Anti-C3b or anti-C5 antibodies conjugated with angiogenesis inhibitors and uses thereof

Abstract

The present invention relates to a fusion protein comprising an anti-C3b antibody or an anti-C5 antibody and an angiogenesis inhibitor, and a composition for treating eye diseases, particularly macular degeneration, using the same. The protein can not only inhibit complement-related pathways, but also effectively regulate angiogenesis. Therefore, the fusion protein dimer can be effectively used for the treatment and prevention of complement-related diseases, particularly eye diseases such as macular degeneration, and therefore has high industrial applicability.

Description

[Technical field]

[0001] The present invention relates to fusion proteins comprising an anti-C3b antibody or an anti-C5 antibody and an angiogenesis inhibitor, and compositions for using same to treat ocular diseases, particularly macular degeneration. [Background technology]

[0002] Macular degeneration (Age-related Macular Degeneration, AMD) is the leading cause of blindness in people over 50 years of age. It is an eye disease that reduces central vision due to damage to the macula. Macular degeneration is broadly classified as wet or dry macular degeneration. In the early stages, dry macular degeneration occurs, with abnormal extracellular deposits called drusen appearing under the macula and pigmentary changes occurring in the retinal pigment epithelium (RPE). Wet macular degeneration causes choroidal neovascularization, pigment epithelial detachment, macular edema, retinal hemorrhage, and retinal exudation, leading to blindness due to the death of retinal nerve cells.

[0003] The complement system, on the other hand, is an important component of innate immunity against microbial infections and comprises a group of proteins that are normally present in serum in an inactive state. These proteins are activated through the classical, lectin and alternative pathways. Molecules on the surface of microorganisms activate this pathway, leading to the formation of a protease complex known as C3 convertase.

[0004] Activation of the complement pathway generates biologically active fragments of complement proteins, such as C3a and C5a and anaphylatoxins such as the C5b-9 membrane attack complex (MAC), which mediate inflammatory responses in leukocyte chemotaxis, activation of macrophages, neutrophils, platelets, mast cells, and endothelial cells, increased vascular permeability, cell lysis, and tissue damage.

[0005] Furthermore, recent studies have demonstrated that complement components C3 and C5 are major components of drusen in AMD patients (Mulling, RF et al., FASEB J., 14, 835-46, 2000). In addition, Japanese Patent No. 4897690 reports that complement is involved in some eye diseases. However, to date, there is an increasing need for drugs that effectively treat eye diseases, especially macular degeneration, and research into therapeutic agents for macular degeneration continues. Summary of the Invention [Problem to be solved by the invention]

[0006] Therefore, the present inventors have conducted research to effectively treat and prevent eye diseases, particularly macular degeneration, and have found that a fusion protein that blocks the complement-related pathway and the neovascularization pathway can be used as a therapeutic agent for macular degeneration, thereby completing the present invention. [Means for solving the problem]

[0007] In one aspect of the invention, a fusion protein is provided comprising an antibody fragment that specifically binds to C3b (complement component 3b) or C5 (complement component 5) and a protein that specifically binds to VEGF (vascular endothelial growth factor).

[0008] In another embodiment of the present invention, a fusion protein dimer is provided in which two fusion proteins are linked to each other.

[0009] In yet another aspect of the invention, a polynucleotide encoding the fusion protein is provided.

[0010] In yet yet another aspect of the invention, there is provided a vector containing the polynucleotide.

[0011] In yet another aspect of the invention, there is provided a transformed host cell into which the vector has been introduced.

[0012] In yet another aspect of the present invention, there is provided a pharmaceutical composition for treating or preventing an eye disease, comprising the fusion protein or the fusion protein dimer as an active ingredient. Effect of the Invention

[0013] The fusion protein comprising the antibody fragment specifically binding to C3b or C5 according to the present invention and the protein specifically binding to VEGF can not only effectively inhibit the complement-related mechanism, but also effectively inhibit angiogenesis.Therefore, it can effectively treat or prevent eye diseases caused by the complement system and eye diseases caused by angiogenesis.Therefore, the fusion protein can be effectively used to effectively treat macular degeneration, particularly both dry macular degeneration and wet macular degeneration. [Brief description of the drawings]

[0014] [Figure 1a] FIG. 1 shows the results of identifying the prepared MOR09611, MOR09675, S77, eculizumab, PRO236, and PRO237 by SDS-PAGE. [Figure 1b] FIG. 1 shows the results of identifying prepared PRO238, PRO239, PRO240, PRO241, PRO242, and PRO243 by SDS-PAGE. [Figure 1c] FIG. 1 shows the results of identifying prepared PRO017, KNP-301, and aflibercept by SDS-PAGE. [Diagram 2] FIG. 1 is a schematic diagram showing an example of a bispecific antibody of the present invention. [Figure 3a] 3a and 3b are graphs showing the results obtained by measuring the binding affinity of KNP-301, PRO236, PRO237, PRO238, PRO239, PRO241, PRO242, S77, eculizumab, MOR09611, and MOR09675 to human C3b by ELISA. [Figure 3b]3a and 3b are graphs showing the results obtained by measuring the binding affinity of KNP-301, PRO236, PRO237, PRO238, PRO239, PRO241, PRO242, S77, eculizumab, MOR09611, and MOR09675 to human C3b by ELISA. [Figure 4] 1 is a graph showing the results obtained by measuring the binding affinity of PRO239, eculizumab, and PRO017 to human C5 by ELISA. [Figure 5a] 5a and 5b are graphs showing the results obtained measuring the binding affinity of aflibercept, PRO236, PRO237, PRO238, PRO017, PRO239, PRO241, and PRO242 to human VEGF165 by ELISA. [Figure 5b] 5a and 5b are graphs showing the results obtained measuring the binding affinity of aflibercept, PRO236, PRO237, PRO238, PRO017, PRO239, PRO241, and PRO242 to human VEGF165 by ELISA. [Figure 6a] 6a to 6c are graphs showing the results of measuring the alternative complement pathway inhibitory effects of KNP-301, PRO236, PRO237, PRO239, PRO241, PRO242, S77, eculizumab, MOR09611, and MOR09675 by hemolysis assay (AH50). [Figure 6b] 6a to 6c are graphs showing the results of measuring the alternative complement pathway inhibitory effects of KNP-301, PRO236, PRO237, PRO239, PRO241, PRO242, S77, eculizumab, MOR09611, and MOR09675 by hemolysis assay (AH50). [Figure 6c] 6a to 6c are graphs showing the results of measuring the alternative complement pathway inhibitory effects of KNP-301, PRO236, PRO237, PRO239, PRO241, PRO242, S77, eculizumab, MOR09611, and MOR09675 by hemolysis assay (AH50). [Figure 7a]7a to 7c are graphs showing the results obtained by measuring the classical complement pathway inhibitory effects of KNP-301, PRO236, PRO237, PRO239, PRO241, PRO242, S77, eculizumab, MOR09611, and MOR09675 by hemolysis assay (CH50). [Figure 7b] 7a to 7c are graphs showing the results obtained by measuring the classical complement pathway inhibitory effects of KNP-301, PRO236, PRO237, PRO239, PRO241, PRO242, S77, eculizumab, MOR09611, and MOR09675 by hemolysis assay (CH50). [Figure 7c] 7a to 7c are graphs showing the results obtained by measuring the classical complement pathway inhibitory effects of KNP-301, PRO236, PRO237, PRO239, PRO241, PRO242, S77, eculizumab, MOR09611, and MOR09675 by hemolysis assay (CH50). [Figure 8a] 1 is a graph showing the results obtained by measuring the VEGF signaling inhibitory effects of aflibercept, PRO236, and PRO237 using reporter cells. [Figure 8b] 1 is a graph showing the results obtained by measuring the VEGF signaling inhibitory effects of PRO238, PRO239, and PRO241 using reporter cells. [Figure 8c] 1 is a graph showing the results obtained by measuring the VEGF signaling inhibitory effects of PRO242 and PRO017 using reporter cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Fusion proteins comprising antibody fragments that specifically bind to C3b or C5 In one aspect of the invention, a fusion protein is provided comprising an antibody fragment that specifically binds to C3b (complement component 3b) or C5 (complement component 5) and a protein that specifically binds to VEGF (vascular endothelial growth factor).

[0016] As used herein, the term "C3b (complement component 3b)" refers to a fragment of C3 formed by cleaving complement component C3 by C3 convertase, and can be used interchangeably with "complement C3b protein." In particular, C3b plays a central role in the complement system, binding to cell surfaces and acting as a marker. In addition, it has an opsonizing effect that allows phagocytes with C3b receptors to recognize it and cause cytotoxicity. In addition, it activates C5 and forms the membrane attack complex (MAC) (kubi immunology, WH Freeman and Company, New York).

[0017] As used herein, the terms "complement component," "complement protein," or "complement component protein" refer to molecules involved in the activation of the complement system. Classical pathway components include, for example, C1q, C1r, C1s, C2, C3, C4, C5, C6, C7, C8, C9, and the C5b-9 complex. Alternative pathway components include, for example, factor B, factor D, properdin, factor H, and factor I.

[0018] As used herein, the term "antibody fragment that specifically binds to C3b" may be used interchangeably with "anti-C3b antibody" and refers to an antibody that specifically binds to complement C3b protein or an antigen-binding fragment thereof.

[0019] The antibody or antigen-binding fragment thereof that specifically binds to C3b specifically binds to complement component C3b and blocks activation of the complement pathway. Therefore, it can be used to treat diseases associated with activation of the complement pathway, such as macular degeneration-related diseases (including, for example, AMD, North Carolina macular dystrophy, Sorsby fundus dystrophy, Stargardt's disease, pattern dystrophy, Best's disease, dominant drusen and Malattia Leventinese (radial drusen), extramacular changes occurring before and after macular degeneration and / or dysfunction, retinal detachment, choroidal degeneration, retinal degeneration, photoreceptor degeneration, RPE degeneration, mucopolysaccharidoses, rod-cone dystrophy, cone-rod dystrophy, cone degeneration, etc.).

[0020] An "antibody that specifically binds to C3b" comprises at least two heavy chains and two light chains, linked by disulfide bonds. The heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region, and the light chain consists of a light chain variable region (VL) and a light chain constant region. The VH and VL regions are further subdivided into hypervariable regions called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs).

[0021] "Antigen-binding fragment of an antibody that specifically binds C3b" refers to one or more fragments of an intact antibody that retain the ability to specifically bind to a given antigen, i.e., C3b. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include Fab, scFv, F(ab')2, diabodies, triabodies, sdAbs, and VFv. H Examples include H.

[0022] In one embodiment, an antibody that specifically binds to C3b or an antigen-binding fragment thereof may comprise a heavy chain variable region comprising an HCDR1 of SEQ ID NO:20, an HCDR2 of SEQ ID NO:21, and an HCDR3 of SEQ ID NO:22, and a light chain variable region comprising an LCDR1 of SEQ ID NO:23, an LCDR2 of SEQ ID NO:24, and an LCDR3 of SEQ ID NO:25. Additionally, the antibody may comprise a heavy chain variable region comprising an HCDR1 of SEQ ID NO:28, an HCDR2 of SEQ ID NO:29, and an HCDR3 of SEQ ID NO:30, and a light chain variable region comprising an LCDR1 of SEQ ID NO:31, an LCDR2 of SEQ ID NO:32, and an LCDR3 of SEQ ID NO:33. Additionally, the antibody may comprise a heavy chain variable region comprising an HCDR1 of SEQ ID NO:36, an HCDR2 of SEQ ID NO:37, and an HCDR3 of SEQ ID NO:38, and a light chain variable region comprising an LCDR1 of SEQ ID NO:39, an LCDR2 of SEQ ID NO:40, and an LCDR3 of SEQ ID NO:41.

[0023] In another embodiment, an antibody that specifically binds to C3b, or an antigen-binding fragment thereof, may comprise a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 26 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 27. Additionally, the antibody may comprise a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 34 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 35. Additionally, the antibody may comprise a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 42 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 43.

[0024] The anti-C3b antibody may refer to any antibody known to those skilled in the art without being limited thereto. As another example, the antibody may be the anti-C3b antibody or a fragment thereof disclosed in U.S. Patent Application Publication No. 2010-0291106 or U.S. Patent No. 8,377,437.

[0025] As used herein, the term "C5 (complement component 5)" can be used interchangeably with "complement C5 protein" and is a protein that is cleaved and released into C5a and C5b fragments by C5 convertase. C5a is an anaphylatoxin that releases neutrophils and cytokines that mediate inflammation, and C5b combines with C6, C7, C8, and C9 to form a membrane attack complex, thereby destroying cell membranes. It is known that when the concentration of C5a in the body is kept high, the expression of IL-17 and IL-22 is increased, and the increased expression of IL-17 and IL-22 acts as an inflammatory factor and induces VEGF and angiogenesis (Liu B. et al., J.Transl.Med., 2011;9:1-12).

[0026] As used herein, the term "antibody fragment that specifically binds to C5" may be used interchangeably with "anti-C5 antibody" and refers to an antibody that specifically binds to complement C5 protein or an antigen-binding fragment thereof.

[0027] The antibody or antigen-binding fragment thereof that specifically binds to C5 specifically binds to complement component C5 and blocks activation of the complement pathway, and can therefore be used to treat diseases associated with activation of the complement pathway, such as macular degeneration-related diseases.

[0028] An "antibody that specifically binds to C5" comprises at least two heavy chains and two light chains, which are linked by disulfide bonds. The heavy chains and light chains are as described above.

[0029] "Antigen-binding fragment of an antibody that specifically binds to C5" refers to one or more fragments of an intact antibody that retain the ability to specifically bind to a given antigen, i.e., C5. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include Fab, scFv, F(ab')2, diabodies, triabodies, sdAbs, and VFv. H Examples include H.

[0030] In one embodiment, an antibody that specifically binds to C5 or an antigen-binding fragment thereof may comprise a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 44, an HCDR2 of SEQ ID NO: 45, and an HCDR3 of SEQ ID NO: 46, and a light chain variable region comprising an LCDR1 of SEQ ID NO: 47, an LCDR2 of SEQ ID NO: 48, and an LCDR3 of SEQ ID NO: 49.

[0031] In another embodiment, an antibody that specifically binds to C5, or an antigen-binding fragment thereof, may comprise a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:50 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:51.

[0032] The anti-C5 antibody may refer to any antibody known to those skilled in the art, without being limited thereto. As another example, the antibody may be the anti-C5 antibody or a fragment thereof disclosed in U.S. Patent No. 6,355,245 or U.S. Patent Application Publication No. 2019-0177436.

[0033] As used herein, the term "VEGF (vascular endothelial growth factor)" refers to vascular endothelial growth factor, which causes angiogenesis by binding to its receptor, VEGFR (VEGF receptor). VEGF plays a role in inducing the proliferation, migration, and differentiation of endothelial cells by activating various signal transduction cascades. Under pathological conditions, VEGF induces abnormal angiogenesis and promotes the proliferation of tumor cells and retinal cells and vascular leakage, thereby causing tumor growth and metastasis, diabetic retinopathy, and age-related macular degeneration.

[0034] Furthermore, in the present specification, VEGF or VEGF family proteins are collectively referred to as "VEGF". VEGF family proteins may have activity equivalent to or similar to that of VEGF. In this case, "activity" may mean, for example, specific binding to a VEGF receptor, and this specific binding can be measured by a method known to those skilled in the art.

[0035] The VEGF family protein may be one or more selected from the group consisting of VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, PlGF (placental growth factor) and recombinant VEGF. Preferably, the VEGF may be VEGF-A, VEGF-B, or PlGF, which are preferentially required for angiogenesis.

[0036] As used herein, the term "PlGF (placental growth factor)" refers to a transmembrane protein encoded on chromosome 2p21-p16. PlGF acts as a selective ligand for VEGFR-1 and can promote angiogenesis. PlGF is at least 40% identical in amino acid composition to VEGF. In one embodiment, PlGF may be PlGF-1 or PlGF-2.

[0037] As used herein, the term "recombinant VEGF" refers to VEGF that has been recombined by alternative exon splicing. Recombinant VEGF may be, for example, but is not limited to, VEGF111, VEGF121, VEGF145, VEGF148, VEFG165, VEGF183, VEGF189, or VEGF206 according to amino acid numbering.

[0038] As used herein, the term "protein that specifically binds to VEGF" refers to an antibody that specifically binds to the extracellular domain of VEGF or a VEGF receptor.

[0039] As used herein, the term "VEGF receptor" refers to a receptor that binds to VEGF, and the biological functions of VEGF are mediated through VEGFR-1 (Flt-1), VEGFR-2 (KDR / Flk-1), and VEGFR-3 (Flt-4). VEGFR consists of seven extracellular immunoglobulin (Ig)-like domains, a transmembrane (TM) domain, a regulatory juxtamembrane domain, an intracellular tyrosine kinase domain, and several other tyrosine residues. Specifically, the extracellular immunoglobulin (Ig)-like domains of VEGFR-1 and VEGFR-2 are known to be VEGF-binding domains.

[0040] In the present invention, the VEGF receptor may be, but is not limited to, VEGF receptor 1 (VEGFR-1) or VEGF receptor 2 (VEGFR-2).

[0041] As used herein, the term "extracellular domain of a VEGF receptor" refers to the domain of a VEGF receptor that binds to VEGF, specifically, the portion of the extracellular domain that contains the VEGF ligand, excluding the transmembrane and cytoplasmic regions of the VEGF receptor.

[0042] Specifically, the extracellular domain of the VEGF receptor may be a fragment of the VEGF receptor that binds to VEGF. Furthermore, the extracellular domain of the VEGF receptor may inhibit angiogenesis by binding to VEGF-A, VEGF-B, or PlGF. In this case, one embodiment of the extracellular domain of the VEGF receptor may include the amino acid sequence of SEQ ID NO: 52. Furthermore, the extracellular domain of the VEGF receptor may be in a form in which a part of the extracellular domain of the VEGF receptor including SEQ ID NO: 52 is truncated or altered.

[0043] As used herein, the term "antibody that specifically binds to VEGF" refers to an antibody or a fragment thereof that specifically binds to VEGF and induces an antigen-antibody reaction, and is also called an anti-VEGF antibody.

[0044] Antibody is a general term for a molecule capable of specific antigen-antibody binding to VEGF. Furthermore, the antibody can be used in any form as long as the antibody contains an antigen-binding domain capable of specific binding to VEGF. The antibody or a fragment thereof may be Fab (antigen-binding fragment), F(ab)2, scFv (single-chain variable fragment), di-scFv, sdAb (single-domain antibody), chimeric antibody, humanized antibody, human antibody, or variants thereof.

[0045] The anti-VEGF antibody may comprise any one of the variable sites selected from the group consisting of aflibercept, bevacizumab, ranibizumab, ramucirumab, brolucizumab, faricimab, KSI-301, vanucizumab, BI-836880, HuMab G6-31, B20-4.1, BAT-5906, nabicixizumab, zilpakimab, hPV-19 and AT-001. Preferably, the anti-VEGF antibody may comprise the variable region of aflibercept, bevacizumab, ranibizumab, brolucizumab, KSI-301, vanucizumab, BI-836880 or BAT-5906.

[0046] At this time, aflibercept refers to a recombinant humanized fusion protein that inhibits VEGF-A and PlGF in blood vessels. Aflibercept can be directly injected into the eye. Bevacizumab is an antibody that is an angiogenesis inhibitor that inhibits blood vessel growth by inhibiting VEGF-A in blood vessels. For the treatment of macular degeneration, bevacizumab can be directly injected into the eye. Ranibizumab is an Fab that is effective in treating wet macular degeneration by inhibiting angiogenesis. Ramucirumab is an antibody that inhibits angiogenesis mediators or VEGF receptor 2. Brolucizumab is an scFv that binds to VEGF-A, inhibits angiogenesis, and treats wet macular degeneration. Faricimab is a bispecific antibody that inhibits VEGF-A and angiopoietin-2.

[0047] Additionally, KSI-301 is an antibody effective in treating wet macular degeneration. Vanucizumab is a bispecific humanized monoclonal antibody that inhibits VEGF-A and angiopoietin-2. BI-836880 is a humanized bispecific nanobody that inhibits VEGF and angiopoietin-2. HuMab G6-31 is a Fab fragment that inhibits human VEGF. B20-4.1 is an scFv fragment that inhibits human VEGF.

[0048] Furthermore, BAT-5906 is an antibody effective in treating wet macular degeneration. Nabicixizumab is an anti-DLL4 / VEGF bispecific antibody. Zilpakimab is an anti-DLL4 / VEGF bispecific antibody, also called ABT-165. hPV-19 is an anti-VEGF antibody with anti-angiogenic and anti-tumor activity. AT-001 is an antibody that inhibits angiogenesis by blocking human VEGF receptor 3.

[0049] In one embodiment, the anti-VEGF antibody may comprise the variable region of BI-836880. Specifically, the antibody may comprise a heavy chain variable region comprising CDR1 of SEQ ID NO:88, CDR2 of SEQ ID NO:89, and CDR3 of SEQ ID NO:90.

[0050] In one embodiment, the anti-VEGF antibody may comprise the variable region of bevacizumab. Specifically, the antibody may comprise a heavy chain variable region comprising HCDR1 of SEQ ID NO: 91, HCDR2 of SEQ ID NO: 92, and HCDR3 of SEQ ID NO: 93, and a light chain variable region comprising LCDR1 of SEQ ID NO: 94, LCDR2 of SEQ ID NO: 95, and LCDR3 of SEQ ID NO: 96.

[0051] In one embodiment, the anti-VEGF antibody may comprise the variable region of ranibizumab. Specifically, the antibody may comprise a heavy chain variable region comprising HCDR1 of SEQ ID NO: 97, HCDR2 of SEQ ID NO: 98, and HCDR3 of SEQ ID NO: 99, and a light chain variable region comprising LCDR1 of SEQ ID NO: 100, LCDR2 of SEQ ID NO: 101, and LCDR3 of SEQ ID NO: 102.

[0052] In one embodiment, the anti-VEGF antibody may comprise the variable region of ramucirumab. Specifically, the antibody may comprise a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 103, an HCDR2 of SEQ ID NO: 104, and an HCDR3 of SEQ ID NO: 105, and a light chain variable region comprising an LCDR1 of SEQ ID NO: 106, an LCDR2 of SEQ ID NO: 107, and an LCDR3 of SEQ ID NO: 108.

[0053] In one embodiment, the anti-VEGF antibody may comprise the variable region of faricimab. Specifically, the antibody may comprise a heavy chain variable region comprising HCDR1 of SEQ ID NO: 109, HCDR2 of SEQ ID NO: 110, and HCDR3 of SEQ ID NO: 111, and a light chain variable region comprising LCDR1 of SEQ ID NO: 112, LCDR2 of SEQ ID NO: 113, and LCDR3 of SEQ ID NO: 114.

[0054] In one embodiment, the anti-VEGF antibody may comprise the variable region of KSI-301. Specifically, the antibody may comprise a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 115, an HCDR2 of SEQ ID NO: 116, and an HCDR3 of SEQ ID NO: 117, and a light chain variable region comprising an LCDR1 of SEQ ID NO: 118, an LCDR2 of SEQ ID NO: 119, and an LCDR3 of SEQ ID NO: 120.

[0055] In one embodiment, the anti-VEGF antibody may comprise the variable region of vanucizumab. Specifically, the antibody may comprise a heavy chain variable region comprising HCDR1 of SEQ ID NO: 121, HCDR2 of SEQ ID NO: 122, and HCDR3 of SEQ ID NO: 123, and a light chain variable region comprising LCDR1 of SEQ ID NO: 124, LCDR2 of SEQ ID NO: 125, and LCDR3 of SEQ ID NO: 126.

[0056] In one embodiment, the anti-VEGF antibody may comprise the variable regions of BAT-5906. Specifically, the antibody may comprise a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 127, an HCDR2 of SEQ ID NO: 128, and an HCDR3 of SEQ ID NO: 129, and a light chain variable region comprising an LCDR1 of SEQ ID NO: 130, an LCDR2 of SEQ ID NO: 131, and an LCDR3 of SEQ ID NO: 132.

[0057] In one embodiment, the anti-VEGF antibody may comprise the variable region of nabicixizumab. Specifically, the antibody may comprise a heavy chain variable region comprising HCDR1 of SEQ ID NO: 133, HCDR2 of SEQ ID NO: 134, and HCDR3 of SEQ ID NO: 135, and a light chain variable region comprising LCDR1 of SEQ ID NO: 136, LCDR2 of SEQ ID NO: 137, and LCDR3 of SEQ ID NO: 138.

[0058] In one embodiment, the anti-VEGF antibody may comprise the variable region of zilpakimab. Specifically, the antibody may comprise a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 139, an HCDR2 of SEQ ID NO: 140, and an HCDR3 of SEQ ID NO: 141, and a light chain variable region comprising an LCDR1 of SEQ ID NO: 142, an LCDR2 of SEQ ID NO: 143, and an LCDR3 of SEQ ID NO: 144.

[0059] In one embodiment, the anti-VEGF antibody may comprise the variable regions of hPV-19. Specifically, the antibody may comprise a heavy chain variable region comprising HCDR1 of SEQ ID NO: 145, HCDR2 of SEQ ID NO: 146, and HCDR3 of SEQ ID NO: 147, and a light chain variable region comprising LCDR1 of SEQ ID NO: 148, LCDR2 of SEQ ID NO: 149, and LCDR3 of SEQ ID NO: 150.

[0060] In one embodiment, the anti-VEGF antibody may comprise the variable region of AT-001. Specifically, the antibody may comprise a heavy chain variable region comprising HCDR1 of SEQ ID NO: 151, HCDR2 of SEQ ID NO: 152, and HCDR3 of SEQ ID NO: 153, and a light chain variable region comprising LCDR1 of SEQ ID NO: 154, LCDR2 of SEQ ID NO: 155, and LCDR3 of SEQ ID NO: 156.

[0061] In one embodiment, the anti-VEGF antibody may comprise a heavy chain of SEQ ID NO: 157 and a light chain of SEQ ID NO: 158; a heavy chain variable region of SEQ ID NO: 159 and a light chain of SEQ ID NO: 160; a heavy chain of SEQ ID NO: 161 and a light chain of SEQ ID NO: 162; a heavy chain of SEQ ID NO: 163 and a light chain of SEQ ID NO: 164; a heavy chain of SEQ ID NO: 165 and a light chain of SEQ ID NO: 166; a heavy chain of SEQ ID NO: 167 and a light chain of SEQ ID NO: 168; a heavy chain of SEQ ID NO: 169 and a light chain of SEQ ID NO: 170; a heavy chain of SEQ ID NO: 171 and a light chain of SEQ ID NO: 172; a heavy chain of SEQ ID NO: 173 and a light chain of SEQ ID NO: 174; a heavy chain variable region of SEQ ID NO: 175 and a light chain variable region of SEQ ID NO: 176; or a heavy chain variable region of SEQ ID NO: 177 and a light chain variable region of SEQ ID NO: 178.

[0062] The fragment of the anti-VEGF antibody may be an scFv (single chain variable fragment). In this case, the scFv refers to a form in which the heavy chain variable region and the light chain variable region are linked by a peptide linker. Specifically, the scFv may include a variable region including CDR1 of SEQ ID NO: 179, CDR2 of SEQ ID NO: 180, CDR3 of SEQ ID NO: 181, CDR4 of SEQ ID NO: 182, CDR5 of SEQ ID NO: 183, and CDR6 of SEQ ID NO: 184. Furthermore, the scFv may include the amino acid sequence of SEQ ID NO: 185. In this case, one embodiment of the scFv may be brolucizumab.

[0063] The anti-VEGF antibody may comprise the variable region of HuMab G6-31 or B20-4.1. Specifically, the antibody may comprise a heavy chain variable region comprising HCDR1 of SEQ ID NO: 186, HCDR2 of SEQ ID NO: 187, and HCDR3 of SEQ ID NO: 188, and a light chain variable region comprising LCDR1 of SEQ ID NO: 189, LCDR2 of SEQ ID NO: 190, and LCDR3 of SEQ ID NO: 191. Furthermore, the antibody may comprise a heavy chain variable region comprising HCDR1 of SEQ ID NO: 192, HCDR2 of SEQ ID NO: 193, and HCDR3 of SEQ ID NO: 194, and a light chain variable region comprising LCDR1 of SEQ ID NO: 195, LCDR2 of SEQ ID NO: 196, and LCDR3 of SEQ ID NO: 197.

[0064] The antibody that specifically binds to VEGF can refer to any antibody known to those skilled in the art, without being limited thereto. As another example, the antibody can be an anti-VEGF antibody or a fragment thereof disclosed in U.S. Patent No. 9,527,925, U.S. Patent No. 8,268,314, or U.S. Patent Application Publication No. 2019-0167790.

[0065] Linkers and Fc variants The antibody fragment that specifically binds to C3b or C5 and the protein that specifically binds to VEGF may be linked by a linker, which may be, but is not limited to, a peptide linker, an immunoglobulin fragment, or a combination thereof.

[0066] The linker connects two proteins. An embodiment of the linker may include 1 to 50 amino acids, albumin or a fragment thereof, an Fc domain of an immunoglobulin, and the like. In this case, the Fc domain of an immunoglobulin refers to a protein that includes the heavy chain constant region 2 (CH2) and the heavy chain constant region 3 (CH3) of an immunoglobulin, but does not include the variable regions of the heavy and light chains of the immunoglobulin and the light chain constant region 1 (CH1). The immunoglobulin may be IgG, IgA, IgE, IgD, or IgM, and may preferably be IgG1. In this case, the Fc domain of a wild-type immunoglobulin G1 may have the amino acid sequence of SEQ ID NO: 68. As used herein, the Fc domain may refer to a region including the CH2 and CH3 domains, excluding the hinge region.

[0067] Furthermore, the Fc domain of an immunoglobulin may be not only a wild-type Fc domain, but also an Fc domain variant. Furthermore, as used herein, the term "Fc domain variant" may refer to a form that differs from the wild-type Fc domain in terms of glycosylation pattern, a form that has high glycosylation compared to the wild-type Fc domain, or a form that has low glycosylation compared to the wild-type Fc domain, or a deglycosylated form. Furthermore, an unglycosylated Fc domain is included herein. The Fc domain or these variants may be adjusted to have an adjusted number of sialic acids, fucosylation, or glycosylation through the culture conditions or genetic engineering of the host.

[0068] Furthermore, the glycosylation of the Fc domain of an immunoglobulin can be modified by conventional methods, such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms. Furthermore, the Fc domain variant may be in a form in which the Fc regions of each of immunoglobulins IgG, IgA, IgE, IgD, or IgM are mixed. Furthermore, the Fc domain variant may be in a form in which some amino acids in the Fc domain are replaced with other amino acids.

[0069] As used herein, the term "Fc domain variant" refers to an Fc domain in which the glycosylation of the wild-type Fc domain has been altered, the sequence between the Fc domains has been mixed, or some amino acids of the wild-type Fc domain have been deleted, altered, substituted, and / or added. The deletion, alteration, substitution, and / or addition of some amino acids of the wild-type Fc domain can be performed by methods known to those skilled in the art. In one embodiment, the Fc domain variant may have some amino acid sequences of the wild-type Fc domain substituted and / or added.

[0070] The "amino acid" introduced by substitution and / or addition may be any one selected from the group consisting of lysine (K), alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine ​​(C), glutamine (Q), glutamic acid (E), glycine (G), histidine (H), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y) and valine (V).

[0071] The Fc domain mutations may be for modulating the activity or function of the antibody. In one embodiment, the Fc domain mutations may be for modulating the effector function of the antibody or the antibody cytotoxic activity.

[0072] In one embodiment, the Fc domain variant may comprise a DANG mutation, where "DANG mutation" refers to the D265A / N297G mutation that eliminates effector function of human IgG1 or mouse IgG2a.

[0073] Effector functions mediated by the Fc region of an IgG molecule include C1q binding, complement-dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptor, BCR), etc. Generally, these effector functions require association of the Fc region with a binding domain (e.g., an antibody variable domain).

[0074] Effector functions can be altered by substitution of the amino acid sequence of a non-mutated Fc region, and Fc regions with altered effector functions can be designed, for example, by modifying C1q binding and / or FcR binding, thereby altering CDC activity and / or ADCC activity. That is, "DANG mutation" means that the effector function mediated by the Fc region is removed from the IgG molecule, preventing undesirable reduction in effector function during antibody production.

[0075] An embodiment of the Fc domain variant may be a substitution of D27A, N59G, D118E, L120M, D27A / N59G, or D27A / N59G / D118E / L120M in the amino acid sequence of SEQ ID NO: 68. Furthermore, lysine (K) may be added to position 209 of the amino acid sequence of SEQ ID NO: 68. Specifically, an embodiment of the Fc domain variant may have any one of the amino acid sequences of SEQ ID NOs: 62 to 67.

[0076] The fusion protein may have a structure in which an antibody fragment that specifically binds to C3b or C5 and a protein that specifically binds to VEGF are linked, or a protein that specifically binds to VEGF and an antibody fragment that specifically binds to C3b or C5 are linked, respectively, to their N-terminus and C-terminus using the Fc domain as a linker. The link between the N-terminus or C-terminus of the Fc domain and the antibody fragment that specifically binds to C3b or C5, or the protein that specifically binds to VEGF, may be achieved by a linker peptide, as the case may be.

[0077] Fusion protein structure Specifically, the fusion protein has the following structural formula (I) or (II): N'-X-[linker (1)]n-Fc domain or its variant-[linker (2)]mY-C'(I) N'-Y-[linker (1)]n-Fc domain or its variant-[linker (2)]mX-C'(II) It may also consist of In structural formulas (I) and (II), N' is the N-terminus of the fusion protein, C' is the C-terminus of the fusion protein; X is an antibody fragment that specifically binds to C3b or C5; Y is a protein that specifically binds to VEGF, Linkers (1) and (2) are peptide linkers; n and m are each independently 0 or 1.

[0078] The antibody fragment that specifically binds to C3b or C5, the protein that specifically binds to VEGF, and the Fc domain are each as described above.

[0079] As used herein, the term "fusion protein" refers to a recombinant protein in which two or more proteins or domains that perform a specific function within the protein are linked together so that each protein or domain performs its own function. A linker peptide, which generally has a flexible structure, can be inserted between the two or more proteins or domains.

[0080] In this case, the peptide linker (1) may consist of 5 to 80 consecutive amino acids, 7 to 70 consecutive amino acids, or 10 to 60 consecutive amino acids, or 12 to 50 amino acids. In one embodiment, the peptide linker (1) may consist of 30 amino acids. Furthermore, the peptide linker (1) may contain at least one cysteine. Specifically, it may contain 1, 2, or 3 cysteines. Furthermore, the peptide linker (1) may be derived from an immunoglobulin hinge, and may further contain (G4S)n (wherein n is an integer of 1 to 10). In one embodiment, the peptide linker (1) may be a peptide linker consisting of any one of the amino acid sequences of SEQ ID NOs: 53 to 57.

[0081] The peptide linker (2) may consist of 1 to 50 consecutive amino acids, or 3 to 30 consecutive amino acids, or 5 to 20 amino acids. In one embodiment, the peptide linker (2) may be (G4S)n (wherein n is an integer of 1 to 10). In this case, n in (G4S)n may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the peptide linker (2) may be a peptide linker consisting of any one of the amino acid sequences of SEQ ID NOs: 58 to 61.

[0082] In one embodiment, the antibody fragment may be a Fab or scFv that specifically binds to C3b.

[0083] Specifically, a Fab or scFv that specifically binds to C3b may comprise a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 20, an HCDR2 of SEQ ID NO: 21, and an HCDR3 of SEQ ID NO: 22, and a light chain variable region comprising an LCDR1 of SEQ ID NO: 23, an LCDR2 of SEQ ID NO: 24, and an LCDR3 of SEQ ID NO: 25.

[0084] Furthermore, a Fab or scFv that specifically binds to C3b may comprise a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 28, an HCDR2 of SEQ ID NO: 29, and an HCDR3 of SEQ ID NO: 30, and a light chain variable region comprising an LCDR1 of SEQ ID NO: 31, an LCDR2 of SEQ ID NO: 32, and an LCDR3 of SEQ ID NO: 33.

[0085] Furthermore, a Fab or scFv that specifically binds to C3b may comprise a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 36, an HCDR2 of SEQ ID NO: 37, and an HCDR3 of SEQ ID NO: 38, and a light chain variable region comprising an LCDR1 of SEQ ID NO: 39, an LCDR2 of SEQ ID NO: 40, and an LCDR3 of SEQ ID NO: 41.

[0086] In one embodiment, a Fab or scFv that specifically binds to C3b may comprise a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 26 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 27. Additionally, a Fab or scFv that specifically binds to C3b may comprise a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 34 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 35. Additionally, a Fab or scFv that specifically binds to C3b may comprise a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 42 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 43.

[0087] In one embodiment, the antibody fragment may be a Fab or scFv that specifically binds to C5.

[0088] Specifically, a Fab or scFv that specifically binds to C5 may comprise a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 44, an HCDR2 of SEQ ID NO: 45, and an HCDR3 of SEQ ID NO: 46, and a light chain variable region comprising an LCDR1 of SEQ ID NO: 47, an LCDR2 of SEQ ID NO: 48, and an LCDR3 of SEQ ID NO: 49.

[0089] In one embodiment, a Fab or scFv that specifically binds to C5 may comprise a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:50 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:51.

[0090] The fusion protein of the present invention may include a fusion protein containing a light chain variable region and a light chain constant region of an anti-C3b antibody, a heavy chain variable region and a heavy chain constant region (CH1) of an anti-C3b antibody, an Fc domain, and a protein that specifically binds to VEGF. In this case, at least one protein that specifically binds to VEGF may be contained in the anti-C3b antibody. Furthermore, the protein that specifically binds to VEGF may be bound to the C-terminus of the anti-C3b antibody.

[0091] Furthermore, the fusion protein of the present invention may include a fusion protein containing the light chain variable region and light chain constant region of an anti-C5 antibody, the heavy chain variable region and heavy chain constant region (CH1) of an anti-C5 antibody, an Fc domain, and a protein that specifically binds to VEGF. In this case, at least one protein that specifically binds to VEGF may be contained in the anti-C5 antibody. Furthermore, the protein that specifically binds to VEGF may be bound to the C-terminus of the anti-C5 antibody.

[0092] In one embodiment, the fusion protein may comprise a polypeptide of SEQ ID NO:2 and a polypeptide of SEQ ID NO:9; a polypeptide of SEQ ID NO:4 and a polypeptide of SEQ ID NO:10; a polypeptide of SEQ ID NO:6 and a polypeptide of SEQ ID NO:11; a polypeptide of SEQ ID NO:8 and a polypeptide of SEQ ID NO:12; a polypeptide of SEQ ID NO:13; a polypeptide of SEQ ID NO:14; a polypeptide of SEQ ID NO:15; or a polypeptide of SEQ ID NO:16.

[0093] Specifically, SEQ ID NOs: 2, 4, and 6 are amino acid sequences of fusion proteins containing the light chain variable region and the light chain constant region of an anti-C3b antibody, respectively. Furthermore, SEQ ID NOs: 9, 10, and 11 are amino acid sequences containing i) the heavy chain variable region and the heavy chain constant region (CH1) of an anti-C3b antibody, ii) an Fc domain, and iii) a protein that specifically binds to VEGF, respectively.

[0094] Furthermore, SEQ ID NO: 8 is the amino acid sequence of a fusion protein comprising the light chain variable region and light chain constant region of an anti-C5 antibody, and SEQ ID NO: 12 is the amino acid sequence comprising i) the heavy chain variable region and heavy chain constant region (CH1) of an anti-C5 antibody, ii) an Fc domain, and iii) a protein that specifically binds to VEGF.

[0095] In this case, the combination of SEQ ID NOs: 2 and 9, the combination of SEQ ID NOs: 4 and 10, the combination of SEQ ID NOs: 6 and 11, and the combination of SEQ ID NOs: 8 and 12 are linked by disulfide (-SS-) bonds and held together by non-covalent interactions.

[0096] Specifically, SEQ ID NOs: 13, 14, and 15 are each amino acid sequences comprising an scFv comprising: i) a protein that specifically binds to VEGF, ii) an Fc domain, iii) a heavy chain variable region of an anti-C3b antibody, and iv) a light chain variable region of an anti-C3b antibody.

[0097] Furthermore, SEQ ID NO: 16 is an amino acid sequence comprising i) a protein that specifically binds to VEGF, ii) an Fc domain, iii) a heavy chain variable region of an anti-C5 antibody, and iv) an scFv comprising a light chain variable region of an anti-C5 antibody.

[0098] In another embodiment, the fusion protein comprises a polypeptide having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a polypeptide of SEQ ID NO:2 and a polypeptide of SEQ ID NO:9; a polypeptide of SEQ ID NO:4 and a polypeptide of SEQ ID NO:10; a polypeptide of SEQ ID NO:6 and a polypeptide of SEQ ID NO:11; a polypeptide of SEQ ID NO:8 and a polypeptide of SEQ ID NO:12; a polypeptide of SEQ ID NO:13; a polypeptide of SEQ ID NO:14; a polypeptide of SEQ ID NO:15; or a polypeptide of SEQ ID NO:16. In this case, the identity is, for example, percent homology and can be determined by homology comparison software such as the BlastN software of the National Center for Biotechnology Information (NCBI).

[0099] Fusion Protein Dimer In another aspect of the present invention, a fusion protein dimer is provided in which two fusion proteins, comprising an antibody fragment that specifically binds to C3b or C5 and a protein that specifically binds to VEGF, are bound to each other.

[0100] In this case, the bond between the fusion proteins constituting the dimer may be achieved by, but is not limited to, a disulfide bond formed by cysteines present in the linker. The fusion proteins constituting the dimer may be the same or different from each other. Preferably, the dimer may be a homodimer.

[0101] Polynucleotides encoding fusion proteins In another aspect of the present invention, a polynucleotide is provided that encodes a fusion protein comprising an antibody fragment that specifically binds to C3b or C5 and a protein that specifically binds to VEGF. Specifically, the polynucleotide may comprise a polynucleotide of SEQ ID NO:77 and a polynucleotide of SEQ ID NO:70; a polynucleotide of SEQ ID NO:78 and a polynucleotide of SEQ ID NO:72; a polynucleotide of SEQ ID NO:79 and a polynucleotide of SEQ ID NO:74; a polynucleotide of SEQ ID NO:80 and a polynucleotide of SEQ ID NO:76; a polynucleotide of SEQ ID NO:81; a polynucleotide of SEQ ID NO:82; a polynucleotide of SEQ ID NO:83; or a polynucleotide of SEQ ID NO:84. In the polynucleotide, one or more nucleotides may be mutated by substitution, deletion, insertion, or a combination thereof. When the nucleotide sequence is prepared by chemical synthesis, a synthesis method known in the art, for example, the synthesis method described in Engels and Uhlmann (Angew Chem IntEd Engl., 37:73-127, 1988), may be used. Such methods include the triester, phosphite, phosphoramidite and H-phosphate methods, PCR and other autoprimer methods, oligonucleotide synthesis on solid supports, and the like.

[0102] According to one embodiment, the polynucleotide may comprise a nucleic acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identity to a polynucleotide of SEQ ID NO:77 and a polynucleotide of SEQ ID NO:70; a polynucleotide of SEQ ID NO:78 and a polynucleotide of SEQ ID NO:72; a polynucleotide of SEQ ID NO:79 and a polynucleotide of SEQ ID NO:74; a polynucleotide of SEQ ID NO:80 and a polynucleotide of SEQ ID NO:76; a polynucleotide of SEQ ID NO:81; a polynucleotide of SEQ ID NO:82; a polynucleotide of SEQ ID NO:83; or a polynucleotide of SEQ ID NO:84.

[0103] The polynucleotide may further comprise a nucleic acid encoding a signal sequence or leader sequence. As used herein, the term "signal sequence" refers to a signal peptide that induces secretion of a target protein. The signal peptide is translated and then cleaved in the host cell. Specifically, a signal sequence is an amino acid sequence that causes translocation of a protein across the endoplasmic reticulum (ER) membrane.

[0104] Signal sequences are well known in the art for their properties. Such signal sequences typically contain 16-30 amino acid residues, but may contain more or fewer than these amino acid residues. A typical signal peptide is composed of three regions: an N-terminal region; a central hydrophobic region; and a more polar C-terminal region. The central hydrophobic region contains 4-12 hydrophobic residues that anchor the signal sequence as the immature polypeptide translocates across the membrane lipid bilayer.

[0105] After initiation, the signal sequence is cleaved in the lumen of the ER by a cellular enzyme commonly known as a signal peptidase. At this time, the signal sequence may be a secretory signal sequence of tPa (tissue plasminogen activator), HSV gDs (signal sequence of glycoprotein D of herpes simplex virus), or growth hormone. Preferably, a secretory signal sequence used in higher eukaryotic cells, including mammals, can be used.

[0106] A vector carrying a polynucleotide encoding a fusion protein In another aspect of the invention, there is provided a vector comprising the polynucleotide.

[0107] A vector can be introduced into a host cell and recombined and inserted into the genome of the host cell. Alternatively, a vector is understood to be a nucleic acid means that contains a polynucleotide sequence capable of replicating autonomously as an episome. Vectors include linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors and analogs thereof. Examples of viral vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, etc.

[0108] Specifically, examples of vectors include plasmid DNA, phage DNA, etc., and include commercially developed plasmids (pUC18, pBAD, pIDTSAMRT-AMP, etc.), Escherichia coli-derived plasmids (pYG601BR322, pBR325, pUC118, pUC119, etc.), Bacillus subtilis-derived plasmids (pUB110, pTP5, etc.), yeast-derived plasmids (YEp13, YEp24, YCp50, etc.), phage DNA (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP, etc.), animal virus vectors (retrovirus, adenovirus, vaccinia virus, etc.), and insect virus vectors (baculovirus, etc.). Vectors show different protein expression levels and modifications depending on the host cell, so it is preferable to select and use the host cell most suitable for the purpose.

[0109] As used herein, the term "gene expression" or "expression" of a target protein is understood to mean the transcription of a DNA sequence, the translation of an mRNA transcript, and the secretion of a fusion protein product or a fragment thereof. A useful expression vector may be RcCMV (Invitrogen, Carlsbad) or a variant thereof. The expression vector may include a human cytomegalovirus (CMV) promoter to promote continuous transcription of the target gene in mammalian cells and a bovine growth hormone polyadenylation signal sequence to increase the steady-state level of RNA after transcription.

[0110] Transformed cells expressing the fusion protein In another aspect of the invention, there is provided a transformed host cell into which the vector has been introduced.

[0111] Host cells for transformed cells can include, but are not limited to, prokaryotic cells, eukaryotic cells, and cells of mammalian, plant, insect, fungal, or cellular origin. Examples of prokaryotic cells include Escherichia coli. Examples of eukaryotic cells include yeast. Examples of mammalian cells include CHO cells, F2N cells, CSO cells, BHK cells, Bowes melanoma cells, HeLa cells, 911 cells, AT1080 cells, A549 cells, HEK293 cells, HEK293T cells, and the like. However, mammalian cells are not limited thereto, and any cells known to those skilled in the art to be usable as mammalian host cells can be used.

[0112] Furthermore, when introducing an expression vector into a host cell, the CaCl2 precipitation method, the Hanahan method, the efficiency of which is increased by using a reducing agent such as dimethyl sulfoxide (DMSO) in the CaCl2 precipitation method, electroporation, calcium phosphate precipitation, protoplast fusion, the agitation method using silicon carbide fibers, Agrobacterium-mediated transformation, transformation methods using PEG, dextran sulfate, or lipofectamine, and desiccation / inhibition-mediated transformation can be used.

[0113] As described above, to optimize the properties of the fusion protein as a therapeutic agent or for other purposes, the glycosylation pattern of the fusion protein (e.g., sialic acid, fucosylation, glycosylation) can be adjusted by manipulating glycosylation-related genes present in the host cell via methods known to those of skill in the art.

[0114] Methods for Producing Fusion Proteins In another aspect of the present invention, a method for producing a fusion protein comprising an antibody fragment that specifically binds to C3b or C5 and a protein that specifically binds to VEGF is provided, the method comprising the step of culturing a transformed cell. Specifically, the production method may comprise the steps of: i) culturing the transformed cell to obtain a culture product; and ii) recovering the fusion protein from the culture product.

[0115] The method for culturing the transformed cells can be carried out using methods that are widely known in the art. In particular, the culturing can be carried out in a batch process or continuously in a fed-batch or repeated fed-batch process.

[0116] Use of the fusion protein or dimer thereof In another aspect of the present invention, there is provided a pharmaceutical composition for treating or preventing an eye disease, comprising as an active ingredient a fusion protein containing an antibody fragment that specifically binds to C3b or C5 and a protein that specifically binds to VEGF, or a fusion protein dimer in which two fusion proteins are bound to each other.

[0117] The fusion proteins and fusion protein dimers are described above.

[0118] As used herein, the term "ocular disease" may refer to a general term for diseases occurring in the eye. Ocular disease may refer to ocular diseases caused or aggravated by complement activity or angiogenesis, or ocular diseases that include excessive angiogenesis as the main condition. Ocular disease may be any one selected from the group consisting of age-related macular degeneration (AMD), geographic atrophy (GA), choroidal neovascularization (CNV), uveitis, diabetic retinopathy and other ischemia-related retinopathies, diabetic macular edema, pathological myopia, von Hippel-Lindau disease, ocular histoplasmosis, central retinal vein occlusion (CRVO), corneal neovascularization, and retinal neovascularization.

[0119] The preferred dosage of the pharmaceutical composition varies depending on the condition and weight of the patient, the severity of the disease, the form of the drug, the route and duration of administration, and can be appropriately selected by those skilled in the art. In the pharmaceutical composition for treating or preventing eye disease of the present invention, the active ingredient can be included in any amount (effective amount) according to the use, dosage form, purpose of preparation, etc., as long as the active ingredient shows activity in treating eye disease or can show a therapeutic effect on macular degeneration in particular. The conventional effective amount will be determined within the range of 0.001 wt% to 20.0 wt% based on the total weight of the composition. As used herein, the term "effective amount" refers to the amount of the active ingredient that can induce an effect of improving or treating the condition of eye disease, in particular, an effect of improving or treating the condition of macular degeneration. Such an effective amount can be determined experimentally within the scope of common sense of a person skilled in the art.

[0120] As used herein, the term "treatment" can be used to include both therapeutic and prophylactic treatment. In this context, prevention can be used to mean alleviating or reducing a condition or disease in a subject. In one embodiment, the term "treatment" includes both any form of application or administration to treat a disease in a mammal, including a human. Furthermore, the term includes inhibiting or slowing the progression of a disease or disease, and includes the meaning of restoring or repairing a damaged or lost function; stimulating an inefficient process; or alleviating a significant disease, to partially or completely alleviate the disease.

[0121] Pharmacokinetic parameters such as bioavailability and underlying parameters such as clearance rate may also affect efficacy. Thus, "enhanced efficacy" (e.g., improved efficacy) may be due to improved pharmacokinetic parameters and improved efficacy, which can be measured by comparing parameters such as clearance rate and treatment or amelioration of ocular disease in experimental animals or human subjects.

[0122] As used herein, the term "therapeutically effective amount" or "pharmaceutical effective amount" refers to an amount of a compound or composition effective to prevent or treat a disease of interest, sufficient to treat the disease at a reasonable benefit / risk ratio applicable to any medical treatment, and without causing side effects. The level of the effective amount can be determined according to factors including the patient's health condition, the type and severity of the disease, the activity of the drug, the patient's sensitivity to the drug, the mode of administration, the time of administration, the route of administration and the rate of excretion, the duration of treatment, drugs used in combination or simultaneously, and other factors well known in the medical field. In one embodiment, a therapeutically effective amount refers to an amount of drug effective to treat an ocular disease.

[0123] At this time, the pharmaceutical composition may further comprise a pharma- ceutically acceptable carrier. The pharma-ceutically acceptable carrier may be any carrier, so long as the carrier is a non-harmful substance suitable for delivery to a patient. Distilled water, alcohol, fat, wax and inert solids may be included as carriers. Pharmaceutically acceptable auxiliary agents (buffers, dispersants) may also be included in the pharmaceutical composition.

[0124] Specifically, by including a pharma- ceutical composition in addition to an active ingredient, a pharma- ceutical composition can be prepared in a parenteral dosage form according to an administration route using a conventional method known in the art. In this case, the term "pharma- ceutical acceptable" means that the carrier does not inhibit the activity of the active ingredient and does not have toxicity beyond that compatible with the subject to which it is applied (prescribed).

[0125] When the pharmaceutical composition is prepared as a parenteral preparation, it can be formulated with a suitable carrier according to a method known in the art into the form of injection, transdermal patch, nasal inhalant, and suppository.When it is formulated as an injection, sterilized water, ethanol, polyol, such as glycerol or propylene glycol, or their mixture can be used as a suitable carrier, and isotonic liquid, such as Ringer's solution, triethanolamine-containing phosphate buffered saline (PBS), sterile water for injection or 5% dextrose, etc., can be preferably used.The formulation of pharmaceutical compositions is known in the art, and can be specifically referred to Remington's Pharmaceutical Sciences (19th edition, 1995), etc. This document is considered to be a part of this specification.

[0126] The preferred dosage of the pharmaceutical composition may be in the range of 0.01 μg / kg to 10 g / kg or 0.01 mg / kg to 1 g / kg per day, depending on the patient's condition, weight, sex, age, severity of the patient, and route of administration. Administration may be once a day or divided into several times a day. Such dosage should not be construed as limiting the scope of the present invention in any aspect.

[0127] The subjects to which the pharmaceutical composition can be applied (prescribed) are mammals and humans, with humans being particularly preferred. In addition to the active ingredient, the pharmaceutical composition of the present invention may further comprise any compound or natural extract known to have a therapeutic effect on eye diseases, particularly macular degeneration.

[0128] In another aspect of the present invention, there is provided use of a fusion protein comprising an antibody fragment that specifically binds to C3b or C5 and a protein or a dimer thereof that specifically binds to VEGF, for the manufacture of a medicament for the treatment or prevention of an eye disease.

[0129] In another aspect of the invention, there is provided a method for treating or preventing an eye disease, the method comprising administering to a subject a fusion protein comprising an antibody fragment that specifically binds C3b or C5 and a protein or dimer that specifically binds VEGF.

[0130] The fusion protein, the fusion protein dimer in which two fusion proteins are bound to each other, and the eye disease are as described above.In this case, the subject may be a subject suffering from an eye disease.Furthermore, the subject may be a mammal, and preferably a human.

[0131] The administration route, dosage and frequency of administration of the fusion protein or fusion protein dimer can be carried out in various ways and amounts according to the patient's condition and the presence or absence of side effects, so that the fusion protein or fusion protein dimer can be administered to the subject in various ways and amounts. The optimal administration method, dosage and frequency of administration can be selected within an appropriate range by those skilled in the art. Furthermore, the fusion protein or fusion protein dimer can be administered in combination with other drugs or bioactive substances that are known to have a therapeutic effect on the disease to be treated, or can be formulated in the form of a combined preparation with other drugs. EXAMPLES

[0132] The present invention will now be described in more detail with reference to the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

[0133] Preparation Example 1. Preparation of fusion protein

[0134] [Table 1]

[0135] [SEQ ID NO: 1] is the heavy chain sequence of human anti-C3b antibody MOR09611.

[0136] [SEQ ID NO: 2] is the light chain sequence of human anti-C3b antibody MOR09611.

[0137] [SEQ ID NO: 3] is the heavy chain sequence of human anti-C3b antibody MOR09675.

[0138] [SEQ ID NO: 4] is the light chain sequence of human anti-C3b antibody MOR09675.

[0139] [SEQ ID NO: 5] is composed of the heavy chain variable region sequence of human anti-C3b antibody S77, the human IgG1 CH1 region sequence, and human IgG1 Fc with effector function removed by DANG mutations (D265A, N297G).

[0140] [SEQ ID NO: 6] is the light chain sequence of human anti-C3b antibody S77.

[0141] [SEQ ID NO: 7] is composed of the heavy chain variable region sequence of the human anti-C5 (anti-C5) antibody eculizumab, the human IgG1 CH1 region sequence, and the human IgG1 Fc DANG.

[0142] [SEQ ID NO: 8] is the light chain sequence of the human anti-C5 antibody eculizumab.

[0143] [SEQ ID NO: 9] is composed of the heavy chain variable region sequence of human anti-C3b antibody MOR09611, human IgG1 CH1 region sequence, human IgG1 Fc DANG, linker GGGGSGGGGS (SEQ ID NO: 59), and the VEGF binding site of aflibercept.

[0144] [SEQ ID NO: 10] is composed of the heavy chain variable region sequence of human anti-C3b antibody MOR09675, human IgG1 CH1 region sequence, human IgG1 Fc DANG, linker GGGGSGGGGS, and the VEGF binding site of aflibercept.

[0145] [SEQ ID NO: 11] is composed of the heavy chain variable region sequence of human anti-C3b antibody S77, human IgG1 CH1 region sequence, human IgG1 Fc DANG, linker GGGGSGGGGS, and the VEGF binding site of aflibercept.

[0146] [SEQ ID NO: 12] is composed of the heavy chain variable region sequence of the human anti-C5 antibody eculizumab, the human IgG1 CH1 region sequence, human IgG1 Fc DANG, the linker GGGGSGGGGS, and the VEGF binding site of aflibercept.

[0147] [SEQ ID NO: 13] is composed of the VEGF binding site of aflibercept, the linker GGGGS (SEQ ID NO: 58), human IgG1 Fc DANG, the linker GGGSGGGGSGGGGGS (SEQ ID NO: 60), the heavy chain variable region sequence of human anti-C3b antibody MOR09611, the linker GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 61), and the light chain variable region sequence of MOR09611.

[0148] [SEQ ID NO: 14] is composed of the VEGF binding site of aflibercept, the linker GGGGS, human IgG1 Fc DANG, the linker GGGSGGGGSGGGGGS, the heavy chain variable region sequence of the human anti-C3b antibody MOR09675, the linker GGGGSGGGGSGGGGSGGGGS, and the light chain variable region sequence of MOR09675.

[0149] [SEQ ID NO: 15] is composed of the VEGF binding site of aflibercept, the linker GGGGS, human IgG1 Fc DANG, the linker GGGSGGGGSGGGGGS, the heavy chain variable region sequence of human anti-C3b antibody S77, the linker GGGGSGGGGSGGGGSGGGGS, and the light chain variable region sequence of S77.

[0150] [SEQ ID NO: 16] is composed of the VEGF binding site of aflibercept, the linker GGGGS, human IgG1 Fc DANG, the linker GGGGSGGGSGGGGGS, the light chain variable region sequence of the human anti-C5 antibody eculizumab, the linker GGGGSGGGSGGGGGS, and the heavy chain variable region sequence of eculizumab.

[0151] [SEQ ID NO: 17] is composed of human IgG1 Fc DANG.

[0152] [SEQ ID NO: 18] is composed of the extracellular domain region (20-283) of human CRIg protein, the linker GGGGS, and human IgG1 Fc DANG.

[0153] [SEQ ID NO: 19] consists of the sequence of aflibercept.

[0154] MOR09611 (SEQ ID NOs: 1 and 2) is a human anti-C3b antibody.

[0155] MOR09675 (SEQ ID NOs: 3 and 4) is a human anti-C3b antibody.

[0156] S77 (SEQ ID NOs: 5 and 6) is a human anti-C3b antibody.

[0157] Eculizumab (SEQ ID NOs: 7 and 8) is a human anti-C5 antibody.

[0158] PRO236 (SEQ ID NOs: 2 and 9) is an effector-function-depleted bispecific antibody in which the VEGF binding sites of the human anti-C3b antibody MOR09611 and aflibercept are located at the N-terminus and C-terminus, respectively.

[0159] PRO237 (SEQ ID NOs: 4 and 10) is an effector-function-depleted bispecific antibody in which the VEGF binding sites of the human anti-C3b antibody MOR09675 and aflibercept are located at the N-terminus and C-terminus, respectively.

[0160] PRO238 (SEQ ID NOs: 6 and 11) is an effector-function-depleted bispecific antibody in which the VEGF binding sites of the human anti-C3b antibody S77 and aflibercept are located at the N-terminus and C-terminus, respectively.

[0161] PRO239 (SEQ ID NOs: 8 and 12) is an effector-function-depleted bispecific antibody in which the VEGF binding sites of the human anti-C5 antibodies eculizumab and aflibercept are located at the N-terminus and C-terminus, respectively.

[0162] PRO240 (SEQ ID NO: 13) is an effector-function-depleted bispecific antibody in which the VEGF-binding site of aflibercept and the single-chain variable region fragment (single-chain variable fragment, scFv) sequence of the human anti-C3b antibody MOR09611 are located at the N-terminus and C-terminus, respectively.

[0163] PRO241 (SEQ ID NO: 14) is an effector-function-depleted bispecific antibody in which the VEGF binding site of aflibercept and a single chain variable region fragment sequence of the human anti-C3b antibody MOR09675 sequence are located at the N-terminus and C-terminus, respectively.

[0164] PRO242 (SEQ ID NO: 15) is an effector-function-depleted bispecific antibody in which the VEGF-binding site of aflibercept and the single-chain variable region fragment sequence of the human anti-C3b antibody S77 are located at the N- and C-termini, respectively.

[0165] PRO243 (SEQ ID NO: 16) is an effector-function-depleted bispecific antibody in which the VEGF-binding site of aflibercept and the single chain variable region fragment sequence of the human anti-C5 antibody eculizumab are located at the N- and C-termini, respectively.

[0166] KNP-301 (sequence number 18) is an effector function-removed Fc fusion protein dimer in which the extracellular domain region of human CRIg and the VEGF-binding site of aflibercept are located at the N-terminus and C-terminus, respectively.

[0167] [SEQ ID NO: 1] Anti-C3b (MOR09611) HC [ka]

[0168] [SEQ ID NO: 2] Anti-C3b (MOR09611) LC [ka]

[0169] [SEQ ID NO: 3] Anti-C3b (MOR09675) HC [ka]

[0170] [SEQ ID NO: 4] Anti-C3b (MOR09675) LC [ka]

[0171] [SEQ ID NO: 5] Anti-C3b(S77)HC-hu IgG1 Fc DANG [ka]

[0172] [SEQ ID NO: 6] Anti-C3b (S77) LC [ka]

[0173] [SEQ ID NO: 7] Anti-C5 (eculizumab) HC-hu IgG1 Fc DANG [ka]

[0174] [SEQ ID NO: 8] Anti-C5 (eculizumab) LC [ka]

[0175] [SEQ ID NO: 9] Anti-C3b (MOR09611) HC-hu IgG1 Fc DANG-VEGF binder [ka]

[0176] [SEQ ID NO: 10] Anti-C3b (MOR09675) HC-hu IgG1 Fc DANG-VEGF binder [ka]

[0177] [SEQ ID NO: 11] Anti-C3b(S77)HC-hu IgG1 Fc DANG-VEGF binder [ka]

[0178] [SEQ ID NO: 12] Anti-C5 (eculizumab) HC-hu IgG1 Fc DANG-VEGF binder [ka]

[0179] [SEQ ID NO: 13] VEGF binder-hu IgG1 Fc DANG-anti-C3b (MOR09611) scFv [ka]

[0180] [SEQ ID NO: 14] VEGF binder-hu IgG1 Fc DANG-anti-C3b (MOR09675) scFv [ka]

[0181] [SEQ ID NO: 15] VEGF binder-hu IgG1 Fc DANG-anti-C3b(S77)scFv [ka]

[0182] [SEQ ID NO: 16] VEGF binder-hu IgG1 Fc DANG-anti-C5 (eculizumab) scFv [ka]

[0183] [SEQ ID NO: 17] hu IgG1 Fc DANG [ka]

[0184] [SEQ ID NO: 18] hu CRIg-hu IgG1 Fc DANG-VEGF binder [ka]

[0185] [SEQ ID NO: 19] VEGF binder-hu IgG1 Fc [ka]

[0186] Preparation Example 2. Protein Preparation

[0187] 2. Preparation Example 2.1. Vector Composition and Plasmid Maxipreps The reagents and equipment used are described in Tables 2 and 3 below.

[0188] [Table 2]

[0189] [Table 3]

[0190] The synthetic DNA fragment was amplified by PCR, and the PCR product was gel purified. The pTT5 vector was cut with restriction enzymes EcoRI and BamHI, and then gel purified. Each PCR product and the linear vector were ligated using an infusion kit. The resulting vector was transformed into ECOS101 DH5α competent cells, and the cells were cultured on 2xYT agar plates containing 100 μg / ml ampicillin. All operation processes were performed according to standard transformation protocols. Positive recombinant products were identified by colony PCR, and sequence verification sequencing was performed on the recombinant plasmids. A single colony was selected and the seed culture was inoculated into 5 mL of 2xYT medium containing 100 μg / mL ampicillin. The culture was shaken at 37°C for 8 hours.

[0191] The seed culture was then diluted in 200 mL of 2xYT selective medium at a ratio of 1:1,000. The culture was incubated at 37°C for 16 hours with shaking. The bacterial cells were harvested by centrifugation at 4,700 rpm for 10 minutes at 4°C. The bacterial pellet was resuspended in 12 mL of RES-EF buffer. Then, 12 mL of LYS-EF buffer was added, the closed tube was mixed thoroughly by vigorously inverting, and then incubated at room temperature for 5 minutes. 12 mL of NEU-EF buffer was added to the lysate and mixed thoroughly and quickly by vigorously inverting. Before pouring the lysate into the NucleoBond® Xtra column filter, a homogenous suspension of the precipitate was prepared by inverting the lysate tube three times to prevent clogging of the filter.

[0192] The Nucleobond® Extra column filter and Nucleobond® Extra column were then washed with 10 mL of Filter Wash Buffer FIL-EF. The Nucleobond® Extra column filter was removed by pulling or inverting the column. The Nucleobond® Extra column was washed with 90 mL of Wash Buffer ENDO.

[0193] The Nucleobond® Extra column was washed with 45 mL of wash buffer WASH-EF. Plasmid DNA was eluted with 15 mL of elution buffer ELU. The eluate was collected in a 50 mL centrifuge tube. 10.5 mL of room temperature isopropanol was added to precipitate the eluted plasmid DNA. After vortexing, the mixture was left for 2 minutes.

[0194] Then, 5 mL of 70% ethanol was added to the pellet. Using a pipette tip, the ethanol was carefully and completely removed from the tube. The pellet was dried at room temperature (20-25°C). The DNA pellet was then dissolved in 1,000 μL of purified water.

[0195] Preparative Example 2.2. Cell Transfection and Protein Expression The materials and reagents used are listed in Table 4 below.

[0196] [Table 4]

[0197] The 293F seed strain containing complete medium was maintained in an incubator shaker at 130 rpm, 37°C and 8% CO2. 6 cells / ml~0.4×10 6 Cells were cultured at a density of 2.6 × 10 cells / mL and the medium was changed every 2–3 days. 24 h before transfection, freshly subcultured 293F cells were cultured at a density of 2.6 × 10 6 The cells were cultured in an incubator shaker at 130 rpm, 37° C. and 8% CO2.

[0198] On the day of transfection, use fresh medium to reduce the cell density to 5.0 × 10 6The transfection reagent was adjusted to 1000 cells / mL. The adjustment was made in a 3 L shaker flask with a total volume of 1 L. 0.4 mg of HC plasmid and 0.6 mg of LC plasmid were diluted with 50 mL of OPTI MEM I and filtered through a 0.22 μm filter. Then, 2 mg of PEI was diluted with 50 mL of OPTI MEM I to prepare the transfection reagent.

[0199] The diluted PEI was added to the DNA mixture and then immediately mixed. It was then incubated at room temperature for 15 minutes. 6 The DNA-PEI mixture was added to 293F cells prepared at 1000 cells / mL. The cells were then continuously cultured for 24 hours in an incubator shaker at 130 rpm, 37°C and 8% CO2. At 24 hours after transfection, 10% peptone was added to 1 / 20 of the culture solution, resulting in a final concentration of 0.5%. The cells were then cultured in an incubator shaker at 130 rpm, 37°C and 8% CO2. Cell density / viability was measured and recorded daily during the period 2-5 days after transfection. Cells were harvested for purification 7 days after transfection or when cell viability was less than 70%.

[0200] Preparative Example 2.3. Protein Purification The reagents, buffer compositions, and equipment used for protein purification are listed in Tables 5 to 7 below.

[0201] [Table 5]

[0202] [Table 6]

[0203] [Table 7]

[0204] Proteins were purified using a MabSelect Sure column. Specifically, the supernatant was collected by centrifugation at 2,000×g for 20 min at 4°C. The supernatant was then filtered through a Zaltopore 2 filter. The clarified supernatant was loaded onto a 5 mL MabSelect Sure column equilibrated with Buffer A. The column was then washed with Buffer A until the A280 absorbance reached baseline. The column was further washed with 10 CV of Buffer B. The column was further washed with 10 CV of Buffer A. The bound protein was eluted with 6 CV of Buffer C, and 1 / 6 volume of Buffer D was added to neutralize the eluted material. SDS-PAGE and SEC-HPLC analysis were then performed.

[0205] The protein was then purified using an SEC column: the supernatant was loaded onto an SEC column equilibrated with the final buffer, and the protein was eluted with the final buffer.

[0206] As a result, as shown in Figures 1a to 1c, purified fusion protein dimers MOR09611, MOR09675, S77, eculizumab, PRO236, PRO237, PRO238, PRO239, PRO240, PRO241, PRO242, PRO243, PRO017, KNP-301, and aflibercept were identified by SDS-PAGE.

[0207] Experimental Example 1. Identification of binding affinity of fusion proteins to complement proteins and VEGF by ELISA To determine whether the fusion protein dimers of one embodiment inhibit the complement pathway, it was determined by enzyme immunoassay whether KNP-301, MOR09611, MOR09675, PRO236, PRO237, PRO238, PRO239, PRO241, PRO242, S77, and eculizumab bind to C3b protein, and whether PRO239, eculizumab, and PRO017 bind to C5.

[0208] Specifically, human C3b protein was immobilized on a plate, and KNP-301, MOR09611, MOR09675, PRO236, PRO237, PRO238, PRO239, PRO241, PRO242, S77, and eculizumab were bound to it. Next, anti-human immunoglobulin G antibody and anti-horseradish peroxidase (HRP) antibody were sequentially bound to it. Furthermore, human C5 protein was immobilized on a plate, and PRO239, eculizumab, and PRO017 were bound to human C5.

[0209] As a result, as shown in Figures 3a and 3b, KNP-301, MOR09611, MOR09675, PRO236, PRO237, PRO238, PRO241, PRO242, and S77 were found to bind to human C3b in a concentration-dependent manner. Furthermore, as shown in Figure 4, PRO239 and eculizumab were found to bind to human C5 in a concentration-dependent manner.

[0210] To determine whether the fusion protein according to one embodiment has an anti-VEGF effect, the binding of the fusion protein to human VEGF165 protein was determined by enzyme immunoassay, and the binding affinity of PRO236, PRO237, PRO238, PRO239, PRO241, PRO242, and PRO017 to human VEGF165 was measured by ELISA.

[0211] Specifically, human VEGF165 protein was immobilized on a plate, and PRO236, PRO237, PRO238, PRO239, PRO241, PRO242, PRO017, and aflibercept (control) were bound to the plate. Next, anti-human immunoglobulin G antibody and anti-horseradish peroxidase (HRP) antibody were sequentially bound to the plate.

[0212] As a result, as shown in Figures 5a and 5b, it was found that aflibercept having anti-VEGF, PRO236, PRO237, PRO238, PRO239, PRO241, and PRO242 bound to human VEGF165 in a concentration-dependent manner.

[0213] Experimental Example 2. Analysis of the effect of inhibiting the alternative complement pathway by hemolysis analysis To determine whether the fusion protein dimer according to one embodiment inhibits the alternative complement pathway, KNP-301, MOR09611, MOR09675, PRO236, PRO237, PRO239, PRO241, PRO242, S77, and eculizumab were subjected to a hemolysis assay (AH50). Specifically, 0.5 mL of rabbit red blood cells were washed with TBS using a centrifuge at 400×g for 10 minutes. After repeating this process twice, the rabbit red blood cells were washed again with GVB EGTA buffer using centrifugation at 400×g for 10 minutes. The concentration of rabbit red blood cells was then adjusted to 1×10 using GVB EGTA buffer. 9 Adjusted to cells / mL.

[0214] To analyze AH50 by sensitized red blood cells, 12% human C1q-depleted serum was added to the 96-well plate (50 μL / well). In addition, KNP-301, MOR09611, MOR09675, PRO236, PRO237, PRO239, PRO241, PRO242, S77, and eculizumab were added at various concentrations. After incubation at 4°C for 30 minutes, rabbit red blood cells were added (2 × 10 6 Cells were cultured at 37°C for 1.5 h and centrifuged at 600 x g for 10 min. 110 μL of supernatant was collected and OD415 value was measured.

[0215] The alternative complement pathway inhibitory effects of KNP-301, MOR09611, MOR09675, PRO236, PRO237, PRO239, PRO241, PRO242, S77, and eculizumab were identified by AH50 hemolytic assay.

[0216] As a result, as shown in Figures 6a to 6c, it was found that KNP-301, MOR09611, MOR09675, PRO236, PRO237, PRO239, PRO241, PRO242, S77, and eculizumab inhibited hemolysis by the alternative complement pathway in a concentration-dependent manner.

[0217] Experimental Example 3. Analysis of the effect of inhibiting the classical complement pathway by hemolysis analysis To determine whether the fusion protein dimers according to one embodiment do not inhibit the alternative complement pathway, KNP-301, MOR09611, MOR09675, PRO236, PRO237, PRO239, PRO241, PRO242, S77, and eculizumab were subjected to a hemolytic assay (CH50).

[0218] Specifically, sheep Ab-sensitized red blood cells were centrifuged in TBS at 400 × g for 10 min, and this process was repeated twice, after which the sheep red blood cells were washed in GVB++ buffer by centrifugation at 400 × g for 10 min. The sheep sensitized red blood cells were then washed at 1 × 10 9 The concentration was adjusted to 100 / mL.

[0219] To analyze CH50 by sensitized red blood cells, 4.5% human factor B-depleted serum was added to 96-well plates (50 μL / well), and then treated with KNP-301, MOR09611, MOR09675, PRO236, PRO237, PRO239, PRO241, PRO242, S77, and eculizumab at various concentrations. After incubation at 4°C for 30 min, sensitized sheep red blood cells were added (2.5 × 10 6 Cells were incubated at 37° C. for 30 min. 110 μL of supernatant was collected by centrifugation at 600×g for 10 min, and OD415 was measured.

[0220] As a result, as shown in Figures 7a to 7c, it was found that KNP-301, MOR09611, MOR09675, PRO236, PRO237, PRO241, PRO242, and S77 do not inhibit hemolysis by the classical complement pathway, whereas PRO239 and eculizumab inhibit hemolysis.

[0221] Experimental Example 4. Analysis of the efficacy of fusion protein dimers using VEGF reporter cells To determine whether the fusion protein dimer of one embodiment effectively inhibits VEGF protein, it was determined whether it inhibits the binding between VEGF and the VEGF receptor.

[0222] The VEGF signaling inhibitory effect of aflibercept, PRO236, PRO237, PRO238, PRO239, PRO241, and PRO242 was identified using reporter cells. Specifically, using VEGF reporter cells (GA3001, Promega, USA) that generate luminescent light by receptor-mediated signaling upon binding of VEGF, it was confirmed by the degree of luminescence whether aflibercept, PRO236, PRO237, PRO238, PRO239, PRO241, PRO242, and PRO017 inhibit the binding of VEGF to the VEGF receptor.

[0223] As a result, as shown in Figures 8a to 8c, it was found that aflibercept, PRO236, PRO237, PRO238, PRO239, PRO241, and PRO242 inhibited VEGF receptor-mediated signaling in a concentration-dependent manner.

Claims

1. A fusion protein comprising an antibody fragment that specifically binds to C3b (complement component 3b) or C5 (complement component 5), and a protein that specifically binds to VEGF (vascular endothelial growth factor).

2. The fusion protein of claim 1 , wherein the antibody fragment that specifically binds to C3b or C5 and the protein that specifically binds to VEGF are linked by a linker.

3. The fusion protein of claim 2 , wherein the linker is a peptide linker, an immunoglobulin fragment, or a combination thereof.

4. The fusion protein of claim 3 , wherein the immunoglobulin fragment comprises a DANG mutation.

5. The fusion protein of claim 3, wherein the immunoglobulin fragment comprises any one of the amino acid sequences of SEQ ID NOs: 62 to 68.

6. The fusion protein of claim 1 , wherein the protein that specifically binds to VEGF comprises an antibody or a fragment thereof that specifically binds to VEGF, or the extracellular domain of a VEGF receptor.

7. The fusion protein of claim 6 , wherein the VEGF receptor is VEGF receptor 1 or VEGF receptor 2.

8. The fusion protein of claim 7, wherein the extracellular domain of the VEGF receptor comprises the amino acid sequence of SEQ ID NO:

52.

9. The following structural formula (I) or (II): N'-X-[linker (1)]n-Fc domain or variant thereof-[linker (2)]m-Y-C' (I) N'-Y-[linker (1)]n-Fc domain or variant thereof-[linker (2)]m-X-C'(II) It consists of In structural formulas (I) and (II), N' is the N-terminus of the fusion protein; C' is the C-terminus of the fusion protein; X is the antibody fragment that specifically binds to C3b or C5; Y is the protein that specifically binds to VEGF, the linkers (1) and (2) are peptide linkers, n and m are each independently 0 or 1; The fusion protein of claim 1.

10. The fusion protein according to claim 9, wherein the linker (1) comprises any one of the amino acid sequences of SEQ ID NOs: 53 to 57.

11. The fusion protein of claim 9, wherein the linker (2) comprises any one of the amino acid sequences of SEQ ID NOs: 58 to 61.

12. 10. The fusion protein of claim 9, wherein X is a Fab or scFv that specifically binds to C3b or C5.

13. The Fab or scFv that specifically binds to C3b is i) a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 20, an HCDR2 of SEQ ID NO: 21, and an HCDR3 of SEQ ID NO: 22, and a light chain variable region comprising an LCDR1 of SEQ ID NO: 23, an LCDR2 of SEQ ID NO: 24, and an LCDR3 of SEQ ID NO: 25; ii) a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 28, an HCDR2 of SEQ ID NO: 29, and an HCDR3 of SEQ ID NO: 30, and a light chain variable region comprising an LCDR1 of SEQ ID NO: 31, an LCDR2 of SEQ ID NO: 32, and an LCDR3 of SEQ ID NO: 33; or iii) a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 36, an HCDR2 of SEQ ID NO: 37, and an HCDR3 of SEQ ID NO: 38, and a light chain variable region comprising an LCDR1 of SEQ ID NO: 39, an LCDR2 of SEQ ID NO: 40, and an LCDR3 of SEQ ID NO: 41; The fusion protein of claim 12, comprising:

14. The Fab or scFv that specifically binds to C3b is i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 26 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 27; ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 34 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 35; or iii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 42 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 43 The fusion protein of claim 13, comprising:

15. The Fab or scFv that specifically binds to C5 is a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 44, an HCDR2 of SEQ ID NO: 45, and an HCDR3 of SEQ ID NO: 46; or A light chain variable region comprising an LCDR1 of SEQ ID NO: 47, an LCDR2 of SEQ ID NO: 48, and an LCDR3 of SEQ ID NO:

49. The fusion protein of claim 12, comprising:

16. The fusion protein of claim 15, wherein the Fab or scFv that specifically binds to C5 comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 50 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

51.

17. The fusion protein of claim 1, comprising a polypeptide of SEQ ID NO:2 and a polypeptide of SEQ ID NO:9; a polypeptide of SEQ ID NO:4 and a polypeptide of SEQ ID NO:10; a polypeptide of SEQ ID NO:6 and a polypeptide of SEQ ID NO:11; a polypeptide of SEQ ID NO:8 and a polypeptide of SEQ ID NO:12; a polypeptide of SEQ ID NO:13; a polypeptide of SEQ ID NO:14; a polypeptide of SEQ ID NO:15; or a polypeptide of SEQ ID NO:

16.

18. A fusion protein dimer in which two fusion proteins according to any one of claims 1 to 17 are bound to each other.

19. The fusion protein dimer of claim 18, which is a homodimer.

20. A polynucleotide encoding the fusion protein of any one of claims 1 to 17.

21. A vector comprising the polynucleotide of claim 20.

22. A host cell transformed with the vector of claim 21.

23. A pharmaceutical composition comprising the fusion protein of claim 1 as an active ingredient.

24. A pharmaceutical composition comprising the fusion protein dimer described in claim 19 as an active ingredient.

25. A pharmaceutical composition for treating or preventing an eye disease, wherein the eye disease is any one selected from the group consisting of age-related macular degeneration (AMD), geographic atrophy (GA), choroidal neovascularization (CNV), uveitis, diabetic retinopathy and other ischemia-related retinopathies, diabetic macular edema, pathological myopia, von Hippel-Lindau disease, ocular histoplasmosis, central retinal vein occlusion (CRVO), corneal neovascularization, and retinal neovascularization, as described in claim 23.

26. A pharmaceutical composition for treating or preventing an eye disease, wherein the eye disease is any one selected from the group consisting of age-related macular degeneration (AMD), geographic atrophy (GA), choroidal neovascularization (CNV), uveitis, diabetic retinopathy and other ischemia-related retinopathies, diabetic macular edema, pathological myopia, von Hippel-Lindau disease, ocular histoplasmosis, central retinal vein occlusion (CRVO), corneal neovascularization, and retinal neovascularization.

27. 24. The pharmaceutical composition of claim 23, further comprising a pharmaceutically acceptable carrier.

28. The pharmaceutical composition of claim 24, further comprising a pharmaceutically acceptable carrier.

29. Use of the fusion protein of claim 1 for the manufacture of a medicament for treating or preventing an eye disease.

30. Use of the fusion protein dimer described in claim 19 for the manufacture of a medicament for treating or preventing an eye disease.

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