Dual-targeting antibody that specifically binds to interleukin-17A and tumor necrosis factor-α

Abstract

The present invention relates to a dual-target antibody that specifically binds to interleukin-17A (IL-17A) and tumor necrosis factor-α (TNF-α), and uses thereof. The dual-target antibody or antigen-binding fragment thereof of the present invention has the following advantages: it shows high specificity for interleukin-17A and tumor necrosis factor-α, shows excellent neutralizing ability compared with existing single-target antibodies, and also rapidly inhibits inflammation and immune responses by simultaneously inhibiting interleukin-17 (IL-17) and tumor necrosis factor-α, thereby reducing the dosage and enhancing the therapeutic effect.

Description

Technical Field

[0001] The present invention relates to a dual-targeting antibody that specifically binds to interleukin-17A (IL-17A) and tumor necrosis factor-α (TNF-α) and uses thereof. Background Art

[0002] Tumor necrosis factor-alpha (TNF-α) is an inflammatory cytokine produced by activated macrophages, CD4+ T cells, NK cells, and other immune cells. It regulates inflammatory responses by regulating immune cells. TNF-α induces hemorrhagic necrosis and apoptosis in some tumor cells, promotes T-cell proliferation, and promotes B-cell differentiation and proliferation, thereby activating immune responses (Biotherapy. 3(2):103-11, 1991). TNF-α is an important cytokine for normal immune responses, but when imbalances such as overexpression of TNF-α occur in the body, various autoimmune reactions occur. Furthermore, as a proinflammatory cytokine, it increases the production of other cytokines at the site of autoimmune reactions, accelerating their symptoms (Annu Rev Immunol. 14:397-440, 1996; Inflamm Bowel Dis. 5(2):119-33, 1999).

[0003] To treat and alleviate such autoimmune diseases, various tumor necrosis factor-α inhibitors have been developed, with representative examples being infliximab (trade name: Remicade), adalimumab (trade name: Humira), and etanercept (trade name: Enbrel). However, 10% to 30% of patients with autoimmune diseases are ineffective against tumor necrosis factor-α inhibitors, and approximately 60% of patients who respond to tumor necrosis factor-α inhibitors experience decreased efficacy, requiring dose increases or alternative therapies (Rheumatology (Oxford). 46(7):1153-6, 2007; Ann Rheum Dis. 65(6):746-52, 2006).

[0004] Interleukin-17 (IL-17, interleukin-17A) is a representative inflammatory cytokine produced by Th cells, known as T helper 17 cells (J Exp Med. 1; 183(6): 2593-603, 1996). Interleukin-17 consists of six families: interleukin-17A, interleukin-17B (IL-17B), interleukin-17C (IL-17C), interleukin-17D (IL-17D), interleukin-17E (IL-17E), and interleukin-17F (IL-17F). In particular, interleukin-17F has the highest homology with interleukin-17A. Interleukin-17A and interleukin-17F form homodimers or heterodimers that bind to each other. Interleukin-17, which forms a dimer, binds to the receptor interleukin-17R (IL-17R). Interleukin-17R has five subtypes: interleukin-17RA (IL-17RA), interleukin-17RB (IL-17RB), interleukin-17RC (IL-17RC), interleukin-17RD (IL-17RD), and interleukin-17RE (IL-17RE). When interleukin-17 binds to the receptor, it activates a signaling system that induces chemokines, which in turn attract immune cells such as monocytes and neutrophils to the site of inflammation. Interleukin-17 works together with tumor necrosis factor-α and interleukin-1 (IL-1), and their signal transduction systems have been confirmed in the pathogenesis of various autoimmune diseases (J Invest Dermatol. 131(3):677-87, 2011; N Engl J Med. 27; 361(9):888-98, 2009; J Invest Dermatol. 133(1):17-26, 2013).

[0005] Interleukin-17 inhibitors have been developed and approved for use as pharmaceuticals for the treatment and alleviation of interleukin-17-mediated autoimmune diseases. In January 2015, the U.S. Food and Drug Administration (FDA) approved secukinumab (Cosentyx), an interleukin-17 inhibitory monoclonal antibody, for the treatment of severe plaque psoriasis.

[0006] Interestingly, in a Phase III clinical trial of secukinumab in patients with ankylosing spondylitis refractory to tumor necrosis factor-α inhibitors, ASAS20 response rates ranged from 45.5% to 58.8%, depending on the dosing route. This compares to ASAS20 response rates of 60% and 66.3% in patients not previously treated with tumor necrosis factor-α inhibitors, respectively, suggesting that secukinumab is effective in patients with active ankylosing spondylitis, regardless of prior exposure to tumor necrosis factor-α inhibitors (2014 ACR / ARHP annual meeting, ABSTRACT NUMBER: 819). However, the ASAS20 non-response rates remain at 40% and the ASAS40 non-response rates at 60%, indicating a need for therapeutic agents that can improve these conditions.

[0007] For this reason, considering the excellent efficacy of tumor necrosis factor-α inhibitors and interleukin-17 inhibitors for ankylosing spondylitis and axial spondylitis, there is a desire for combination therapy that enhances efficacy and addresses side effects (Rheumatology (Oxford). 55(suppl 2):ii38-ii42, 2016). Furthermore, it has been reported that patients who experience adverse reactions to conventional single-target antibodies for anti-tumor necrosis factor-α or anti-interleukin-17 therapy may need to switch to bispecific drugs (Rheumatology (Oxford). 2017 Oct 12).

[0008] These results suggest that, compared to inhibiting a single cytokine, simultaneous inhibition of TNF-α and IL-17, while maintaining levels that do not compromise the normal immune system, can rapidly suppress inflammation and immune responses, providing an effective and sustained treatment approach. Furthermore, this treatment approach is expected to be effective in patients who are unresponsive to TNF-α inhibitors or those who are resistant. Combination therapies exist, where immunosuppressants against TNF-α and IL-17 are administered separately. However, considering the inconvenience of increased dosing frequency and dosage, as well as the increased production costs associated with producing each agent, the use of dual antibodies, which promise minimal dosing and reduced costs, is effective.

[0009] Thus, the present inventors, through their efforts to develop a dual-targeting antibody that specifically binds to interleukin-17A and tumor necrosis factor-α, prepared an excellent dual-targeting antibody that specifically inhibits both interleukin-17A and tumor necrosis factor-α. They also confirmed that this antibody exhibits the desired therapeutic effect while reducing the occurrence of side effects, thereby completing the present invention.

[0010] The above information described in this Background section is only for enhancement of understanding of the background of the invention and thus may not contain information that forms the known prior art to ordinary technicians in the technical field to which the invention belongs. Summary of the Invention

[0011] The present invention aims to provide a dual-targeting antibody that specifically binds to interleukin-17A and tumor necrosis factor-α, comprising an antibody or an antigen-binding fragment thereof that specifically binds to interleukin-17A and an antibody or an antigen-binding fragment thereof that specifically binds to tumor necrosis factor-α.

[0012] Another object of the present invention is to provide a polynucleotide encoding the aforementioned dual-targeting antibody or antigen-binding fragment thereof.

[0013] Another object of the present invention is to provide a vector comprising the above-mentioned polynucleotide, a cell transformed by the above-mentioned vector, and a method for preparing the above-mentioned dual-targeting antibody or an antigen-binding fragment thereof.

[0014] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating autoimmune diseases, comprising the above-mentioned dual-targeting antibody or antigen-binding fragment thereof.

[0015] Another object of the present invention is to provide an antibody-drug conjugate comprising the above-mentioned dual-targeting antibody or antigen-binding fragment thereof.

[0016] To achieve the above objectives, the present invention provides a dual-targeting antibody that specifically binds to interleukin-17A and tumor necrosis factor-α, comprising an antibody or an antigen-binding fragment thereof that specifically binds to interleukin-17A and an antibody or an antigen-binding fragment thereof that specifically binds to tumor necrosis factor-α, characterized in that the antibody or antigen-binding fragment thereof that specifically binds to interleukin-17A comprises: (i) a heavy chain complementary determining region 1 of SEQ ID NO:1 or SEQ ID NO:8, a heavy chain complementary determining region 2 selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:9, and SEQ ID NO:11, and a heavy chain complementary determining region 3 selected from the group consisting of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:10; and (ii) a light chain complementary determining region 1 of SEQ ID NO:12, a light chain complementary determining region 2 of SEQ ID NO:13, and a light chain complementary determining region 3 of SEQ ID NO:14.

[0017] Furthermore, the present invention provides a polynucleotide encoding the aforementioned dual-targeting antibody or an antigen-binding fragment thereof.

[0018] Furthermore, the present invention provides a vector comprising the above-mentioned polynucleotide.

[0019] Furthermore, the present invention provides cells transformed with the above-mentioned vector.

[0020] Furthermore, the present invention provides a method for preparing the aforementioned dual-targeting antibody or antigen-binding fragment thereof, comprising: step (a) culturing the aforementioned cells; and step (b) recovering the dual-targeting antibody or antigen-binding fragment thereof that specifically binds to interleukin-17A and tumor necrosis factor-α from the cultured cells.

[0021] Furthermore, the present invention provides a method for preventing or treating an autoimmune disease, comprising administering to an individual a pharmaceutical composition for preventing or treating an autoimmune disease comprising the dual-targeting antibody or an antigen-binding fragment thereof, or the dual-targeting antibody or an antigen-binding fragment thereof.

[0022] Furthermore, the present invention provides an antibody-drug conjugate comprising the aforementioned dual-targeting antibody or antigen-binding fragment thereof and a drug bound thereto. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This figure shows the purification of the diabody candidate SDA-0070 by gel filtration chromatography (GFC) and fast protein liquid chromatography (FPLC), followed by analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

[0024] Figures 2a to 2c The diagram shows that SDA-0070 is a diabody analyzed by enzyme-linked immunosorbent assay (ELISA) and shows specific binding only to interleukin-17A and tumor necrosis factor-α and no non-specific binding.

[0025] Figure 3 Graph showing the results of dual-binding ELISA of interleukin-17A and tumor necrosis factor-α for SDA-0070.

[0026] Figures 4a to 4c The graph shows the evaluation of the neutralizing ability of SDA-0070 in human HT-29 cells.

[0027] Figure 5a and Figure 5b The graph shows the evaluation of the neutralizing ability of SDA-0070 in HEK-Blue derived cells.

[0028] Figure 6 This is a graph showing the evaluation of equivalence by evaluating the neutralization ability of SDA-0070 according to the purification method in human HT-29 cells.

[0029] Figures 7a to 7c Figure 2 is a graph evaluating the efficacy equivalence of transient expression product of SDA-0070 and bulk product expressed in stabilized cells in human HT-29 cells.

[0030] Figure 8a and Figure 8b Figure 2 is a graph evaluating the efficacy equivalence of transient expression products of SDA-0070 and bulk products expressed in HEK-Blue-derived cells.

[0031] Figure 9 Graph showing the results of CD64 binding studies used to analyze antibody-dependent cell-mediated cytotoxicity (ADCC) of SDA-0070.

[0032] Figure 10 Graph showing the results of CD16a binding studies used to analyze the antibody-dependent cell-mediated cytotoxicity of SDA-0070.

[0033] Figure 11 Graph showing the results of bovine serum albumin (BSA) binding studies used to analyze the antibody-dependent cell-mediated cytotoxicity of SDA-0070.

[0034] Figure 12 The results of a study showing the ability of SDA-0070 to simultaneously bind to interleukin-17A and tumor necrosis factor-α.

[0035] Figure 13 This figure shows the results of evaluating the ability of SDA-0070 to simultaneously neutralize interleukin-17A and tumor necrosis factor-α in commercial rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) cells.

[0036] Figure 14 This figure shows the results of evaluating the neutralizing ability of SDA-0070 against interleukin-17A in commercially available rheumatoid arthritis fibroblast-like synoviocytes.

[0037] Figure 15 This figure shows the results of evaluating the neutralizing ability of SDA-0070 against tumor necrosis factor-α in commercial rheumatoid arthritis fibroblast-like synoviocytes.

[0038] Figures 16a to 16c Graphs showing the results of evaluating the neutralizing abilities of Humira, SDA-0070, and LY3114062 against tumor necrosis factor-α and interleukin-17A, respectively, in rheumatoid arthritis fibroblast-like synoviocytes derived from patients.

[0039] Figure 17The graph shows the results of confirming the neutralizing ability of SDA-0070 against tumor necrosis factor-α and interleukin-17A in C57BL / 6 mice. DETAILED DESCRIPTION

[0040] Unless otherwise defined, the meaning of all technical terms and scientific terms used in this specification is the same as that commonly understood by those of ordinary skill in the art to which the present invention belongs. Generally, the nomenclature used in this specification is well known in the art and is commonly used in the art.

[0041] The dual-targeting antibody of the present invention more effectively inhibits the inflammatory cytokines tumor necrosis factor-α and interleukin-17A, thereby exhibiting efficacy in suppressing autoimmune diseases. It possesses the ability to bind to both tumor necrosis factor-α and interleukin-17A. In particular, it exhibits excellent specificity, completely ignoring non-target antigenic proteins and specifically binding only to human tumor necrosis factor-α and human interleukin-17A or interleukin-17A / F. This demonstrates superior efficacy compared to antibodies targeting only tumor necrosis factor-α (Humira), antibodies targeting only interleukin-17A (Secukinumab), and the existing dual antibody targeting tumor necrosis factor-α and interleukin-17A (LY3114062).

[0042] Therefore, in one aspect of the present invention, a dual-targeting antibody that specifically binds to interleukin-17A and tumor necrosis factor-α comprises an antibody or an antigen-binding fragment thereof that specifically binds to interleukin-17A and an antibody or an antigen-binding fragment thereof that specifically binds to tumor necrosis factor-α, wherein the antibody or antigen-binding fragment thereof that specifically binds to interleukin-17A comprises: (i) a heavy chain complementary determining region 1 of SEQ ID NO: 1 or SEQ ID NO: 8, a heavy chain complementary determining region 2 selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 9, and SEQ ID NO: 11, and a heavy chain complementary determining region 3 selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 10; and (ii) a light chain complementary determining region 1 of SEQ ID NO: 12, a light chain complementary determining region 2 of SEQ ID NO: 13, and a light chain complementary determining region 3 of SEQ ID NO: 14.

[0043] The term "dual-targeting antibody" in the present invention refers to an antibody that has binding or antagonistic ability to more than one target, and refers to a form in which multiple antibodies that have binding or antagonistic ability to two different targets are bound together, or an antibody that has binding ability to one target is bound to a substance that has antagonistic ability to another target.

[0044] The interleukin-17A protein, which is one target of the dual-targeting antibody of the present invention, can be derived from any species, for example, primates such as humans and monkeys, or rodents such as mice and rats. Interleukin-17 is associated with abnormal expression in the immune system associated with autoimmune diseases such as rheumatoid arthritis (RA), psoriasis (PsA), lupus erythematosus, Crohn's disease, multiple sclerosis, systemic sclerosis, and Behçet's disease.

[0045] The term "antibody" in the present invention includes polyclonal antibodies and monoclonal antibodies, and includes not only intact forms having two full-length light chains and two full-length heavy chains, but also fragments of antibody molecules.

[0046] A complete antibody has a structure with two full-length light chains and two full-length heavy chains, each light chain connected to the heavy chain by a disulfide bond. The constant region of the heavy chain has γ, μ, α, δ and ε types, and has subclasses of γ1, γ2, γ3, γ4, α1 and α2. The constant region of the light chain has κ and λ types. The basic four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light chains (L) and two identical heavy chains (H). The light chain has a variable region (VL) at the N-terminus, followed by a constant region at its other end. The heavy chain has a variable region (VH) at the N-terminus, followed by three constant regions (CH) for the α chain and γ chain respectively, and four constant regions for the μ and ε isomers. The term "variable" indicates that the sequence of a specific part of the variable region varies greatly between antibodies. The variable region mediates antigen binding and determines the specificity of a specific antibody for a specific antigen. Variability in both the light and heavy chain variable regions is concentrated in the hypervariable regions (HVRs), specifically three segments called complementarity determining regions (CDRs). The more highly conserved portions of the variable regions are called framework regions (FRs). The heavy and light chain variable regions, abstracted from the N-terminus to the C-terminus, have the structure of framework region 1, complementarity determining region 1, framework region 2, complementarity determining region 2, framework region 3, complementarity determining region 3, and framework region 4.

[0047] As used herein, the term "heavy chain" refers to a full-length heavy chain comprising a variable region domain heavy chain variable region having an amino acid sequence sufficient to confer specificity to an antigen, and three constant region domains: reconnected constant region 1, reconnected constant region 2, and reconnected constant region 3, and fragments thereof.

[0048] Furthermore, the term "light chain" as used in this specification refers to a full-length light chain and fragments thereof comprising a variable region domain light chain variable region having an amino acid sequence sufficient to confer specificity to an antigen and a constant region domain light chain constant region (CL).

[0049] Antibody antigen-binding fragments or antibody fragments refer to fragments with antigen-binding function, including Fab, F(ab')2, and Fv.

[0050] An "Fv" fragment is an antibody fragment that contains the complete antibody recognition and binding site. This region consists of one heavy chain variable domain and one chain variable domain, for example, a dimer that is firmly, virtually covalently bound by a scFv.

[0051] A "Fab" fragment contains the variable and constant domains of the light chain and the variable and first constant domain (CH1) of the heavy chain. A F(ab')2 antibody fragment typically comprises a pair of Fab fragments covalently linked near their carboxyl termini by hinge cysteines.

[0052] "Single-chain Fv" or "scFv" antibody fragments comprise the heavy and light chain variable domains of an antibody, present within a single polypeptide chain. The Fv polypeptide may further comprise a polypeptide linker between the heavy and light chain variable domains, enabling the scFv to form the desired structure for antigen binding.

[0053] In one embodiment, the antibodies of the present invention are in the form of Fv (e.g., scFv) or intact antibodies. Furthermore, the heavy chain constant region can be selected from one of the subtypes γ, μ, α, δ, or ε. For example, the constant region is γ1 (immunoglobulin G1 (IgG1)), γ3 (immunoglobulin G3 (IgG3)), or γ4 (immunoglobulin G4 (IgG4)). The light chain constant region can be κ or λ.

[0054] The antibodies of the present invention include monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single-chain Fvs (scFV), single-chain antibodies, Fab fragments, F(ab') fragments, disulfide-bonded Fvs (sdFV), and anti-idiotypic (anti-Id) antibodies or epitope-binding fragments of the above antibodies, but are not limited thereto.

[0055] Monoclonal antibodies are antibodies obtained from a substantially homogeneous group of antibodies, i.e., antibodies that are identical except for possible naturally occurring mutations that may be present in trace amounts in the individual antibodies comprising the group. Monoclonal antibodies are highly specific, being induced against a single antigenic site.

[0056] The "humanized" forms of non-human (e.g., mouse) antibodies are chimeric antibodies containing minimal sequence derived from non-human immunoglobulins. In most cases, humanized antibodies are human immunoglobulins (recipient antibodies) in which residues from the recipient's hypervariable regions are substituted with residues from a hypervariable region of a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, having the desired specificity, affinity, and capacity.

[0057] The above-mentioned "human antibody" refers to the utilization of molecules derived from human immunoglobulins to form antibodies comprising complementary determining regions and structural regions, all of which are composed of human immunoglobulins. Human antibodies include phage display libraries and can be prepared by various techniques well known in the art. Human antibodies are modified to react with antigen test inoculations to produce antibodies, and can be prepared by administering antigens to transgenic animals such as immunized transgenic mice (xenomouse) whose endogenous loci do not work. Compared to existing ixekizumab, which is a humanized antibody, the antibodies of the present invention are human antibodies.

[0058] A portion of the heavy and / or light chain is identical or homologous to the corresponding sequence in antibodies derived from a particular species or a particular antibody class or subclass, whereas the remaining chain(s) comprise a "chimeric" antibody (immunoglobulin, also including fragments of the above antibodies that exhibit the desired biological activity).

[0059] As used herein, "antibody variable domain" refers to the light and heavy chain portions of an antibody molecule comprising the amino acid sequences of the complementarity determining regions and the framework regions. The heavy chain variable region refers to the variable domain of the heavy chain. The light chain variable region refers to the variable domain of the light chain.

[0060] "Complementarity determining regions" refer to the amino acid residues in the antibody variable domain that are required for antigen binding. Each variable domain generally has three complementary determining regions identified as complementary determining region 1, complementary determining region 2, and complementary determining region 3.

[0061] In the dual-targeting antibody or antigen-binding fragment thereof of the present invention according to one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to the above-mentioned interleukin-17A may comprise: the heavy chain complementary determining region 1 of SEQ ID NO: 1, the heavy chain complementary determining region 2 of SEQ ID NO: 2, and the heavy chain complementary determining region 3 of SEQ ID NO: 3; the heavy chain complementary determining region 1 of SEQ ID NO: 1, the heavy chain complementary determining region 2 of SEQ ID NO: 4, and the heavy chain complementary determining region 3 of SEQ ID NO: 5; the heavy chain complementary determining region 1 of SEQ ID NO: 1, the heavy chain complementary determining region 2 of SEQ ID NO: 4, and the heavy chain complementary determining region 3 of SEQ ID NO: 6; the heavy chain complementary determining region 1 of SEQ ID NO: 1, the heavy chain complementary determining region 2 of SEQ ID NO: 4, and the heavy chain complementary determining region 3 of SEQ ID NO: 7; the heavy chain complementary determining region 1 of SEQ ID NO: 8, the heavy chain complementary determining region 2 of SEQ ID NO: 9, and the heavy chain complementary determining region 3 of SEQ ID NO: 10; or the heavy chain complementary determining region 1 of SEQ ID NO: 8, the heavy chain complementary determining region 2 of SEQ ID NO: 9, and the heavy chain complementary determining region 3 of SEQ ID NO: 10. The heavy chain complementarity determining region 2 of SEQ ID NO: 11 and the heavy chain complementarity determining region 3 of SEQ ID NO: 10.

[0062] "Framework regions" are variable domain residues excluding the complementarity determining region residues. Each variable domain typically has four framework regions identified as framework region 1, framework region 2, framework region 3, and framework region 4.

[0063] In the dual-targeting antibody or antigen-binding fragment thereof of the present invention according to one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to the above-mentioned interleukin-17A may comprise a heavy chain variable region framework region selected from the group consisting of SEQ ID NO: 15 to SEQ ID NO: 26. Specifically, it may include: a heavy chain variable region framework region selected from the group consisting of framework region 1 of SEQ ID NO: 15, framework region 2 of SEQ ID NO: 16, framework region 3 of SEQ ID NO: 17, and framework region 4 of SEQ ID NO: 18; a heavy chain variable region framework region selected from the group consisting of framework region 1 of SEQ ID NO: 19, framework region 2 of SEQ ID NO: 16, framework region 3 of SEQ ID NO: 20, and framework region 4 of SEQ ID NO: 21; a heavy chain variable region framework region selected from the group consisting of framework region 1 of SEQ ID NO: 22, framework region 2 of SEQ ID NO: 23, framework region 3 of SEQ ID NO: 24, and framework region 4 of SEQ ID NO: 25; and a heavy chain variable region framework region selected from the group consisting of framework region 1 of SEQ ID NO: 22, framework region 2 of SEQ ID NO: 26, framework region 3 of SEQ ID NO: 24, and framework region 4 of SEQ ID NO: 25.

[0064] In another embodiment of the dual-targeting antibody or antigen-binding fragment thereof of the present invention, the antibody or antigen-binding fragment thereof that specifically binds to interleukin-17A may comprise a light chain variable region framework region selected from the group consisting of SEQ ID NO: 27 to SEQ ID NO: 33. Specifically, the light chain variable region framework region may comprise: framework region 1 of SEQ ID NO: 27, framework region 2 of SEQ ID NO: 28, framework region 3 of SEQ ID NO: 29, and framework region 4 of SEQ ID NO: 30; or framework region 1 of SEQ ID NO: 31, framework region 2 of SEQ ID NO: 32, framework region 3 of SEQ ID NO: 33, and framework region 4 of SEQ ID NO: 30.

[0065] In the dual-targeting antibody or antigen-binding fragment thereof of the present invention comprising the aforementioned complementarity determining regions 1 to 3 and framework regions, the antibody or antigen-binding fragment thereof that specifically binds to interleukin-17A may comprise a heavy chain variable region selected from the group consisting of SEQ ID NO: 34 to SEQ ID NO: 39. Furthermore, the antibody or antigen-binding fragment thereof of the present invention may comprise a light chain variable region such as SEQ ID NO: 40 or SEQ ID NO: 41.

[0066] In the dual-targeting antibody or antigen-binding fragment thereof of the present invention according to one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to the above-mentioned interleukin-17A may comprise the following heavy chain variable region and light chain variable region: (i) a heavy chain variable region of SEQ ID NO: 34 and a light chain variable region of SEQ ID NO: 40; (ii) a heavy chain variable region of SEQ ID NO: 35 and a light chain variable region of SEQ ID NO: 41; (iii) a heavy chain variable region of SEQ ID NO: 36 and a light chain variable region of SEQ ID NO: 41; (iv) a heavy chain variable region of SEQ ID NO: 37 and a light chain variable region of SEQ ID NO: 41; (v) a heavy chain variable region of SEQ ID NO: 38 and a light chain variable region of SEQ ID NO: 40; or (vi) a heavy chain variable region of SEQ ID NO: 39 and a light chain variable region of SEQ ID NO: 41.

[0067] "Tumor necrosis factor-α", which is one target of the dual-targeting antibody of the present invention, is an inflammatory cytokine produced in immune cells such as activated macrophages, CD4+ T cells, and NK cells, and plays a role in regulating inflammatory responses by regulating immune cells. The above-mentioned tumor necrosis factor-α protein can be derived from all species, for example, it can be derived from primates such as humans and monkeys, or it can be derived from rodents such as mice and rats. Immune activation of tumor necrosis factor-α mediates autoimmune diseases such as rheumatoid arthritis (RA), ankylosing spondylitis, and inflammatory bowel disease (psoriasis).

[0068] In the dual-targeting antibodies of the present invention, the antibody or antigen-binding fragment thereof that specifically binds to tumor necrosis factor-α can be used without limitation. An antibody having the property of binding to tumor necrosis factor-α to inhibit the activity of tumor necrosis factor-α can be used, preferably one selected from the group consisting of adalimumab (product name: Humira), golimumab (product name: Simponi), certolizumab pegol (product name: Cimicifuga) and infliximab (product name: Remicade) or a variant thereof, but is not limited thereto.

[0069] In the dual-targeting antibody of the present invention, preferably, the antibody or antigen-binding fragment thereof that specifically binds to tumor necrosis factor-α comprises: (i) the heavy chain complementary determining region 1 of SEQ ID NO: 54, the heavy chain complementary determining region 2 of SEQ ID NO: 55, and the heavy chain complementary determining region 3 of SEQ ID NO: 56, and the light chain complementary determining region 1 of SEQ ID NO: 57, the light chain complementary determining region 2 of SEQ ID NO: 58, and the light chain complementary determining region 3 of SEQ ID NO: 59; (ii) the heavy chain complementary determining region 1 of SEQ ID NO: 60, the heavy chain complementary determining region 2 of SEQ ID NO: 61, and the heavy chain complementary determining region 3 of SEQ ID NO: 62, and the light chain complementary determining region 1 of SEQ ID NO: 63, the light chain complementary determining region 2 of SEQ ID NO: 64, and the light chain complementary determining region 3 of SEQ ID NO: 65; (iii) the heavy chain complementary determining region 1 of SEQ ID NO: 66, the heavy chain complementary determining region 2 of SEQ ID NO: 67, and the light chain complementary determining region 3 of SEQ ID NO: NO:68, and the light chain complementary determining region 1 of SEQ ID NO:69, the light chain complementary determining region 2 of SEQ ID NO:70, and the light chain complementary determining region 3 of SEQ ID NO:71; or (iv), the heavy chain complementary determining region 1 of SEQ ID NO:72, the heavy chain complementary determining region 2 of SEQ ID NO:73, and the heavy chain complementary determining region 3 of SEQ ID NO:74, and the light chain complementary determining region 1 of SEQ ID NO:75, the light chain complementary determining region 2 of SEQ ID NO:76, and the light chain complementary determining region 3 of SEQ ID NO:77. More preferably, it comprises: (i) the heavy chain variable region of SEQ ID NO: 78 and the light chain variable region of SEQ ID NO: 79; (ii) the heavy chain variable region of SEQ ID NO: 80 and the light chain variable region of SEQ ID NO: 81; (iii) the heavy chain variable region of SEQ ID NO: 82 and the light chain variable region of SEQ ID NO: 83; or (iv) the heavy chain variable region of SEQ ID NO: 84 and the light chain variable region of SEQ ID NO: 85, but is not limited thereto.

[0070] The sequences of antibodies that specifically bind to tumor necrosis factor-α are shown in Table 1 (heavy chain complementary determining regions and heavy chain variable regions) and Table 2 (light chain complementary determining regions and light chain variable regions).

[0071] Table 1

[0072]

[0073]

[0074] Table 2

[0075]

[0076]

[0077] In one embodiment of the present invention, a linker was introduced at the 3'-end of the nucleic acid sequence of the anti-tumor necrosis factor-α antibody heavy chain (SEQ ID NO: 86), followed by ligation of the optimized anti-interleukin-17A heavy chain variable region and light chain variable region scFv nucleic acid sequence (SEQ ID NO: 87). This was then used to express a dual-targeting antibody that simultaneously inhibits the activities of tumor necrosis factor-α and interleukin-17A in HEK293F cells.

[0078] The above-mentioned diabodies are antibodies that can bind to two different antigens (target proteins) and can be prepared by genetic engineering or any other method. The diabodies of the present invention can be in the form of multiple antibodies connected by a linker.

[0079] The aforementioned linker refers to a linker that can connect two different fusion partners (e.g., biological polymers) via hydrogen bonds, electrostatic interactions, van der Waals forces, disulfide bonds, salt bridges, hydrophobic interactions, covalent bonds, and the like. Specifically, it may have at least one cysteine residue capable of participating in at least one disulfide bond under physiological conditions or other standard peptide conditions (e.g., peptide purification conditions, peptide storage conditions). In addition to simply connecting the fusion partners, it may also function as a spacer of a specified size between the fusion partners or as a hinge that provides flexibility or rigidity to the fusion. The aforementioned linker may be a non-peptide linker or a peptide linker, and may also include all linkers that directly connect via peptide bonds, disulfide bonds, and the like.

[0080] The peptide linker may include a plurality of amino acid sequences or an amino acid sequence in the form of a repeated motif.

[0081] The non-peptide linker can be a biodegradable polymer such as polyethylene glycol (PEG) homopolymer, polypropylene glycol homopolymer, ethylene glycol-propylene glycol copolymer, polyoxyethylene polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinyl ethyl ether, lipopolymer, chitin, hyaluronic acid, or a combination thereof. Specifically, it can be a polyethylene glycol homopolymer, and its derivatives well known in the art and derivatives that can be easily prepared using the state of the art are also within the scope of the present invention.

[0082] The site to be directly or indirectly linked via the linker is not particularly limited, and may be an Fc portion, Fab', F(ab')2, Fab, Fv, or the like.

[0083] In the present invention, the dual-targeting antibody comprises: the heavy chain and light chain of an antibody that specifically binds to interleukin-17A; and the heavy chain and light chain of an antibody that specifically binds to tumor necrosis factor-α. Preferably, the C-termini of the heavy chain constant regions of the antibody that specifically binds to interleukin-17A and the antibody that specifically binds to tumor necrosis factor-α are linked to each other. Preferably, the C-termini of the heavy chain constant region of the antibody that specifically binds to tumor necrosis factor-α is linked to the antigen-binding fragment of the antibody that specifically binds to interleukin-17A, but the present invention is not limited thereto.

[0084] In the dual-targeting antibody of the present invention, the connection between the above-mentioned antibodies or the connection between the above-mentioned antibody-fragment can be connected by various methods such as linker binding, direct chemical binding or genetic fusion. Preferably, the connection is through a linker (SEQ ID NO: 88), but is not limited thereto.

[0085] At the same time, the present invention is characterized in that the antibody fragment is a single-chain variable fragment (scFv).

[0086] In a specific embodiment of the present invention, the inventors prepared the interleukin-17A antigen protein by cloning the interleukin-17A gene, reacted it with the library phage to obtain scFv-phage that specifically binds to interleukin-17A, and repeated the panning process of infecting it in Escherichia coli three times to amplify it.

[0087] The present inventors then screened monoclonal antibodies from the polyclonal phage antibody panel with high binding capacity in the third round of panning and further screened for monoclonal antibodies using enzyme-linked immunosorbent assay (ELISA). Sequence analysis of the monoclonal phage screened in this manner confirmed the complementarity-determining region patterns and polypeptide sequences of the heavy and light chain variable regions of the six monoclonal antibodies (Tables 4 and 5).

[0088] SDA-0070, a bispecific antibody targeting interleukin-17A and tumor necrosis factor-α, was prepared by cloning the scFv of 7H3C11FW from the six aforementioned monoclonal phage clones screened for the 3'-end nucleic acid sequence of the Humira heavy chain, an anti-tumor necrosis factor-α monoclonal antibody. The bispecific antibody, SDA-0070, was confirmed to bind specifically to tumor necrosis factor-α and human interleukin-17A or interleukin-17A / F using an enzyme-linked immunosorbent assay. The antibody demonstrated significantly superior specific binding and neutralizing potency compared to single-antibodies targeting interleukin-17A or tumor necrosis factor-α, as well as to existing tumor necrosis factor-α and interleukin-17A bispecific antibody (LY3114062).

[0089] Therefore, it was confirmed that the dual-targeting antibody of the present invention that specifically binds to interleukin-17A and tumor necrosis factor-α has strong antigen-binding ability to both interleukin-17A and tumor necrosis factor-α.

[0090] As long as the properties of the antibodies or antibody fragments of the present invention are maintained, antibodies or antibody fragments with mutations in the variable region are also within the scope of the invention. Examples of this include antibodies with conservative amino acid substitutions in the variable region. Conservative substitutions refer to replacements with other amino acid residues that have similar properties to the original amino acid sequence. For example, lysine, arginine, and histidine have similar properties due to their basic side chains, while aspartic acid and glutamic acid have similar properties due to their acidic side chains. Furthermore, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan have similar properties due to their uncharged polar side chains; alanine, valine, leucine, threonine, isoleucine, proline, phenylalanine, and methionine have similar properties due to their nonpolar side chains; and tyrosine, phenylalanine, tryptophan, and histidine have similar properties due to their aromatic side chains. Therefore, it is obvious to a person skilled in the art that even if amino acid substitutions occur within a group having similar properties as described above, no different changes in properties will be shown. Therefore, as long as the properties of the antibodies of the present invention are maintained, antibodies mutated by conservative substitutions in the variable region are also included in the scope of protection of the present invention.

[0091] If variations with biologically equivalent activity as described above are considered, the polypeptide sequences of the antibodies or antibody fragments of the present invention include sequences that show substantial identity to the sequences described in the sequence numbers. Substantial identity as described above means that when the sequences of the present invention described above are aligned with any other sequence to maximize their correspondence, when the aligned sequences are analyzed using algorithms commonly used in the art, there is at least 61% homology, more preferably 70% homology, even more preferably 80% homology, and most preferably 90% homology, preferably sequences that are 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical. Alignment methods for sequence comparison are well known in the art. The National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) is available at the National Center for Biotechnology Information (NCBI) and can be used on the Internet in conjunction with sequence analysis programs such as blastp, blasm, blastx, tblastn, and tblastx. The Basic Local Alignment Search Tool can be accessed at www.ncbi.nlm.nih.gov / BLAST / . Methods for sequence homology comparison using this program can be identified at www.ncbi.nlm.nih.gov / BLAST / blast_help.html.

[0092] In yet another aspect, the present invention relates to a polynucleotide encoding the above-mentioned dual-targeting antibody or an antigen-binding fragment thereof.

[0093] The polynucleotide encoding the dual-targeting antibody or antigen-binding fragment thereof of the present invention can be isolated for recombinant production of the antibody or antigen-binding fragment thereof. The isolated polynucleotide can be inserted into a replicable vector for additional cloning (amplification of deoxyribonucleic acid (DNA)) or additional expression. Based on this, in another aspect, the present invention relates to a vector comprising the above-mentioned polynucleotide.

[0094] "Polynucleotide" encompasses a variety of nucleic acids, such as deoxyribonucleic acid (genomic deoxyribonucleic acid (gDNA) and complementary deoxyribonucleic acid (cDNA)) and ribonucleic acid (RNA) molecules. Nucleotides, the basic building blocks of nucleic acids, include not only natural nucleotides but also analogs with modified sugar or base moieties. The sequences of the polynucleotides encoding the heavy and light chain variable regions of the present invention may be modified. Such modifications include additions, deletions, or non-conservative or conservative substitutions of nucleotides.

[0095] In one embodiment, the polynucleotide encoding the dual-targeting antibody or antigen-binding fragment thereof of the present invention, for example, the polynucleotide encoding the heavy chain variable region of the antibody or antigen-binding fragment thereof that specifically binds to interleukin-17A can be selected from the group consisting of SEQ ID NO: 42 to SEQ ID NO: 47, the polynucleotide encoding the light chain variable region can be SEQ ID NO: 48 or SEQ ID NO: 49, the polynucleotide encoding the heavy chain variable region of the antibody or antigen-binding fragment thereof that specifically binds to tumor necrosis factor-α can be SEQ ID NO: 86, and the polynucleotide encoding the light chain region can be SEQ ID NO: 89.

[0096] It should be understood that the nucleic acids of the present invention also include nucleotide sequences that exhibit substantial identity to the aforementioned nucleotide sequences. Substantial identity means that, when the nucleotide sequence of the present invention is aligned with any other sequence to maximize correspondence, the nucleotide sequence exhibits at least 80% identity, more preferably at least 90% identity, and most preferably at least 95% identity, preferably nucleotide sequences exhibiting greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99% identity when the aligned sequences are analyzed using algorithms commonly used in the art.

[0097] Deoxyribonucleic acids encoding the above-mentioned antibodies can be easily isolated or synthesized using conventional procedures (e.g., using deoxyribonucleic acids encoding the heavy chain variable region and light chain variable region of the antibody and oligonucleotide probes that can specifically bind). For additional cloning or expression of the above-mentioned deoxyribonucleic acids, various vectors can be used. Vector components generally include one or more of the following, but are not limited thereto: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0098] The term "vector" as used herein refers to a means for expressing a target gene in a host cell, and includes plasmid vectors, cosmid vectors, bacteriophage vectors, and vectors such as adenoviral vectors, retroviral vectors, and adeno-associated viral vectors. In the above-mentioned vectors, the nucleic acid encoding the antibody is operably linked to a promoter.

[0099] "Operably linked" refers to the functional binding between a nucleic acid expression regulatory sequence (e.g., a promoter, a signal sequence, or an array of transcriptional regulatory factor binding sites) and other nucleic acid sequences, whereby the regulatory sequence regulates the transcription and / or translation of the other nucleic acid sequences.

[0100] When prokaryotic cells are used as hosts, the gene usually contains a strong promoter capable of transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter and T7 promoter, etc.), a ribosome binding site for initiating translation, and a transcription / translation termination sequence. Furthermore, for example, when eukaryotic cells are used as hosts, promoters derived from the genome of mammalian cells (e.g., metallothionein promoter, β-actin promoter, human hemoglobin promoter, and human myocreatine promoter) or promoters derived from mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, herpes simplex virus (HSV) tk promoter, mouse mammary tumor virus (MMTV) promoter, human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, Moloney virus promoter, Epstein-Barr virus (EBV) promoter, and Rous sarcoma virus (RSV) promoter) can be used. These promoters generally have a polyadenylation sequence as a transcription termination sequence.

[0101] Depending on the situation, the vector can also be fused with other sequences to facilitate purification of the antibodies expressed from the vector. Examples of fused sequences include glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA), and hexahistidine (6xHis; Quiagen, USA).

[0102] The above-mentioned vector contains an antibiotic resistance gene commonly used in the art as a selection marker, for example, there are resistance genes to ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and tetracycline.

[0103] In another aspect, the present invention relates to cells transformed with the aforementioned vectors. The cells used to produce the dual-targeting antibodies of the present invention can be prokaryotes, yeast, or higher eukaryotic cells, but are not limited thereto.

[0104] Bacillus strains such as Escherichia coli, Bacillus subtilis, and Bacillus thuringiensis, prokaryotic host cells such as Streptomyces, Pseudomonas (e.g., Pseudomonas putida), Proteus mirabilis, and Staphylococcus (e.g., Staphylococcus carnosus) can be used.

[0105] In addition, animal cells have attracted much attention as host cells. Useful host cell lines include, but are not limited to, COS-7, BHK, CHO, CHO-S, CHOK1, DXB-11, DG-44, CHO / -DHFR, CV1, COS-7, HEK293, BHK, TM4, VERO, HELA, MDCK, BRL 3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC 5, FS4, 3T3, RIN, A549, PC12, K562, PER.C6, SP2 / 0, NS-0, U20S or HT1080.

[0106] In another aspect, the present invention relates to a method for preparing the above-mentioned dual-targeting antibody or its antigen-binding fragment, comprising: step (a), culturing the transformed cells; and step (b), recovering the above-mentioned dual-targeting antibody or its antigen-binding fragment from the obtained cell culture fluid.

[0107] The cells can be cultured in a variety of culture media, including commercially available culture media. The culture media may also contain appropriate concentrations of any other necessary supplements known to those skilled in the art. Culture conditions such as temperature and pH, which are tailored to the host cells selected for expression, will be readily apparent to those skilled in the art.

[0108] When recovering the above-mentioned antibody or antigen-binding fragment thereof, for example, the cell culture fluid can be centrifuged or ultrafiltered to remove impurities, and the product can be purified using affinity chromatography, etc. Additional purification techniques can be used, such as anion or cation exchange chromatography, hydrophobic interaction chromatography, hydroxyapatite column chromatography, etc.

[0109] In another aspect of the present invention, the present invention relates to an antibody-drug conjugate comprising the above-mentioned dual-targeting antibody or antigen-binding fragment thereof and a drug bound thereto.

[0110] The above-mentioned drugs include chemical substances, radionuclides, immunotherapeutic agents, cytokines, chemokines, toxins, biological agents and enzyme inhibitors, etc. For example, the antibodies or fragments thereof of the present invention can be directly or indirectly bound to anticancer agents, and the above-mentioned anticancer agents can be exemplified by acivicin, aclarubicin, acodazole, ciprofloxacin, adolesin, alanosin, aldesleukin, allopurinol sodium, hexamethylmelamine, aminoglutethimide, aminafide, polyinosin, amsacrine, androgen, serpentin, aphidicolin, leucine oncolytic enzyme, asparaginase, 5-azacitidine, azathioprine, bacillus Calmette-Guérin (BCG), triazine benzamide, β-2-deoxythioguanosine, bisantrene hydrochloride, bleomycin sulfate, busulfan, buthionine sulfoxide, BWA773U82, BW502U83 / HCl, BW 7U85 mesylate, ceracemide, carbetamid, carboplatin, carmustine, chlorambucil, chloroquinoxaline-sulfonamide, chlorozotocin, chromomycin A3, cisplatin, cladribine, corticosteroids, Corynebacterium brevis, CPT-11, clinatoxin, ancitabine, cyclophosphamide, cytarabine, sodium bromoanisole, dabis maleate, dacarbazine, actinomycin D, daunorubicin hydrochloride, deazauridine, dexrazoxane, dianhydrodulcitol, diazaquinone, dibromodulcitol, didemnin B, ethyl dithiocarbamate, diglycol aldehyde, dihydro-5-azacytidine, doxorubicin, echinocin, edatrexate, edilfosine, eflornithine, Elliott's solution, elsamitrucin, epirubicin, esorubicin, estramustine phosphate, estrogen, etanercept, amifostine, etoposide, fadrozole, fazarabine, fenretinide, filgrastim, finasteride, flavone acetate, floxuridine, fludarabine phosphate, 5'-fluorouracil, Fluosol TM, flutamide, gallium nitrate, gemcitabine, goserelin acetate, hepsulfam, hexamethylenebisacetamide, homoharringtonine, hydrazine sulfate, 4-hydroxyandrostenedione, hydroxyurea, idarubicin hydrochloride, ifosfamide, 4-ebomidol, iproplatin, isotretinoin, folinate calcium, leuprolide acetate, levamisole, daunorubicin liposomes, liposomal doxorubicin, lomustine, lonidamine, maytansine, nitrogen mustard hydrochloride, melphalan, meprobamate, merbarone, 6-mercaptopurine, sodium thioethanesulfonate, methanol extract of BCG, methotrexate, N-methylformamide, mifepristone, mitoguanidine, mitomycin-C, mitotane, mitoxantrone hydrochloride, monocyte / macrophage colony-stimulating factor, nabilone, nafoxidine, neocarzinostatin , octreotide acetate, ormaplatin, oxaliplatin, paclitaxel, pala, pentostatin, piperazine dione, pipobroman, pirarubicin, piroxine, pyroxantrone hydrochloride, PIXY-321, plicamycin, porfimer sodium, prednimustine, procarbazine, progesterone, pyraclostrobin, razoxane, sargramostim, semustine, spirogermanium, spiromustine, streptozocin, streptozocin, sulfonylchloride , suramin sodium, tamoxifen, taxotere, tegafur, teniposide, terephthalate, thioguanine, thiotepa, thymidine injection, thiazofurine, topotecan, toremifene, tretinoin, trifluoperazine hydrochloride, trifluridine, trimetrexate, tumor necrosis factor, uramustine, vinblastine sulfate, vincristine sulfate, vindesine, vinorelbine, vinblastine, Yoshi 864, daunorubicin, cytarabine, etoposide, melphalan, taxotere, and taxol.

[0111] In another aspect of the present invention, the present invention relates to a composition for preventing or treating an autoimmune disease, comprising the aforementioned dual-targeting antibody or antigen-binding fragment thereof, or the aforementioned antibody-drug conjugate as an active ingredient. For example, the present invention may be a pharmaceutical composition for preventing or treating an autoimmune disease, comprising: (a) a pharmaceutically effective amount of the dual-targeting antibody or antigen-binding fragment thereof, or the aforementioned antibody-drug conjugate of the present invention; and (b) a pharmaceutically acceptable carrier.

[0112] In another aspect of the present invention, the present invention relates to a method for preventing or treating autoimmune diseases, which comprises the step of administering to an individual a required effective amount of the dual-targeting antibody or antigen-binding fragment thereof or the above-mentioned antibody-drug conjugate of the present invention.

[0113] The above-mentioned autoimmune diseases can be selected from the group consisting of rheumatoid arthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, multiple sclerosis, interstitial fibrosis, lupus erythematosus, glomerulonephritis, Crohn's disease, inflammatory bowel disease, autoimmune eye disease, childhood arthritis, Behçet's disease, interleukin-1 receptor antagonist deficiency (DIRA), tumor necrosis factor receptor-associated periodic syndrome (TRAPS), neonatal multisystem inflammatory disease (NOMID), familial Mediterranean fever (FMF) and cryoporphyrin-associated periodic syndrome (CAPS), but are not limited thereto.

[0114] "Prevention" refers to all actions to inhibit or delay the progression of autoimmune diseases by administering the composition of the present invention, and "treatment" refers to inhibiting the development, alleviating or eliminating autoimmune diseases.

[0115] The pharmaceutically acceptable carriers included in the composition of the present invention are carriers commonly used in the formulation of pharmaceutical compositions, including, but not limited to, lactose, glucose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above ingredients, the composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, and preservatives.

[0116] In the present invention, "subject" refers to a mammal, preferably a human, suffering from or at risk of symptoms or diseases that can be alleviated, suppressed or treated by administering the dual-target antibody of the present invention.

[0117] In the present invention, "administration" refers to the administration of a substance specified by an individual primer by an appropriate method. The administration route of the composition comprising the dual-targeting antibody of the present invention can also be administered by any conventional route that can reach the target tissue. The administration route can be intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, and rectal administration, but is not limited thereto. Preferably, when administered orally, because the protein is digestible, the oral composition is formulated in a manner that encapsulates the active agent or protects it from degradation in the stomach. In addition, the pharmaceutical composition of the present invention can be administered by any device that allows the active substance to move to the target cells.

[0118] The appropriate dosage of the composition of the present invention varies depending on factors such as the formulation method, administration method, patient's age, weight, sex, pathological condition, diet, administration time, administration lens, metabolic rate and reaction sensitivity. A skilled doctor can easily determine and prescribe a dosage that is effective for the desired treatment or prevention. For example, the daily dose of the pharmaceutical composition of the present invention is 0.0001 mg / kg to 100 mg / kg, for example, it can be administered at a dose of 1 mg / kg to 2 g / kg. The above-mentioned antibody or antigen-binding fragment thereof can be administered once or several times. In this specification, the term "pharmaceutically effective amount" refers to an amount sufficient to prevent or treat autoimmune diseases.

[0119] The composition of the present invention can be administered as a single therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with existing therapeutic agents.

[0120] The compositions of the present invention can be formulated using pharmaceutically acceptable carriers and / or excipients according to methods readily implemented by those skilled in the art, thereby preparing the compositions in unit dosage form or by adding them to a multi-dose solvent. In this case, the dosage form can be a solution, suspension, or emulsion in an oily or aqueous medium, or can be in the form of an extract, powder, suppository, powder, granule, tablet, or capsule, and can also include a dispersant or stabilizer.

[0121] Example

[0122] The present invention will be described in more detail below by way of examples. These examples are intended only to illustrate the present invention, and the scope of the present invention is not limited to these examples.

[0123] Example 1: Expression and purification of interleukin-17 antigen

[0124] 1-1: Preparation of interleukin-17A protein expression vector

[0125] To clone the extracellular domain gene of interleukin-17A, polymerase chain reaction (PCR) was performed using human interleukin-17A cDNA (Sinobiological, Beijing, China: SEQ ID NO: 50) as a template and the primers listed in Table 3. The amplified PCR product was inserted into the N293F vector to create an interleukin-17A-N293F vector expressing a protein fused to a polyhistidine tag (His) at the C-terminus of interleukin-17A. DH5α Escherichia coli cells were transformed with the prepared interleukin-17A-N293F vector, and ampicillin-resistant strains were selected. Insertion of the interleukin-17A-His gene (SEQ ID NO: 51) was confirmed by cleavage with the Sfi I and Xho I restriction enzymes and sequencing. A vector expressing mouse interleukin-17A-His was also prepared in a similar manner.

[0126] Table 3

[0127]

[0128]

[0129] 1-2: Expression and purification of interleukin-17A antigen

[0130] To produce human interleukin-17A-His (hIL-17A-His) or mouse interleukin-17A-His (mIL-17A-His) antigen, 5 × 10 5 HEK293F cells were seeded with Freestyle media (Gibco, cat. A13835) and cultured for one day. The interleukin-17A-N293F vector prepared in 1-1 was then transfected into the HEK293F cells using polyethylenimine (PEI, Aldrich, cat. 408727). Plasmid and polyethylenimine were mixed at a 1:2 (w / w) ratio to form a polyplex, which was then transfected into the cells. Cell culture fluids cultured for 7 days were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane for treatment with anti-His horseradish peroxidase (HRP) antibody. Expression was confirmed using an ECL substrate.

[0131] The expressed human interleukin-17A-His and mouse interleukin-17A-His culture fluids were separated and purified using Ni-NTA columns, and then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the proteins were analyzed using CBR-250 staining.

[0132] Example 2: Screening of interleukin-17 antibodies

[0133] 2-1: Biopanning

[0134] To screen for candidate antibodies that specifically bind to human interleukin-17A, the Ymax-nABL library (YBiologics, South Korea) consisting of phage display technology and naive cDNA from human B cells was used.

[0135] The human interleukin-17A-His prepared in Example 1 was coated onto an immunosorbent tube and the library phage was added. After reacting at room temperature for 2 hours, the scFv-phage that specifically bound to the antigen was eluted by washing with 1× phosphate-buffered saline (PBST) and 1× phosphate-buffered saline (PBS). The eluted scFv-phage was reinfected with Escherichia coli and amplified by panning to obtain a phage pool. The phage amplified in the first round of panning was used as the target, and the number of phosphate-buffered saline (PBS) washes was increased. Second and third rounds of panning were repeated using the same method as the first round.

[0136] When the CFU of the human interleukin-17A phage antibody library derived from the first to third rounds of panning was confirmed, the output CFU of the third round of panning was confirmed to have increased approximately 10-fold compared to the output CFU from the first to second rounds of panning.

[0137] 2-2: Screening for antibodies specific to interleukin-17A

[0138] To determine the antigen specificity of the positive poly-ScFv-phage antibody pools obtained through each round of panning, a multi-phage enzyme-linked immunosorbent assay (ELISA) was performed. Direct ELISAs (direct ELISAs) were performed using phage pools obtained from each round of panning, using immunoplates coated with the human interleukin-17A-His antigen used for panning and the mouse interleukin-17A-His antigen to confirm cross-binding with the mouse antigen. The results showed that binding to the human interleukin-17A-His antigen increased primarily in the third round of multi-ScFv-phage, confirming an increase in anti-interleukin-17A phage antibodies in the positive phage pools from the third round of panning.

[0139] E. coli containing single colonies were randomly selected from the positive phage pool from the third round of panning and cultured in 96-deep-well plates treated with 2xYT / C medium at mid-log phase at 37°C and 300 rpm to induce helper phage infection. The cells were pelleted by centrifugation at 6000 rpm for 10 minutes, and only the eluted single scFv-phage present in the supernatant was recovered.

[0140] Human interleukin-17A-His and bovine serum albumin were coated at 0.4 μg / ml on Maxisorb 96-well ELISA plates (Nunc, Denmark). After blocking with phosphate-tween buffer containing 3% skim milk, the single scFv-phage obtained above was added to each antigen-coated well. The ELISA plate was washed three times with 0.05% phosphate-tween buffer. Anti-M13 horseradish peroxidase (HRP) antibody diluted in 0.05% phosphate-tween buffer was added to the wells and incubated for 1 hour. After color development with o-phenylenediamine dihydrochloride (OPD) substrate for 10 minutes, the reaction was terminated with 2N H2SO4. The absorbance of the stained plate was measured at 490 nm using a SpectraMax ELISA reader (Molecular Devices, USA). As a result, dozens of single scFv-phage clones were screened for binding signals to human interleukin-17A-his that were at least three times stronger than the signal binding to bovine serum albumin used as a control antigen.

[0141] For the above-mentioned monoclonal clones screened, phagemid DNA was isolated using a DNA purification kit (Qiagen, Germany). Specific clones with different sequences were confirmed by analyzing the base sequence of the isolated DNA, the sequence of the heavy chain, and the sequence of the light chain. Afterwards, affinity maturation was performed for the purpose of changing the heavy chain for the purpose of enhancing affinity, thereby screening for scFv specific to interleukin-17A. The sequences of antibodies specific to interleukin-17A are shown in Table 4 (heavy chain complementary determining region and heavy chain variable region) and Table 5 (light chain complementary determining region and light chain variable region) below.

[0142] Table 4

[0143]

[0144]

[0145]

[0146] Table 5

[0147]

[0148]

[0149]

[0150] Example 3: Preparation of diabodies targeting interleukin-17A and tumor necrosis factor-α

[0151] 3-1: Production of double antibodies

[0152] In order to prepare a diabody that is a fusion of Humira, which is an anti-interleukin-17A antibody and an anti-tumor necrosis factor-α antibody, the scFv of 7H3C11FW in the anti-interleukin-17A antibody monoclonal prepared in Example 2 above was linked to the 3'-end of the Humira heavy chain nucleic acid sequence and named SDA-0070.

[0153] Humira (adalimumab), an anti-tumor necrosis factor-α antibody, was produced using a synthetic gene based on the amino acid sequence comprising the heavy chain variable region (SEQ ID NO: 90) and the amino acid sequence comprising the light chain variable region (SEQ ID NO: 91). Subsequently, a linker was introduced to the 3'-terminus of the nucleic acid sequence of the Humira heavy chain, an anti-tumor necrosis factor-α antibody (SEQ ID NO: 86), followed by ligation of an optimized scFv nucleic acid sequence (SEQ ID NO: 87) utilizing the anti-interleukin-17A heavy chain variable region and light chain variable region. The amino acid sequence of the diabody heavy chain produced in this manner is represented by SEQ ID NO: 92.

[0154] In order to produce and prepare this diabody as a protein, HEK293F was used as a production cell for temporary expression. The plasmid construct prepared by inserting the nucleic acid sequence of the above-mentioned diabody into a vector named N293F was mixed with polyethyleneimine to form an artificial synthetic polymer. After being treated with HEK293F cells for transfection, the cells were cultured in Freestyle (Gibco, USA) medium for 6 days. The culture medium was centrifuged at 8000 rpm to remove cell debris, and the pure culture medium was obtained using a bottle top filter (Millipore, Steritop-GP Filter Unit. Cat. No. SCGPS01RE).

[0155] 3-2: Antibody Purification

[0156] To isolate and purify the tumor necrosis factor-α / interleukin-17A diabody present in the culture medium, affinity purification was performed using Protein A resin (KANEKA, Japan) as the first purification step. After adding 4 ml of Protein A to an empty column (Bio-Rad, BR731-1550), 100 ml of Dulbecco's phosphate buffer (DPBS) (LB001-02) was filled and washed. Culture medium was added to the resin-filled column and flowed into it at a flow rate of 1 ml / min (Bio-Rad, EP-1 Econo pump, USA). After washing with 150 ml of Dulbecco's phosphate buffer, elution was performed with 10 ml of 0.1 M glycine-HCl (pH 3.3). The eluate was neutralized by adding 10% 1M Tris-HCl (pH 9.0), and the buffer was replaced with Dulbecco's phosphate buffer using an Amicon Ultra-15 (Millipore, UF C901096). This process was repeated approximately three times, and when the concentration reached approximately 1 ml, the concentration was stopped and the concentration was confirmed. Concentration was determined by ultraviolet (UV) quantification using Epoch (Biotek, USA).

[0157] In order to remove high molecular weight substances, gel filtration chromatography was performed using Superdex200 resin (GE healthcare, USA) as a second purification. 2CV equilibration was performed using Dulbecco's phosphate buffer (welgene, South Korea) as the mobile phase. After filtering the first purified protein using a 0.22 μm syringe filter (Millipore, SLGP033RB), 10 ml was loaded onto the sample loop. The loaded protein was added to the column, and the fractions corresponding to the target molecule were pooled. The combined fractions were concentrated using Amicon Ultra-15 (Millipore, UFC905096) and sterilized by filtration using a 0.22 μm syringe filter (Millipore, SLGP033RB).

[0158] 3-3: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high performance liquid chromatography (HPLC)

[0159] The purified protein was prepared by mixing it with a reducing or non-reducing buffer. In order to separate the proteins, a 12% acrylamide separation gel (TransLab, South Korea) was prepared and installed in a fraction collector (BioRad, USA), and 1X electrophoresis buffer was filled to load 2 μg of sample. The separation gel was made to contain a hole containing a size marker (BioRad, USA) for the sampled protein. After the loading was completed, the electrodes were connected to the fraction collector from (-) to (+) direction, and electrophoresis was performed at 200 V until the brilliant blue dye of the loading buffer reached the end of the gel. After electrophoresis, the separation gel was stained with Coomassie brilliant blue stain, and after decolorization with decolorization buffer, the residual reagent was removed with distilled water (DW), and the marker and purified protein separation form ( Figure 1 After purification by gel filtration chromatography, the antibody was analyzed and confirmed by size exclusion (SE)-high performance liquid chromatography.

[0160] Example 4: Analysis of Binding Specificity of Diabodies

[0161] To confirm the antigen-specific binding specificity of the diabody, an enzyme-linked immunosorbent assay was performed. Human interleukin-17A, mouse interleukin-17A (R&D system, USA), marmoset interleukin-17A (Beijing Sino Biological Science and Technology Co., Ltd., China), macaque interleukin-17A, human tumor necrosis factor-α (Peprotech, USA), human interleukin-17F (R&D system, USA), human interleukin-17A / F (R&D system, USA), and bovine serum albumin were coated in 96-well plates at 15 nM at 37°C. After blocking the uncoated portion with 0.05% 3% skim milk / phosphate Tween buffer, 66 nM, 6.6 nM, and 0.6 nM interleukin-17A antibodies were added to the plate and reacted at 37°C for 1 hour. Anti-human Fc-horseradish peroxidase (Pierce, USA) was diluted 1:3000 and reacted for 1 hour. Tetramethylbenzidine (TMB) substrate (BD, USA) was then treated for 10 minutes to develop color, and the reaction was terminated with 2N H2SO4. The color-developed plate was then measured for absorbance at 450 nm using a Sunrise ELISA reader (TECAN, Switzerland).

[0162] As a result, unlike the human tumor necrosis factor-α single-target antibody, the diabody not only binds to human tumor necrosis factor-α, but also binds to human interleukin-17A, maintaining binding to marmoset, macaque, and human interleukin-17A / F heterodimers ( Figure 2a ). In addition, no binding to non-target antigen proteins such as interleukin-17B, interleukin-17C, interleukin-17D, interleukin-17E, human RAGE-his, human ErbB2-his, human VEGF-his, human CD93-his, and human HMGB1-his belonging to the interleukin-17 ligand family is shown ( Figure 2b and Figure 2c ) That is, it was confirmed that the antibody of the present invention is a very specific antibody that enhances binding only to human tumor necrosis factor-α and human interleukin-17A or interleukin-17A / F.

[0163] Example 5: Evaluation of the Binding Ability of Diabodies to Two Antigens Using ELISA

[0164] To analyze the ability of the diabody to simultaneously bind to interleukin-17A and tumor necrosis factor-α, an enzyme immunoassay was used in which the target antigen tumor necrosis factor-α was coated and the remaining target antigen interleukin-17A was used for detection.

[0165] Tumor necrosis factor-α (Peprotech, USA) was diluted to 0.26 μg / ml in Dulbecco's phosphate buffer (Welgene, Korea), and 100 μl was added to each well of a 96-well plate (Corning, USA) and incubated at 4°C for 16 hours for coating. The washing buffer used was PBS-T (0.1% Tween 20), and skimmilk (BD Biosciences, USA) was diluted to 3% in PBS-T for blocking. Antibody samples were diluted 10-fold from 6.6 nM to 660 fM for a total of five dilutions. 100 μl of the diluted antibody samples were applied to each well of a 96-well plate and reacted at 25°C for 1 hour. Interleukin-17A-His (Sigma Biosciences, S0995) was diluted to 2.25 μg / ml and treated at 100 μl per well. After reacting at 25°C for 1 hour, anti-His-horseradish peroxidase (Thermofisher Scientific, USA) was diluted 1:5000 as a secondary antibody and treated at 100 μl per well. The cells were reacted at 25°C for 1 hour. Tetramethylbenzidine (Sigma Biosciences, S0995) was used. Galdrich, USA) was used as substrate, 100 μl was added to each well and reacted for 20 minutes, then the substrate reaction was terminated with 2.5N H2SO4 and the substrate was detected by Multiskan TM The absorbance was measured at 450 nm using a GO Microplate Spectrophotometer (Thermofisher Scientific, USA).

[0166] As a result, no binding reaction occurred with the single antibody, but the double antibody showed simultaneous binding reaction of tumor necrosis factor-α and interleukin-17A according to the concentration ( Figure 3 and Table 6).

[0167] Table 6

[0168] EC50 of each antibody against tumor necrosis factor-α and interleukin-17A antigens

[0169] SDA-0070 Humira secukinumab EC50 (pM) 133.7 - -

[0170] Example 6: Evaluation of the efficacy of SDA-0070 cation exchange chromatography (CEX) purification using HT-29 cell analysis

[0171] HT-29 (Homo sapiens colorectal adenocarcinoma) (Korea Cell Line Bank, South Korea) used in this experiment is a cell line derived from human colorectal adenocarcinoma and is known to secrete various immune cytokines and chemokines, such as CXCL-1 (human GRO-α), in response to interleukin-17 and tumor necrosis factor-α. The cells were cultured three or more times in RPMI1640 (Hyclone, USA) medium supplemented with 10% fetal bovine serum (Gibco, USA), 1% antibiotics, and anti-antibodies (penicillin / streptomycin / anti-mycoplasma; Gibco, USA).

[0172] The neutralization ability test was performed on three types: the neutralization ability for interleukin-17A alone, the neutralization ability for tumor necrosis factor-α alone, and the neutralization ability for interleukin-17A and tumor necrosis factor-α at the same time. Interleukin-17A or tumor necrosis factor-α was diluted alone or mixed to prepare dilutions, and the antibody sample was diluted 2 times each time from 736pM to 0.36pM, and finally treated with a total of 12 concentrations. The diluted antibodies and the previously prepared antigen dilutions were mixed in a 96-well plate and reacted at 37°C for 1 hour. The HT-29 cells recovered by trypsin-ethylenediaminetetraacetic acid (EDTA) treatment of the plate after the antigen-antibody reaction were plated at 7.5×10 per well. 4 Cells were seeded and cultured at 37°C in a 5% CO2 environment for 48 hours. After 48 hours, the cell-depleted culture medium was recovered. The CXCL-1 concentration in the recovered culture medium was measured using the Human CXCL-1 ELISA kit (R&D Systems, USA) to evaluate the antibody's ability to neutralize the antigen.

[0173] The results of the neutralization test comparing SDA-0070 showed that the dual-antibody showed superior neutralization ability against interleukin-17A and tumor necrosis factor-α compared to the single-target antibody. Furthermore, the ability to neutralize both interleukin-17A and tumor necrosis factor-α also showed a trend of being superior ( Figures 4a to 4c Table 7 shows the results of the inhibitory ability evaluation of the SDA-0070 clone using HT-29 cells.

[0174] Table 7

[0175]

[0176] Example 7: Evaluation of the efficacy of SDA-0070 CEX purified product using reporter cell assay

[0177] HEK-Blue expressing interleukin-17RA and interleukin-17RC, which are interleukin-17 receptors TM Interleukin-17 cell line (Invivogen, USA) and HEK-Blue expressing tumor necrosis factor receptor I (TNFRI) and tumor necrosis factor receptor II (TNFRII) as tumor necrosis factor-α receptors TM The ability to neutralize interleukin-17A and tumor necrosis factor-α was evaluated using a tumor necrosis factor-α cell line (Invivogen, USA). When culturing HEK-Blue cells, DMEM-HG (Hyclone, USA) containing 10% fetal bovine serum (Gibco, USA), 1% antibiotics and anti-antibiotics (penicillin / streptomycin / antimycoplasma; Gibco, USA) was used. Zeocin (Invivogen, USA) was used as a selective antibiotic to maintain the expression of each receptor and SEAP. TM HEK-Blue is also used as a mixed antibiotic product in interleukin-17 cell lines. TM Selection (invivogen, USA). All cells used in the experiment were subcultured more than 3 times. When the cells were recovered from the culture plate, cell digestion solution (accutase) (Merck Millipore, Germany) was used. Antigen dilutions were prepared in such a way that the final concentration of interleukin-17A (R&D, USA) became 0.6 ng / ml and the final concentration of tumor necrosis factor-α (R&D, USA) became 0.4 ng / ml. Each antibody sample was diluted 2 times each time from 736 pM to 0.36 pM, and finally treated with a total of 12 concentrations. The diluted antibodies and antigen dilutions were mixed in a 96-well plate and reacted at a temperature of 37°C for 1 hour. In the plate where the antigen-antibody reaction was completed, 5×10 4 Cell seeding (seeding) uses HEK-Blue recovered from cell digestion fluid TM Interleukin-17 cells and HEK-Blue T M Tumor necrosis factor-α cells were cultured at 37°C in a 5% CO2 environment for 24 hours. After 24 hours, the culture medium was collected to remove the cells. QUAN TI-Blue was added to the collected culture medium. TMThe culture medium (Invivogen, USA) was added and incubated at 37°C. The reaction was carried out using Multiskan for 20 to 60 minutes depending on the reaction rate of the substrate produced. TM The absorbance was measured at 622 nm using a GO Microplate Spectrophotometer (Thermofisher scientific, USA).

[0178] Comparative test results of the HEK-Blue neutralization ability of the SDA-0070_CEX purified product showed superior efficacy compared to secukinumab, a single-targeting antibody for interleukin-17, and similar efficacy to Humira, a single-targeting antibody for tumor necrosis factor-α ( Figure 5a and Figure 5b Table 8 shows the IC values of SDA-0070 using HEK-Blue cell line. 50 result.

[0179] Table 8

[0180]

[0181] Example 8: Evaluation of the Efficacy Equivalence of Substances According to the Purification Method by Analyzing HT-29 Cells

[0182] 8-1: Cation exchange chromatography purification

[0183] In order to improve the purification process of the diabody, a cation exchange chromatography column (Thermofisher Scientific Poros, USA) was used in addition to the existing gel filtration chromatography column (GE Healthcare, UK).

[0184] After cation exchange chromatography resin (Thermo, the U.S.) is filled into the column, the sodium acetate (sodium acetate) buffer of mobile phase A is utilized to be connected to the AKTA Pure L (GE healthcare, the U.S.) of equilibrium, and the mobile phase A of mobile 5CV is carried out equilibrium. The purified product is dialyzed for the first time using mobile phase A, filtered and injected by a syringe filter (Millipore, the U.S.) of 0.22 μm. Non-adherent protein is removed by the mobile phase A of mobile 5CV, and the protein loaded is eluted according to the mobile phase B (mobile phase A+1M NaCl) of the concentration gradient. Eluent is added to a dialysis bag (Spectrumlabs) and dialyzed twice at intervals of at least 4 hours using Dulbecco's phosphate buffer (welgene, Korea). For the protein dialyzed, amicon Ultra-15 (Millipore, UFC905096) is utilized to be concentrated into more than 20 mg / mL, and sterilized and filtered using a syringe filter (Millipore, SLGP033RB) of 0.22 μm.

[0185] 8-2: Comparison of neutralization ability according to purification method

[0186] The ability of antibody samples purified using a gel filtration column (GE Healthcare, UK) and a cation exchange column (Thermofisher Scientific Poros, USA) to simultaneously neutralize interleukin-17A and tumor necrosis factor-α in HT-29 cells was compared to evaluate whether differences in purification methods affect efficacy.

[0187] For the test, cells cultured after subculturing HT-29 cell line (Human colorectal adenocarcinoma) (Korea Cell Bank, Korea) for more than three times in RPMI1640 (Hyclone, USA) medium containing 10% fetal bovine serum (Gibco, USA), 1% antibiotics, and anti-antibiotics (penicillin / streptomycin / antimycoplasma; Gibco, USA) were used.

[0188] Interleukin-17A (R&D Systems, USA) and tumor necrosis factor-α (R&D Systems, USA) dilutions were prepared and the antibody samples were diluted 4-fold from 736 pM to 0.7 pM, for a total of 6 concentrations. The diluted samples were mixed with the antigen dilutions in a 96-well plate and reacted at 37°C for 1 hour. HT-29 cells were recovered by trypsin-ethylenediaminetetraacetic acid treatment on the plate after the antigen-antibody reaction was completed and plated at 7.5×10 cells per well. 4 Cells were seeded and cultured at 37°C in a 5% CO2 environment for 48 hours. After 48 hours, the cell-depleted culture medium was recovered. The CXCL-1 concentration in the recovered culture medium was measured using the Human CXCL-1 ELISA kit (R&D, USA) to evaluate the neutralizing ability of the antibodies against each antigen.

[0189] The results of the test on the ability of multiple antibody samples to simultaneously neutralize interleukin-17A and tumor necrosis factor-α on HT-29 cells confirmed that the ability to simultaneously neutralize interleukin-17A and tumor necrosis factor-α was maintained at a similar level between the samples purified by gel filtration chromatography column and the samples purified by cation exchange chromatography column ( Figure 6 Table 9 below evaluates the efficacy of gel filtration chromatography and cation exchange chromatography purification using HT-29 cells.

[0190] Table 9

[0191] name <![CDATA[IC 50 (pM)]]> Humira 63.19 Secukinumab 960.7 SDA-0070_GFC 18.2 SDA-0070_CEX 19.56

[0192] Example 9: Evaluation of Efficacy Equivalence Between HEK293 and CHO-S Derived Samples Using HT-29 Cell Assays

[0193] Neutralization ability in HT-29 cells was evaluated using the antibody sample SDA-0070_CEX produced using the HEK293 transient expression system and the sample SDA-0070_RD1601 produced using the CHO-S cell line system and purified by secondary cation exchange chromatography. The efficacy equivalence was compared according to the production cell line.

[0194] For this test, HT-29 cells were subcultured three or more times in RPMI1640 (Hyclone, USA) medium containing 10% fetal bovine serum (Gibco, USA), 1% antibiotics, and anti-antibodies (penicillin / streptomycin / antimycoplasma; Gibco, USA). Neutralization assays were performed to determine the neutralization ability of interleukin-17A alone, tumor necrosis factor-α alone, and both interleukin-17A and tumor necrosis factor-α. For the interleukin-17A neutralization assay, interleukin-17A (R&D, USA) was diluted to a final concentration of 3.75 ng / ml. For the tumor necrosis factor-α neutralization assay, tumor necrosis factor-α (R&D, USA) was diluted to a final concentration of 1.17 ng / ml. To test the simultaneous neutralization ability of interleukin-17A and tumor necrosis factor-α, interleukin-17A was diluted to a final concentration of 2.5 ng / ml, and tumor necrosis factor-α was diluted to a final concentration of 0.39 ng / ml. The antibody sample was diluted 2-fold each time from 736 pM to 0.36 pM, and finally treated with a total of 12 concentrations. The diluted antibody and antigen dilutions were mixed in a 96-well plate and reacted at 37°C for 1 hour. HT-29 cells were recovered by trypsin-ethylenediaminetetraacetic acid treatment on the plate after the antigen-antibody reaction was completed and inoculated. The cells were cultured at 37°C and 5% CO2 for 48 hours, and the culture medium was recovered. The CXCL-1 concentration in the recovered culture medium was measured using the ELISA kit Human CXCL-1 (R&D, USA) to evaluate the neutralization ability of the antibody against the antigen.

[0195] The results of the comparative test on the neutralization ability between the sample SDA-0700_CEX produced by the HEK293 transient expression system and the sample SDA-0070_RD1601 produced by the CHO-S cell line of the same clone showed no difference in the neutralization ability against interleukin-17 and tumor necrosis factor-α ( Figures 7a to 7c Table 10 below compares the efficacy of production systems using HT-29 cells.

[0196] Table 10

[0197]

[0198] Example 10: Evaluating the Efficacy of HEK293 and CHO-S Derived Samples by Analyzing Reporter Cells

[0199] Neutralization abilities of the antibody samples SDA-0070_CEX produced using the HEK293 cell line transient expression system and SDA-0070_RD1601 produced using the CHO-S cell line system were evaluated in HEK-Blue-derived cells, and the efficacy equivalence was compared based on the production cell lines.

[0200] HEK-Blue expressing interleukin-17RA and interleukin-17RC, which are interleukin-17 receptors TM Interleukin-17 cell line (Invivogen, USA) and HEK-Blue expressing tumor necrosis factor receptor I and tumor necrosis factor receptor II as tumor necrosis factor-α receptors TM The ability to neutralize interleukin-17A and tumor necrosis factor-α was evaluated using a tumor necrosis factor-α cell line (Invivogen, USA). When culturing HEK-Blue cells, DMEM-HG (hyclone, USA) containing 10% fetal bovine serum (Gibco, USA), 1% antibiotics and anti-antibiotics (penicillin / streptomycin / antimycoplasma; Gibco, USA) was used. Zeocin (Invivogen, USA) was used as a selective antibiotic to maintain the expression of each receptor and SEAP. TM HEK-Blue is also used as a mixed antibiotic product in interleukin-17 cell lines. TM Selection (invivogen, USA, hb-sel). All cells used in the experiment were subcultured more than 3 times. When the cells were recovered from the culture dish, a cell digestion solution (Merck Millipore, Germany) was used. Antigen dilutions were prepared in such a way that the final concentration of interleukin-17A (R&D, USA, 7955-IL-025 / CF) became 0.6 ng / ml and the final concentration of tumor necrosis factor-α (R&D, USA, 210-TA-020 / CF) became 0.4 ng / ml. Each antibody sample was diluted 2 times each time from 736 pM to 0.36 pM, and finally treated with a total of 12 concentrations. The diluted antibodies and antigen dilutions were mixed in a 96-well plate and reacted at 37°C for 1 hour. In the plate where the antigen-antibody reaction is complete, HEK-Blue interleukin-17 cells and HEK-Blue tumor necrosis factor-α cells recovered by cell digestion are inoculated and cultured at 37°C in a 5% CO2 environment for 24 hours. After 24 hours, the culture medium without cells is recovered. QUANTI-Blue is added to the recovered culture medium. TMThe solution was added to the culture medium (Invivogen, USA) and incubated at 37°C. The reaction rate of the substrate was measured within 20 to 60 minutes using a spectrophotometer (thermofisher scientific, USA, Multiskan TM The absorbance was measured at 622 nm using a GO Microplate Spectrophotometer.

[0201] The results of the comparative test on the neutralization ability between the sample SDA-0070_CEX produced by the HEK293 transient expression system and the sample SDA-0070_RD1601 produced by the CHO-S cell line of the same clone showed no difference in the efficacy of the two cell-derived proteins ( Figure 8a 、 Figure 8b Table 11 below compares the efficacy of each protein using HEK-Blue derived cells.

[0202] Table 11

[0203]

[0204] Example 11: Affinity Evaluation of Antibodies to Human FcRn Antigen

[0205] The affinity of multiple antibodies for human or monkey FcRn (Sinobiological, Beijing Sino-Biotech Co., Ltd., China) antigens was measured using the Octet QK analyzer (Fortebio Inc., USA). The Octet analyzer utilizes the BLI (Bio-Layer Interferometry) principle to measure changes in the thickness of the protein layer bound to the biosensor surface, enabling real-time monitoring of protein binding to other biomolecules.

[0206] Human or monkey FcRn-His antigen bound to a histidine residue was immobilized at the C-terminus of the protein using a nickel-coated Ni-NTA biosensor (ForteBio Inc, 18-5101). The antibodies Humira and SDA-0070 prepared at different concentrations were then bound to determine affinity.

[0207] The affinity survey results of all antibodies for human FcRn showed that the binding rate (Kon) of SDA-0070 at pH 6.0 was 1.6×10 6 1 / Ms, showing a similar binding rate to Humira with normal immunoglobulin G structure, and a dissociation rate (Kdis) lower than 1×10 -31 / s. KD reflects the minimal difference in dissociation rate (Kdis). The KD of Humira is 6.4×10 -10 M level, the KD of SDA-0070 was 5.0×10 -10 At a pH of 7.4, the dissociation rate (Kdis) of SDA-0070 was 5.83×10, which was similar to that of Humira. -3 1 / s. Furthermore, the results of monkey FcRn analysis were similar to those of human FcRn. Despite the structural differences of the diabody, the FcRn affinity was similar to that of immunoglobulin G (IgG) in normal results. Table 12 below shows the kinetic parameters between SDA-0070 and human, monkey, and FcRn.

[0208] Table 12

[0209]

[0210] Example 12: Binding Evaluation of SDA-0070 for Antibody-Dependent Cell-Mediated Cytotoxicity of Tooth Decay Proteins

[0211] To measure the binding capacity of SDA-0070 in antibody-dependent cell-mediated cytotoxicity (ADC C), an indirect enzyme-linked immunosorbent assay (Indirect-ELISA) was performed. Antibody-dependent cell-mediated cytotoxicity is a phenomenon in which NK cells recognize the Fc region of antibodies bound to the surface of target cells and exhibit cytotoxicity against these cells. CD16, CD64, and other proteins located on the surface of NK cells bind to the Fc region of the antibody to trigger activation signals, thereby inducing apoptosis in infected cells.

[0212] To evaluate the binding of SDA-0070 to CD64 and CD16, an IgG1-type antibody (Sigma Aldrich, USA) and Humira (Abbvie, USA) were used as positive control antibodies. An IgG4 antibody (Sigma Aldrich, USA) was also used as a negative control antibody. Recombinant human FcγRIIIA / CD16a protein (R&D systems, USA), recombinant human FcγRI / CD64 protein (R&D systems, USA), and bovine serum albumin (Sigma Aldrich, USA) were added to a 96-well plate (NUNC, 96-well plate bottom) at a concentration of 100 μl per well using Dulbecco's Phosphate Buffered Saline (DPBS) for at least 12 hours before coating. The antibody panel to be tested for binding was diluted 4-fold from 1380 nM and treated with 100 μl of each well for 2 hours. The cells were treated with a secondary antibody, Peroxidase-AffiniPure F(ab')2Fragment Goat Anti-Human IgG, F(ab')2Fragment Specific (Jackson Immune Research, USA), and reacted for 1 hour. Color development was confirmed with tetramethylbenzidine treatment, and the substrate reaction was terminated with H2SO4 treatment. The fluorescence was then detected using a spectrophotometer (Thermo Scientific, USA) at 450 nm.

[0213] The results of measuring the degree of binding of the Fc portion of the antibody group to NK cell-derived CD64 showed that the binding ability of SDA-0070 was at the same level as that of the Humira and immunoglobulin G1 control groups ( Figure 9 ).

[0214] Furthermore, regarding the binding ability of the antibody group to CD16a, the immunoglobulin G1 control antibody was the best, followed by SDA-0070 and Humira antibodies, which were similar ( Figure 10 ). It was confirmed that SDA-0070 and the control antibody did not react with bovine serum albumin ( Figure 11 ).

[0215] Example 13: Analysis of the ability of SDA-0070 to simultaneously bind to interleukin-17A and tumor necrosis factor-α

[0216] As described below, the simultaneous binding of SDA-0070 to tumor necrosis factor-α and interleukin-17A was confirmed in real time using OCTET.

[0217] The Octet QK analysis device (Fortebio Inc, USA) was used to bind the antibody to the AHC biosensor (ForteBio Inc, USA) via the Fc region. SDA-0070 was diluted in 1× Kinetics buffer (Fortebio Inc, USA) at a concentration of 10 ug / ml and loaded onto the AHC biosensor for binding. The unbound state of SDA-0070 bound to the sensor was confirmed by the stabilization time. Depending on the conditions, only 20 nM tumor necrosis factor-α (Peprotech, USA) or Kinetics buffer was treated to compare tumor necrosis factor-α binding in real time. Afterwards, only 15 nM interleukin-17A (R&D systems, USA) or Kinetics buffer was treated to compare interleukin-17A binding in real time according to the conditions.

[0218] As a result, similar to the results confirmed by using ELISA, SDA-0070 was confirmed to recognize and bind to two antigens simultaneously through real-time result data ( Figure 12 ).

[0219] Example 14: Neutralization Ability Analysis of Anti-IL-17A and Anti-TNF-α Antibodies

[0220] 2×10 4Commercially available rheumatoid arthritis fibroblast-like synoviocytes (Cell Application, USA), known for their ability to express hIL-6, an inflammatory cytokine, induced by tumor necrosis factor-α or interleukin-17A, were seeded and cultured for 24 hours at 37°C in 5% CO2. In this example, SDA-0070 was evaluated in combination with Humira (Abbvie, USA), a single-antibody targeting tumor necrosis factor-α; secukinumab (Novartis, Switzerland), a single-antibody targeting interleukin-17A; and LY3114062 (Eli Lilly, WO2014137961, USA), a dual-antibody targeting tumor necrosis factor-α and interleukin-17A. At 1.87 ng / ml of human tumor necrosis factor-α (R&D system, USA) and 18.7 ng / ml of human interleukin-17A (R&D system, USA), the anti-interleukin-17A antibody, secukinumab, was diluted 4-fold each time from 17664 pM and mixed. SDA-0070, LY3114062, or Humira, an anti-tumor necrosis factor-α antibody, was diluted in stages from 4416 pM and mixed, and the mixtures were reacted at 37°C for 1 hour.

[0221] The mixture was treated with the rheumatoid arthritis fibroblast-like synoviocytes cultured above and cultured together for 24 hours. Afterwards, only the culture medium was removed and processed using an enzyme-linked immunosorbent assay (ELISA) kit (R&D system, USA) for hIL-6 measurement. The hIL-6 expression level was measured using a spectrophotometer (Multiskan GO, Thermo Scientific, USA).

[0222] The results confirmed that the SDA-0070 diabody showed a more excellent interleukin-6 (IL-6) secretion inhibitory ability compared to the single anti-interleukin-17 antibody or single anti-tumor necrosis factor-α antibody treatment groups ( Figures 13 to 15 , Tables 13 to 15).

[0223] Table 13

[0224] Evaluation of the ability of commercial rheumatoid arthritis fibroblast-like synoviocytes to simultaneously neutralize interleukin-17A and tumor necrosis factor-α

[0225] Humira SDA-0070 LY3114062 Secukinumab <![CDATA[IC 50 (pM)]]> 44.10 13.72 14.85 1614

[0226] Table 14

[0227] Evaluation results of the neutralization ability of interleukin-17A in commercial rheumatoid arthritis fibroblast-like synoviocytes

[0228] Humira SDA-0070 LY3114062 Secukinumab <![CDATA[IC 50 (pM)]]> - 312.4 371.5 519.1

[0229] Table 15

[0230] Evaluation of the Neutralization Ability of Tumor Necrosis Factor-α in Commercial Rheumatoid Arthritis Fibroblast-like Synoviocytes

[0231] Humira SDA-0070 LY3114062 Secukinumab <![CDATA[IC 50 (pM)]]> 31.75 30.65 18.57 -

[0232] Example 15: Evaluation of the Interleukin-17A and Tumor Necrosis Factor-α Neutralizing Ability of SDA-0070 Using Patient-Derived Rheumatoid Arthritis Fibroblast-Like Synoviocytes

[0233] In this example, rheumatoid arthritis-fibroblast-like synoviocytes (RA-FLS) from patients with rheumatoid arthritis, which are not commercially available RA-FLS, were isolated and cultured to evaluate the activity of SDA-0070. Synovial tissue was minced into small pieces and reacted in Dulbecco's modified Eagle's medium (WelGENE Inc., South Korea) supplemented with 1 mg / ml type 2 collagenase (Worthington Biochemical Corporation). The cells were resuspended in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% P / S, filtered through a 40 μm cell strainer, and adhered to a 100 mm culture dish. Only attached cells were recultured and used.

[0234] Rheumatoid arthritis fibroblast-like synoviocytes (RA03) isolated and cultured from patients were cultured at a rate of 5×10 4 cells / ml were seeded in 24-well plates and cultured at 37°C. 3 , 10 2 , 10 1 , 10 0 ,、10 -1 , 10 -2 and 1 0-3pM treatment of Humira, SDA-0070, and LY3114062 was performed. One hour later, 10 ng / ml of tumor necrosis factor-α (R&D systems, USA) and 50 ng / ml of interleukin-17A (R&D systems, USA) were simultaneously treated. After 48 hours, the cell culture medium was recovered. The recovered culture medium was centrifuged at 3500 rpm for 5 minutes to obtain the supernatant, which was stored in an ultra-low temperature freezer and used for enzyme-linked immunosorbent assay experiments. To determine the concentration of interleukin-6 in the cell culture medium, human interleukin-6 enzyme-linked immunosorbent assay (Human IL-6 ELISA) (R&D systems, USA) was used for analysis.

[0235] In rheumatoid arthritis fibroblast-like synoviocytes (RA03), the IC values of the antibody-treated group were confirmed under conditions of TNF-α + IL-17A-induced IL-6 production. 50 Value (pM unit). As a result, when viewing IC in descending order 50 The values of SDA-0070, LY3114062, and Humira were analyzed to be 0.3694 pM, 1.420 pM, and 1.966 pM, respectively. It was confirmed that SDA-0070, as a dual antibody, was the most effective in inhibiting both tumor necrosis factor-α and interleukin-17A, approximately 3.8 times more effective than LY3114062 and approximately 5 times more effective than Humira, a single-targeted antibody against tumor necrosis factor-α. Figures 16a to 16c ).

[0236] Example 16: Determination of Interleukin-17A and Tumor Necrosis Factor-α Functional Inhibition Capacity in C57BL / 6 Mice

[0237] In vivo experiments were conducted to confirm changes in serum KC concentrations induced by interleukin-17A / tumor necrosis factor-α stimulation following administration of the diabody.

[0238] Six-week-old male C57 / BL6 mice were injected with phosphate-buffered saline, SDA-0070 (40 μg / mouse), LY3114062 (40 μg / mouse), secukinumab (30 μg / mouse), and Humira (30 μg / mouse). One hour later, 0.5 μg / mouse of tumor necrosis factor-α (R&D systems, USA) and 6 μg / mouse of interleukin-17A (R&D systems, USA) were subcutaneously injected, and blood was collected 4 hours later. The collected blood was centrifuged at 5000 rpm for 10 minutes to obtain serum, which was stored in an ultra-low temperature freezer. KC (CXCL1) concentrations were measured using the Quantikine ELISA Mouse CXCL1 / KCImmunoassay kit (R&D systems, USA).

[0239] Mouse serum diluted in assay diluent was treated in a microplate coated with anti-mouse KC antibody and allowed to react at room temperature for 2 hours. After washing five times with wash buffer, 100 μl of mouse KC conjugate was treated and allowed to react at room temperature for 2 hours. After washing five more times with wash buffer, 100 μl of substrate solution was added and allowed to react for 30 minutes. The reaction was terminated with stop solution, and the OD was measured at 450 nm using a spectrophotometer.

[0240] As a result, compared with the positive control group (TNF-α+IL-17A combination only), the groups treated with antibodies all showed statistically significantly lower KC levels, and among them, it was confirmed that the SDA-0070 treatment group had the lowest KC level. Compared with the Humira treatment group, the SDA-0070 treatment group had a p=0.0012, which showed statistically significant and excellent efficacy. Compared with the LY3114062 treatment group, the SDA-0070 treatment group had a p=0.0138, which showed statistically excellent efficacy. That is, the results of confirming the changes in serum KC concentration caused by interleukin-17A / tumor necrosis factor-α stimulation by the administration of the double antibody into the body confirmed that SDA-0070 had excellent neutralization ability against both interleukin-17A and tumor necrosis factor-α antigens ( Figure 17 ).

[0241] Industrial applicability

[0242] The dual-targeted antibodies or antigen-binding fragments thereof of the present invention exhibit high specificity for interleukin-17A and tumor necrosis factor-α. Compared with existing single-targeted antibodies, they not only exhibit excellent neutralization ability but also simultaneously inhibit interleukin-17 and tumor necrosis factor-α, thereby rapidly suppressing inflammation and immune responses, thereby having the advantages of reducing the dosage and enhancing the therapeutic effect.

[0243] The specific parts of the present invention described in detail above are merely preferred embodiments, and the scope of the present invention is not limited thereto. Such specific embodiments are obvious to those skilled in the art. Therefore, the essential scope of the present invention is defined by the scope of the claims and their equivalents.

[0244] Sequence Listing Free Text

[0245] Attach electronic documents. <110> Y Biotech Co., Ltd. <120> Dual-targeting antibody specifically binding to interleukin-17A and tumor necrosis factor-α <130> KHP212111222.4 <160> 92 <170> KopatentIn 2.0 <210> 1 <211> 5 <212> PRT <213> Artificial sequence <220> <223> H-CDR1 <400> 1 Ser Tyr Thr Met His 1 5 <210> 2 <211> 16 <212> PRT <213> Artificial sequence <220> <223> H-CDR2 <400> 2 Ile Ser Phe Asp Gly Arg Ser Lys Leu Tyr Gly Asp Ser Val Arg Asp 1 5 10 15 <210> 3 <211> 11 <212> PRT <213> Artificial sequence <220> <223> H-CDR3 <400> 3 Arg Gly Arg Glu Gly Glu Asp Ala Phe Asp Leu 1 5 10 <210> 4 <211> 16 <212> PRT <213> Artificial sequence <220> <223> H-CDR2 <400> 4 Ile Ser Phe Asp Gly Arg Ser Lys Leu Tyr Gly Asp Ser Val Lys Gly 1 5 10 15 <210> 5 <211> 11 <212> PRT <213> Artificial sequence <220> <223> H-CDR3 <400> 5 Gly Ser Val Arg Gly Glu Ala Ala Phe Asp Leu 1 5 10 <210> 6 <211> 11 <212> PRT <213> Artificial sequence <220> <223> H-CDR3 <400> 6 Gly Ser Lys Leu Gly Glu Asp Ala Phe Asp Leu 1 5 10 <210> 7 <211> 11 <212> PRT <213> Artificial sequence <220> <223> H-CDR3 <400> 7 Gly Ser Arg Ile Gly Glu Asp Ala Phe Asp Leu 1 5 10 <210> 8 <211> 5 <212> PRT <213> Artificial sequence <220> <223> H-CDR1 <400> 8 Asp His Ala Met His 1 5 <210> 9 <211> 18 <212> PRT <213> Artificial sequence <220> <223> H-CDR2 <400> 9 Ser Leu Ile Ser Gly Asp Gly Gly Ala Thr Tyr Tyr Ala Asp Ser Val 1 5 10 15 Lys Gly <210> 10 <211> 15 <212> PRT <213> Artificial sequence <220> <223> H-CDR3 <400> 10 His Phe Ser Asp Ser Arg Gly Arg Ser Asp Val Pro Phe Asp Ile 1 5 10 15 <210> 11 <211> 18 <212> PRT <213> Artificial sequence <220> <223> H-CDR2 <400> 11 Gly Leu Ile Gly Pro Asp Gly Gly Ala Thr Tyr Tyr Ala Asp Ser Val 1 5 10 15 Lys Gly <210> 12 <211> 11 <212> PRT <213> Artificial sequence <220> <223> L-CDR1 <400> 12 Ser Gly Asp Asn Leu Arg Thr Lys Tyr Val Ser 1 5 10 <210> 13 <211> 7 <212> PRT <213> Artificial sequence <220> <223> L-CDR2 <400> 13 Gln Asp Thr Arg Arg Pro Ser 1 5 <210> 14 <211> 10 <212> PRT <213> Artificial sequence <220> <223> L-CDR3 <400> 14 Met Thr Trp Asp Val Asp Thr Thr Ser Met 1 5 10 <210> 15 <211> 30 <212> PRT <213> Artificial sequence <220> <223> FR1 <400> 15 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Ala Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Gly 20 25 30 <210> 16 <211> 15 <212> PRT <213> Artificial sequence <220> <223> FR2 <400> 16 Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Thr Leu 1 5 10 15 <210> 17 <211> 32 <212> PRT <213> Artificial sequence <220> <223> FR3 <400> 17 Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Met Leu Tyr Leu Lys 1 5 10 15 Ile Ser Asp Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 <210> 18 <211> 11 <212> PRT <213> Artificial sequence <220> <223> FR4 <400> 18 Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 1 5 10 <210> 19 <211> 30 <212> PRT <213> Artificial sequence <220> <223> FR1 <400> 19 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Gly 20 25 30 <210> 20 <211> 32 <212> PRT <213> Artificial sequence <220> <223> FR3 <400> 20 Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Ser Leu Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 <210> twenty one <211> 11 <212> PRT <213> Artificial sequence <220> <223> FR4 <400> twenty one Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> twenty two <211> 30 <212> PRT <213> Artificial sequence <220> <223> FR1 <400> twenty two Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp 20 25 30 <210> 23 <211> 13 <212> PRT <213> Artificial sequence <220> <223> FR2 <400> 23 Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 1 5 10 <210> 24 <211> 32 <212> PRT <213> Artificial sequence <220> <223> FR3 <400> 24 Arg Phe Ile Ile Ser Arg Asp Asn Ser Lys Asn Ser Leu Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 <210> 25 <211> 11 <212> PRT <213> Artificial sequence <220> <223> FR4 <400> 25 Trp Gly Gln Gly Thr Leu Ile Thr Val Ser Ser 1 5 10 <210> 26 <211> 13 <212> PRT <213> Artificial sequence <220> <223> FR2 <400> 26 Trp Val Arg Gln Ala Pro Gly Asn Gly Leu Glu Trp Val 1 5 10 <210> 27 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> FR1 <400> 27 Ser Tyr Glu Leu Thr Gln Ala Pro Ser Leu Ser Val Ser Pro Gly Gln 1 5 10 15 Thr Ala Asn Ile Ile Cys 20 <210> 28 <211> 15 <212> PRT <213> Artificial sequence <220> <223> FR2 <400> 28 Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Leu Leu Val Ile Tyr 1 5 10 15 <210> 29 <211> 32 <212> PRT <213> Artificial sequence <220> <223> FR3 <400> 29 Gly Ile Pro Ala Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr 1 5 10 15 Leu Thr Ile Ser Gly Thr Gln Thr Arg Asp Glu Ser Thr Tyr Tyr Cys 20 25 30 <210> 30 <211> 11 <212> PRT <213> Artificial sequence <220> <223> FR4 <400> 30 Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 1 5 10 <210> 31 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> FR1 <400> 31 Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln 1 5 10 15 Thr Ala Ser Ile Thr Cys 20 <210> 32 <211> 15 <212> PRT <213> Artificial sequence <220> <223> FR2 <400> 32 Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr 1 5 10 15 <210> 33 <211> 32 <212> PRT <213> Artificial sequence <220> <223> FR3 <400> 33 Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr 1 5 10 15 Leu Thr Ile Ser Gly Thr Gln Ala Met Asp Glu Ala Asp Tyr Tyr Cys 20 25 30 <210> 34 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 34 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Ala Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Gly Ser Tyr 20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Thr Leu Ile Ser Phe Asp Gly Arg Ser Lys Leu Tyr Gly Asp Ser Val 50 55 60 Arg Asp Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Met Leu Tyr 65 70 75 80 Leu Lys Ile Ser Asp Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Gly Arg Glu Gly Glu Asp Ala Phe Asp Leu Trp Gly Gln 100 105 110 Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 35 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 35 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Gly Ser Tyr 20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Thr Leu Ile Ser Phe Asp Gly Arg Ser Lys Leu Tyr Gly Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ser Val Arg Gly Glu Ala Ala Phe Asp Leu Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 36 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 36 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Gly Ser Tyr 20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Thr Leu Ile Ser Phe Asp Gly Arg Ser Lys Leu Tyr Gly Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ser Lys Leu Gly Glu Asp Ala Phe Asp Leu Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 37 <211> 120 <212> PRT <213> Artificial sequence <220> <223> VH <400> 37 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Gly Ser Tyr 20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Thr Leu Ile Ser Phe Asp Gly Arg Ser Lys Leu Tyr Gly Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ser Arg Ile Gly Glu Asp Ala Phe Asp Leu Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 38 <211> 124 <212> PRT <213> Artificial sequence <220> <223> VH <400> 38 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp His 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Leu Ile Ser Gly Asp Gly Gly Ala Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Ile Ile Ser Arg Asp Asn Ser Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg His Phe Ser Asp Ser Arg Gly Arg Ser Asp Val Pro Phe Asp 100 105 110 Ile Trp Gly Gln Gly Thr Leu Ile Thr Val Ser Ser 115 120 <210> 39 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 39 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp His 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Asn Gly Leu Glu Trp Val 35 40 45 Gly Leu Ile Gly Pro Asp Gly Gly Ala Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Ile Ile Ser Arg Asp Asn Ser Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg His Phe Ser Asp Ser Arg Gly Arg Ser Asp Val Pro Phe Asp 100 105 110 Ile Trp Gly Gln Gly Thr Leu Ile Thr Val Ser Ser 115 120 <210> 40 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 40 Ser Tyr Glu Leu Thr Gln Ala Pro Ser Leu Ser Val Ser Pro Gly Gln 1 5 10 15 Thr Ala Asn Ile Ile Cys Ser Gly Asp Asn Leu Arg Thr Lys Tyr Val 20 25 30 Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Leu Leu Val Ile Tyr 35 40 45 Gln Asp Thr Arg Arg Pro Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Thr Arg 65 70 75 80 Asp Glu Ser Thr Tyr Tyr Cys Met Thr Trp Asp Val Asp Thr Thr Ser 85 90 95 Met Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 41 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 41 Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln 1 5 10 15 Thr Ala Ser Ile Thr Cys Ser Gly Asp Asn Leu Arg Thr Lys Tyr Val 20 25 30 Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr 35 40 45 Gln Asp Thr Arg Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Met Thr Trp Asp Val Asp Thr Thr Ser 85 90 95 Met Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 42 <211> 360 <212> DNA <213> Artificial sequence <220> <223> VH <400> 42 caggtgcagc tggtggagtc tgggggaggc gtggcccagc ctgggaggtc cctcagactc 60 tcctgtgcag cctctggatt cgccttcggt agttacacta tgcactgggt ccgccaggcg 120 ccaggcaagg gactggagtg ggtgacactt atatcgtttg atggacgtag caagctttac 180 ggagactccg tgagggaccg attcaccatc tccagagaca attccaagaa catgctgtat 240 ctgaaaataa gtgacctgcg atctgaggac acggccgtgt attactgtgc gagacggggg 300 agggagggtg aagatgcttt cgatctctgg ggccaaggga caatggtcac cgtctcctca 360 360 <210> 43 <211> 360 <212> DNA <213> Artificial sequence <220> <223> VH <400> 43 caggtgcagc tggtggagtc tgggggaggc gtggtgcagc ctgggaggtc cctcagactc 60 tcctgtgcag cctctggatt cgccttcggt agttacacta tgcactgggt ccgccaggcg 120 ccaggcaagg gactggagtg ggtgacactt atatcgtttg atggacgtag caagctttac 180 ggagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cagcctgtat 240 ctgcagatga acagcctgcg agccgaggac acggccgtgt attactgtgc gagagggtct 300 gtgcggggtg aagctgcttt cgatctctgg ggccaaggga cactggtcac cgtctcctca 360 360 <210> 44 <211> 360 <212> DNA <213> Artificial sequence <220> <223> VH <400> 44 caggtgcagc tggtggagtc tgggggaggc gtggtgcagc ctgggaggtc cctcagactc 60 tcctgtgcag cctctggatt cgccttcggt agttacacta tgcactgggt ccgccaggcg 120 ccaggcaagg gactggagtg ggtgacactt atatcgtttg atggacgtag caagctttac 180 ggagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cagcctgtat 240 ctgcagatga acagcctgcg agccgaggac acggccgtgt attactgtgc gagagggagt 300 aagttgggtg aagatgcttt cgatctctgg ggccaaggga cactggtcac cgtctcctca 360 360 <210> 45 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> VH <400> 45 caggtgcagc tggtggagtc tgggggaggc gtggtgcagc ctgggaggtc cctcagactc 60 tcctgtgcag cctctggatt cgccttcggt agttacacta tgcactgggt ccgccaggcg 120 ccaggcaagg gactggagtg ggtgacactt atatcgtttg atggacgtag caagctttac 180 ggagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cagcctgtat 240 ctgcagatga acagcctgcg agccgaggac acggccgtgt attactgtgc gagaggttcg 300 cgtattggtg aagatgcttt cgatctctgg ggccaaggga cactggtcac cgtctcctca 360 360 <210> 46 <211> 372 <212> DNA <213> Artificial sequence <220> <223> VH <400> 46 caggtgcagc tggtggagtc tgggggaggc gtggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cgtctggatt cacctttgat gatcatgcca tgcactgggt ccgtcaagct 120 ccagggaagg gtctggagtg ggtctctctt attagcggtg atggtggtgc cacatactat 180 gcagactctg tgaagggccg gttcatcatc tccagagaca acagcaaaaa ctccctgtat 240 ctgcaaatga acagtctgag agccgaggac acggccgtgt attactgtgc gagacatttt 300 tctgatagtc gtggtcgctc cgatgttcct tttgatatct ggggccaagg gacactgatc 360 accgtctcct ca 372 <210> 47 <211> 372 <212> DNA <213> Artificial sequence <220> <223> VH <400> 47 caggtgcagc tggtggagtc tgggggaggc gtggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cgtctggatt cacctttgat gatcatgcca tgcactgggt ccgtcaagct 120 ccagggaatg gtctggagtg ggtcggcctg attggtcctg atggtggtgc cacatactat 180 gcagactctg tgaagggccg gttcatcatc tccagagaca acagcaaaaa ctccctgtat 240 ctgcaaatga acagtctgag agccgaggac acggccgtgt attactgtgc gagacatttt 300 tctgatagtc gtggtcgctc cgatgttcct tttgatatct ggggccaagg gacactgatc 360 accgtctcct ca 372 <210> 48 <211> 324 <212> DNA <213> Artificial sequence <220> <223> VL <400> 48 tcctatgagc tgacacaggc accctcactg tccgtgtcgc caggacagac agccaacatc 60 atctgctctg gagataactt gcgtactaaa tatgtttctt ggtatcagca gaagccaggc 120 cagtcccctt tattggtcat ctatcaggac accaggcggc cctcaggcat ccctgcgcga 180 ttctcaggct ccaactcggg gaacacagcc actctgacca tcagcgggac ccagactaga 240 gatgaatcta cctattactg tatgacgtgg gacgtcgaca ctacctcgat gattttcggc 300 ggagggacca agctgaccgt ccta 324 <210> 49 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> VL <400> 49 tcctatgagc tgacacagcc cccctcagtg tccgtgtcgc caggacagac agccagcatc 60 acctgctctg gagataactt gcgtactaaa tatgtttctt ggtatcagca gaagccaggc 120 cagtcccctg tgttggtcat ctatcaggac accaggcggc cctcaggcat ccctgagcga 180 ttctcaggct ccaactcggg gaacacagcc actctgacca tcagcgggac ccaggctatg 240 gatgaagctg actattactg tatgacgtgg gacgtcgaca ctacctcgat gattttcggc 300 ggagggacca agctgaccgt ccta 324 <210> 50 <211> 396 <212> DNA <213> Artificial Sequence <220> <223> hIL-17A <400> 50 ggaatcacaa tcccacgaaa tccaggatgc ccaaattctg aggacaagaa cttcccccgg 60 actgtgatgg tcaacctgaa catccataac cggaatacca ataccaatcc caaaaggtcc 120 tcagattact acaaccgatc cacctcacct tggaatctcc accgcaatga ggaccctgag 180 agatatccct ctgtgatctg ggaggcaaag tgccgccact tgggctgcat caacgctgat 240 gggaacgtgg actaccacat gaactctgtc cccatccagc aagagatcct ggtcctgcgc 300 agggagcctc cacactgccc caactccttc cggctggaga agatactggt gtccgtgggc 360 tgcacctgtg tcaccccgat tgtccaccat gtggcc 396 <210> 51 <211> 420 <212> DNA <213> Artificial sequence <220> <223> IL-17A-His <400> 51 ggaatcacaa tcccacgaaa tccaggatgc ccaaattctg aggacaagaa cttcccccgg 60 actgtgatgg tcaacctgaa catccataac cggaatacca ataccaatcc caaaaggtcc 120 tcagattact acaaccgatc cacctcacct tggaatctcc accgcaatga ggaccctgag 180 agatatccct ctgtgatctg ggaggcaaag tgccgccact tgggctgcat caacgctgat 240 gggaacgtgg actaccacat gaactctgtc cccatccagc aagagatcct ggtcctgcgc 300 agggagcctc cacactgccc caactccttc cggctggaga agatactggt gtccgtgggc 360 tgcacctgtg tcaccccgat tgtccaccat gtggcccatc atcatcatca tcaccatcac 420 420 <210> 52 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Primers <400> 52 ggaatcacaa tcccacgaaa t 21 <210> 53 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Primers <400> 53 ggccacatgg tggacaatcg g 21 <210> 54 <211> 5 <212> PRT <213> Artificial sequence <220> <223> H-CDR1 <400> 54 Asp Tyr Ala Met His 1 5 <210> 55 <211> 17 <212> PRT <213> Artificial sequence <220> <223> H-CDR2 <400> 55 Ala Ile Thr Trp Asn Ser Gly His Ile Asp Tyr Ala Asp Ser Val Glu 1 5 10 15 Gly <210> 56 <211> 14 <212> PRT <213> Artificial sequence <220> <223> H-CDR3 <400> 56 Ala Lys Val Ser Tyr Leu Ser Thr Ala Ser Ser Leu Asp Tyr 1 5 10 <210> 57 <211> 11 <212> PRT <213> Artificial sequence <220> <223> L-CDR1 <400> 57 Arg Ala Ser Gln Gly Ile Arg Asn Tyr Leu Ala 1 5 10 <210> 58 <211> 7 <212> PRT <213> Artificial sequence <220> <223> L-CDR2 <400> 58 Ala Ala Ser Thr Leu Gln Ser 1 5 <210> 59 <211> 9 <212> PRT <213> Artificial sequence <220> <223> L-CDR3 <400> 59 Gln Arg Tyr Asn Arg Ala Pro Tyr Thr 1 5 <210> 60 <211> 8 <212> PRT <213> Artificial sequence <220> <223> H-CDR1 <400> 60 Gly Phe Ile Phe Ser Ser Tyr Ala 1 5 <210> 61 <211> 8 <212> PRT <213> Artificial sequence <220> <223> H-CDR2 <400> 61 Met Ser Tyr Asp Gly Ser Asn Lys 1 5 <210> 62 <211> 19 <212> PRT <213> Artificial sequence <220> <223> H-CDR3 <400> 62 Ala Arg Asp Arg Gly Ile Ala Ala Gly Gly Asn Tyr Tyr Tyr Tyr Gly 1 5 10 15 Met Asp Val <210> 63 <211> 7 <212> PRT <213> Artificial sequence <220> <223> L-CDR1 <400> 63 Ser Gln Ser Val Tyr Ser Tyr 1 5 <210> 64 <211> 3 <212> PRT <213> Artificial sequence <220> <223> L-CDR2 <400> 64 Asp Ala Ser 1 <210> 65 <211> 10 <212> PRT <213> Artificial sequence <220> <223> L-CDR3 <400> 65 Gln Gln Arg Ser Asn Trp Pro Pro Phe Thr 1 5 10 <210> 66 <211> 8 <212> PRT <213> Artificial sequence <220> <223> H-CDR1 <400> 66 Gly Tyr Val Phe Thr Asp Tyr Gly 1 5 <210> 67 <211> 9 <212> PRT <213> Artificial sequence <220> <223> H-CDR2 <400> 67 Ile Asn Thr Tyr Ile Gly Glu Pro Ile 1 5 <210> 68 <211> 11 <212> PRT <213> Artificial sequence <220> <223> H-CDR3 <400> 68 Ala Arg Gly Tyr Arg Ser Tyr Ala Met Asp Tyr 1 5 10 <210> 69 <211> 6 <212> PRT <213> Artificial sequence <220> <223> L-CDR1 <400> 69 Gln Asn Val Gly Thr Asn 1 5 <210> 70 <211> 3 <212> PRT <213> Artificial sequence <220> <223> L-CDR2 <400> 70 Ser Ala Ser 1 <210> 71 <211> 9 <212> PRT <213> Artificial sequence <220> <223> L-CDR3 <400> 71 Gln Gln Tyr Asn Ile Tyr Pro Leu Thr 1 5 <210> 72 <211> 8 <212> PRT <213> Artificial sequence <220> <223> H-CDR1 <400> 72 Gly Phe Ile Phe Ser Asn His Trp 1 5 <210> 73 <211> 10 <212> PRT <213> Artificial sequence <220> <223> H-CDR2 <400> 73 Ile Arg Ser Lys Ser Ile Asn Ser Ala Thr 1 5 10 <210> 74 <211> 11 <212> PRT <213> Artificial sequence <220> <223> H-CDR3 <400> 74 Ser Arg Asn Tyr Tyr Gly Ser Thr Tyr Asp Tyr 1 5 10 <210> 75 <211> 6 <212> PRT <213> Artificial sequence <220> <223> L-CDR1 <400> 75 Gln Phe Val Gly Ser Ser 1 5 <210> 76 <211> 3 <212> PRT <213> Artificial sequence <220> <223> L-CDR2 <400> 76 Tyr Ala Ser 1 <210> 77 <211> 9 <212> PRT <213> Artificial sequence <220> <223> L-CDR3 <400> 77 Gln Gln Ser His Ser Trp Pro Phe Thr 1 5 <210> 78 <211> 121 <212> PRT <213> Artificial sequence <220> <223> VH <400> 78 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Thr Trp Asn Ser Gly His Ile Asp Tyr Ala Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Ser Tyr Leu Ser Thr Ala Ser Ser Leu Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 79 <211> 107 <212> PRT <213> artificial sequence <220> <223> VL <400> 79 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Arg Tyr Asn Arg Ala Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 80 <211> 126 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 80 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser Ser Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Asn Gly Leu Glu Trp Val 35 40 45 Ala Phe Met Ser Tyr Asp Gly Ser Asn Lys Lys Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Gly Ile Ala Ala Gly Gly Asn Tyr Tyr Tyr Tyr Gly 100 105 110 Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 81 <211> 108 <212> PRT <213> artificial sequence <220> <223> VL <400> 81 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Tyr Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro 85 90 95 Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys 100 105 <210> 82 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 82 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Val Phe Thr Asp Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Thr Tyr Ile Gly Glu Pro Ile Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Tyr Arg Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser 115 <210> 83 <211> 107 <212> PRT <213> artificial sequence <220> <223> VL <400> 83 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Ala Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Tyr Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ile Tyr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 84 <211> 119 <212> PRT <213> artificial sequence <220> <223> VH <400> 84 Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Met Lys Leu Ser Cys Val Ala Ser Gly Phe Ile Phe Ser Asn His 20 25 30 Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val 35 40 45 Ala Glu Ile Arg Ser Lys Ser Ile Asn Ser Ala Thr His Tyr Ala Glu 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ala 65 70 75 80 Val Tyr Leu Gln Met Thr Asp Leu Arg Thr Glu Asp Thr Gly Val Tyr 85 90 95 Tyr Cys Ser Arg Asn Tyr Tyr Gly Ser Thr Tyr Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Leu Thr Val Ser 115 <210> 85 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 85 Asp Ile Leu Leu Thr Gln Ser Pro Ala Ile Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Phe Val Gly Ser Ser 20 25 30 Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Glu Ser Met Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Thr Val Glu Ser 65 70 75 80 Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Ser His Ser Trp Pro Phe 85 90 95 Thr Phe Gly Ser Gly Thr Asn Leu Glu Val Lys 100 105 <210> 86 <211> 1347 <212> DNA <213> Artificial sequence <220> <223> VH <400> 86 gaagtgcagc tggtggagag cggcggcggc ctggtgcagc ccggccggag cctgcggctg 60 agctgcgccg ccagcggctt caccttcgac gactacgcca tgcactgggt gcggcaggcc 120 cccggcaagg gcctggagtg ggtgagcgcc atcacctgga acagcggcca catcgactac 180 gccgacagcg tggagggccg gttcaccatc agccgggaca acgccaagaa cagcctgtac 240 ctgcagatga acagcctgcg ggccgaggac accgccgtgt actactgcgc caaggtgagc 300 tacctgagca ccgccagcag cctggactac tggggccagg gcaccctggt caccgtctct 360 agcgctagca ccaagggccc atcggtcttc cccctggcac cctcctccaa gagcacctct 420 gggggcacag cggccctggg ctgcctggtc aaggactact tccccgaacc ggtgacggtg 480 tcgtggaact caggcgccct gaccagcggc gtgcacacct tcccggctgt cctacagtcc 540 tcaggactct actccctcag cagcgtggtg accgtgccct ccagcagcct gggcacccag 600 acctacatct gcaacgtgaa tcacaagccc agcaacacca aggtggacaa gaaagttgag 660 cccaaatctt gtgacaaaac tcacacatgc ccaccgtgcc cagcacctga actcctgggg 720 ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc 780 cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac 840 tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac 900 aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc 960 aaggagtaca agtgcaaggt ctccaacaaa gccctcccag cccccatcga gaaaaccatc 1020 tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggat 1080 gagctgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac 1140 atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc 1200 gtgctggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg 1260 tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 1320 acgcagaaga gcctctccct gtctccg 1347 <210> 87 <211> 744 <212> DNA <213> Artificial Sequence <220> <223> scFv <400> 87 caggtgcagc tggtggagtc tgggggaggc gtggtgcagc ctgggaggtc cctcagactc 60 tcctgtgcag cctctggatt cgccttcggt agttacacta tgcactgggt ccgccaggcg 120 ccaggcaagt gcctggagtg ggtgacactt atatcgtttg atggacgtag caagctttac 180 ggagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cagcctgtat 240 ctgcagatga acagcctgcg agccgaggac acggccgtgt attactgtgc gagagggtct 300 gtgcggggtg aagctgcttt cgatctctgg ggccaaggga cactggtcac cgtctcctca 360 ggcggcggtg gatccggcgg aggaggctcc ggaggtggcg gaagcggtgg cggaggatct 420 tcctatgagc tgacacagcc cccctcagtg tccgtgtcgc caggacagac agccagcatc 480 acctgctctg gagataactt gcgtactaaa tatgtttctt ggtatcagca gaagccaggc 540 cagtcccctg tgttggtcat ctatcaggac accaggcggc cctcaggcat ccctgagcga 600 ttctcaggct ccaactcggg gaacacagcc actctgacca tcagcgggac ccaggctatg 660 gatgaagctg actattactg tatgacgtgg gacgttgaca ctacctcgat gattttcggc 720 tgcgggacca agctgaccgt ccta 744 <210> 88 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Linker <400> 88 ggaggcggag gttctggcgg cggcggctcc ggtggaggtg gctca 45 <210> 89 <211> 642 <212> DNA <213> Artificial Sequence <220> <223> VL <400> 89 gacatccaga tgacccagtc tcccagcagc ctgagcgcca gcgtgggcga ccgggtgacc 60 atcacctgcc gggccagcca gggcatccgg aactacctgg cctggtacca gcagaagccc 120 ggcaaggccc ccaagctgct gatctacgcc gccagcaccc tgcagagcgg cgtgcccagc 180 cggttcagcg gcagcggcag cggcaccgac ttcaccctga ccatcagcag cctgcagccc 240 gaggacgtgg ccacctacta ctgccagcgg tacaaccggg ccccctacac cttcggccag 300 ggcaccaagg tggaaatcaa aagaaccgtg gctgcaccat ctgtcttcat cttcccgcca 360 tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420 cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 480 gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 540 ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600 ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gt 642 <210> 90 <211> 449 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 90 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Thr Trp Asn Ser Gly His Ile Asp Tyr Ala Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Ser Tyr Leu Ser Thr Ala Ser Ser Leu Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro <210> 91 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 91 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Arg Tyr Asn Arg Ala Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 92 <211> 712 <212> PRT <213> Artificial Sequence <220> <223> SDA-0070 <400> 92 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Thr Trp Asn Ser Gly His Ile Asp Tyr Ala Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Ser Tyr Leu Ser Thr Ala Ser Ser Leu Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 450 455 460 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 465 470 475 480 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Gly Ser Tyr 485 490 495 Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Val 500 505 510 Thr Leu Ile Ser Phe Asp Gly Arg Ser Lys Leu Tyr Gly Asp Ser Val 515 520 525 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Ser Leu Tyr 530 535 540 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 545 550 555 560 Ala Arg Gly Ser Val Arg Gly Glu Ala Ala Phe Asp Leu Trp Gly Gln 565 570 575 Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 580 585 590 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Tyr Glu Leu 595 600 605 Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln Thr Ala Ser Ile 610 615 620 Thr Cys Ser Gly Asp Asn Leu Arg Thr Lys Tyr Val Ser Trp Tyr Gln 625 630 635 640 Gln Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr Gln Asp Thr Arg 645 650 655 Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn 660 665 670 Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met Asp Glu Ala Asp 675 680 685 Tyr Tyr Cys Met Thr Trp Asp Val Asp Thr Thr Ser Met Ile Phe Gly 690 695 700 Cys Gly Thr Lys Leu Thr Val Leu 705 710

Claims

1. A dual-targeting antibody that specifically binds to interleukin-17A and tumor necrosis factor-α, characterized in that it comprises an antigen-binding fragment of an antibody that specifically binds to interleukin-17A and the heavy and light chains of an antibody that specifically binds to tumor necrosis factor-α, the C-terminus of the heavy-chain constant region of the antibody that specifically binds to the above-mentioned tumor necrosis factor-α is connected to the antigen-binding fragment of the antibody that specifically binds to interleukin-17A, and the antigen-binding fragment of the antibody that specifically binds to interleukin-17A comprises: (i) heavy-chain complementarity-determining region 1 of SEQ ID NO:1, heavy-chain complementarity-determining region 2 of SEQ ID NO:4, and heavy-chain complementarity-determining region 3 of SEQ ID NO:5; and (ii) light-chain complementarity-determining region 1 of SEQ ID NO:12, light-chain complementarity-determining region 2 of SEQ ID NO:13, and light-chain complementarity-determining region 3 of SEQ ID NO:14, the antigen-binding fragment of the antibody that specifically binds to interleukin-17A is a single-chain variable fragment, the antibody that specifically binds to tumor necrosis factor-α comprises: heavy-chain complementarity-determining region 1 of SEQ ID NO:54, heavy-chain complementarity-determining region 2 of SEQ ID NO:55, and heavy-chain complementarity-determining region 3 of SEQ ID NO:56, and light-chain complementarity-determining region 1 of SEQ ID NO:57, light-chain complementarity-determining region 2 of SEQ ID NO:58, and light-chain complementarity-determining region 3 of SEQ ID NO:

59.

2. The dual-targeting antibody that specifically binds to interleukin-17A and tumor necrosis factor-α according to claim 1, characterized in that the antigen-binding fragment of the antibody that specifically binds to the above-mentioned interleukin-17A comprises a heavy-chain variable-region framework region selected from the group consisting of SEQ ID NOs: 15 to 26.

3. The dual-targeting antibody that specifically binds to interleukin-17A and tumor necrosis factor-α according to claim 2, characterized in that the antigen-binding fragment of the antibody that specifically binds to the above-mentioned interleukin-17A comprises a heavy-chain variable-region framework region selected from the group consisting of the following framework regions: framework region 1 of SEQ ID NO:15, framework region 2 of SEQ ID NO:16, framework region 3 of SEQ ID NO:17, and framework region 4 of SEQ ID NO:18; framework region 1 of SEQ ID NO:19, framework region 2 of SEQ ID NO:16, framework region 3 of SEQ ID NO:20, and framework region 4 of SEQ ID NO:21; framework region 1 of SEQ ID NO:22, framework region 2 of SEQ ID NO:23, framework region 3 of SEQ ID NO:24, and framework region 4 of SEQ ID NO:25; and Framework region 1 of SEQ ID NO:22, framework region 2 of SEQ ID NO:26, framework region 3 of SEQ ID NO:24, and framework region 4 of SEQ ID NO:

25.

4. The dual-targeting antibody that specifically binds to interleukin-17A and tumor necrosis factor-α according to claim 1, wherein, the antigen-binding fragment of the antibody that specifically binds to the above-mentioned interleukin-17A comprises a light chain variable region framework region selected from the group consisting of SEQ ID NO:27 to SEQ ID NO:

33.

5. The dual-targeting antibody that specifically binds to interleukin-17A and tumor necrosis factor-α according to claim 4, wherein, the antigen-binding fragment of the antibody that specifically binds to the above-mentioned interleukin-17A comprises the following light chain variable region framework regions: framework region 1 of SEQ ID NO:27, framework region 2 of SEQ ID NO:28, framework region 3 of SEQ ID NO:29, and framework region 4 of SEQ ID NO:30; or framework region 1 of SEQ ID NO:31, framework region 2 of SEQ ID NO:32, framework region 3 of SEQ ID NO:33, and framework region 4 of SEQ ID NO:

30.

6. The dual-targeting antibody that specifically binds to interleukin-17A and tumor necrosis factor-α according to claim 1, wherein, the antigen-binding fragment of the antibody that specifically binds to the above-mentioned interleukin-17A comprises: the heavy chain variable region of SEQ ID NO:35; and the light chain variable region of SEQ ID NO:

41.

7. The dual-targeting antibody that specifically binds to interleukin-17A and tumor necrosis factor-α according to claim 1, wherein, the above-mentioned antibody that specifically binds to tumor necrosis factor-α comprises: the heavy chain variable region of SEQ ID NO:78 and the light chain variable region of SEQ ID NO:

79.

8. The dual-targeting antibody that specifically binds to interleukin-17A and tumor necrosis factor-α according to claim 7, wherein, the above-mentioned antibody that specifically binds to tumor necrosis factor-α is adalimumab with the product name Humira.

9. The dual-targeting antibody that specifically binds to interleukin-17A and tumor necrosis factor-α according to claim 1, wherein, the antibody-fragments are connected by a linker.

10. A polynucleotide, wherein, encoding the dual-targeting antibody according to any one of claims 1 to 9.

11. A vector, wherein, comprising the polynucleotide according to claim 10.

12. A cell, wherein, transformed by the vector according to claim 11.

13. A method for preparing the dual-targeting antibody according to any one of claims 1 to 9, wherein, comprising: step (a), culturing the cell according to claim 12; and Step (b), recovering the dual-targeting antibody that specifically binds to interleukin-17A and tumor necrosis factor-α from the obtained cell culture medium.

14. A pharmaceutical composition for preventing or treating autoimmune diseases, characterized in that it contains the dual-targeting antibody described in any one of claims 1 to 9 as an active ingredient.

15. Use of the dual-targeting antibody described in any one of claims 1 to 9 in the preparation of a pharmaceutical composition for preventing or treating autoimmune diseases, wherein the autoimmune disease is rheumatoid arthritis.

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