RU2804963C2 - Humanized anti-il17a antibody and its application - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: monoclonal antibody that binds to IL17A is proposed. The present invention also discloses a nucleic acid encoding said antibody, an expression vector and a host cell containing the sequence of said nucleic acid, and a method for producing the antibody. In addition, the invention is related to an antibody-drug conjugate, pharmaceutical composition and kit, which are used for treatment of psoriasis.

EFFECT: said monoclonal antibody specifically binds IL17A protein with high affinity, has superior or equivalent ability to block IL17A and IL17A/F-induced inflammatory cytokine secretion by human HFF epidermal fibroblasts compared to comparable drugs, and has improved in vivo efficacy.

22 cl, 9 dwg, 5 tbl, 7 ex

Description

TECHNICAL FIELD

[0001] The present invention relates to the field of immunotherapy for autoimmune diseases, and relates to a drug based on a humanized monoclonal antibody against IL17A and its use.

BACKGROUND OF THE ART

[0002] IL17A, also commonly referred to as IL-17, is an inflammatory cytokine consisting of 155 amino acids with a molecular weight of 35 kDa. It is mainly secreted by T helper 17 cells, in addition to T helper 17 cells, CD4+, CD8+ T and γδ-T cells can also secrete IL-17. The IL-17 family includes six members, IL17A, IL-17B, IL-17C, IL-17D (IL-27), IL-17E (IL-25) and IL-17F (Gu, Wu et al. 2013), among of which IL17A and IL-17F are the most important members. With 55% amino acid homology, IL17A and IL-17F can form homodimers or heterodimers linked by disulfide bonds (Dubin and Kolls 2009). When bound to various IL-17RA-expressing cells of the IL-17 receptor family, such as macrophages, dendritic cells, hematopoietic cells, osteoblasts, fibroblasts, etc., the IL17A/A or IL-17A/F dimer can activate NFκB, C /EBPs, MAPK and other signaling pathways in receptor cells that induce these cells to secrete inflammatory factors and chemokines (IL-6, IL-8, CXCL1, etc.), recruit neutrophils and mediate the development of inflammatory reactions (Mitra, Raychaudhuri, etc. . 2014). The occurrence and development of many inflammation-related autoimmune diseases such as psoriasis, psoriatic arthritis, rheumatoid arthritis and ankylosing spondylitis are closely related to the IL-17 pathway (Wang, Suzuki et al. 2017), with a significant increase in IL-17 expression levels in serum patients (Marinoni, Ceribelli et al. 2014), resulting in a persistent inflammatory response. Additionally, IL-17 can also directly affect endothelial cells, epithelial cells, fibroblasts and keratinocytes of the skin, enhancing the release of multiple inflammatory factors and causing pathological changes in the skin (Mitra, Raychaudhuri et al. 2014, Brembilla, Senra et al. 2018). Thus, blocking IL-17 pathways provides the potential to inhibit the autoimmune disease process.

[0003] Currently marketed antibody drugs targeting IL-17 are COSENTYX®/Secukinumab (US 7807155 B2) from Novartis and Taltz®/Ixekizumab (US 7838638 B2/CN 101326195 B) from Eli Lilly, underlying mechanism Their actions are as follows: through their binding to IL17A and IL17A/F, the binding of IL17A to the receptor (IL-17RA/C) is inhibited, subsequently blocking the release of inflammatory factors and chemokines, thereby effectively alleviating autoimmune diseases (Fala 2016, Liu, Lu et al. 2016. Secukinumab is approved for the treatment of plaque psoriasis, psoriatic arthritis and ankylosing spondylitis. Ixekizumab is approved for the treatment of plaque psoriasis and psoriatic arthritis. The humanized anti-IL17A monoclonal antibody of the present invention is able to specifically bind IL17A protein with high affinity, has a strong ability to block the binding of IL17A, IL17A /F with the IL17RA receptor and has superior or equivalent ability to block EL17A, IL17A/F, preventing the induction of inflammatory cytokine secretion by human epidermal fibroblasts HFF, compared to comparable drugs; in a mouse model of psoriasis, the antibody also demonstrates significantly better in vivo pharmacodynamic activity than comparable drugs, with significantly lower post-dose PASI scores; This humanized antibody exhibits favorable pharmacokinetic profiles in cynomolgus monkeys, including rapid absorption after subcutaneous injection, long half-life, and better drug exposure, laying the foundation for longer dosing cycles. The antibody of the present invention is intended to be used for, but not limited to, the treatment of psoriasis.

Short description

[0004] The present invention satisfies the above need by developing an IL17A binding antibody with a novel structure. The present invention discloses a humanized monoclonal antibody that specifically binds IL17A with high affinity and has a strong ability to block the binding of IL17A, IL17A/F to the IL17RA receptor. Compared with other comparable drugs, the antibody of the present invention has superior or equivalent ability to block IL17A and IL17A/F, preventing the induction of inflammatory cytokine secretion by human HFF epidermal fibroblasts; in a mouse model of psoriasis, the antibody also demonstrated significant superior pharmacological activity in vivo compared to comparable drugs and significantly reduced post-dose PASI scores; In a pharmacokinetic study in cynomolgus monkeys, the humanized antibody demonstrated superior pharmacokinetic characteristics, including rapid absorption after subcutaneous injection, long half-life, better drug exposure, etc., laying the foundation for longer dosing cycles. The humanized monoclonal antibody of the present invention can be used for the treatment of psoriasis.

[0005] In one aspect, the present invention provides an isolated anti-IL17A antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising a heavy chain CDR1 region having the amino acid sequence set forth in SEQ ID NO: 13, a heavy chain CDR2 region having the amino acid sequence shown in SEQ ID NO: 14, and the heavy chain CDR3 region having the amino acid sequence shown in SEQ ID NO: 15; and a light chain variable region comprising a light chain CDR1 region having the amino acid sequence specified in SEQ ID NO: 10, a light chain CDR2 region having the amino acid sequence specified in SEQ ID NO: 11, and a light chain CDR3 region having the amino acid sequence , specified in SEQ ID NO: 12. (6 CDRs common to the mouse M069 antibody and the humanized H069 antibody are identical in mouse and human.)

[0006] In one embodiment, said anti-IL17A antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 22, or an amino acid sequence of at least 90%, 92%, 95 %, 98% or 99% sequence identity to SEQ ID NO: 22; and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 23, or an amino acid sequence at least 90%, 92%, 95%, 98%, or 99% identical to the sequence of SEQ ID NO: 23. (Sequence amino acids of the variable regions of the heavy chain and light chain of the humanized antibody H069)

[0007] In one embodiment, the anti-IL17A antibody or antigen-binding fragment thereof is a humanized antibody or a chimeric antibody.

[0008] In one embodiment, said anti-IL17A antibody further comprises a heavy chain constant region and a light chain constant region, preferably said heavy chain constant region is an IgG1 heavy chain constant region having the amino acid sequence set forth in SEQ ID NO: 24, or an amino acid sequence that is at least 90%, 92%, 95%, 98%, or 99% identical to the sequence of SEQ ID NO: 24; and/or said light chain constant region is a human kappa light chain constant region having the amino acid sequence set forth in SEQ ID NO: 25, or an amino acid sequence of at least 90%, 92%, 95%, 98%, or 99% sequence identical to SEQ ID NO: 25. (Heavy chain amino acid sequence and light chain constant region of humanized antibody H069).

[0009] In one embodiment, said anti-IL17A antibody further comprises a signal peptide linked to a heavy chain variable region and/or a signal peptide linked to a light chain variable region, preferably said signal peptide linked to a heavy chain variable region is the sequence amino acids specified in SEQ ID NO: 20 or an amino acid sequence at least 90%, 92%, 95%, 98% or 99% identical to the sequence of SEQ ID NO: 20; and/or said signal peptide attached to the light chain variable region is the amino acid sequence set forth in SEQ ID NO: 21 or an amino acid sequence at least 90%, 92%, 95%, 98%, or 99% identical to the sequence SEQ ID NO: 21. (Amino acid sequences of the signal peptides of the heavy chain and light chain of the humanized antibody H069).

[0010] In one embodiment, said anti-IL17A antibody or antigen-binding fragment thereof is an IgG antibody, preferably an IgG1 antibody.

[0011] In one embodiment, said anti-IL17A antibody or antigen-binding fragment thereof is a monoclonal antibody.

[0012] In one embodiment, the binding affinity K _D of said anti-IL17A antibody or antigen-binding fragment thereof to recombinant human IL17A protein is 0.1-10E-11 M, preferably 0.5-5E-11 M, and more preferably 2 ,88E-11 M.

[0013] In one embodiment, the binding affinity K _D of said anti-IL17A antibody or antigen-binding fragment thereof to recombinant human IL17A/F protein is 0.1-10E-10 M, preferably 0.5-5E-10 M, or more preferably 5.37E-10 M.

[0014] In one embodiment, said antigen-binding fragment is an Fv, Fab, Fab', Fab'-SH, F(ab')2, Fd fragment, Fd' fragment, single chain antibody molecule, or single domain antibody; wherein said single chain antibody molecule is preferably a scFv, di-scFv, tri-scFv, diabody or scFab.

[0015] In another aspect, the present invention provides an antibody-drug conjugate comprising an anti-IL17A antibody or antigen-binding fragment thereof as described herein and an additional therapeutic agent, wherein preferably said anti-IL17A antibody or antigen-binding fragment thereof is coupled to an additional therapeutic agent via a linker.

[0016] In yet another aspect, the present invention provides a nucleic acid coding for an anti-IL17A antibody or an antigen-binding fragment thereof described herein.

[0017] In one embodiment, said nucleic acid comprises the nucleotide sequence set forth in SEQ ID NO: 30 encoding a heavy chain variable region and/or the nucleotide sequence set forth in SEQ ID NO: 31 encoding a light chain variable region.

[0018] In another aspect, the present invention provides an expression vector containing the specified nucleic acid described herein.

[0019] In another aspect, the present invention provides a host cell containing the specified nucleic acid described herein or the specified expression vector described herein.

[0020] In yet another aspect, the present invention provides a method for producing an anti-IL17A antibody or an antigen-binding fragment thereof described herein, comprising growing said host cell as described herein under conditions suitable for expressing the antibody and isolating expressed antibody from the culture fluid.

[0021] In yet another aspect, the present invention provides a pharmaceutical composition comprising an anti-IL17A antibody or an antigen-binding fragment thereof described herein, or an antibody-drug conjugate described herein, or said nucleic acid described herein document, or the specified expression vector described herein, and a pharmaceutically acceptable carrier.

[0022] In yet another aspect, the present invention provides an anti-IL17A antibody or antigen-binding fragment thereof described herein, or an antibody-drug conjugate described herein, or a pharmaceutical composition described herein, for the treatment of psoriasis .

[0023] In yet another aspect, the present invention provides a method for treating psoriasis, comprising administering to a subject in need thereof a therapeutically effective amount of an anti-IL17A antibody or antigen-binding fragment thereof described herein, or an antibody-drug conjugate described herein. herein, or the pharmaceutical composition described herein, thereby treating psoriasis.

[0024] In yet another aspect, the present invention provides the use of an anti-IL17A antibody or an antigen-binding fragment thereof described herein, or an antibody-drug conjugate described herein or a pharmaceutical composition described herein, for the production of a drug. drugs for the treatment of psoriasis.

[0025] In yet another aspect, the present invention provides a pharmaceutical composition comprising an anti-IL17A antibody or an antigen-binding fragment thereof described herein, or an antibody-drug conjugate described herein, or a pharmaceutical composition described herein , and one or more additional therapeutic agents.

[0026] In yet another aspect, the present invention provides a kit comprising an anti-IL17A antibody or an antigen-binding fragment thereof described herein, or an antibody-drug conjugate described herein, or a pharmaceutical composition described herein, and preferably further comprising an administration device.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In FIG. 1 shows that IL17A-M069 blocks IL17A-induced IL-6 secretion by HFF cells.

[0028] In FIG. Figure 2 shows the binding of IL17A-H069 to recombinant human IL17A protein.

[0029] In FIG. 3 shows the binding of IL17A-H069 to recombinant human IL17A/F protein.

[0030] In FIG. 4 shows cross-species cross-linking between IL17A-H069 and mouse IL17A protein.

[0031] In FIG. 5 shows that IL17A-H069 blocks the binding of IL17RA to the IL17A protein.

[0032] In FIG. 6 shows that IL17A-H069 blocks the binding of IL17RA to the IL17A/F protein.

[0033] In FIG. 7 shows that IL17A-H069 blocks IL17A-induced IL-6 secretion by HFF cells.

[0034] In FIG. 8 shows the effect of IL17A-H069 on the psoriasis area and PASI in a mouse model of psoriasis.

[0035] In FIG. 9 shows a drug concentration versus time curve following a single subcutaneous administration of IL17A-H069 in cynomolgus monkeys.

DETAILED DESCRIPTION

[0036] Various aspects of the present invention relate to an isolated anti-IL17A antibody or antigen-binding fragment thereof, an antibody-drug conjugate containing said antibody or antigen-binding fragment thereof, a nucleic acid, and an expression vector encoding said antibody or antigen-binding fragment thereof. fragment, and a host cell containing said nucleic acid or expression vector, a method for producing said anti-IL17A antibody or an antigen-binding fragment thereof, a pharmaceutical composition containing said anti-IL17A antibody or an antigen-binding fragment thereof, and a method of using said anti-IL17A antibody or an antigen-binding fragment thereof for the treatment of psoriasis.

[0037] Definitions

[0038] Unless otherwise specified, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present invention relates. For purposes of the present invention, the following terms are defined in accordance with the meanings commonly understood in the art.

[0039] As used herein and in the accompanying claims, the singular forms “one,” “other,” and “said” include the plural designation of the subject matter unless the context clearly indicates otherwise.

[0040] The term "antibody" refers to an immunoglobulin molecule and refers to any form of antibody that exhibits the desired biological activity. These include, but are not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies and multispecific antibodies (eg, bispecific antibodies), and even antibody fragments. In general, full-length antibody structures preferably comprise four polypeptide chains, two heavy (H) chains and two light (L) chains, typically linked by disulfide bonds. Each heavy chain contains a heavy chain variable region and a heavy chain constant region. Each light chain contains a light chain variable region and a light chain constant region. In addition to this conventional full-length antibody structure, this structure also includes other derivative forms.

[0041] The heavy chain variable region and the light chain variable region can be further subdivided into more conserved regions (called framework regions (FR)) and hypervariable regions (called complementarity determining regions (CDR)), separated by intervals.

[0042] The term “complementarity determining region” (CDR, for example, CDR1, CDR2 and CDR3) refers to those amino acid residues in the specified variable region of an antibody whose presence is necessary for antigen binding. Each variable region typically contains three CDR regions, designated CDR1, CDR2 and CDR3. Each complementarity determining region may contain amino acid residues from a “complementarity determining region” according to Kabat numbering (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD , 1991) and/or residues from the “hypervariable loop” (Chothia and Lesk; J MolBiol 196: 901-917 (1987)).

[0043] The term “framework” or “FR” residues means residues in a specified variable region other than CDR residues as defined herein.

[0044] Each heavy chain variable region and light chain variable region typically contains 3 CDRs and up to 4 FRs, with said CDRs and FRs arranged from amino terminus to carboxyl terminus in the following order, for example: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.

[0045] The complementarity determining region (CDR) and framework region (FR) of the specified antibody can be determined using the Kabat system (Kabat et al.: Sequences of Proteins of Immunological Interest, 5th edition, US Department of Health and Human Services, PHS, NIH, NIH Publication No. 91-3242, 1991).

[0046] The term “constant region” refers to that sequence of amino acids in the light and heavy chains of an antibody that is not directly involved in the binding of the antibody to an antigen, but influences various effector functions, such as antibody-induced cytotoxicity.

[0047] According to the antigenic differences in the constant region amino acid sequence, the antibody heavy chain can be classified into one of five classes: α, β, ε, γ, and μ. When it forms a full-length light chain antibody, it can be classified into one of five classes: IgA, IgD, IgE, IgG and IgM, which can further be classified into five subclasses (isotypes) such as IgG1, IgG2, IgG3, IgG4 , IgAn IgA2. Based on the amino acid sequence of its constant region, the light chain of an antibody can be classified as κ and λ.

[0048] "Antigen-binding antibody fragment" means a portion of an intact antibody molecule that retains at least some of the binding specificity of the parent antibody and typically includes at least a portion of the antigen-binding region or variable region (e.g., one or more CDRs) of the parent antibody antibody. Examples of antigen binding fragments include Fv fragment, Fab, Fab', Fab'-SH, F(ab')2, Fd, Fd', single chain antibody molecules (e.g. scFv, di-scFv or tri-scFv, diagelo or scFab ), single domain antibodies, but are not limited to them.

[0049] The term “antibody fragment” refers to a partial-length antibody molecule that retains at least some of the biological properties of the original antibody, including an Fc fragment, in addition to, but not limited to, the fragments described above as “antigen-binding fragments.”

[0050] The term "antibody-drug conjugate" or "ADC" refers to a binding protein, such as an antibody or an antigen-binding fragment thereof, that is chemically linked to one or more chemical agents (also referred to herein as an "agent") which may optionally be a therapeutic agent or a cytotoxic agent. In one preferred embodiment, the ADC includes an antibody, cytotoxic or therapeutic drug, and a linker by which said drug can be bound or conjugate with said antibody. ADCs can typically contain from 1 to 8 drugs conjugate with the antibody, including 2, 4, 6 or 8 drugs. Non-limiting examples of drugs that may be included in ADCs include mitotic inhibitors, antitumor antibiotics, immunomodulators, gene therapy vectors, alkylating agents, anti-IL17A agents, antimetabolites, boron agents, chemotherapeutic protective agents, hormones, antihormonal agents, corticosteroids, photoactive agents therapeutic agents, oligonucleotides, radionuclide agents, topoisomerase inhibitors, tyrosine kinase inhibitors and radiosensitizers.

[0051] The term "chimeric antibody" refers to an antibody in which a portion of said heavy chain and/or light chain is derived from one particular source or species and the remaining portion is derived from another source or species. Said “chimeric antibody” may also be a functional fragment described above. "Humanized antibodies" are a subset of "chimeric antibodies".

[0052] The term “humanized antibody” or “humanized antigen-binding fragment” as used herein means an antibody or antibody fragment that: (i) is obtained from a non-human source (for example, a transgenic mouse having a heterologous immune system) and based on human germline sequence; or (ii) is a chimeric antibody in which the variable region is of non-human origin and the constant region is of human origin; or (iii) a CDR graft in which said variable region CDR is of non-human origin, one or more variable region framework regions are of human origin, and the constant region, if any, is of human origin. The goal of “humanization” is to eliminate the immunogenicity of antibodies of non-human origin in the human body while maintaining the highest possible affinity. As a template for humanization, it is advisable to select the human backbone sequence that is most similar to the backbone sequence of the antibody originating from a non-human source. In some cases, it may be necessary to replace one or more amino acids in the human framework sequence with corresponding residues in the non-human construct to avoid loss of affinity.

[0053] The term "monoclonal antibody" refers to an antibody obtained from an essentially homogeneous population of antibodies, i.e. each individual antibody within a given population is identical, except for possible mutations (eg natural mutations) which may be present in very small quantities. Thus, the term "monoclonal" indicates the nature of the specified antibody in question, i.e., and not a mixture of unrelated antibodies. Unlike polyclonal antibody preparations, which typically include different antibodies against different epitopes, all monoclonal antibodies in monoclonal antibody preparations are directed against a single epitope of the antigen. In addition to its specificity, monoclonal antibody preparations have the advantage that they are usually not contaminated with other antibodies. The term "monoclonal" should not be understood as requiring the production of the specified antibody by any particular method.

[0054] Said antibody "specifically binds" to a target antigen, such as a tumor-associated peptide target antigen (in this case, IL17A), i.e. binds said antigen with sufficient affinity to enable the use of said antibody as a therapeutic agent by targeting a cell or tissue expressing said antigen and does not significantly cross-react with other proteins or does not significantly cross-react with proteins other than homologs and variants of the target proteins mentioned above (for example, mutant forms, splice variants, or truncated forms of the protein resulting from hydrolysis).

[0055] The term "binding affinity" refers to the strength of the sum of non-covalent interactions between the individual binding sites of a molecule and its binding partners. Unless otherwise specified, “binding affinity” as used herein refers to intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (eg, antibody and antigen). As used herein, the term "K _D " refers to the equilibrium dissociation constant of the antibody-antigen interaction. As used herein, the term " _kon " refers to the rate constant at which an antibody binds to an antigen. As used herein, the term " _koff " refers to the rate constant at which an antibody dissociates from the antibody/antigen complex. "K _D ", "binding rate constant k _on " and "dissociation rate constant k _off " are commonly used to describe the affinity between a molecule (such as an antibody) and its binding partner (such as an antigen). Affinity, i.e., the degree of strength at which a ligand binds to a specific protein. Binding affinity is influenced by non-covalent intermolecular interactions such as hydrogen bonds, electrostatic interactions, hydrophobic and van der Waals forces between two molecules. In addition, the binding affinity between a ligand and its target molecule can be influenced by the presence of other molecules. Affinity can be analyzed by traditional methods known in the art, including the ELISA described herein.

[0056] The term "epitope" includes any cluster of protein determinants that specifically binds to an antibody or T-cell receptor. Epitope determinant clusters typically consist of reactive surface groups of the molecule (eg, amino acid or sugar side chains or combinations thereof) and often have specific three-dimensional structural features as well as specific charge characteristics.

[0057] The term “isolated” antibody is an antibody that has been identified and isolated from components of the cell where the antibody is expressed. Isolated antibodies include antibodies in situ within recombinant cells where at least one component is not present in the antibody's natural environment. However, typically, the isolated antibody is obtained through at least one purification step.

[0058] “Sequence identity” between two polypeptide or nucleic acid sequences indicates the number of residues that are identical between said sequences as a percentage of the total number of residues, and is calculated based on the size of the smaller of the molecules being compared. When calculating percent identity, the compared sequences are compared to obtain the maximum match between the specified sequences, with gaps in the comparison (if any) resolved using a specific algorithm. Preferred computer program methods for determining identity between two sequences include, but are not limited to, the GCG program suites, including GAP, BLASTP, BLASTN, and FASTA (Altschul et al., 1990, J. Mol. Biol. 215: 403-410). The above procedures are publicly available from the International Center for Biotechnology Information (NCBI) and other sources. The well-known Smith-Waterman algorithm can also be used to determine identity.

[0059] The term "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. Preferred are natural human FcR sequences and preferably receptors that bind to IgG antibodies (gamma receptors), which include FcyRI, FcyRII and FcyRIII isoforms, as well as variants of these receptors. All other FcRs are included under the term "FcR". The term also includes the neonatal receptor (FcRn), which is responsible for the transport of maternal IgG to the fetus (Guyer et al., Journal of Immunology 117: 587 (1976) and Kim et al., Journal of Immunology 24: 249 (1994)).

[0060] The term "neonatal Fc receptor", abbreviated as "FcRn", binds to the Fc region of an IgG antibody. The neonatal Fc receptor (FcRn) plays an important role in the metabolic fate of IgG-like antibodies in vivo. FcRn functions to rescue IgG from the lysosomal degradation pathway, thereby reducing its serum clearance and prolonging its half-life. Thus, the FcRn binding/IgG characteristics in vitro indicate its pharmacokinetic properties in the bloodstream in vivo.

[0061] The term "effector function" refers to that biological activity attributable to the Fc region of an antibody, which varies from isotype to isotype. Examples of antibody effector function include C1q binding and complement dependent cytotoxicity (CDC), Fc receptor binding, antibody dependent cell mediated cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex mediated antigen uptake by antigen presenting cells, suppression cell surface receptors (eg, B cell receptors) and B cell activation.

[0062] The term "effector cell" refers to a cell that expresses one or more FcRs and performs effector functions. In one aspect, said effector cells express less FcγRIII and perform ADCC effector functions. Examples of human cells that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils. Effector cells can be isolated from natural sources, such as blood. Effector cells are typically lymphocytes associated with the effector phase and function to produce cytokines (helper T cells), kill cells infected by pathogens (cytotoxic T cells), or secrete antibody (differentiated B cells).

[0063] “Immune cells” include cells that are of hematopoietic origin and play a role in the immune response. Immune cells include: lymphocytes such as B cells and T cells; natural killer cells; and myeloid cells such as monocytes, macrophages, eosinophils, mast cells, basophils and granulocytes.

[0064] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig binds to Fcγ receptors present on certain cytotoxic cells (eg, NK cells, neutrophils and macrophages), allowing these cytotoxic effector cells to specifically bind to target cells bearing antigens, and subsequent destruction of said target cells using, for example, a cytotoxin. In vitro ADCC assays, such as the in vitro ADCC assays documented in US Patent No. 5,500,362 or 5,821,337 or US Patent No. 6,737,056 (Presta), can be performed to evaluate the ADCC activity of a target antibody. Useful effector cells for use in such cases include PBMCs and NK cells.

[0065] “Complement-dependent cytotoxicity” or “CDC” refers to the lysis of target cells in the presence of complement. The classical pathway of complement activation is initiated by the binding of the first component of the complement system (C1q) to an antibody (of the appropriate subclass), which binds to the corresponding antigen. To assess complement activation, a CDC assay is performed, such as the CDC assay described in Gazzano-Santoro et al., J. Immunol Methods 202: 163 (1996). For example, US Patent No. 6,194,551 B1 and WO 1999/51642 describe polypeptide variants containing an altered Fc region amino acid sequence (Fc region variant polypeptides) and polypeptide variants having increased or decreased C1q binding.

[0066] The terms “COSENTYX bioequivalent” and “Taltz bioequivalent” refer to antibodies prepared according to the COSENTYX and Taltz structures, respectively.

[0067] Amino acid sequence of the specified antibody according to the present invention

[0068] The present invention used recombinant human IL17A protein to immunize mice, and then prepared the antibody clone IL17A-M069, which specifically binds to recombinant human IL17A protein by screening a phage display library. The nucleotide sequence encoding the heavy and light chain variable regions of the IL17A-M069 scFv antibody was then introduced by PCR into pSTEP2 vectors containing the nucleotide sequence encoding the mouse IgG1 constant region or the mouse kappa light chain constant region, respectively, and grown for expression. Highly purified antibodies were obtained by protein A column purification. ELISA showed that the mouse antibody was able to block IL17A-induced IL-6 secretion by HFF cells.

[0069] Then, using the classical humanized CDR transplantation method, human antibody light chain or heavy chain variable regions whose sequence is closer to the specified mouse light or heavy chain variable region sequence, humanized light chain variable region ( _VL ) were selected as a template. and a heavy chain variable region (V _H ) in turn by inserting each of the three CDRs (Table 1) of the light chain or heavy chain of a rabbit antibody into the variable regions of said human antibody. Because key sites of the mouse antibody framework are required to maintain stability of CDR activity, the key sites were mutated back to the corresponding mouse antibody sequence. IL17A-H069 light chain/heavy chain expression vectors were prepared by total gene synthesis, transfected into deficient CHO-K1-GS cells and cultured for expression, clones having the highest degree of antibody expression were selected for further growth, resulting in IL17A antibody -H069 highly purified and of high quality.

[0070] Nucleic acids according to the present invention

[0071] The present invention also relates to nucleic acid molecules encoding antibodies or fragments thereof according to the present invention. The sequences of these nucleic acid molecules include, but are not limited to, SEQ ID NOs: 2-7, 26-33, 36-37, 40-41 and 43.

[0072] The nucleic acid molecules of the present invention are not limited to the sequences described herein, but also include variants thereof. The variants in the present invention may be described with reference to their physical hybridization properties. One skilled in the art will appreciate that using nucleic acid hybridization techniques, nucleic acids can be used to identify their complements, as well as their equivalents and homologues. It is also understood that hybridization can occur at a complementarity of less than 100%. However, with appropriate selection of conditions, hybridization techniques can be used to distinguish between specified DNA sequences based on structural relevance to the DNA sequence of a particular probe. For guidance on such conditions, see Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1989 and Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Sedman, J. G., Smith, J. A. and Struhl, K. eds. (1995). Current Protocols in Molecular Biology. New York: John Wiley and Sons.

[0073] Recombinant vectors and expression

[0074] The present invention also provides recombinant constructs containing one or more nucleotide sequences of the present invention. Said recombinant construct of the present invention is produced by inserting said nucleic acid molecule encoding said antibody of the present invention into a vector such as a plasmid, phagemid, phage or viral vector.

[0075] Antibodies provided herein can be produced by recombinant expression of the nucleotide sequence encoding the light and heavy chains or portions thereof in a host cell. To recombinantly express said antibody, said host cell may be transfected with one or more recombinant expression vectors carrying a nucleotide sequence encoding the light and/or heavy chains or portions thereof, such that said light and heavy chains are expressed in said host cell. Standard recombinant DNA methodology is used to produce nucleic acids encoding heavy and light chains, to incorporate said nucleic acids into recombinant expression vectors, and to introduce said vectors into host cells, for example, Sambrook, Fritsch and Maniatis (eds.), Molecular Cloning; A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), Ausubel, F.M. et al. (eds.) Current Protocols in Molecular Biology, Greene Publishing Associates, (1989) and described in US Patent No. 4,816,397 to Boss et al.

[0076] Suitable host cells include prokaryotic and eukaryotic cells. Examples of prokaryotic host cells are bacteria, and examples of eukaryotic host cells are yeast, insect or mammalian cells. It should be understood that the design of an expression vector, including the choice of regulatory sequence, is determined by a number of factors, such as the choice of host cell, the level of expression of the desired protein, and whether expression is constitutive or inducible.

[0077] Bacterial expression

[0078] An expression vector for use in bacteria is constructed by inserting a structural DNA sequence encoding the desired antibody along with appropriate translation initiation and termination signals and functional promoters into the actual reading frame. The vector contains one or more phenotypic selection markers and an origin of replication to ensure vector persistence and allow amplification in the host as needed. Suitable prokaryotic hosts for transformation include several species of E. coli, Bacillus subtilis, Salmonella typhimurium, as well as Pseudomonas, Streptomyces and Staphylococcus.

[0079] Said bacterial vector may be based on, for example, a phage, plasmid or phagemid. These vectors may contain selection markers and bacterial origins of replication that are derived from commercially available plasmids, which typically contain elements of the well-known cloning vector pBR322 (ATCC 37017). After transforming the appropriate host strain and growing the host strain to the appropriate cell density, the selected promoter is derepressed/induced by an appropriate method (eg, temperature change or chemical induction), and the cells are cultured for some more time. The cells are usually collected by centrifugation, disrupted by physical or chemical methods, and the resulting crude extract is stored for further purification.

[0080] In a bacterial system, a variety of expression vectors can be advantageously selected according to the intended use of the expressed protein. For example, when large quantities of such proteins need to be produced for antibody production or for screening a peptide library, for example, a vector may be required that allows high-level expression of the product fusion protein for easy purification.

[0081] Mammalian cell expression and purification

[0082] Preferred regulatory sequences for expression in mammalian host cells include viral elements that allow high-level protein expression in mammalian cells, such as cytomegalovirus (CMV)-derived promoters and/or enhancers (e.g., CMV promoter/enhancer), simian virus 40 (SV40) promoters and/or enhancers (eg, SV40 promoter/enhancer), adenovirus promoters and/or enhancers (eg, adenovirus major late promoter (AdMLP)), and polyoma virus promoters and/or enhancers. For further description of viral regulatory elements and their sequences, see, for example, US Pat. No. 5,168,062, Stinski, US Pat. No. 4,510,245, Bell et al., and US Pat. No. 4,968,615, Schaffner et al. These recombinant expression vectors may also include a point origin of replication and selection marker (see, for example, US Patents No. 4,399,216, No. 4,634,665 and No. 5,179,017, Axel et al.), suitable selection markers include genes that confer resistance to drugs, such as G418, hygromycin or methotrexate, to cells -hosts into which the specified vector is introduced. For example, the dihydrofolate reductase (DHFR) gene confers resistance to methotrexate, while the neo gene confers resistance to G418.

[0083] Transfection of the specified expression vector into host cells can be carried out using standard methods such as electroporation, calcium phosphate precipitation and DEAE-dextran transfection.

[0084] Suitable mammalian host cells for expression of the antibody provided herein include Chinese hamster ovary cells (CHO cells) [including dhfr-CHO cells described by Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, using the DHFR selection marker as described, for example, in R.J. Kaufman and R.A. Sharp (1982) Mol. Biol. 159:601-621], NSO myeloma cells, COS cells and SP2 cells.

[0085] Antibodies of the present invention can be isolated and purified from recombinant cell culture by known methods, including ammonium sulfate or ethanol precipitation, acid extraction, Protein A affinity chromatography, Protein G affinity chromatography, anion or cation exchange chromatography, phosphocellulose chromatography , hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, lectin chromatography, but not limited to. High performance liquid chromatography (HPLC) can also be used for purification. See, for example, Colligan, Current Protocols in Immunology, or Current Protocols in Protein Science, John Wiley & Sons, NY, N.Y., (1997-2001), for example, chapters 1, 4, 6, 8, 9, and 10, each of which is incorporated herein by reference in its entirety.

[0086] Characteristics and functions of the antibody of the present invention

[0087] Characteristic analysis and functional analysis of the humanized antibody IL17A-H069 according to the present invention were carried out. The analysis showed that the antibody of the present invention has the following advantages:

[0088] (1) Better specific binding to recombinant human IL17A protein than biosimilar COSENTYX (EC ₅₀ of humanized antibody IL17A-H069 is 46 ng/ml, while EC ₅₀ of biosimilar COSENTYX is 74.8 ng/ml) (Example 4.1. 1).

[0089] (2) Better specific binding to the recombinant human IL17A/F dimeric protein than the COSENTYX biosimilar (EC ₅₀ of the humanized IL17A-H069 antibody is 36.3 ng/ml; while the EC ₅₀ of the COSENTYX biosimilar is 63.9 ng/ml) (Example 4.1.2).

[0090] (3) Good binding affinity (much better than biosimilar COSENTYX; 2.88E-11 M vs. 9.55-11 M) and favorable association coefficient (much faster than biosimilar COSENTYX; 6.71E+05 M ^-1 s ^-1 versus 1.78E+05 M ^-1 s ^-1 ) with recombinant human IL17A protein, good binding affinity (higher than biosimilar COSENTYX; 5.37E-10 M versus 1.10-09 M) and a favorable association coefficient (faster than biosimilar COSENTYX; 1.44E+05 M ^-1 s ^-1 versus 8.00E+04 M ^-1 s ^-1 ) (Example 4.1.3).

[0091] (4) No cross-linking activity with mouse mIL17A protein (Example 4.1.4).

[0092] (5) Effectively binds recombinant human IL17A protein and effectively inhibits the binding of IL17A protein to the IL17RA receptor, much better than the COSENTYX biosimilar but comparable to the Taltz biosimilar ( _IC50 : 0.50 μg/ml vs. 2.99 μg/ ml versus 0.50 μg/ml; maximum degree of inhibition: 85.4% versus 73.5% versus 89.5% (Example 4.2.1).

[0093] (6) Effectively binds recombinant human IL17A/F dimeric protein and effectively inhibits the binding of IL17A protein to the IL17RA receptor, better than COSENTYX biosimilar and Taltz biosimilar ( _IC50 : 1.02 μg/ml vs. 1.2 μg/ml vs. 1.35 μg/ml; maximum degree of inhibition: 92.3%, 87.9% and 75%) (Example 4.2.2).

[0094] (7) Blocks IL17A, IL17A/F-induced IL-6 secretion by HFF cells; IL17A neutralizing activity is much higher than the COSENTYX biosimilar (EC ₅₀ 0.19 μg/ml vs. 0.22 μg/ml, maximum neutralization rate 94.6% vs 51.6%) and IL17A neutralizing activity similar to the Taltz biosimilar (EC ₅₀ 0.20 μg/ml versus 0.19 μg/ml, maximum degree of neutralization 90.3% versus 95.9%); IL17A/F neutralization activity is also higher than that of the biosimilar COSENTYX (EC ₅₀ 1.19 μg/ml versus 2.25 μg/ml, maximum degree of neutralization 85.0% versus 79.5%); and IL17A/F neutralization activity is better than the Taltz biosimilar at low concentrations, but similar to the Taltz biosimilar at high concentrations ( _EC50 : 0.83 μg/ml vs. 1.10 μg/ml, maximum neutralization degree: 72.90 % versus 76.3%) (Versus 5.1).

[0095] (8) When tested in a mouse model of psoriasis, the antibody of the present invention effectively alleviates the progression of psoriasis and reduces symptoms with significantly greater efficacy than the Taltz biosimilar. (Example 6).

[0096] (9) Pharmacokinetically, the antibody of the present invention has faster absorption after subcutaneous injection and a longer half-life (Example 7).

[0097] Applications

[0098] The antibody of the present invention can be used to treat colorectal cancer. The antibody of the present invention can also be used to produce a drug for the treatment of these diseases.

[0099] Pharmaceutical compositions

[00100] Antibodies of the present invention can be prepared with at least one other agent (eg, a stable compound) to form a pharmaceutical composition comprising an antibody of the present invention and one or more pharmaceutically acceptable carriers, diluents or excipients. Said pharmaceutical composition may optionally contain additional therapeutic agents.

[00101] Sets

[00102] The present invention also relates to a pharmaceutical kit and a kit containing one or more containers, wherein said containers contain the above pharmaceutical compositions according to the present invention. Such containers may be accompanied by specifications in a form prescribed by the government agency regulating the manufacture, use, or distribution of the drug or biological product, which reflect the approval of its administration to humans by the specified agency of the country in which the product is manufactured, used, or distributed.

[00103] Receipt and storage

[00104] The pharmaceutical composition of the present invention can be prepared by a method known in the art, for example, by conventional methods of mixing, dissolving, granulating, lozenges, grinding, emulsifying, encapsulating, pouring or lyophilizing.

[00105] Once prepared, pharmaceutical compositions containing the compounds of the present invention, formulated in a dosage form in a suitable carrier, can be placed in appropriate containers and labeled for the treatment of the specified condition. Such labeling should include the amount, frequency, and route of administration of the drug.

[00106] Combinations

[00107] The pharmaceutical composition containing the antibodies of the present invention described above may also be combined with one or more other therapeutic agents, such as antineoplastic agents, as long as the resulting combination does not cause unacceptable side effects.

EXAMPLES

[00108] The present invention will be better understood with reference to the following non-limiting experimental examples. All experimental methods in the following examples, unless otherwise noted, are conventional methods. Experimental materials used in the following examples, unless otherwise noted, were purchased from conventional biochemical reagent distributors.

[00109] Example 1: Anti-IL17A Antibody Screening

[00110] 1.1 Immunization of mice

[00111] Mice were immunized with IL17A protein according to the method described by StGroth and others (de StGroth and Scheidegger 1980) with suitable modifications. Recombinant human IL17A protein (from SinoBiological, Inc, cat. no. 10247-H07B) was used to immunize mice. The amino acid sequence of the PL7A protein (UniprotKB Q16552) is the sequence of Met1-Ala155 (SEQ ID NO: 1). Recombinant human IL17A protein was mixed with aluminum phosphate adjuvant (the first and fourth immunizations were given with additional Freund's complete adjuvant CFA as an emulsion in phosphate-buffered saline, and mice were immunized subcutaneously at multiple sites at 50 μg per dose of the mixture for 4 times at intervals of 2 weeks, 3 weeks, and 3 weeks, respectively. Starting with the third immunization, blood was collected seven days after each immunization through the medial canthal plexus of the eyes. Anti-mouse IL17A antibody titer in serum was measured by ELISA using a recombinant coating The serum titer after the fourth immunization reached the desired level (ELISA, OD>1.0) after a dilution of 1:8000, and these mice were given a booster dose of 25 μg intravenous recombinant human IL17A protein 75 days after the fourth immunization. After 4 days, the mice were sacrificed, and the spleen tissue was removed and frozen in liquid nitrogen.

[00112] 1.2 Construction and Screening of Antibody Phage Display Library

[00113] RNA was extracted from mouse spleen tissue using TriPure Isolation Reagent (from Roche, Cat. No. 11,667,165,001), and cDNA was prepared by reverse transcription of RNA using a reverse transcription kit (from Invitrogen, Cat. No. 18080-051). 2 primer pairs were designed to amplify the mouse antibody light chain variable region sequence and 1 primer pair to amplify the heavy chain variable region sequence according to the method described in Jones and Bendig 1991. These sequences encoding the mouse antibody light and heavy chain variable regions were combined with the nucleotide sequence encoding the scFv by overlapping extension PCR, then the two nucleotide sequences were joined using a linker (SEQ ID NO: 2) to combine with the nucleotide sequence encoding the scFv; then enzymatically ligated into the phage vector pComb3x (Sino Biological, Inc.) using SƒiI restriction endonuclease (Fermentas), and electrotransformed into X-Blue competent cells to produce a mouse scFv antibody phage display library; the size of the library is (sic). A phage library enriched with positive antibodies against IL17A was obtained by screening using ELISA using the phage antibody sorting method (Aitken 2002). ScFv antibodies that specifically bound to recombinant human IL17A protein were expressed by individual phage colonies from the enrichment library and tested for their binding to recombinant human IL17A protein by ELISA. The nucleotide sequence of the scFv antibody of one colony was sequenced as SEQ ID NO: 3, and the antibody, named IL17A-M069, was obtained from this colony after the steps in Example 1.3.

[00114] 1.3 Production of mouse monoclonal antibody against IL17A

[00115] A nucleotide sequence encoding a heavy chain variable region (SEQ ID NO: 4) with a heavy chain signal peptide sequence (SEQ ID NO: 43) and a mouse IgG1 heavy chain constant region sequence (SEQ ID NO: 6) of said scFv antibody, amplified and assembled by overlap extension PCR, introduced into HindIII+XbaI digested pSTEP2 vector (Fermentas), thereby obtaining a full heavy chain expression vector (SEQ ID NO: 36); similarly, the nucleotide sequence encoding the light chain variable region (SEQ ID NO: 5) with the light chain signal peptide sequence (SEQ ID NO: 29) and the mouse kappa light chain constant region sequence (SEQ ID NO: 7) of the specified scFv antibody was amplified and assembled by overlap extension PCR and introduced into the HindIII+XbaI restriction enzyme digested pSTEP2 vector (Fermentas), thereby obtaining a full light chain expression vector (SEQ ID NO: 37). Primers for assembly of the heavy chain signal peptide, heavy chain variable region, and heavy chain constant region of IgG1 are as follows:

[00116] Primers for assembly of the light chain signal peptide, light chain variable region, and mouse kappa light chain constant region are as follows:

[00117] 293E cells (ATCC) were subcultured in SCD4-4-TC2 medium (SinoBiological, Inc.) in a 200 ml flask per flask with an initial seeding density of 0.3~0.4*10 ⁶ cells/ml, and the flask was cultured in a rocking chair with CO ₂ at a rotation speed of 175 rpm at 37°C, until the cell density reaches 1.5 ~ 3 * 10 ⁶ cells/ml. Then, the plasmids encoding the light chain and the heavy chain were mixed in a 1:1 ratio, and 100 μg of mixed plasmid DNA and 800 μl of TF2 transfection reagent were added to a culture flask, which was then cultured in a shaker with a rotation speed of 175 rpm at 37 °C for 7 days for subsequent collection. The culture liquid was centrifuged at 4000 rpm for 25 min, the supernatant was collected and storage buffer was added. After equilibrating the Protein A chromatography column with 1/5 volume of the supernatant using 5-10 column volumes of phosphate-buffered saline, the filtered supernatant was applied to the specified chromatography column and again equilibrated with 5-10 column volumes, then the contents of the column were eluted using sodium acetate buffer for sample collection. The sample was neutralized with Tris, resulting in a highly purified monoclonal antibody in a neutral solution.

[00118] Example 2: Functional Analysis of Anti-IL17A Mouse Monoclonal Antibody

[00119] 2.1 Mouse IL17A-M069 antibody blocks IL17A-induced IL-6 secretion by HFF cells

[00120] As described in Beerli, Bauer et al. 2014, IL17A stimulates the secretion of IL-6 cytokines by human foreskin fibroblasts HFF in vitro. Anti-IL17A antibody was added to this system to evaluate the neutralizing effect of anti-IL17A antibody on IL17A by determining the secretion of IL-6 by HFF cells. HFF cells (ATCC, SCRC-1041) were inoculated into a 96-well plate at a cell density of 1 x 10 ⁴ per well and grown overnight in DMEM containing 15% FBS. Various concentrations of IL17A-M069 antibody and Taltz biosimilar positive control were added respectively at 10 μl per well the next day, then IL17A protein at a final concentration of 50 ng/ml was added at 10 μl per well. The indicated 96-well plate was incubated at 37°C, 5% CO ₂ in a cell incubator for 48 hours, and empty well B (no cells), negative control M' (cells inoculated, no antibody sample, IL17A added) and M were used (cells were inoculated without antibody sample and without IL17A). After incubation, the supernatant was collected and IL-6 secretion was measured by ELISA. From the DL-6 secretion level in the sample wells and the M' well group, the IL-6 secretion level in the M well group, respectively, was subtracted to calculate the degree of inhibition. Inhibition degree % = (1 - IL-6 secretion in wells with sample) / IL-6 secretion in well group M' × 100%. A standard curve was calculated using statistical software, taking the antibody sample concentration value as the horizontal coordinate and IL-6 secretion as the vertical coordinate. The results are shown in Fig. 1, mouse IL17A-M069 antibody could block IL17A-induced IL-6 secretion by HFF cells, and the maximum degree of inhibition and the average concentration of IL17A-M069 inhibition of IL17A protein were similar to those of the biosimilar Taltz used as a positive control, the maximum degree of inhibition of IL17A- M069 of mouse and biosimilar Taltz was 96.33% and 97.35%, respectively, and the EC ₅₀ value was 243.3 ng/ml and 246.6 ng/ml, respectively. Therefore, IL17A-M069 is a suitable antibody with good in vitro activity, and subsequent humanization modification and functional analysis were carried out with IL17A-M069.

[00121] Example 3: Humanization and production of anti-IL17A antibody IL17A-M069

[00122] Based on the results of the functional analysis of the mouse IL17A-M069 antibody in Example 2, humanization and production were carried out accordingly.

[00123] 3.1 Determination of CDR sequences of the light and heavy chains of the anti-IL17A antibody IL17A-M069

[00124] The amino acid sequence of the heavy chain and light chain variable regions of the IL17A-M069-scFv antibody was obtained from the nucleotide sequence of the IL17A-M069-scFv antibody determined in Example 1.3, see SEQ ID NO: 8/9.

[00125] The amino acid sequence of each of the three light and heavy chain CDRs of the mouse IL17A-M069-scFv antibody was determined with reference to the Kabat index (Abhinandan and Martin 2008, Dondelinger, Filee et al. 2018) and the IMGT numbering system (Lefranc 2014), see Table 1 and SEQ ID NO: 10-15. The above corresponding three light chain and heavy chain CDRs were transplanted into subsequent steps and retained in the final humanized antibody IL17A-H069, see Examples 3.2 and 3.3.

[00126] 3.2 Transplantation of mouse CDR antibodies

[00127] Humanization of the mouse antibody was accomplished using the classical CDR transplantation humanization method (Kettleborough, Saldanha et al. 1991). The human light or heavy chain variable regions of the antibody whose sequences are most similar to the reported mouse light or heavy chain variable region sequences (>50% similarity) were selected as a template, and the sequences of each of the three CDRs (SEQ ID NO: 10-15 ) from the mouse light or heavy chain was introduced into the variable region of a human antibody, resulting in humanized amino acid sequences of the light chain variable region ( _VL ) or the heavy chain variable region ( _VH ), respectively. The human antibody template for the IL17A-M069 light chain variable region was antibody IGKV4-1*01, which has 75.2% homology to the IL17A-M069 light chain, and the human antibody template for the variable region was IGHV1-69-2*01, which has 65.3% homology with IL17A-M069 heavy chain.

[00128] 3.3 Back mutations in the humanized variable region framework region

[00129] Because some key amino acids in the mouse framework region are required to maintain CDR activity, the key amino acids were back-mutated to the corresponding amino acid sequences of the mouse antibody, the following sites were back-mutated: according to the Kabat index system in the light chain, position 48 was back-mutated to V, position 49 was backmutated to D, and position 87 was backmutated to F; however, in the heavy chain, position 24 was back-mutated to A, and position 43 was back-mutated to H. The humanized antibody IL17A-H069 was produced by humanized CDR transplantation and back mutations in the framework region, and the amino acid sequences of its heavy and light chains are shown in SEQ ID NO: 16/17, respectively; the amino acid sequences of its heavy and light chains in form containing signal peptides are shown respectively in SEQ ID NO: 18/19, wherein the sequences of the signal peptides connected to the heavy/light chains are shown in SEQ ID NO: 20/21; the variable region sequences of the heavy chain/light chain of the humanized antibody are shown in SEQ ID NO: 22/23; the sequences of the IgG1 heavy chain constant region/human kappa light chain constant region of the humanized antibody are given in SEQ ID NO: 24/25), respectively.

[00130] 3.4 Preparation of Humanized Monoclonal Antibody IL17A-H069

[00131] A nucleotide sequence (SEQ ID NO: 27) encoding the light chain and signal peptide of the antibody IL17A-H069, which contains the following nucleotide sequence encoding the light chain signal peptide (SEQ ID NO: 29), a light chain variable region (SEQ ID NO: 31) of the humanized antibody and the human kappa light chain constant region (SEQ ID NO: 33) concatenated in this order were amplified by PCR and introduced into a self-developed vector pGS (KpnI+XbaI) by infusion, and the correct plasmid sequence verified by sequencing. Similarly, the nucleotide sequence (SEQ ID NO: 26) encoding the heavy chain of the IL17A-H069 antibody containing the signal peptide, which contains the following nucleotide sequence encoding the heavy chain signal peptide (SEQ ID NO: 28), the heavy chain variable region of the humanized antibody ( SEQ ID NO: 30) and the human IgG1 heavy chain constant region (SEQ ID NO: 32) concatenated in this order were amplified by PCR and introduced into a GS vector (NheI+NotI), which was then verified to contain the correct lung gene chains by infusion, and the correct assembly of vectors expressing both the light and heavy chains of IL17A-H069 was verified by sequencing. These expression vectors are eukaryotic expression vectors containing the GS gene as a selection marker and the light and heavy chains of the antibody as expression elements. These expression vectors were transfected into CHO-K1-GS deficient cells and IL17A-H069 high expression cell lines were obtained by MSX screening. Clones with high antibody expression were selected by ELISA, and these high expression cell lines were selected taking into account both the growth status of the cells and key characteristics of the quality of the antibody as a drug. Serum-free suspension cultures were used to grow the IL17A-H069-producing CHO cell line, resulting in IL17A-H069 antibodies of high purity and quality.

[00132] Example 4: Antigen Binding Affinity Analysis of Humanized Antibody IL17A-H069

[00133] 4.1 Humanized Antibody Binding Affinity Analysis for IL17A Protein

[00134] 4.1.1 Binding of IL17A-H069 to recombinant human IL17A protein

[00135] Recombinant human IL17A protein (SinoBiological, Inc.) at various concentrations was coated on a 96-well plate overnight at 4°C in an amount of 100 μl per well. The plate was washed the next day and blocked at room temperature for 1 hour. After incubation with 100 μL of 2 μg/mL biosimilar COSENTYX (SinoCelltech Co., Ltd.) and IL17A-H069 (SinoCelltech Co., Ltd.), respectively, the plate was washed to remove unbound antibody, then incubated with goat anti-human IgG Fc/HRP and washed several times, and then added chromogenic substrate solution until color appeared. OD ₄₅₀ was measured after color formation was complete. Using the concentration of recombinant human IL17A protein as the horizontal coordinate and the OD ₄₅₀ value as the vertical coordinate, graphPad Prism 6.0 software was used to analyze the data and generate a dose-effectiveness curve from which the mean effective concentration EC ₅₀ value was calculated.

[00136] The results shown in FIG. 2 demonstrate that the EC ₅₀ value of binding of the COSENTYX biosimilar to the recombinant human IL17A protein is 74.8 ng/ml, R ² =0.9993; the EC ₅₀ value of IL17A-H069 binding to recombinant human IL17A protein is 46 ng/ml, R ² =0.9958. This indicates that the ability of IL17A-H069 to bind to recombinant human IL17A protein is slightly better than the biosimilar COSENTYX.

[00137] 4.1.2 Binding of IL17A-H069 to recombinant human IL17A/IL17F protein

[00138] Recombinant human IL17A/F dimeric protein (SinoBiological, Inc., CT047-HNAE) at various concentrations was coated on a 96-well plate overnight at 4°C at 100 μl per well. The plate was washed the next day and blocked at room temperature for 1 hour. After incubation with 100 μL of 2 μg/mL biosimilar COSENTYX (SinoCelltech Co., Ltd.) and IL17A-H069 (SinoCelltech Co., Ltd.), respectively, the plate was washed to remove unbound antibody, then incubated with goat anti-human IgG Fc/HRP and washed several times, and then added chromogenic substrate solution until color appeared. OD450 was measured after color formation was complete. Using the recombinant human IL17A/F protein concentration as the horizontal coordinate and the OD ₄₅₀ value as the vertical coordinate, graphPad Prism 6.0 software was used to analyze the data and generate a dose-effectiveness curve from which the mean effective concentration EC ₅₀ value was calculated.

[00139] The results shown in FIG. 3 demonstrate that the EC ₅₀ value of binding of the COSENTYX biosimilar to the recombinant human IL17A/F protein is 63.9 ng/ml, R ² =0.9999; the EC ₅₀ value of IL17A-H069 binding to the recombinant human IL17A/F protein is 36.3 ng/ml, R ² =1.0. This indicates that the ability of IL17A-H069 to bind to the recombinant human IL17A/F dimeric protein is slightly better than that of the biosimilar COSENTYX.

[00140] 4.1.3 Binding affinity test of IL17A-H069 with recombinant human IL17A protein and recombinant human IL17A/TL17F protein

[00141] Affinities of IL17A-H069 at various concentrations (0.42 nM, 0.90 nM, 1.74 nM and 3.47 nM) and the positive control COSENTYX (Norvatis, SHM12) at various concentrations (0.90 nM, 1 .74 nM, 3.47 nM, 6.94 nM, and 13.9 nM) with biotinylated IL7A or IL17A/F proteins were determined, respectively, using the Octet Biomolecular Interaction Assay system. The results in Table 2 show that the binding affinity value K _D of IL17A-H069 to recombinant human IL17A protein was 2.88E-11 M, the association rate constant kop was 6.71E+05 M ^-1 s ^-1 and the dissociation rate constant k _off was 1.93E-05 m ^-1 ; while the binding affinity value of K _D COSENTYX to IL17A protein was 9.55E-11 M, the association rate constant kop was 1.78E+05 M ^-1 s ^-1 and the dissociation rate constant k _off was 1.70E-05 s ^-1 . The binding affinity K _{D of} IL17A-H069 to the recombinant human IL17A/F protein was 5.37E-10 M, the association rate constant k _on was 1.44E+0.5M ^-1 s ^-1 and the dissociation rate constant k _off was 7.72E -05 s ^-1 ; while the binding affinity value of K _D COSENTYX to IL17A/F protein was 1.10E-09 M, the association rate constant kop was 8.00E+04 M ^-1 s ^-1 and the dissociation rate constant k _off was 8.79E- 05 s ^-1 . The results demonstrate that IL17A-H069 binds to the IL17A protein with higher affinity than COSENTYX, the affinity of IL17A-H069 is approximately 3.32 times higher than that of COSENTYX, and IL17A-H069 has a higher association rate, so that IL17A-H069 has stronger binding activity to IL17A protein than COSENTYX; IL17A-H069 binds to the IL17A/F protein with stronger affinity than COSENTYX, IL17A-H069 has approximately 2.05 times the affinity of COSENTYX, and IL17A-H069 has a higher association rate, so IL17A-H069 has stronger binding activity with IL17A/F protein than COSENTYX.

[00142] 4.1.4 Determination of species-specific cross-reactivity of IL17A-H069 with mouse IL17A protein

[00143] Recombinant human EL17A protein (Sino Biological, Inc.) and mouse mIL17A protein (Sino Biological, Inc.) at various concentrations, respectively, were applied to a 96-well plate in an amount of 100 μl per well overnight at 4°C. The plate was washed the next day, blocked at room temperature for 1 hour. 100 μl of 2 μg/ml IL17A-H069 (Sino Biological, Inc.), COSENTYX (Norvatis, SUM 12) as a positive control and H7N9-R1 antibody were added ( SinoCelltech Co., Ltd.) as a negative control and incubated accordingly. The plate was washed to remove unbound antibody. The plate was incubated with goat anti-human IgG Fc/HRP (Sino Biological, Inc.) and then washed several times, and chromogenic substrate solution was added until color appeared. After color formation was complete, OD ₄₅₀ was measured. The results shown in Fig. 4 demonstrate that IL17A-H069 did not cross-link with the mouse mIL17A protein.

[00144] 4.2 IL17A-H069 blocks the binding of IL17A protein and IL17A/F protein to the IL17RA receptor

[00145] 4.2.1 IL17A-H069 blocks IL17A protein from binding to the IL17RA receptor

[00146] IL17A protein at a concentration of 0.4 μg/ml was applied to a 96-well plate in an amount of 100 μl per well overnight at 4°C. The plate was washed the next day, blocked at room temperature for 1 hour. 100 μl of 2 μg/ml biotinylated IL17RA-His-biotin protein (Sino Biological, Inc.) was added to each well, then various concentrations of IL17A-H069 (SinoCelltech Co) were added to each well. ., Ltd.), COSENTYX (Norvatis, SHM12) as a positive control, Taltz (Eli Lilly) as a positive control, and H7N9-R1 antibody (SinoCelltech Co., Ltd.) as a negative control, respectively, and incubated. The plate was washed to remove unbound antibody. The plate was incubated with streptavidin/HRP (Beijing ZSGB-Bio Co., Ltd., SA-5004), then washed several times, and a chromogenic substrate solution was added until color appeared. After completion of color formation, OD ₄₅₀ was measured in each studied group in duplicate.

[00147] Taking the antibody concentration as the horizontal coordinate and the inhibition rate % as the vertical coordinate, graphPad Prism 6.0 software was used for data analysis and plotting, and IC ₅₀ values were calculated. Inhibition rate % = (OD _control - OD _sample ) / OD _control × 100%, where OD _control denotes the OD value of the well with only the added protein IL17RA-His-biotin, but without the addition of antibody, OD _sample denotes the OD value of the well as with the added protein IL17RA-His-biotin and with added antibody.

[00148] The results shown in FIG. 5 demonstrate that biotinylated IL17RA protein can effectively bind to plated recombinant human IL17A protein, and the IL17A-H069 antibody can inhibit the binding of IL17A protein to the IL17RA receptor with a significantly better profile as represented by its inhibition curve than COSENTYX used as a positive control, but the profile of IL17A-H069 in inhibiting the binding of the IL17A protein to the IL17RA receptor was close to the profile of Taltz, used as a positive control. The IC ₅₀ values of IL17A-H069, COSENTYX, and Taltz were 0.50 μg/ml, 2.99 μg/ml, and 0.50 μg/ml, respectively, and the maximum inhibition rates were 85.4%, 73.5%, and 89 .5%, respectively.

[00149] 4.2.2 IL17A-H069 blocks IL17A/F protein from binding to the IL17RA receptor

[00150] IL17RA-Fc protein (Sino Biological, Inc.) at a concentration of 5 μg/ml was applied to a 96-well plate in an amount of 100 μl per well overnight at 4°C. The plate was washed the next day, blocked at room temperature for 1 hour. 100 μl of 0.8 μg/ml IL17A/F-biotin protein (Sino Biological, Inc.) was added to each well, then various concentrations of IL17A-H069 (SinoCelltech Co., Ltd.), COSENTYX biosimilar (SinoCelltech Co., Ltd.) as a positive control, Taltz biosimilar (SinoCelltech Co., Ltd.) as a positive control, and H7N9-R1 antibody (SinoCelltech Co., Ltd.) as a positive control. as a negative control, respectively, and incubated. The plate was washed to remove unbound antibody. The plate was incubated with streptavidin/HRP (Beijing ZSGB-Bio Co., Ltd., SA-5004) and then washed several times, and chromogenic substrate solution was added until color appeared. OD ₄₅₀ was measured after color appearance had stabilized.

[00151] Taking the antibody concentration as the horizontal coordinate and the degree of inhibition % as the vertical coordinate, graphPad Prism 6.0 software was used to analyze the data and plot the curve, and the IC ₅₀ values were calculated. Inhibition rate % = (OD _control - OD _sample ) / OD _control × 100%, where OD _control denotes the OD value of the well with only the added IL17A/F-biotin protein without the addition of antibody, OD _sample denotes the OD value of the well as with the added IL17A protein/ F-biotin and added antibody.

[00152] The results shown in FIG. 6 demonstrate that IL17A/F-biotin protein can effectively bind to the coating of recombinant human IL17RA-Fc protein, and the addition of IL17A-H069 antibody can effectively inhibit the binding of IL17A/F protein to IL17RA-Fc receptor. IL17A-H069 has a stronger effect of inhibiting the binding of IL17A/F protein to the IL17RA-Fc receptor than the COSENTYX biosimilar used as a positive control and the Taltz biosimilar used as a positive control. The IC ₅₀ values of IL17A-H069, COSENTYX biosimilar and Taltz biosimilar were 1.02 µg/ml, 1.20 µg/ml and 1.35 µg/ml, respectively, and the maximum inhibition rates were 92.3%, 87.9% and 75%, respectively.

[00153] Example 5: Functional Analysis of Humanized Antibody IL17A-H069

[00154] 5.1 IL17A-H069 blocks IL17A- or IL17A/F-induced IL-6 secretion by HFF cells

[00155] HFF cells were inoculated into a 96-well plate at a cell density of 1 x 10 ⁴ per well and grown overnight in DMEM containing 15% FBS. Various concentrations of IL17A-H069 (SinoCelltech Co., Ltd.) and COSENTYX ( Norvatis) as a positive control or Taltz (Eli Lilly) as a positive control were respectively added at 10 μl per well the next day. Then, 10 μl of IL17A protein (Sino Biological, Inc. 12047-HNAS) at a final concentration of 50 ng/ml or IL17A/F protein (Sino Biological, Inc. CT047-HNAE) at a final concentration of 1 μg/ml was added to each well, respectively. . The 96-well plate was incubated at 37°C, 5% CO ₂ in a cell incubator for 48 hours, and control well B (no cells), negative control M (with added cells, without added antibody sample, with added IL17A or IL17A) were used /F) and M' (with added cells, without added antibody sample, and without added EL17A or IL17A/F). After incubation, the supernatant was collected and IL-6 secretion was measured by ELISA. From the IL-6 secretion value in the sample wells and the M group wells, the IL-6 secretion value in the M' group wells, respectively, was subtracted to calculate the degree of inhibition. Inhibition rate % = (1 - IL-6 secretion in sample wells) / IL-6 secretion in M group wells × 100%. A standard curve was calculated using statistical software, taking the antibody sample concentration as the horizontal coordinate and IL-6 secretion as the vertical coordinate, and using a 4-parameter logistic regression equation to construct a standard "S" curve to calculate the half maximum effective concentration (EC ₅₀ ) sample of the specified antibody.

[00156] In the measurements described above, as shown in FIG. 7 and Table 3, the activity of IL17A-H069 in neutralizing IL17A ( _EC50 : 0.19 μg/ml, maximum neutralization rate: 94.6%) was much higher than that of COSENTYX used as a positive control ( _EC50 : 0.22 μg/ml, maximum neutralization rate: 51.6%) (Figure 7A); Compared with Taltz used as a positive control, the IL17A neutralizing activity of IL17A-H069 ( _EC50 : 0.20 μg/ml, maximum neutralization rate: 90.3%) was close to the neutralizing activity of Taltz ( _EC50 : 0, 19 μg/ml, maximum neutralization rate: 95.9%) (Figure 7C); IL17A-H069 activity in neutralizing IL17A/F (EC ₅₀ : 1.19 μg/ml, maximum neutralization rate: 85.0%) was also slightly higher than that of COSENTYX (EC ₅₀ : 2.25 μg/ml, maximum neutralization rate neutralization: 79.5%) (Fig. 7B). In comparison with Taltz used as a positive control, the IL17A/F neutralizing activity of IL17A-H069 at high concentration ( _EC50 : 0.83 μg/ml, maximum neutralization rate: 72.90%) was close to Taltz ( _EC50 : 1.10 μg/mL, maximum neutralization rate: 76.3%), and superior to Taltz, used as a positive control, at low concentrations (Figure 7D). In conclusion, IL17A-H069 has better biological activity in neutralizing IL17A and IL17A/F.

[00157] Example 6: In Vivo Efficacy of Humanized Antibody in Mice

[00158] 6.1 In vivo efficacy of IL17A-H069 in the psoriasis (PsO) model of immune-reconstituted hPBMC mice

[00159] Using hPBMC from 3 donors, a total of 60 B-NDG mice (Biocytogen Pharmaceuticals (Beijing) Co., Ltd.) with a humanized immune system were generated (20 mice per hPBMC donor). Peripheral blood was collected after one week and the percentage of human-derived cells was measured using cell cytometry. 20 mice had percentages of human-derived cells in the range of 0.04-1.5%, 31 mice had percentages in the range of 1.5-7%, and 8 mice had percentages >7%. Mice with a percentage of 1.5-7% were selected to create an IMQ (imiquimod)-induced psoriasis mouse model from which the efficacy of IL17A-H069 was assessed.

[00160] Selected mice were grouped according to this strategy: 5 non-model mice were grouped into the G1 normal control group; 5 mice with model psoriasis, which were not administered the drug, were combined into group G2, i.e., the psoriasis model group; 7 psoriasis mice treated with IL17A-H069 were grouped into G3; and 7 psoriasis mice treated with the Taltz positive control were combined into group G4. The backs of all mice were shaved to create an open area of approximately 2 cm × 3 cm, and 100 mg of IMQ cream was applied to the back of each mouse in groups G2, G3 and G4, and 10 mg of IMQ cream was applied to the ears of each mouse in groups G2, G3 and G4 daily for 10 days. The PASI values of the mice were recorded daily. According to the PASI scoring criterion, as shown in Table 4, mice were scored 0–4 for the degree of erythema, desquamation, and thickening of the dorsal skin on the posterior side of the lesion, respectively, and the three scores were summed to form a total score. For groups G3 and G4, antibody administration began on day 1 of IMQ cream application at a dose of 10 μg/kg weekly.

[00161] The results are shown in FIG. 8. Compared with mice in the normal control group, the PASI score was significantly higher in mice in the psoriasis model group, reflecting that this mouse model can characterize psoriasis to some extent; from day 5, PASI scores were significantly lower in the IL17A-H069 group compared with the psoriasis model group; and compared with the Taltz control group, IL17A-H069 showed higher in vivo efficacy in reducing psoriasis scores in mice, thus IL17A-H069 is effective in alleviating psoriasis onset (PsO) and reducing psoriasis symptoms in a mouse model of psoriasis.

[00162] Example 7: Pharmacokinetics of a Humanized Antibody in Vivo

[00163] 7.1 Pharmacokinetics of single subcutaneous administration of IL17A-H069 in cynomolgus monkeys

[00164] In this example, single subcutaneous injections of IL17A-H069 antibody were administered to cynomolgus monkeys at a dosage of 1 mg/kg. Serum was collected before administration and after 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 24 hours, 48 hours, 3 days, 4 days, 7 days, 10 days, 14 days, 17 days, 21 days, 24 days , 28 days, 31 days and 35 days after administration, respectively. The developed ELISA method was used to measure IL17A-H069 drug concentration in mouse serum, and pharmacokinetic parameters were calculated using Phoenix-WinNonlin Non-Compartmental Analysis (NCA) software (Pharsight) 6.4. The dynamic pattern of changes in the drug profile in vivo after a single subcutaneous administration of IL17A-H069 was studied to study the dynamic changes of the drug in the body after a single subcutaneous administration of IL17A-H069.

[00165] Changes in IL17A-H069 drug concentration over time are shown in Table 6 and FIG. 9. There was no significant gender difference in _Cmax and AUC _last between female and male mice (data not shown), and the half-life t1 _/2 of IL17A-H069 was 353.66 hours, the _Tmax value was 34 hours. In terms of exposure in vivo, the AUC _{of last} IL17A-H069 was 3846.86 h*μg/ml.

[00166] At a dosage of 1 mg/kg, IL17A-H069 has a shorter T _max and longer t _1/2 so that IL17A-H06 exhibits superior pharmacokinetics, including rapid absorption after subcutaneous injection, long half-life and better drug exposure and etc., thereby laying the foundation for a longer dosing cycle.

Literature Cited

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Beerli, R.R., M. Bauer, A. Fritzer, L.B. Rosen, R.B. Buser, M. Hanner, M. Maudrich, M. Nebenfuehr, J. A. S. Toepfer, S. Mangold, A. Bauer, S.M. Holland, S.K. Browne and A. Meinke (2014). "Mining the human autoantibody repertoire: isolation of potent IL17A-neutralizing monoclonal antibody from a patient with thymoma." mAbs 6(6): 1608–1620.

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LIST OF SEQUENCES

<110> SinoCellTech Ltd.

<120> HUMANIZED ANTI-IL17A ANTIBODY AND ITS APPLICATION

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Met Thr Pro Gly Lys Thr Ser Leu Val Ser Leu Leu Leu Leu Leu Ser

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Leu Glu Ala Ile Val Lys Ala Gly Ile Thr Ile Pro Arg Asn Pro Gly

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Cys Pro Asn Ser Glu Asp Lys Asn Phe Pro Arg Thr Val Met Val Asn

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Leu Asn Ile His Asn Arg Asn Thr Asn Thr Asn Pro Lys Arg Ser Ser

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Asp Tyr Tyr Asn Arg Ser Thr Ser Pro Trp Asn Leu His Arg Asn Glu

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Asp Pro Glu Arg Tyr Pro Ser Val Ile Trp Glu Ala Lys Cys Arg His

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Leu Gly Cys Ile Asn Ala Asp Gly Asn Val Asp Tyr His Met Asn Ser

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Val Pro Ile Gln Gln Glu Ile Leu Val Leu Arg Arg Glu Pro Pro His

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Cys Pro Asn Ser Phe Arg Leu Glu Lys Ile Leu Val Ser Val Gly Cys

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Thr Cys Val Thr Pro Ile Val His His Val Ala

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<223> The nucleotide sequence of the linker used for

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<223> Nucleotide sequence of the mouse scFv antibody used in

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gacattgtga tgtcacagtc tccatcctcc ctggctatgt cagtaggaca gaaggtcact 60

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gaatctgggg tccctgatcg cttcataggc agtggatctg ggacagattt cagtcttacc 240

atcagcagtg tgcaggctga ggacctggca gattacttct gtcagcaaca ttataccact 300

ccattcacgt tcggctcggg gaccaagctg gaaataaaat ctagtggtgg cggtggttcg 360

ggcggtggtg gaggtggtag ttctagatct tcccaggccc accttcaaca gtctggggct 420

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actgactatg aaatgcactg ggtgaaacag acacctgtgc atggcctgga atggattgga 540

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ctgactgcag acaagtcctc cagtacagcc tacatggagc tcagcagcct gacatctgag 660

gactctgctg tctattactg tacaagaggg gatcacgacg gaaggactga ctactggggc 720

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<223> Nucleotide sequence of the variable region of the heavy chain

mouse IL17A-M069 antibodies

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tcctgcaagg ctttgggcta cacatttact gactatgaaa tgcactgggt gaaacagaca 120

cctgtgcatg gcctggaatg gattggagtt attcatccag gaggtggtgg tacggcctac 180

aatcagaagt tcaagggcaa ggccacactg actgcagaca agtcctccag tacagcctac 240

atggagctca gcagcctgac atctgaggac tctgctgtct attactgtac aagaggggat 300

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<223> Nucleotide sequence of the light chain variable region

mouse IL17A-M069 antibodies

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gacattgtga tgtcacagtc tccatcctcc ctggctatgt cagtaggaca gaaggtcact 60

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tggtaccagc agaaaccagg acagtctcct aaacttctgg tagactttgc atccactagg 180

gaatctgggg tccctgatcg cttcataggc agtggatctg ggacagattt cagtcttacc 240

atcagcagtg tgcaggctga ggacctggca gattacttct gtcagcaaca ttataccact 300

ccattcacgt tcggctcggg gaccaagctg gaaataaaa 339

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<223> Nucleotide sequence of the constant region of the heavy chain of IgG1

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gccaaaacga cacccccatc tgtctatcca ctggcccctg gatctgctgc ccaaactaac 60

tccatggtga ccctgggatg cctggtcaag ggctatttcc ctgagccagt gacagtgacc 120

tggaactctg gatccctgtc cagcggtgtg cacaccttcc cagctgtcct gcagtctgac 180

ctctacactc tgagcagctc agtgactgtc ccctccagca cctggcccag cgagaccgtc 240

acctgcaacg ttgcccaccc ggccagcagc accaaggtgg acaagaaaat tgtgcccagg 300

gattgtggtt gtaagccttg catatgtaca gtcccagaag tatcatctgt cttcatcttc 360

cccccaaagc ccaaggatgt gctcaccatt actctgactc ctaaggtcac gtgtgttgtg 420

gtagacatca gcaaggatga tcccgaggtc cagttcagct ggtttgtaga tgatgtggag 480

gtgcacacag ctcagacgca accccgggag gagcagttca acagcacttt ccgctcagtc 540

agtgaacttc ccatcatgca ccaggactgg ctcaatggca aggagttcaa atgcagggtc 600

aacagtgcag ctttccctgc ccccatcgag aaaaccatct ccaaaaccaa aggcagaccg 660

aaggctccac aggtgtacac cattccacct cccaaggagc agatggccaa ggataaagtc 720

agtctgacct gcatgataac agacttcttc cctgaagaca ttactgtgga gtggcagtgg 780

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<212> DNA

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cgacataaca gctatacctg tgaggccact cacaagacat caacttcacc cattgtcaag 300

agcttcaaca ggaatgagtg ttaaa 325

<210> 8

<211> 118

<212>PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence of the variable region of the heavy chain

mouse IL17A-M069 antibodies

<400> 8

Gln Ala His Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala

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Ser Val Lys Leu Ser Cys Lys Ala Leu Gly Tyr Thr Phe Thr Asp Tyr

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Glu Met His Trp Val Lys Gln Thr Pro Val His Gly Leu Glu Trp Ile

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Gly Val Ile His Pro Gly Gly Gly Gly Thr Ala Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

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Thr Arg Gly Asp His Asp Gly Arg Thr Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Leu Thr Val Ser Ser

115

<210> 9

<211> 113

<212>PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence of the light chain variable region

mouse IL17A-M069 antibodies

<400> 9

Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Met Ser Val Gly

1 5 10 15

Gln Lys Val Thr Met Asn Cys Lys Ser Asn Gln Ser Leu Leu Asn Arg

20 25 30

Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Ser Pro Lys Leu Leu Val Asp Phe Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ile Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Thr

65 70 75 80

Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln

85 90 95

His Tyr Thr Thr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile

100 105 110

Lys

<210> 10

<211> 14

<212>PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence CDR1 of the light chain of the antibody IL17A-M069

mouse / humanized antibody IL17A-H069

<400> 10

Gln Ser Leu Leu Asn Arg Ser Asn Gln Lys Asn Tyr Leu Ala

1 5 10

<210> 11

<211> 7

<212>PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence CDR2 of the light chain of the antibody IL17A-M069

mouse / humanized antibody IL17A-H069

<400> 11

Phe Ala Ser Thr Arg Glu Ser

15

<210> 12

<211> 9

<212>PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence CDR3 of the light chain of the antibody IL17A-M069

mouse / humanized antibody IL17A-H069

<400> 12

Gln Gln His Tyr Thr Thr Pro Phe Thr

15

<210> 13

<211> 10

<212>PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence CDR1 of the heavy chain of the antibody IL17A-M069

mouse / humanized antibody IL17A-H069

<400> 13

Gly Tyr Thr Phe Thr Asp Tyr Glu Met His

1 5 10

<210> 14

<211> 17

<212>PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence CDR2 of the heavy chain of the antibody IL17A-M069

mouse / humanized antibody IL17A-H069

<400> 14

Val Ile His Pro Gly Gly Gly Gly Thr Ala Tyr Asn Gln Lys Phe Lys

1 5 10 15

Gly

<210> 15

<211> 11

<212>PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence CDR3 of the heavy chain of the antibody IL17A-M069

mouse / humanized antibody IL17A-H069

<400> 15

Thr Arg Gly Asp His Asp Gly Arg Thr Asp Tyr

1 5 10

<210> 16

<211> 447

<212>PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence of the heavy chain of a humanized antibody

IL17A-H069

<400> 16

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Glu Met His Trp Val Gln Gln Ala Pro Gly His Gly Leu Glu Trp Met

35 40 45

Gly Val Ile His Pro Gly Gly Gly Gly Thr Ala Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Gly Asp His Asp Gly Arg Thr Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 17

<211> 220

<212>PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence of the light chain of a humanized antibody

IL17A-H069

<400> 17

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Arg

20 25 30

Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Val Asp Phe Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Phe Cys Gln Gln

85 90 95

His Tyr Thr Thr Pro Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile

100 105 110

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

115 120 125

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

130 135 140

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

145 150 155 160

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

165 170 175

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

180 185 190

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

195 200 205

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 18

<211> 466

<212>PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence of the heavy chain of a humanized antibody

IL17A-H069 containing signal peptide

<400> 18

Met Glu Leu Gly Leu Ser Trp Ile Phe Leu Leu Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys

20 25 30

Pro Gly Ala Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe

35 40 45

Thr Asp Tyr Glu Met His Trp Val Gln Gln Ala Pro Gly His Gly Leu

50 55 60

Glu Trp Met Gly Val Ile His Pro Gly Gly Gly Gly Thr Ala Tyr Asn

65 70 75 80

Gln Lys Phe Lys Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asp

85 90 95

Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Thr Arg Gly Asp His Asp Gly Arg Thr Asp Tyr Trp Gly

115 120 125

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

130 135 140

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

145 150 155 160

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

165 170 175

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

180 185 190

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

195 200 205

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

210 215 220

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

225 230 235 240

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

245 250 255

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

260 265 270

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

275 280 285

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

290 295 300

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

305 310 315 320

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

325 330 335

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

340 345 350

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

355 360 365

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

370 375 380

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

385 390 395 400

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

405 410 415

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

420 425 430

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

435 440 445

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

450 455 460

Pro Gly

465

<210> 19

<211> 239

<212>PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence of the light chain of a humanized antibody

IL17A-H069 containing signal peptide

<400> 19

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val

20 25 30

Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu

35 40 45

Leu Asn Arg Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys

50 55 60

Pro Gly Gln Pro Pro Lys Leu Leu Val Asp Phe Ala Ser Thr Arg Glu

65 70 75 80

Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe

85 90 95

Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Phe

100 105 110

Cys Gln Gln His Tyr Thr Thr Pro Phe Thr Phe Gly Pro Gly Thr Lys

115 120 125

Val Asp Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro

130 135 140

Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu

145 150 155 160

Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp

165 170 175

Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp

180 185 190

Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys

195 200 205

Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln

210 215 220

Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210> 20

<211> 19

<212>PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence of heavy chain signal peptide

humanized antibody IL17A-H069

<400> 20

Met Glu Leu Gly Leu Ser Trp Ile Phe Leu Leu Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys

<210> 21

<211> 19

<212>PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence of light chain signal peptide

humanized antibody IL17A-H069 / mouse antibody IL17A-M069

<400> 21

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser

<210> 22

<211> 118

<212>PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence of the variable region of the heavy chain

humanized antibody IL17A-H069

<400> 22

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Glu Met His Trp Val Gln Gln Ala Pro Gly His Gly Leu Glu Trp Met

35 40 45

Gly Val Ile His Pro Gly Gly Gly Gly Thr Ala Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Gly Asp His Asp Gly Arg Thr Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 23

<211> 113

<212>PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence of the light chain variable region

humanized antibody IL17A-H069

<400> 23

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Arg

20 25 30

Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Val Asp Phe Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Phe Cys Gln Gln

85 90 95

His Tyr Thr Thr Pro Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile

100 105 110

Lys

<210> 24

<211> 329

<212>PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence of the heavy chain constant region

humanized antibody IL17A-H069

<400> 24

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly

325

<210> 25

<211> 107

<212>PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence of the light chain constant region

humanized antibody IL17A-H069

<400> 25

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 26

<211> 1407

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence of the heavy chain of a humanized antibody

IL17A-H069 containing signal peptide

<400> 26

atggagttgg gactgagctg gattttcctt ttggctattt taaaaggtgt ccagtgtgag 60

gtccaacttg tccagtctgg agcagaggtg aagaagcctg gagccacagt gaagatttcc 120

tgtaaggcat ctggctacac cttcacagac tatgagatgc actgggtcca acaggctcct 180

ggccatggat tggagtggat gggagtgatt caccctggag gaggaggcac agcctacaac 240

cagaagttca agggcagggt gaccatcaca gcagacacca gcacagacac agcctatatg 300

gaactgtcct ccctgaggtc tgaggacaca gcagtctact actgtaccag gggagaccat 360

gatggcagga cagactactg gggacaaggc accctggtga cagtgtcctc tgcaagcacc 420

aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgg gggcacagcg 480

gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca 540

ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac 600

tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc 660

aacgtgaatc acaagcccag caacaccaag gtggacaaga aagttgagcc caaatcttgt 720

gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 780

ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcacg 840

tgcgtggtgg tggacgtgag ccacgaagac cccgaggtca agttcaactg gtacgtggac 900

ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 960

cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 1020

tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 1080

gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag 1140

aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 1200

tggggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 1260

gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 1320

aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac ccagaagtcc 1380

ctgtctctga gccctggcta atagtga 1407

<210> 27

<211> 720

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence of the light chain of a humanized antibody

IL17A-H069 containing signal peptide

<400> 27

atgggctggt cctgtatcat cctgttcctg gtggctacag ccacaggagt gcattctgac 60

attgtgatga cccagagccc tgactccctg gctgtgtccc tgggagagag ggctaccatc 120

aactgtaagt ccagccagtc cctgctgaac aggagcaacc agaagaacta cctggcttgg 180

tatcaacaga agcctggaca acctccaaaa ctgctggtgg actttgccag caccagggag 240

tctggagtgc ctgacaggtt ctctggctct ggctctggca cagacttcac cctgaccatc 300

tcctccctcc aagcagagga tgtggctgtc tacttctgtc aacaacacta caccacacca 360

ttcacctttg gacctggcac caaggtggac atcaagcgta cggtggctgc accatctgtc 420

ttcatcttcc cgccatctga tgagcagttg aaatctggaa ctgcctctgt tgtgtgcctg 480

ctgaataact tctatcccag agaggccaaa gtacagtgga aggtggataa cgccctccaa 540

tcgggtaact cccaggagag tgtcacagag caggacagca aggacagcac ctacagcctc 600

agcagcaccc tgacgctgag caaagcagac tacgagaaac acaaagtcta cgcctgcgaa 660

gtcacccatc agggcctgag ctcgcccgtc acaaagagct tcaacagggg agagtgttaa 720

<210> 28

<211> 57

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence of the heavy chain signal peptide

humanized antibody IL17A-H069

<400> 28

atggagttgg gactgagctg gattttcctt ttggctattt taaaaggtgt ccagtgt 57

<210> 29

<211> 57

<212> DNA

<213> Artificial sequence

<220>

<223> Light chain signal peptide nucleotide sequence

humanized antibody IL17A-H069

<400> 29

atgggctggt cctgtatcat cctgttcctg gtggctacag ccacaggagt gcattct 57

<210> 30

<211> 354

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence of the variable region of the heavy chain

humanized antibody IL17A-H069

<400> 30

gaggtccaac ttgtccagtc tggagcagag gtgaagaagc ctggagccac agtgaagatt 60

tcctgtaagg catctggcta caccttcaca gactatgaga tgcactgggt ccaacaggct 120

cctggccatg gattggagtg gatgggagtg attcaccctg gaggaggagg cacagcctac 180

aaccagaagt tcaagggcag ggtgaccatc acagcagaca ccagcacaga cacagcctat 240

atggaactgt cctccctgag gtctgaggac acagcagtct actactgtac caggggagac 300

catgatggca ggacagacta ctggggacaa ggcaccctgg tgacagtgtc ctct 354

<210> 31

<211> 339

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence of the light chain variable region

humanized antibody IL17A-H069

<400> 31

gacattgtga tgacccagag ccctgactcc ctggctgtgt ccctgggaga gagggctacc 60

atcaactgta agtccagcca gtccctgctg aacaggagca accagaagaa ctacctggct 120

tggtatcaac agaagcctgg acaacctcca aaactgctgg tggactttgc cagcaccagg 180

gagtctggag tgcctgacag gttctctggc tctggctctg gcacagactt caccctgacc 240

atctcctccc tccaagcaga ggatgtggct gtctacttct gtcaacaaca ctacaccaca 300

ccattcacct ttggacctgg caccaaggtg gacatcaag 339

<210> 32

<211> 996

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence of the heavy chain constant region

humanized antibody IL17A-H069

<400> 32

gcaagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60

ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120

tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180

ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240

tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300

aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360

ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420

gaggtcacgt gcgtggtggt ggacgtgagc cacgaagacc ccgaggtcaa gttcaactgg 480

tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 540

agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600

gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660

aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 720

ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840

ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 900

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacc 960

cagaagtccc tgtctctgag ccctggctaa tagtga 996

<210> 33

<211> 324

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence of the light chain constant region

humanized antibody IL17A-H069

<400> 33

cgtacggtgg ctgcaccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct 60

ggaactgcct ctgttgtgtg cctgctgaat aacttctatc ccagagaggc caaagtacag 120

tggaaggtgg ataacgccct ccaatcgggt aactcccagg agagtgtcac agagcaggac 180

agcaaggaca gcacctacag cctcagcagc accctgacgc tgagcaaagc agactacgag 240

aaacacaaag tctacgcctg cgaagtcacc catcagggcc tgagctcgcc cgtcacaaag 300

agcttcaaca ggggagagtg ttaa 324

<210> 34

<211> 249

<212>PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence of the mouse scFv antibody used in

construction of antibody IL17A-M069

<400> 34

Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Met Ser Val Gly

1 5 10 15

Gln Lys Val Thr Met Asn Cys Lys Ser Asn Gln Ser Leu Leu Asn Arg

20 25 30

Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Ser Pro Lys Leu Leu Val Asp Phe Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ile Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Thr

65 70 75 80

Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln

85 90 95

His Tyr Thr Thr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile

100 105 110

Lys Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Gly Ser Ser

115 120 125

Arg Ser Ser Gln Ala His Leu Gln Gln Ser Gly Ala Glu Leu Val Arg

130 135 140

Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Leu Gly Tyr Thr Phe

145 150 155 160

Thr Asp Tyr Glu Met His Trp Val Lys Gln Thr Pro Val His Gly Leu

165 170 175

Glu Trp Ile Gly Val Ile His Pro Gly Gly Gly Gly Thr Ala Tyr Asn

180 185 190

Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser

195 200 205

Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val

210 215 220

Tyr Tyr Cys Thr Arg Gly Asp His Asp Gly Arg Thr Asp Tyr Trp Gly

225 230 235 240

Gln Gly Thr Thr Leu Thr Val Ser Ser

245

<210> 35

<211> 18

<212>PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence of the linker used in

constructing a phage antibody library for binding to the mouse scFv antibody

<400> 35

Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Gly Ser Ser Arg

1 5 10 15

Ser Ser

<210> 36

<211> 1386

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence of the mouse antibody IL17A-M069 heavy chain,

containing a signal peptide

<400> 36

atgggctggt ccctgattct gctgttcctg gtggctgtgg ctaccagggt gctgagccag 60

gcccaccttc aacagtctgg ggctgagctg gtgaggcctg gggcttcagt gaagctgtcc 120

tgcaaggctt tgggctacac atttactgac tatgaaatgc actgggtgaa agacacct 180

gtgcatggcc tggaatggat tggagttatt catccaggag gtggtggtac ggcctacaat 240

cagaagttca agggcaaggc cacactgact gcagacaagt cctccagtac agcctacatg 300

gagctcagca gcctgacatc tgaggactct gctgtctatt actgtacaag aggggatcac 360

gacggaagga ctgactactg gggccaaggc accactctca cagtctcctc agccaaaacg 420

acacccccat ctgtctatcc actggcccct ggatctgctg cccaaactaa ctccatggtg 480

accctgggat gcctggtcaa gggctatttc cctgagccag tgacagtgac ctggaactct 540

ggatccctgt ccagcggtgt gcacaccttc ccagctgtcc tgcagtctga cctctacact 600

ctgagcagct cagtgactgt cccctccagc acctggccca gcgagaccgt cacctgcaac 660

gttgcccacc cggccagcag caccaaggtg gacaagaaaa ttgtgcccag ggattgtggt 720

tgtaagcctt gcatatgtac agtcccagaa gtatcatctg tcttcatctt ccccccaaag 780

cccaaggatg tgctcaccat tactctgact cctaaggtca cgtgtgttgt ggtagacatc 840

agcaaggatg atcccgaggt ccagttcagc tggtttgtag atgatgtgga ggtgcacaca 900

gctcagacgc aaccccggga ggagcagttc aacagcactt tccgctcagt cagtgaactt 960

cccatcatgc accaggactg gctcaatggc aaggagttca aatgcagggt caacagtgca 1020

gctttccctg cccccatcga gaaaaccatc tccaaaacca aaggcagacc gaaggctcca 1080

caggtgtaca ccattccacc tcccaaggag cagatggcca aggataaagt cagtctgacc 1140

tgcatgataa cagacttctt ccctgaagac attactgtgg agtggcagtg gaatgggcag 1200

ccagcggaga actacaagaa cactcagccc atcatggaca cagatggctc ttacttcgtc 1260

tacagcaagc tcaatgtgca gaagagcaac tgggaggcag gaaatacttt cacctgctct 1320

gtgttacatg agggcctgca caaccaccat actgagaaga gcctctccca ctctcctggt 1380

aaataa 1386

<210> 37

<211> 721

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence of the light chain of the mouse IL17A-M069 antibody,

containing a signal peptide

<400> 37

atgggctggt cctgtatcat cctgttcctg gtggctacag ccacaggagt gcatagcgac 60

attgtgatgt cacagtctcc atcctccctg gctatgtcag taggacagaa ggtcactatg 120

aactgcaagt ccaatcagag ccttttaaat agaagcaatc aaaagaacta tttggcctgg 180

taccagcaga aaccaggaca gtctcctaaa cttctggtag actttgcatc cactagggaa 240

tctggggtcc ctgatcgctt cataggcagt ggatctggga cagatttcag tcttaccatc 300

agcagtgtgc aggctgagga cctggcagat tacttctgtc agcaacatta taccactcca 360

ttcacgttcg gctcggggac caagctggaa ataaaacggg ctgatgctgc accaactgta 420

tccatcttcc caccatccag tgagcagtta acatctggag gtgcctcagt cgtgtgcttc 480

ttgaacaact tctaccccaa agacatcaat gtcaagtgga agattgatgg cagtgaacga 540

caaaatggcg tcctgaacag ttggactgat caggacagca aagacagcac ctacagcatg 600

agcagcaccc tcacgttgac caaggacgag tatgaacgac ataacagcta tacctgtgag 660

gccactcaca agacatcaac ttcacccatt gtcaagagct tcaacaggaa tgagtgttaa 720

a 721

<210> 38

<211> 461

<212>PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence of the mouse antibody IL17A-M069 heavy chain,

containing a signal peptide

<400> 38

Met Gly Trp Ser Leu Ile Leu Leu Phe Leu Val Ala Val Ala Thr Arg

1 5 10 15

Val Leu Ser Gln Ala His Leu Gln Gln Ser Gly Ala Glu Leu Val Arg

20 25 30

Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Leu Gly Tyr Thr Phe

35 40 45

Thr Asp Tyr Glu Met His Trp Val Lys Gln Thr Pro Val His Gly Leu

50 55 60

Glu Trp Ile Gly Val Ile His Pro Gly Gly Gly Gly Thr Ala Tyr Asn

65 70 75 80

Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser

85 90 95

Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val

100 105 110

Tyr Tyr Cys Thr Arg Gly Asp His Asp Gly Arg Thr Asp Tyr Trp Gly

115 120 125

Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser

130 135 140

Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val

145 150 155 160

Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val

165 170 175

Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala

180 185 190

Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro

195 200 205

Ser Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro

210 215 220

Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly

225 230 235 240

Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile

245 250 255

Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys

260 265 270

Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln

275 280 285

Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln

290 295 300

Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu

305 310 315 320

Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg

325 330 335

Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

340 345 350

Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro

355 360 365

Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr

370 375 380

Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln

385 390 395 400

Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly

405 410 415

Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu

420 425 430

Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn

435 440 445

His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys

450 455 460

<210> 39

<211> 239

<212>PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence of the light chain of the mouse IL17A-M069 antibody,

containing a signal peptide

<400> 39

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Met

20 25 30

Ser Val Gly Gln Lys Val Thr Met Asn Cys Lys Ser Asn Gln Ser Leu

35 40 45

Leu Asn Arg Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys

50 55 60

Pro Gly Gln Ser Pro Lys Leu Leu Val Asp Phe Ala Ser Thr Arg Glu

65 70 75 80

Ser Gly Val Pro Asp Arg Phe Ile Gly Ser Gly Ser Gly Thr Asp Phe

85 90 95

Ser Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Asp Tyr Phe

100 105 110

Cys Gln Gln His Tyr Thr Thr Pro Phe Thr Phe Gly Ser Gly Thr Lys

115 120 125

Leu Glu Ile Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro

130 135 140

Pro Ser Ser Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe

145 150 155 160

Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp

165 170 175

Gly Ser Glu Arg Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp

180 185 190

Ser Lys Asp Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys

195 200 205

Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys

210 215 220

Thr Ser Thr Ser Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

225 230 235

<210> 40

<211> 324

<212>PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence of the heavy chain constant region

mouse IL17A-M069 antibodies

<400> 40

Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala

1 5 10 15

Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu

50 55 60

Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val

65 70 75 80

Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys

85 90 95

Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro

100 105 110

Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu

115 120 125

Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser

130 135 140

Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu

145 150 155 160

Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr

165 170 175

Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn

180 185 190

Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro

195 200 205

Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln

210 215 220

Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val

225 230 235 240

Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val

245 250 255

Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln

260 265 270

Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn

275 280 285

Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val

290 295 300

Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His

305 310 315 320

Ser Pro Gly Lys

<210> 41

<211> 107

<212>PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence of the light chain constant region

mouse IL17A-M069 antibodies

<400> 41

Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu

1 5 10 15

Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe

20 25 30

Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg

35 40 45

Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu

65 70 75 80

Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser

85 90 95

Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

100 105

<210> 42

<211> 19

<212>PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence of heavy chain signal peptide

mouse IL17A-M069 antibodies

<400> 42

Met Gly Trp Ser Leu Ile Leu Leu Phe Leu Val Ala Val Ala Thr Arg

1 5 10 15

Val Leu Ser

<210> 43

<211> 57

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence of the heavy chain signal peptide

mouse IL17A-M069 antibodies

<400> 43

atgggctggt ccctgattct gctgttcctg gtggctgtgg ctaccagggt gctgagc 57

22

<---

Claims (31)

1. Isolated anti-IL17A antibody or its antigen-binding fragment containing a heavy chain variable region comprising a heavy chain CDR1 region having the amino acid sequence specified in SEQ ID NO: 13, a heavy chain CDR2 region having the amino acid sequence specified in SEQ ID NO: 14, and a heavy chain CDR3 region having the amino acid sequence, specified in SEQ ID NO: 15; And a light chain variable region comprising a light chain CDR1 region having the amino acid sequence specified in SEQ ID NO: 10, a light chain CDR2 region having the amino acid sequence specified in SEQ ID NO: 11, and a light chain CDR3 region having the amino acid sequence, specified in SEQ ID NO: 12. 2. Antibody against IL17A or its antigen-binding fragment according to claim 1, containing a heavy chain variable region having the amino acid sequence specified in SEQ ID NO: 22, or an amino acid sequence at least 90%, 92%, 95%, 98% or 99% identical to the sequence of SEQ ID NO: 22; And a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 23, or an amino acid sequence at least 90%, 92%, 95%, 98%, or 99% identical to the sequence of SEQ ID NO: 23. 3. An anti-IL17A antibody or an antigen-binding fragment thereof according to claim 1, characterized in that said anti-IL17A antibody or an antigen-binding fragment thereof is a humanized antibody or a chimeric antibody. 4. An anti-IL17A antibody or an antigen-binding fragment thereof according to any one of paragraphs. 1-3, additionally containing a heavy chain constant region and a light chain constant region, where preferably said heavy chain constant region is an IgG1 heavy chain constant region having the amino acid sequence specified in SEQ ID NO: 24, or at least 90% amino acid sequence, 92%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 24; and/or the light chain constant region is a human kappa light chain constant region having the amino acid sequence set forth in SEQ ID NO: 25, or an amino acid sequence that is at least 90%, 92%, 95%, 98%, or 99% identical to the sequence SEQ ID NO: 25. 5. An anti-IL17A antibody or an antigen-binding fragment thereof according to any one of paragraphs. 1-4, additionally containing a signal peptide attached to a heavy chain variable region and/or a signal peptide attached to a light chain variable region, wherein preferably, said signal peptide attached to the heavy chain variable region is the amino acid sequence set forth in SEQ ID NO: 20, or an amino acid sequence that is at least 90%, 92%, 95%, 98%, or 99% identical sequences SEQ ID NO: 20; and/or said signal peptide attached to the light chain variable region is the amino acid sequence set forth in SEQ ID NO: 21, or an amino acid sequence at least 90%, 92%, 95%, 98%, or 99% identical to the SEQ ID sequence NO: 21. 6. An anti-IL17A antibody or an antigen-binding fragment thereof according to any one of paragraphs. 1-5, characterized in that said anti-IL17A antibody or antigen-binding fragment thereof is an IgG antibody, preferably an IgG1 antibody. 7. An anti-IL17A antibody or an antigen-binding fragment thereof according to any one of paragraphs. 1-6, characterized in that said anti-IL17A antibody or antigen-binding fragment thereof is a monoclonal antibody. 8. An anti-IL17A antibody or an antigen-binding fragment thereof according to any one of paragraphs. 1-7, characterized in that the binding affinity KD of said anti-IL17A antibody or antigen-binding fragment thereof to the recombinant human IL17A protein is 0.1-10E-11 M, preferably 0.5-5E-11 M and more preferably 2, 88E-11 M. 9. An anti-IL17A antibody or an antigen-binding fragment thereof according to any one of paragraphs. 1-7, characterized in that the binding affinity KD of said anti-IL17A antibody or its antigen-binding fragment to the recombinant human IL17A/F protein is 0.1-10E-10 M, preferably 0.5-5E-10 M and more preferably 5.37E-10 M. 10. An anti-IL17A antibody or an antigen-binding fragment thereof according to any one of paragraphs. 1-9, characterized in that said antigen-binding fragment is Fv, Fab, Fab', Fab'-SH, F(ab')2, Fd fragment, Fd' fragment, single chain antibody molecule or single domain antibody; wherein the single chain antibody molecule is preferably a scFv, di-scFv, tri-scFv, diabody or scFab. 11. An antibody-drug conjugate for use in the treatment of psoriasis, containing an anti-IL17A antibody or an antigen-binding fragment thereof according to any one of claims. 1-10 and an additional therapeutic agent, where preferably said anti-IL17A antibody or antigen-binding fragment thereof is linked to said additional therapeutic agent via a linker. 12. Nucleic acid encoding an antibody against IL17A or an antigen-binding fragment thereof according to any one of paragraphs. 1-10. 13. The nucleic acid according to claim 12, containing the nucleotide sequence specified in SEQ ID NO: 30, encoding the heavy chain variable region, and/or the nucleotide sequence specified in SEQ ID NO: 31, encoding the light chain variable region. 14. An expression vector containing the nucleic acid according to claim 12 or 13. 15. A host cell for producing an antibody against IL17A or an antigen-binding fragment thereof according to any one of paragraphs. 1-10, containing the nucleic acid according to claim 12 or 13 or the expression vector according to claim 14. 16. A method for producing an antibody against IL17A or an antigen-binding fragment thereof according to any one of paragraphs. 1-10, comprising growing the host cell of claim 15 under conditions suitable for expression of the antibody and isolating the expressed antibody from the culture fluid. 17. Pharmaceutical composition for use in the treatment of psoriasis, containing an antibody against IL17A or its antigen-binding fragment according to any one of paragraphs. 1-10, or the antibody-drug conjugate of claim 11, and a pharmaceutically acceptable carrier. 18. The use of an antibody against IL17A or an antigen-binding fragment thereof according to any one of paragraphs. 1-10, or the antibody-drug conjugate of claim 11, or the pharmaceutical composition of claim 17 in the treatment of psoriasis. 19. A method of treating psoriasis, comprising administering to a subject in need thereof a therapeutically effective amount of an anti-IL17A antibody or an antigen-binding fragment thereof according to any one of claims. 1-10, or the antibody-drug conjugate of claim 11, or the pharmaceutical composition of claim 17 for the treatment of psoriasis. 20. The use of an antibody against IL17A or an antigen-binding fragment thereof according to any one of paragraphs. 1-10, or the antibody-drug conjugate of claim 11, or the pharmaceutical composition of claim 17 for producing a medicament for the treatment of psoriasis. 21. Pharmaceutical combination for use in the treatment of psoriasis, containing an antibody against IL17A or its antigen-binding fragment according to any one of paragraphs. 1-10, or the antibody-drug conjugate of claim 11, or the pharmaceutical composition of claim 17, and one or more additional therapeutic agents. 22. A kit for use in the treatment of psoriasis, containing an antibody against IL17A or an antigen-binding fragment according to any one of paragraphs. 1-10, or the antibody-drug conjugate of claim 11, or the pharmaceutical composition of claim 17, further comprising an administration device.