about 1, 2, 3, 4, 5, or 6 months) treatment or control of angiogenic diseases, symptoms, or conditions. Such devices can be implanted in the eye or periocular tissue of a subject by a skilled physician. Non-limiting examples of ophthalmic implantation systems or devices for sustained release of the active ingredient 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.

[0209] In another aspect, a method for treating or controlling ocular angiogenesis diseases, symptoms, or conditions includes administering a composition comprising the antibody fusion protein of the present invention to a subject in need.

[0210] In another aspect, a method for treating or controlling ocular angiogenesis disorders, symptoms, or conditions comprises administering to a subject in need of such treatment or control a composition comprising an antibody fusion protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 12-29, 38-41, and 57-62, and their conserved variants. Other antibody fusion proteins comprising the various Ang-2 binding units, VEGF binding units, and IL-6R binding units disclosed herein may also be used in this method.

[0211] In another aspect, a method for treating or controlling ocular angiogenesis diseases, symptoms, or conditions caused by abnormal angiogenesis in the posterior segment of the eye comprises intravitreal injection of a composition comprising an antibody fusion protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 12-29, 38-41, and 57-62, and conservatively modified variants thereof.

[0212] In another embodiment, the disease, symptom, or condition is selected from the group consisting of: macular edema caused by diabetes; uveitis; central and branch retinal vein occlusion; choroidal neovascularization, including neovascular age-related macular degeneration (wet age-related macular degeneration), polypoid choroidal angiopathy (PCV), and myopic choroidal neovascularization; vascular leakage; non-proliferative and proliferative diabetic retinopathy; retinopathy of prematurity; corneal neovascularization; corneal inflammation; and neovascular glaucoma.

[0213] In another aspect, the compositions of the present invention may be applied once a week, once a month, once a year, twice a year, three times a year, four times a year, or at a frequency determined to be appropriate for the treatment or control of anterior segment inflammatory diseases, symptoms, or conditions.

[0214] In another aspect, the antibody fusion protein of the present invention can also be used to treat or control tumors, systemic inflammatory diseases or conditions, or autoimmune diseases such as arthritis. This treatment or control can be achieved, for example, through systemic administration. Dosage and regimens for treating specific diseases or conditions can be determined or recommended by medical personnel.

[0215] Example 1: Expression and purification of the fusion protein of the present invention The fusion protein of the present invention was successfully expressed in Chinese hamster ovary (“CHO”) cells. Most of the proteins produced were purified to >95% purity by a single round of affinity chromatography.

[0216] cDNA encoding the amino acid sequences of the fusion proteins identified in Table 3 was synthesized, and expression vectors for each cDNA were constructed based on the circular pcDNA3.4 vector system. These expression vectors were transiently transfected into CHO cells in a chemically defined culture medium. The resulting proteins were purified by ultrafiltration using a protein-A affinity column, followed by sterile filtration at 0.2 µm to obtain a high-purity stock solution. After one round of affinity chromatography purification, the purity was in the range of 82.9% to 100%, and the analysis was performed by size exclusion chromatography (SEC-HPLC).

[0217] Table 3 Protein yield and purity Example 2: ELISA-ascertained activity against human VEGF-A 165 Combination affinity Used coated recombinant human VEGF-A 165 96-well plates (4 µg / ml, 50 µl per well) were used for ELISA assays at +4°C for 16 hours. After nonspecific blocking with 1% BSA at 25°C for 1 hour, serially diluted test antibodies (“Ab”) were added to the coated wells and incubated at 25°C for 1 hour. The bound Abs were detected using a secondary Ab (goat anti-human IgG1-Fc) bound to HRP, and OD was subsequently read. 450 Value. EB-105 series molecules' effect on human VEGF-A 165 Its binding affinity reaches sub-nanomolar concentrations and is comparable to that of aflibercept, fareximab, and conbercept. See also Figure 6A -C. Aflibercept and Conbercept are two human Fc fusion proteins that bind to VEGF-A, VEGF-B, and PlGF. Faraximab is a bispecific antibody that binds to VEGF-A and Ang-2.

[0218] Example 3: The effects of B21138001-B21138009, as determined by ELISA, on human VEGF-B 167 Combination affinity Using recombinant human VEGF-B 167 Perform the same ELISA procedure. The EB-105 series of molecules are effective against human VEGF-B. 167 Its binding affinity reaches the sub-nanomolar concentration level and is comparable to that of aflibercept and conbercept. See also Figure 7 .

[0219] Example 4: Binding affinity of B21138001-B21138009 to human PlGF determined by ELISA The same ELISA procedure was performed using recombinant human PlGF. The EB-105 series molecules exhibit sub-nanomolar binding affinity for human PlGF, comparable to aflibercept and conbercept. See also Figure 8A And B.

[0220] Example 5: Binding affinity to human IL-6R determined by ELISA The same ELISA procedure was performed using recombinant human IL-6R. The EB-105 series of molecules exhibit sub-nanomolar binding affinity for human IL-6R, comparable to tocilizumab and vorbalizumab. Tocilizumab is a clinical-stage mAb targeting IL-6R. Vorbalizumab is a single-domain antibody targeting IL-6R. B781402 and B781405 are bispecific fusion antibodies inhibiting VEGF-A, VEGF-B, P1GF, and IL-6R. See also... Figure 9A -C.

[0221] Example 6: Binding affinity to human Ang-2 determined by ELISA The same ELISA procedure was performed using recombinant human Ang-2. The EB-105 series molecules exhibit sub-nanomolar binding affinity for human Ang-2, comparable to nevasulamb and fareximab. Nevasulamb is a mAb targeting human Ang-2. Fareximab is a clinical-stage bispecific antibody for inhibiting VEGF-A and Ang-2. See also... Figure 10A -C.

[0222] Example 7: Binding affinity of B21138001, B21138003, B21138004, B21138006, B21138008 and B21138009 to human Ang-1 determined by ELISA The same ELISA procedure was performed using recombinant human Ang-1. B21138001 showed very low binding affinity to human Ang-1. B21138003 and B21138006 showed affinity for human Ang-1 comparable to nevasulimab, but slightly higher than farexilimab. See also Figure 11 .

[0223] Example 8: SPR Biacore Binding Affinity Determination SPR Biacore assays were performed at 25°C using HBS-EP+ as the operating buffer. The assay was performed by immobilizing anti-human IgG (Fc) antibodies onto the surface of a CM5 sensor chip and determining the level of ligand immobilization. The amount of anti-Fc antibody coupled to the CM5 sensor chip was approximately 7,000–14,000 reaction units (RUs). The test antibody was injected as a trap onto the surface of the S-series CM5 sensor chip. The analyte (target protein) was diluted to different concentrations with the operating buffer and injected as an associative phase onto the sensor surface for affinity and kinetic measurements. Binding affinity and / or kinetics were measured using a 1:1 binding model.

[0224] SPR Biacore assays indicated that EB-105 molecules bound to the target protein at picomolar or sub-nanomolar concentrations (Tables 4 and 5). B21138001 and B21138002 bound to human VEGF-A with binding affinity comparable to aflibercept. 165 VEGF-B 167 And PlGF. It also binds to human IL-6R and Ang-2 with binding affinity comparable to tocilizumab and fareximab (Tables 4 and 5). Tocilizumab is a clinical-stage mAb targeting human IL-6R. Fareximab is the first FDA-approved bispecific antibody that binds to VEGF-A and Ang-2 for the treatment of DME.

[0225] Table 4 The binding affinity of B21138001 and its comparator to the target protein was measured by SPR Biacore assay. Table 5 The binding affinity of B21138002 and its comparator to the target protein was measured by SPR Biacore assay. Cell-based in vitro functional evaluation of the antibody fusion protein of the present invention Example 9: VEGF-A165 Inhibition of VEGFR-2 signaling mediated The effect of EB-105 molecules on inhibiting VEGF-A was studied using an engineered HEK-293 cell line. 165 The role of EB-105 in mediated VEGFR-2 signaling, in which the expression of the firefly luciferase gene is controlled under the nuclear factor-activated T cell response element (VEGFR-2-NF-AT). Aflibercept and fareximab were used as two comparative agents. Results indicated that EB-105 molecules dose-dependently inhibited VEGF-2. 165 Its effects on stimulated VEGFR-2 signaling are comparable to those of aflibercept and fareximab. See also Figure 12A -C.

[0226] Serial dilutions of EB-105 molecules, aflibercept, and fareximab were mixed with 60 ng / ml human VEGF-A. 165 The cells were incubated together at room temperature for 30 minutes, and then VEGFR-2 luciferase reporter cells were added to each well to study the effect of the test material on the inhibition of VEGF-A. 165 The role of VEGFR-2 signal transduction mediated by a corresponding IC. 50 The values ​​indicate that EB-105 molecules inhibit VEGF-A in a dose-responsive manner. 165 Its effects on stimulated VEGFR-2 signaling are comparable to those of aflibercept and fareximab. Aflibercept is a human Fc fusion protein containing VEGFR-1-D2 and VEGFR-2-D3. Fareximab is a bispecific antibody that binds to VEGF-A and Ang-2.

[0227] Example 10: Inhibition of Ang-2 / Tie-2 interaction The effect of EB-105 on inhibiting the Ang-2 / Tie-2 interaction was investigated using engineered HEK-293 cells overexpressing the human Tie-2 receptor, with fareximab and nevasumab as two comparative agents, by fluorescence activated cell sorting (FACS) analysis. Nevasumab is an mAb targeting Ang-2. Serial dilutions of EB-105, fareximab, and nevasumab were incubated with 100 ng / ml human Ang-2 at 4°C for 60 min. Tie-2-expressing cells were then added to each well, and FACS analysis was used to investigate the effect of the antibodies on blocking the binding of Ang-2 to the Tie-2 receptor. The results showed that EB-105 exhibited a similar effect to nevasumab in blocking the binding of Ang-2 to the Tie-2 receptor in a dose-responsive manner, and its effect was more potent than that of fareximab. See also Figure 13A And B.

[0228] Example 11: Inhibition of Ang-1 / Tie-2 interaction The effect of EB-105 on Ang-1 / Tie-2 interaction was investigated using engineered HEK-293 cells overexpressing the human Tie-2 (hTie-2) receptor, with fareximab and nevasumob as two comparative agents, via FACS analysis. Serial dilutions of EB-105, fareximab, nevasumob, and hTie-2 were incubated with 2 µg / ml human Ang-1-Fc at 4°C for 60 min. Tie-2-expressing cells were then added to each well to investigate the effect of the test material on blocking the binding of Ang-1 to the Tie-2 receptor. Recombinant hTie-2 was used as a positive control. Similar to fareximab and nevasumab, 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 have shown some activity in inhibiting the binding of Ang-1 to the Tie-2 receptor. See also Figure 14A And B.

[0229] Example 12: Inhibition of IL-6 binding to IL-6R Using FACS analysis, the effect of EB-105 on blocking the binding of IL-6 to IL-6R was investigated using engineered CHO-S cells overexpressing human IL-6R and with tocilizumab and vorbalizumab as two comparative agents. Vorbalizumab is a single-domain antibody (also known as a nanobody) targeting human IL-6R. Serial dilutions of EB-105, tocilizumab, and vorbalizumab were incubated with biotinylated human IL-6 at 4°C for 60 min. Then, IL-6R-expressing CHO-S cells were added to each well to investigate the effect of the test materials on blocking the binding of biotinylated IL-6 to IL-6R. The results showed that EB-105 was comparable to tocilizumab and vorbalizumab in blocking the binding of IL-6 to IL-6R in a dose-responsive manner. See also Figure 15A And B.

[0230] Example 13: Cross-species activity of the lead molecule of EB-105 (B21138002) against target proteins in humans, monkeys, rabbits, and rats. Enzyme-linked immunosorbent assay (ELISA) was performed to assess the cross-species binding affinity of the lead molecule B21138002 for VEGF-A, IL-6R, and Ang-2 in humans, rhesus monkeys, rabbits, and rats. B21138002 bound to VEGF-A in humans, rhesus monkeys, and rabbits at sub-nanomolar concentrations. 165 Ang-2 and IL-6R. B21138002 binds to rat VEGF-A 165 It binds to Ang-2, but not to IL-6R. See Table 6.

[0231] Table 6 The cross-species binding affinity of B21138002 to the target protein, measured by ELISA. Pharmacological studies conducted in two animal models B21138002, the lead antibody fusion protein of the present invention, was formulated in phosphate buffer for intravitreal (IVT) injection, thereby investigating its efficacy and exploratory safety in two animal models: a preretinal neovascularization (PRN) model in Dutch black-banded rabbits and a laser-induced choroidal neovascularization (CNV) model in cynomolgus monkeys, as described below.

[0232] Example 14: Effect of a single IVT injection of B21138002 on PRN-induced vascular leakage in rabbits PRN was induced in Dutch Blackband rabbits via in vitro transfusion (IVT) of DL-α-aminoadipic acid (DL-AAA, 80 mM, 50 µl per eye). After PRN development was confirmed eight weeks following DL-AAA injection (Figure 15, indicated by arrows in the top / first row of composite fundus fluorescein angiography images), each eye received a single IVT injection of 50 µl of the carrier, an equivalent molar amount of aflibercept (EYLEA). ® 0.28 mg per eye), fareximab (VABYSMO) ® The effects of either B21138002 (0.36 mg per eye) or B21138002 (0.5 mg per eye) on inhibiting vascular leakage induced by existing PRNs were investigated. 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 fareximab. Figure 16 and Figure 17 Aflibercept and faraximab are FDA-approved treatments for DME.

[0233] Persistent vascular leakage was observed in the mediator treatment group before IVT injection and at 7 and 14 days after IVT injection (n=12). Eyes receiving aflibercept (n=12), fareximab (n=6), or B21138002 (n=6) showed highly effective inhibition of vascular leakage at 7 and 14 days after treatment. Figure 16 The larger arrows indicate extensive leakage caused by fully developed PRN, while the smaller arrows indicate residual fluorescein leakage in a region of the eye treated with fareximab. Figure 17 Results of quantitative analysis of vascular leakage areas at seven and fourteen days after IVT injection of the mediator and test materials are presented. Compared with pre-drug administration leakage, aflibercept, fareximab, and B21138002 effectively inhibited vascular leakage at seven and fourteen days after IVT injection (p < 0.01).

[0234] Example 15: Effect of a single IVT injection of B21138002 on vascular leakage in laser-induced choroidal neovascularization (CNV) in monkeys The effect of B21138002 on inhibiting vascular leakage was investigated in laser-induced CNV in cynomolgus monkeys using aflibercept and fareximab as two clinical comparative agents. CNV development was confirmed by fundus examination (FFA) twelve days after intensive laser coagulation. Two days later (fourteen days after laser coagulation), each eye received a single IVT injection of 50 µl of the agent, or 0.5 mg of aflibercept, fareximab, or B21138002 per eye. 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 a rabbit study of DL-AAA-induced PRN, B21138002 effectively inhibited leakage caused by CNV lesions, and its apparent efficacy was comparable to that of aflibercept and fareximab. Figures 18 to 20 In the mediator treatment group, CNV-induced vascular leakage persisted before administration and at 1, 2, and 4 weeks after administration (n=6). Eyes receiving aflibercept, fareximab, or B21138002 (n=3 / group) showed near-complete suppression of vascular leakage at 1, 2, and 4 weeks after treatment. Fundus photography showed no significant signs of retinal inflammation 4 weeks after IVT injection of test materials including mediator, aflibercept, fareximab, and B21138002. Figure 18 (The last line is a fundus image).

[0235] Figure 19 The results showed that the percentage of grade IV CNV lesions in the mediator treatment group tended to decrease slightly over time, but this change was not statistically significant. Aflibercept (Eylea) ®It significantly reduced the number of grade IV CNV lesions. Faraximab and B21138002 completely inhibited the development of grade IV CNV lesions at 1, 2, and 4 weeks post-treatment. (CNV lesion grading: Grade I - no superfluorescence; Grade II - superfluorescence staining and no fluorescein leakage; Grade III - early superfluorescence with slight fluorescein leakage limited to the laser burn boundary in late FFA; Grade IV - early superfluorescence with late severe fluorescein leakage outside the laser burn boundary. Grade IV lesions are considered highly clinically relevant.) Figure 20 Results of a quantitative analysis of vascular leakage in laser-induced CNV in monkeys were presented. Before administration and at 1, 2, and 4 weeks post-treatment, the mediator treatment group showed relatively persistent leakage caused by CNV lesions. At 1, 2, and 4 weeks post-treatment, B21138002 significantly inhibited CNV leakage, and its efficacy was comparable to that of aflibercept (Eylea). ® It is comparable to faraximab.

[0236] Nucleic acid and amino acid sequence listing SDTGPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTII (SEQ ID NO:1) DVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK (SEQ ID NO: 2) VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP (SEQ ID NO: 3) QVQLQESGPGLVRPSQTLSLTCTVSGYSITSDHAWSWVRQPPGRGLEWIGYISYSGITTYNPSLKSRVTMLRDTSKNQFSLRLSSVTAADTAVYYCARSLARTTAMDYWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP(SEQ ID NO: 4) DIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQID NO: 5) AQTNFMPMDDLEQRLYEQFILQQGLE (SEQ ID NO: 6) MGAQQKFQPLDELEQTLYEQFMLQQALE (SEQ ID NO: 7) MGAQQKYQPLDELDKTLYDQFMLQQGLE (SEQ ID NO: 8) MGAQHTFQPLDELEETLYYQWLYDQLLE (SEQ ID NO: 9) AQQEECEWDPWTCEHMLE (SEQ ID NO: 10) AQTNIQEECEWDPWTCDHMPGKLE (SEQ ID NO: 11) QVQLQESGPGLVRPSQTLSLTCTVSGYSITSDHAWSWVRQPPGRGLEWIGYISYSGITTYNPSLKSRVTMLRDTSKNQFSLRLSSVTAADTAVYYCARSLARTTAMDYWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGGSGGGGSAQTNFMPMDDLEQRLYEQFILQQGLE (SEQ ID NO: 12) SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 13) QVQLQESGPGLVRPSQTLSLTCTVSGYSITSDHAWSWVRQPPGRGLEWIGYISYSGITTYNPSLKSRVTMLRDTSKNQFSLRLSSVTAADTAVYYCARSLARTTAMDYWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGGSAQTNFMPMDDLEQRLYEQFILQQGLE (SEQ ID NO: 14) SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 15) QVQLQESGPGLVRPSQTLSLTCTVSGYSITSDHAWSWVRQPPGRGLEWIGYISYSGITTYNPSLKSRVTMLRDTSKNQFSLRLSSVTAADTAVYYCARSLARTTAMDYWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGGSGGGGSMGAQQKFQPLDELEQTLYEQFMLQQALE (SEQ ID NO: 16) SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 17) QVQLQESGPGLVRPSQTLSLTCTVSGYSITSDHAWSWVRQPPGRGLEWIGYISYSGITTYNPSLKSRVTMLRDTSKNQFSLRLSSVTAADTAVYYCARSLARTTAMDYWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGGSGGGGSMGAQQKYQPLDELDKTLYDQFMLQQGLE (SEQ ID NO: 18) SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 19) QVQLQESGPGLVRPSQTLSLTCTVSGYSITSDHAWSWVRQPPGRGLEWIGYISYSGITTYNPSLKSRVTMLRDTSKNQFSLRLSSVTAADTAVYYCARSLARTTAMDYWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGGSMGAQQKYQPLDELDKTLYDQFMLQQGLE (SEQ ID NO: 20) SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 21) QVQLQESGPGLVRPSQTLSLTCTVSGYSITSDHAWSWVRQPPGRGLEWIGYISYSGITTYNPSLKSRVTMLRDTSKNQFSLRLSSVTAADTAVYYCARSLARTTAMDYWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGGSGGGGSMGAQHTFQPLDELEETLYYQWLYDQLLE (SEQ ID NO: 22) SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO 23) QVQLQESGPGLVRPSQTLSLTCTVSGYSITSDHAWSWVRQPPGRGLEWIGYISYSGITTYNPSLKSRVTMLRDTSKNQFSLRLSSVTAADTAVYYCARSLARTTAMDYWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGGSGGGGSAQQEECEWDPWTCEHMLE (SEQ ID NO:24) SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 25) QVQLQESGPGLVRPSQTLSLTCTVSGYSITSDHAWSWVRQPPGRGLEWIGYISYSGITTYNPSLKSRVTMLRDTSKNQFSLRLSSVTAADTAVYYCARSLARTTAMDYWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGGSGGGGSAQTNIQEECEWDPWTCDHMPGKLE (SEQ ID NO: 26) SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 27) QVQLQESGPGLVRPSQTLSLTCTVSGYSITSDHAWSWVRQPPGRGLEWIGYISYSGITTYNPSLKSRVTMLRDTSKNQFSLRLSSVTAADTAVYYCARSLARTTAMDYWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGGSAQTNIQEECEWDPWTCDHMPGKLE (SEQ ID NO: 28) SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 29) EVQLVESGGGLVQPGGSLRLSCAASGRTFSSYDFVWVRQAPGKGLEFVSAISVEGEHTYYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCNAVKWSDYGPDNDYWGQGTLVTVSS (SEQ ID NO: 30) EVQLVESGGGLVQPGGSLRLSCAASGLYFSSYDIAWVRQAPGKGLEFVSAIDVSGEHTYYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCNAVKWSDYGPDNDYWGQGTLVTVSS (SEQ ID NO: 31) EVQLQESGGGLVQPGGSLRLSCAASGFTLDAYAIGWFRQAPGKEREGVSTIGKSDGSTCYADSVKGRFTISRDNAKNTVWLQMNSLRAEDTAVYYCAARPWWVGDAPSIAAEYEYDLWGQGTQVTVSS (SEQ ID NO: 32) EVQLQESGGGLVQPGGSLRLSCAASGFTLDEYAIGWFRQAPGKEREGVSCIGKADGSTCYADSVKGRFTISRDNAKNTVWLQMNSLRAEDTAVYYCAARPWWVGDPPSIGAEYEYDLWGQGTQVTVSS (SEQ ID NO: 33) QVQLVESGGGLVQPGGSLRLSCAASGFRFSSYAMSWVRQAPGKGLEWVSKINSGGGITYYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCRDEGPFGSWGQGTQVTVSS (SEQ ID NO: 34) QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSDINSGGSETYYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCRDKGPFGSWGQGTQVTVSS (SEQ ID NO: 35) EVQLVESGGGLVQPGGSLRLSCAASGRTFSSYDIIWVRQAPGKGLEFVSAISTSGEHTYYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCNAVKWSDYGPQNDYWGQGTLVTVSS (SEQ ID NO: 36) EVQLQESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSVIGKSDGSTCYADSVKGRFTISRDNAKNTVWLQMNSLRAEDTAVYYCAARPWWVGDRPSIAAEYEYDLWGQGTQVTVSS (SEQ ID NO: 37) MHSSALLCCLVLLTGVRAQVQLQESGPGLVRPSQTLSLTCTVSGYSITSDHAWSWVRQPPGRGLEWIGYSYSGITTYNPSLKSRVTMLRDTSKNQFSLRLSSVTAADTAVYCARSLARTTAMDYWGQGSLVTVSSASTKGPSVFPLA PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGLYFSSYDIAWVRQAPGKGLEFVSAIDVSGEHTYYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCNAVKWSDYGPDNDYWGQGTLVTVSS (SEQ ID NO: 38) MHSSALLCCLVLLTGVRASDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:39) MHSSALLCCLVLLTGVRAQVQLQESGPGLVRPSQTLSLTCTVSGYSITSDHAWSWVRQPPGRGLEWIGYISYSGITTYNPSLKSRVTMLRDTSKNQFSLRLSSVTAADTAVYYCARSLARTTAMDYWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGGSEVQLQESGGGLVQPGGSLRLSCAASGFTLDAYAIGWFRQAPGKEREGVSTIGKSDGSTCYADSVKGRFTISRDNAKNTVWLQMNSLRAEDTAVYYCAARPWWVGDAPSIAAEYEYDLWGQGTQVTVSS (SEQ ID NO: 40) MHSSALLCCLVLLTGVRASDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:41) EVQLVESGGGLVQAGGSLRLSCAASGDDLSSVDMGWYRQAPGKGLEFVGVISRGGGSTYYVDSVQGRFTISRDNAKNTVYLQMNSLRAEDTAVYYCKTEIPYFDDYWGQGTLVTVSS (SEQ ID NO: 42) EVQLVESGGGLVQAGGSLRLSCAASGESLSSVDMGWYRQAPGKGLEFVGVISRGGESTYYVDSVQGRFTISRDNAKNTVYLQMNSLRAEDTAVYYCKTEIPFGDDYWGQGTLVTVSS (SEQ ID NO: 43) EVQLVESGGGLVQAGGSLRLSCAASGGDLSSVDMGWYRQAPGKGLEFVGVISRTGGSTYYVDSVQGRFTISRDNAKNTVYLQMNSLRAEDTAVYYCKTEIPGGDDYWGQGTLVTVSS (SEQ ID NO: 44) EVQLVESGGGLVQAGGSLRLSCAASGESLSSVDMGWYRQAPGKGLEFVGVISRTGGSTYYVDSVQGRFTISRDNAKNTVYLQMNSLRAEDTAVYYCKTEIPGGDDVWGQGTLVTVSS (SEQ ID NO: 45) EVQLVESGGGLVQPGGSLRLSCAASGGTFIDLDMGWYRQAPGNERQFVGVISRTGGSTYYADSVQGRFTISRDNAKNTLYLQMNSLRAEDTATYYCKTEIPGGDDYWGQGTLVTVSS (SEQ ID NO: 46) EVQLVESGGGLVQPGGSLRLSCAASGGTFHLYDMGWYRQAPGNERQFVGVISRGGGSTYYADSVQGRFTISRDNAKNTLYLQMNSLRAEDTATYYCKTEIPGFQDYWGQGTLVTVSS (SEQ ID NO: 47) EVQLVESGGGLVQPGGSLRLSCAASGPEFIDYDMGWYRQAPGNERQFVGVISRGGGSTYYADSVQGRFTISRDNAKNTLYLQMNSLRAEDTATYYCKTEIPFFDDYWGQGTLVTVSS (SEQ ID NO: 48) EVQLVESGGGLVQPGGSLRLSCAASGGTFIDLDMGWYRQAPGNERQFVGVISRGGGSVYYADSVQGRFTISRDNAKNTLYLQMNSLRAEDTATYYCKTEIPGGPDYWGQGTLVTVSS (SEQ ID NO: 49) EVQLVESGGGLVQPGGSLRLSCAASGGTFIDYDMGWYRQAPGNERQFVGVISRGGGAVYYADSVQGRFTISRDNAKNTLYLQMNSLRAEDTATYYCKTEIPFFDDYWGQGTLVTVSS (SEQ ID NO: 50) QVQLVESGGGLVQPGGSLRLSCAASGPTFSNLDMGWYRQAPGKGLELVGVISRTGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCKTEVPFSDDYWGQGTLVTVSS (SEQ ID NO: 51) QVQLVESGGGLVQPGGSLRLSCAASVPTFGDLDMGWYRQAPGKGLELVGVISRTGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCKTEVPFGDDYWGQGTLVTVSS (SEQ ID NO: 52) QVQLVESGGGLVQPGGSLRLSCAASGDTFSNLDMGWYRQAPGKGLELVGVISRTGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCKTEVPWADDYWGQGTLVTVSS (SEQ ID NO: 53) QVQLVESGGGLVQPGGSLRLSCAASVPTFSDVDMGWYRQAPGKGLELVGVISRTGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCKTEVPFADDYWGQGTLVTVSS (SEQ ID NO: 54) QVQLVESGGGLVQPGGSLRLSCAASVPWFSNLDMGWYRQAPGKGLELVGVISRTGGMTYYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCKTEVPGSDDYWGQGTLVTVSS (SEQ ID NO: 55) QVQLVESGGGLVQPGGSLRLSCAASGATFSNLDMGWYRQAPGKGLELVGVISRTGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCKTEVPFDDDYWGQGTLVTVSS (SEQ ID NO: 56) MHSSALLCCLVLLTGVRAEVQLVESGGGLVQAGGSLRLSCAASGDDLSSVDMGWYRQAPGKGLEFVGVISRGGGSTYYVDSVQGRFTISRDNAKNTVYLQMNSLRAEDTAVYYCKTEIPYFDDYWGQGTLVTVSSGGSGGGSGGGGSGGGGGSGGGGSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSAQTNFMPMDDLEQRLYEQFILQQGLE (SEQ ID NO: 57) MHSSALLCCLVLLTGVRAEVQLVESGGGLVQAGGSLRLSCAASGGDLSSVDMGWYRQAPGKGLEFVGVISRTGGSTYYVDSVQGRFTISRDNAKNTVYLQMNSLRAEDTAVYYCKTEIPGGDDYWGQGTLVTVSSGGSGGGSGGGGSGGGGGSGGGGSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSAQTNFMPMDDLEQRLYEQFILQQGLE (SEQ ID NO: 58) MHSSALLCCLVLLTGVRAAQTNFMPMDDLEQRLYEQFILQQGLEGGGGSGGGGSGGGGSSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGDDLSSVDMGWYRQAPGKGLEFVGVISRGGGSTYYVDSVQGRFTISRDNAKNTVYLQMNSLRAEDTAVYYCKTEIPYFDDYWGQGTLVTVSS (SEQ ID NO: 59) MHSSALLCCLVLLTGVRAAQTNFMPMDDLEQRLYEQFILQQGLEGGGGSGGGGSGGGGSSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGGDLSSVDMGWYRQAPGKGLEFVGVISRTGGSTYYVDSVQGRFTISRDNAKNTVYLQMNSLRAEDTAVYYCKTEIPGGDDYWGQGTLVTVSS (SEQ ID NO: 60) MHSSALLCCLVLLTGVRAEVQLVESGGGLVQPGGSLRLSCAASGLYFSSYDIAWVRQAPGKGLEFVSAIDVSGEHTYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCNAVKWSDYGPDNDYWGQGTLVTVSSGGGGSGGGGSGGGGSSDTGRPFVEMYSEIPEIHMTEGRE LVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHE KDKTHTCPPCPAPELLGGPSVFLFPPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGGSGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGDDLSSVDMGWYRQAPGKGLEFVGVISRGGGSTYYVDSVQGRFTISRDNAKNTVYLQMNSLRAEDTAVYYCKTEIPYFDDYWGQGTLVTVSS (SEQ ID NO: 61) MHSSALLCCLVLLTGVRAEVQLVESGGGLVQAGGSLRLSCAASGDDLSSVDMGWYRQAPGKGLEFVGVISRGGGSTYYVDSVQGRFTISRDNAKNTVYLQMNSLRAEDTAVYYCKTEIPYFDDYWGQGTLVTVSSGGGGSGGGGSGGGGSSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGLYFSSYDIAWVRQAPGKGLEFVSAIDVSGEHTYYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCNAVKWSDYGPDNDYWGQGTLVTVSS (SEQ ID NO: 62) SDHAWS (SEQ ID NO: 63) YISYSGITTYNPSLKS (SEQ ID NO: 64) ARTTAMDY (SEQ ID NO: 65) RASQDISSYLN (SEQ ID NO: 66) YTSRLHS (SEQ ID NO: 67) QQGNTLPYT (SEQ ID NO: 68) EVQLVESGGGLVQPGRSLRLSCAASRFTFDDYAMHWVRQAPGKGLEWVSGISWNSGRIGYADSVKGRFTISRDNAENSLFLQMNGLRAEDTALYYCAKGRDSFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVTYLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 69) DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYGASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFASYYCQQANSFPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQID NO: 70) QVQLQESGPGLVKPSETLSLTCAVSGHSISHDHAWSWVRQPPGEGLEWIGFISYSGITNYNPSLQGRVTISRDNSKNTLYLQMNSLRAEDTAVYYCARSLARTTAMDYWGEGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHAHYTQKSLS LSP (SEQID NO: 71) DIQMTQSPSSLSASVGDSVTITCQASTDISSHLNWYQQKPGKAPELLIYYGSHLLSGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCGQGNRLPYTFGQGTKVEIERTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQID NO: 72) AGCGACACCGGCAGACCCTTCGTGGAGATGTACAGCGAGATCCCCGAGATCATCCACATGACCGAGGGCAGAGAGCTGGTGATCCCCTGCAGAGTGACCAGCCCCAACATCACCGTGACCCTGAAGAAGTTCCCCCTGGACACCCTGATCCCCGACGGCAAGAGAATCATCTGGGACAGCAGAAAGGGCTTCATCATCAGCAACGCCACCTACAAGGAGATCGGCCTGCTGACCTGCGAGGCCACCGTGAACGGCCACCTGTACAAGACCAACTACCTGACCCACAGACAGACCAACACCATCATC (SEQ ID NO: 73) GACGTGGTGCTGAGCCCCAGCCACGGCATCGAGCTGAGCGTGGGCGAGAAGCTGGTGCTGAACTGCACCGCCAGAACCGAGCTGAACGTGGGCATCGACTTCAACTGGGAGTACCCCAGCAGCAAGCACCAGCACAAGAAGCTGGTGAACAGAGACCTGAAGACCCAGAGCGGCAGCGAGATGAAGAAGTTCCTGAGCACCCTGACCATCGACGGCGTGACCAGAAGCGACCAGGGCCTGTACACCTGCGCCGCCAGCAGCGGCCTGATGACCAAGAAGAACAGCACCTTCGTGAGAGTGCACGAGAAG (SEQ ID NO: 74) GTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCG (SEQ ID 75) GACATTCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGAGACAGAGTGACCATCACCTGCAGAGCCAGCCAGGACATCTCCAGCTACCTGAACTGGTATCAGCAGAAACCCGGCAAAGCCCCAAAACTGCTGATCTACTACACCAGTAGACTGCACAGCGGCGTGCCCAGCAGATTCTCAGGAAGCGGCTCCGGAACCGACTTCACCTTCACTATCAGCAGCCTGCAGCCCGAAGATATTGCTACTTACTACTGCCAGCAGGGGAACACCCTGCCCTATACCTTCGGCCAGGGCACCAAGGTGGAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGTTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTGATTCTAGA (SEQ ID NO: 77) AGTGCTCAGACCAATTTTATGCCTATGGATGATCTGGAACAGAGACTGTACGAACAGTTCATCCTCCAACAGGGACTG (SEQ ID NO: 78) ATGGGCGCTCAGCAGAAGTTCCAGCCCCTGGACGAGCTGGAGCAGACCCTGTACGAGCAGTTCATGCTCCAGCAGGCCCTGGAG (SEQ ID NO: 79) ATGGGCGCTCAGCAGAAGTACCAGCCCCTGGACGAGCTGGACAAGACCCTGTACGACCAGTTCATGCTCCAGCAGGGGCTGGAA (SEQ ID NO: 80) ATGGGCGCTCAGCACACCTTCCAGCCCCTGGACGAGCTGGAGGAGACACTGTACTACCAGTGGCTCTACGACCAGCTGCTGGAG (SEQ ID NO: 81) GCTCAGCAAGAAGAGTGTGAATGGGACCCTTGGACCTGTGAACACATGCTGGAA (SEQ ID NO:82) GCTCAGACCAATATTCAAGAAGAATGTGAATGGGACCCTTGGACCTGTGATCACATGCCCGGCAAACTGGAA (SEQ ID NO: 83) GAGGTGCAGCTGGTGGAGAGCGGCGGCGGACTGGTGCAGCCTGGAGGATCTCTGAGGCTGTCCTGCGCCGCTTCTGGCCGGACCTTCTCCAGCTACGACTTTGTTTGGGTGCGGCAGGCCCCTGGCAAGGGACTGGAGTTCGTGTCCGCCATCTCCGTTGAGGGCGAGCACACCTACTATGCTGATAGCGTGAAGGGCCGGTTCACCATCAGCAGGGATAACGCCAAGAATACCCTGTATCTGCAGATGAATAGCCTGCGGGCTGAGGACACCGCCGTGTATTACTGTAACGCCGTGAAGTGGAGCGACTACGGCCCCGACAACGATTACTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCC (SEQ IDNO: 102) GAGGTGCAGCTGGTGGAGAGCGGCGGCGGACTGGTGCAGCCTGGAGGATCTCTGAGGCTGTCCTGCGCCGCTTCTGGCCTTTATTTCTCCAGCTACGACATCGCTTGGGTGCGGCAGGCCCCTGGCAAGGGACTGGAGTTCGTGTCCGCCATCGATGTGTCCGGCGAGCACACCTACTATGCTGATAGCGTGAAGGGCCGGTTCACCATCAGCAGGGATAACGCCAAGAATACCCTGTATCTGCAGATGAATAGCCTGCGGGCTGAGGACACCGCCGTGTATTACTGTAACGCCGTGAAGTGGAGCGACTACGGCCCCGACAACGATTACTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCC (SEQ IDNO: 103) GAGGTGCAGCTGCAGGAGAGCGGCGGCGGACTGGTGCAGCCAGGAGGAAGCCTGCGGCTGTCCTGTGCCGCTTCCGGCTTTACCCTGGATGCGTATGCTATCGGCTGGTTTAGGCAGGCCCCTGGCAAGGAGAGGGAGGGCGTGTCCACGATCGGCAAGTCCGACGGCAGCACCTGTTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCTAAGAATACCGTGTGGCTGCAGATGAACTCCCTGCGGGCTGAGGACACCGCCGTGTACTATTGCGCCGCTCGGCCCTGGTGGGTGGGCGACGCGCCTTCTATCGCTGCCGAGTACGAGTACGATCTGTGGGGCCAGGGCACCCAGGTGACCGTGAGCTCC (SEQ ID NO: 104) GAGGTGCAGCTGCAGGAGAGCGGCGGCGGACTGGTGCAGCCAGGAGGAAGCCTGCGGCTGTCCTGTGCCGCTTCTGGCTTTACCCTGGATGAGTATGCTATCGGCTGGTTTAGGCAGGCCCCTGGCAAGGAGAGGGAGGGCGTGTCCTGTATCGGCAAGGCGGACGGCAGCACCTGTTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCTAAGAATACCGTGTGGCTGCAGATGAACTCCCTGCGGGCTGAGGACACCGCCGTGTACTATTGCGCCGCTCGGCCCTGGTGGGTGGGCGACCCTCCTTCTATCGGTGCCGAGTACGAGTACGATCTGTGGGGCCAGGGCACCCAGGTGACCGTGAGCTCC (SEQ ID NO: 105) CAGGTGCAGCTGGTGGAGAGCGGCGGCGGACTGGTGCAGCCTGGAGGAAGCCTGCGGCTGTCCTGTGCCGCCAGCGGATTCAGGTTTAGCAGCTACGCCATGTCCTGGGTGAGGCAGGCTCCTGGCAAGGGCCTGGAGTGGGTGAGCAAGATCAATAGCGGCGGCGGCATCACCTACTACGCTGACTCCGTGAAGGGCAGGTTTACCATCTCCAGGGATAACGCCAAGAATACCCTGTACCTGCAGATGAATAGCCTGCGGGCTGAGGACACCGCCGTGTATTACTGTCGGGACGAGGGCCCTTTTGGCAGCTGGGGCCAGGGCACCCAGGTGACAGTGAGCTCC (SEQ ID NO: 106) CAGGTGCAGCTGGTGGAGAGCGGCGGCGGACTGGTGCAGCCTGGAGGAAGCCTGCGGCTGTCCTGTGCCGCCAGCGGATTCACCTTTAGCAGCTACGCCATGTCCTGGGTGAGGCAGGCTCCTGGCAAGGGCCTGGAGTGGGTGAGCGACATCAATAGCGGCGGCAGTGAGACCTACTACGCTGACTCCGTGAAGGGCAGGTTTACCATCTCCAGGGATAACGCCAAGAATACCCTGTACCTGCAGATGAATAGCCTGCGGGCTGAGGACACCGCCGTGTATTACTGTCGGGACAAGGGCCCTTTTGGCAGCTGGGGCCAGGGCACCCAGGTGACAGTGAGCTCC (SEQ ID NO: 107) GAGGTGCAGCTGGTGGAGAGCGGCGGCGGACTGGTGCAGCCTGGAGGATCTCTGAGGCTGTCCTGCGCCGCTTCTGGCCGGACCTTCTCCAGCTACGACATCATTTGGGTGCGGCAGGCCCCTGGCAAGGGACTGGAGTTCGTGTCCGCCATCTCCACGTCCGGCGAGCACACCTACTATGCTGATAGCGTGAAGGGCCGGTTCACCATCAGCAGGGATAACGCCAAGAATACCCTGTATCTGCAGATGAATAGCCTGCGGGCTGAGGACACCGCCGTGTATTACTGTAACGCCGTGAAGTGGAGCGACTACGGCCCCCAGAACGATTACTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCC (SEQ IDNO: 108) GAGGTGCAGCTGCAGGAGAGCGGCGGCGGACTGGTGCAGCCAGGAGGAAGCCTGCGGCTGTCCTGTGCCGCTTCCGGCTTTACCCTGGATTACTATGCTATCGGCTGGTTTAGGCAGGCCCCTGGCAAGGAGAGGGAGGGCGTGTCCGTGATCGGCAAGTCCGACGGCAGCACCTGTTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCTAAGAATACCGTGTGGCTGCAGATGAACTCCCTGCGGGCTGAGGACACCGCCGTGTACTATTGCGCCGCTCGGCCCTGGTGGGTGGGCGACAGGCCTTCTATCGCTGCCGAGTACGAGTACGATCTGTGGGGCCAGGGCACCCAGGTGACCGTGAGCTCC (SEQ ID NO: 109) GAGGTGCAGTTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCACTGAGACTCTCCTGTGCAGCCTCTGGAGATGATTTAAGTAGCGTAGACATGGGCTGGTACCGCCAGGCTCCAGGGAAGGGCCTGGAGTTTGTCGGCGTTATTAGCCGTGGTGGTGGTAGCACATACTATGTAGACTCCGTGCAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGCGGGCCGAGGACACGGCCGTGTATTACTGTAAGACAGAAATTCCATATTTTGATGACTACTGGGGCCAGGGGACCCTGGTCACCGTCTCCTCA (SEQ ID NO: 114) GAGGTGCAGTTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCACTGAGACTCTCCTGTGCAGCCTCTGGAGAGTCCTTAAGTAGCGTAGACATGGGCTGGTACCGCCAGGCTCCAGGGAAGGGCCTGGAGTTTGTCGGCGTTATTAGCCGTGGTGGTGAGAGCACATACTATGTAGACTCCGTGCAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGCGGGCCGAGGACACGGCCGTGTATTACTGTAAGACAGAAATTCCATTTGGGGATGACTACTGGGGCCAGGGGACCCTGGTCACCGTCTCCTCA (SEQ ID NO: 115) GAGGTGCAGTTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCACTGAGACTCTCCTGTGCAGCCTCTGGAGGTGATTTAAGTAGCGTAGACATGGGCTGGTACCGCCAGGCTCCAGGGAAGGGCCTGGAGTTTGTCGGCGTTATTAGCAGGACGGGTGGTAGCACATACTATGTAGACTCCGTGCAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGCGGGCCGAGGACACGGCCGTGTATTACTGTAAGACAGAAATTCCAGGGGGGGATGACTACTGGGGCCAGGGGACCCTGGTCACCGTCTCCTCA (SEQ ID NO: 116) GAGGTGCAGTTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCACTGAGACTCTCCTGTGCAGCCTCTGGAGAGTCCTTAAGTAGCGTAGACATGGGCTGGTACCGCCAGGCTCCAGGGAAGGGCCTGGAGTTTGTCGGCGTTATTAGCCGTACGGGTGGTAGCACATACTATGTAGACTCCGTGCAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGCGGGCCGAGGACACGGCCGTGTATTACTGTAAGACAGAAATTCCAGGGGGGGATGACGTTTGGGGCCAGGGGACCCTGGTCACCGTCTCCTCA (SEQ ID NO: 117) GAGGTGCAGTTGGTGGAGAGCGGGGGGGGGCTGGTGCAGCCTGGAGGAAGTTTGAGGTTGAGCTGTGCCGCAAGCGGGGGGACATTCATTGATTTGGACATGGGATGGTACAGGCAGGCACCTGGAAACGAGAGGCAGTTTGTGGGGGTGATTTCCCGGACTGGAGGATCCACATACTACGCTGACAGCGTGCAGGGGAGGTTCACCATCTCCCGGGACAACGCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGGGCTGAGGACACTGCTACCTATTACTGCAAGACCGAGATTCCCGGAGGAGACGACTACTGGGGCCAGGGCACACTGGTGACCGTGAGCTCC (SEQ ID NO: 118) GAGGTGCAGTTGGTGGAGAGCGGGGGGGGGCTGGTGCAGCCTGGAGGAAGTTTGAGGTTGAGCTGTGCCGCAAGCGGGGGGACATTCCATCTGTACGACATGGGATGGTACAGGCAGGCACCTGGAAACGAGAGGCAGTTTGTGGGGGTGATTTCCCGCGGCGGAGGATCCACATACTACGCTGACAGCGTGCAGGGGAGGTTCACCATCTCCCGGGACAACGCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGGGCTGAGGACACTGCTACCTATTACTGCAAGACCGAGATTCCCGGATTTCAGGACTACTGGGGCCAGGGCACACTGGTGACCGTGAGCTCC (SEQ ID NO: 119) GAGGTGCAGTTGGTGGAGAGCGGGGGGGGGCTGGTGCAGCCTGGAGGAAGTTTGAGGTTGAGCTGTGCCGCAAGCGGGCCGGAGTTCATTGATTACGACATGGGATGGTACAGGCAGGCACCTGGAAACGAGAGGCAGTTTGTGGGGGTGATTTCCCGCGGCGGAGGATCCACATACTACGCTGACAGCGTGCAGGGGAGGTTCACCATCTCCCGGGACAACGCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGGGCTGAGGACACTGCTACCTATTACTGCAAGACCGAGATTCCCTTTTTTGACGACTACTGGGGCCAGGGCACACTGGTGACCGTGAGCTCC (SEQ ID NO: 120) GAGGTGCAGTTGGTGGAGAGCGGGGGGGGGCTGGTGCAGCCTGGAGGAAGTTTGAGGTTGAGCTGTGCCGCAAGCGGGGGGACATTCATTGATCTGGACATGGGATGGTACAGGCAGGCACCTGGAAACGAGAGGCAGTTTGTGGGGGTGATTTCCCGCGGCGGAGGATCCGTTTACTACGCTGACAGCGTGCAGGGGAGGTTCACCATCTCCCGGGACAACGCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGGGCTGAGGACACTGCTACCTATTACTGCAAGACCGAGATTCCCGGAGGACCTGACTACTGGGGCCAGGGCACACTGGTGACCGTGAGCTCC (SEQ ID NO: 121) GAGGTGCAGTTGGTGGAGAGCGGGGGGGGGCTGGTGCAGCCTGGAGGAAGTTTGAGGTTGAGCTGTGCCGCAAGCGGGGGGACATTCATTGATTACGACATGGGATGGTACAGGCAGGCACCTGGAAACGAGAGGCAGTTTGTGGGGGTGATTTCCCGCGGCGGAGGAGCTGTTTACTACGCTGACAGCGTGCAGGGGAGGTTCACCATCTCCCGGGACAACGCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGGGCTGAGGACACTGCTACCTATTACTGCAAGACCGAGATTCCCTTTTTTGACGACTACTGGGGCCAGGGCACACTGGTGACCGTGAGCTCC (SEQ ID NO: 122) CAGGTCCAGCTCGTCGAGTCAGGCGGCGGCCTCGTGCAGCCTGGAGGAAGTTTGAGGTTGAGTTGTGCAGCCAGCGGTCCTACATTTTCCAACCTCGACATGGGCTGGTACAGGCAGGCACCCGGCAAAGGGCTGGAGCTGGTGGGCGTTATTTCTCGGACAGGAGGGTCCACATACTACGCCGATTCCGTGAAGGGCAGATTCACCATCAGCAGGGACAATGCTAAGAACACCCTGTACCTGCAGATGAACTCCTTGAGGGCTGAGGACACCGCAGTGTACTACTGCAAGACCGAGGTGCCCTTTTCTGACGATTACTGGGGACAGGGCACACTGGTGACCGTGAGCTCC (SEQ ID NO: 123) CAGGTCCAGCTCGTCGAGTCAGGCGGCGGCCTCGTGCAGCCTGGAGGAAGTTTGAGGTTGAGTTGTGCAGCCAGCGTGCCGACATTTGGGGATCTCGACATGGGCTGGTACAGGCAGGCACCCGGCAAAGGGCTGGAGCTGGTGGGCGTTATTTCTCGGACAGGAGGGTCCACATACTACGCCGATTCCGTGAAGGGCAGATTCACCATCAGCAGGGACAATGCTAAGAACACCCTGTACCTGCAGATGAACTCCTTGAGGGCTGAGGACACCGCAGTGTACTACTGCAAGACCGAGGTGCCCTTTGGGGACGATTACTGGGGACAGGGCACACTGGTGACCGTGAGCTCC (SEQ ID NO: 124) CAGGTCCAGCTCGTCGAGTCAGGCGGCGGCCTCGTGCAGCCTGGAGGAAGTTTGAGGTTGAGTTGTGCAGCCAGCGGGGATACATTTTCCAACCTCGACATGGGCTGGTACAGGCAGGCACCCGGCAAAGGGCTGGAGCTGGTGGGCGTTATTTCTCGGACAGGAGGGTCCACATACTACGCCGATTCCGTGAAGGGCAGATTCACCATCAGCAGGGACAATGCTAAGAACACCCTGTACCTGCAGATGAACTCCTTGAGGGCTGAGGACACCGCAGTGTACTACTGCAAGACCGAGGTGCCCTGGGCGGACGATTACTGGGGACAGGGCACACTGGTGACCGTGAGCTCC (SEQ ID NO: 125) CAGGTCCAGCTCGTCGAGTCAGGCGGCGGCCTCGTGCAGCCTGGAGGAAGTTTGAGGTTGAGTTGTGCAGCCAGCGTGCCGACATTTTCCGATGTGGACATGGGCTGGTACAGGCAGGCACCCGGCAAAGGGCTGGAGCTGGTGGGCGTTATTTCTCGGACAGGAGGGTCCACATACTACGCCGATTCCGTGAAGGGCAGATTCACCATCAGCAGGGACAATGCTAAGAACACCCTGTACCTGCAGATGAACTCCTTGAGGGCTGAGGACACCGCAGTGTACTACTGCAAGACCGAGGTGCCCTTTGCTGACGATTACTGGGGACAGGGCACACTGGTGACCGTGAGCTCC (SEQ ID NO: 126) CAGGTCCAGCTCGTCGAGTCAGGCGGCGGCCTCGTGCAGCCTGGAGGAAGTTTGAGGTTGAGTTGTGCAGCCAGCGTGCCGTGGTTTTCCAACCTCGACATGGGCTGGTACAGGCAGGCACCCGGCAAAGGGCTGGAGCTGGTGGGCGTTATTTCTCGGACAGGAGGGATGACATACTACGCCGATTCCGTGAAGGGCAGATTCACCATCAGCAGGGACAATGCTAAGAACACCCTGTACCTGCAGATGAACTCCTTGAGGGCTGAGGACACCGCAGTGTACTACTGCAAGACCGAGGTGCCCGGCTCGGACGATTACTGGGGACAGGGCACACTGGTGACCGTGAGCTCC (SEQ ID NO: 127) CAGGTCCAGCTCGTCGAGTCAGGCGGCGGCCTCGTGCAGCCTGGAGGAAGTTTGAGGTTGAGTTGTGCAGCCAGCGGGGCGACATTTTCCAACCTCGACATGGGCTGGTACAGGCAGGCACCCGGCAAAGGGCTGGAGCTGGTGGGCGTTATTTCTCGGACAGGAGGGTCCACATACTACGCCGATTCCGTGAAGGGCAGATTCACCATCAGCAGGGACAATGCTAAGAACACCCTGTACCTGCAGATGAACTCCTTGAGGGCTGAGGACACCGCAGTGTACTACTGCAAGACCGAGGTGCCCTTTGATGACGATTACTGGGGACAGGGCACACTGGTGACCGTGAGCTCC (SEQ ID NO: 128) AGCGACCACGCCTGGAGC (SEQ ID NO: 135) TACATCAGCTACAGCGGCATCACCCACCTACAACCCCAGCCTGAAGAGC (SEQ ID NO: 136) GCCAGGACCACCGCCATGGACTAC (SEQ ID NO: 137) TACACCAGCAGGCTGCACAGC (SEQ ID NO: 138) AGGGCCAGCCAGGACATCAGCAGCTACCTGAAC (SEQ ID NO: 139) CAGCAGGGCAACACCCTGCCCTACACC (SEQ ID NO: 140) Reference to electronic sequence listing The contents of the electronic sequence list (076908-8004WO01.xml; size: 242 KB; and creation date: September 7, 2023) are incorporated herein by reference in full.

[0237] Although specific embodiments of the invention have been described in the foregoing, those skilled in the art will understand that many equivalents, modifications, substitutions and variations can be made thereto without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

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

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

3. The antibody fusion protein of claim 2 or its antigen-binding fragment or domain, wherein the Ang-2 binding unit comprises an antibody or bioactive polypeptide capable of substantially binding to Ang-2.

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

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

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

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

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

9. The antibody fusion protein of claim 2 or its antigen-binding fragment or domain, 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 together in tandem; and (c) an Ang-2 binding polypeptide, wherein the C-terminus of the VEGF binding unit is attached 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 attached to the N-terminus of the Ang-2 binding polypeptide.

10. The antibody fusion protein of claim 2 or its antigen-binding fragment or domain, 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 tandem; and (c) an Ang-2 sdAb, wherein the C-terminus of the VEGF binding unit is attached 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 attached to the N-terminus of the Ang-2 sdAb.

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

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

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

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

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

16. The antibody fusion protein of claim 8 or the antigen-binding fragment or domain thereof, comprising a polypeptide having a pair of amino acid sequences selected from the group consisting of: SEQ ID NO: 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 of claim 8 or its antigen-binding fragment or domain, comprising a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 57-62.

18. The antibody fusion protein of claim 2, or its antigen-binding fragment or domain thereof, wherein the antibody fusion protein or its antigen-binding fragment or domain is capable of being applied at approximately 1 × 10⁻⁶. -6 M to approximately 1×10 -12 M or approximately 1×10 -8 M to approximately 1×10 -12 The equilibrium dissociation constant (K) within the M range D It binds to IL-6R, Ang-2 and at least one VEGF family member.

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

20. The isolated nucleic acid molecule according to claim 19, wherein the nucleic acid molecule comprises a sequence selected from the group consisting of SEQ ID NO: 78-134.

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

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

23. A host-vector system comprising an expression vector according to claim 22 in a host cell.

24. A method for producing a substantially purified antibody fusion protein, the method comprising: (a) growing cells of a host-vector system according to claim 23 under conditions that allow for the production of the antibody fusion protein; and (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, symptom, or condition in a subject of need, the cause of which is abnormal angiogenesis or inflammation, wherein the method comprises administering to the subject an amount of a composition of an antibody fusion protein or an antigen-binding fragment or domain thereof according to any one of claims 1 to 18.

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

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

28. The method of claim 27, wherein the composition is administered to the subject in the form of eye drops, lacrimal duct plugs, intraocular injections, retroocular injections, subconjunctival injections, periocular injections, subfascial injections, transscleral injections, intravitreal injections, subretinal injections, or suprachoroidal injections.

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

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

31. A method for treating or controlling at least one systemic disease, symptom, or condition in a subject of need, the cause of which is abnormal angiogenesis or inflammation, wherein the method comprises administering to the subject an amount of a composition of an antibody fusion protein or an antigen-binding fragment or domain thereof according to any one of claims 1 to 18.

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

33. A pharmaceutical composition for treating or controlling at least one disease, symptom, or condition caused by abnormal angiogenesis or inflammation, wherein the composition comprises an antibody fusion protein or an antigen-binding fragment or domain thereof according to any one of claims 1 to 18.

34. The pharmaceutical composition of claim 33, wherein the disease, symptom, or condition is selected from the group consisting of: macular edema caused by diabetes, uveitis, central and branch retinal vein occlusion, choroidal neovascularization, neovascular age-related macular degeneration, polypoid choroidal angiopathy, 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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