Anti-IL-17A Antibody and Its Applications

Abstract

The present invention provides antibodies or functional fragments thereof that specifically bind to IL-17A with high affinity. Nucleic acid molecules encoding the antibodies or functional fragments thereof, expression vectors and host cells expressing the antibodies or functional fragments thereof, and methods for producing the antibodies or functional fragments thereof of the present invention are also provided. The present invention also provides pharmaceutical compositions comprising the antibodies or functional fragments thereof of the present invention, and methods for treating immune dysfunction disorders using the antibodies or functional fragments thereof of the present invention.

Description

Technical Field

[0001] The present invention relates to an antibody that specifically binds to IL-17A and an antigen-binding fragment thereof. More specifically, the present invention relates to an antibody that suppresses the biological activity induced by IL-17A and an antigen-binding fragment thereof, a composition in which the antibody or its antigen-binding fragment is used, and a method for treating related diseases. More specifically, the present invention relates to the treatment of immune pathologies including autoimmune and inflammatory diseases such as rheumatoid arthritis, psoriasis, ankylosing spondylitis, multiple sclerosis, systemic lupus erythematosus (SLE), lupus nephritis, chronic obstructive pulmonary disease, asthma, infectious granuloma, cystic fibrosis, cancer diseases, etc. with an anti-IL-17A antibody or an antigen-binding fragment thereof.

Background Art

[0002] Interleukin 17 (IL-17), also known as CTLA-8 or IL-17A, plays an important cooperative role in the immune system. The IL-17 family includes six types: IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, and IL-17F. All six contain four highly conserved and very important cysteine residues, but their biological effects show significant differences. IL-17A and IL-17F have the most similar homology and biological functions, and are the most studied at present. IL-17A expressed in vivo is an N-terminal signal peptide consisting of 23 amino acids, and mature IL-17A is produced by cleavage. Mature IL-17A is linked by disulfide bonds and generally exists and is secreted in the form of a homodimer. However, it may also form a heterodimer IL-17AF when linked to IL-17F. Generally, IL-17A or IL-17 refers to the IL-17A homodimer protein, which is mainly produced by T helper 17 (Th17) cells. However, it may also be synthesized and secreted by other immune cells such as γδT cells, LTi (Lymphoid Tissue inducer cells), ILCs (Innate Lymphoid Cells), and NKT (Natural Killer T) cells (Non-Patent Document 1). The regulation of IL-17A expression is very complex. According to research, naive CD4+ T cells differentiate into Th17 cells upon induction by cytokines such as IL-6, IL-1β, and TGFβ. At this time, the stability of Th17 cells is weak, the amount of secreted IL-17A is small, and the effect on tissue damage is small. In the presence of IL-23, inflammation induction and tissue damage occur through routes such as promoting Th17 cell stability, continuous secretion of IL-17A, upregulation of the expression of inflammatory cytokines (IL-22, CSF-2, IFN-γ), and downregulation of the expression of anti-inflammatory cytokines (IL-2, IL-27, IL-12) (Non-Patent Document 2). Therefore, when IL-23 is abnormally expressed in tissues, the IL-17A pathway plays an important role in tissue damage.

[0003] IL-17 is generally secreted in a fixed position and exerts its effects by binding to the IL-17 receptor (IL-17R) on the surface of target cells in local tissues. The IL-17R family includes five types: IL-17RA, IL-17RB, IL-17RC, IL-17RD, and IL-17RE, and is widely expressed on multiple types of cell membranes (Non-Patent Document 3). IL-17 mainly binds to the IL-17RA / IL-17RC complex on the surface of cells derived from non-hematopoietic cells (e.g., epithelial cells, stromal cells) to exert its effects (Non-Patent Document 4), promoting the secretion of cytokines in cells (e.g., IL-6, G-CSF, GM-CSF, IL-10, TGF-β, TNF-α), chemokines (including IL-8, CXCL1, MCP-1), and prostaglandins (e.g., PGE2), inducing the aggregation of neutrophils and macrophages, releasing reactive oxygen species (ROS), and causing tissue damage (Non-Patent Document 5).

[0004] For example, autoimmune diseases such as psoriasis, rheumatoid arthritis, ankylosing spondylitis, Crohn's disease, and multiple sclerosis strongly threaten human health. According to research, disorders in the secretion of IL-17 are closely related to the onset and development of these diseases. Antibodies targeting IL-17 can effectively alleviate the symptoms of autoimmune diseases by inhibiting the IL-17-IL-17R signaling pathway (Non-Patent Document 6). Cosentyx (secukinumab) developed by Novartis is the world's first IL-17 monoclonal antibody and has obtained permission for use in indications including moderate to severe plaque psoriasis, providing an advanced biotherapy option for many psoriasis patients. However, the development of anti-IL-17 antibodies with different structures, more excellent therapeutic effects, and a wider range of indications has great demand and significant meaning in the treatment of autoimmune-related diseases such as psoriasis, rheumatoid arthritis, and ankylosing spondylitis, as well as other diseases related to IL-17.

Prior Art Documents

Non-Patent Documents

[0005]

Non-Patent Document 1

Non - Patent Document 3

Non - Patent Document 4

Non - Patent Document 5

Non - Patent Document 6

Summary of the Invention

[0006] The present invention provides an antibody or an antigen - binding fragment thereof that specifically binds to human IL - 17A and contains a complementarity - determining region (CDR) sequence selected from at least one of SEQ ID NO:1 - 24, 60 - 65.

[0007] In one embodiment, the antibody or the antigen - binding fragment thereof according to the present invention contains a heavy - chain CDR domain selected from at least one of SEQ ID NO:1 - 3, 4 - 6, 7 - 9, 10 - 12 or 60 - 62, and / or a light - chain CDR domain selected from at least one of SEQ ID NO:13 - 15, 16 - 18, 19 - 21, 22 - 24 or 63 - 65.

[0008] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention comprises a heavy chain variable region (VH), wherein the heavy chain variable region comprises an HCDR1 selected from SEQ ID NO: 1, 4, 7, 10 or 60, an HCDR2 selected from SEQ ID NO: 2, 5, 8, 11 or 61, and an HCDR3 selected from SEQ ID NO: 3, 6, 9, 12 or 62.

[0009] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention comprises a heavy chain variable region (VH), and the HCDR1, HCDR2, and HCDR3 amino acid sequences contained in the heavy chain variable region are selected from any one of the following groups A to E.

[0010] [Table A]

[0011] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention comprises a light chain variable region (VL), wherein the light chain variable region comprises an LCDR1 selected from SEQ ID NO: 13, 16, 19, 22 or 63, an LCDR2 selected from SEQ ID NO: 14, 17, 20, 23 or 64, and an LCDR3 selected from SEQ ID NO: 15, 18, 21, 24 or 65.

[0012] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention comprises a light chain variable region (VL), and the LCDR1, LCDR2, and LCDR3 amino acid sequences contained in the light chain variable region are selected from any one of the following groups F to J.

[0013] [Table B]

[0014] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), and is characterized in that the six CDR amino acid sequences contained in the variable region are selected from any one of the following groups I to VI.

[0015] [Table C]

[0016] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), and is characterized in that the amino acid sequence of the heavy chain variable region is selected from SEQ ID NO: 25, 26, 27, 28, 33, 35, 38 or 40, and / or the amino acid sequence of the light chain variable region (VL) is selected from SEQ ID NO: 29, 30, 31, 32, 34, 36, 38, 39 or 41.

[0017] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), and is characterized in that the variable region amino acid sequence is selected from any one of the following groups 1 to 7.

[0018] [Table D]

[0019] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention comprises a light chain (LC) and / or a heavy chain (HC), and is characterized in that the amino acid sequence of the heavy chain (HC) is selected from SEQ ID NO: 42, 44, 46 or 49, and / or the amino acid sequence of the light chain (LC) is selected from SEQ ID NO: 43, 45, 47, 48 or 50.

[0020] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention comprises a light chain (LC) and a heavy chain (HC), wherein the amino acid sequence of the light chain is shown in SEQ ID NO: 43, and the amino acid sequence of the heavy chain is shown in SEQ ID NO: 42 or 44, or the amino acid sequence of the light chain is shown in SEQ ID NO: 45, and the amino acid sequence of the heavy chain is shown in SEQ ID NO: 44, or the amino acid sequence of the light chain is shown in SEQ ID NO: 47 or 48, and the amino acid sequence of the heavy chain is shown in SEQ ID NO: 46, or the amino acid sequence of the light chain is shown in SEQ ID NO: 50, and the amino acid sequence of the heavy chain is shown in SEQ ID NO: 49, which is characterized by this.

[0021] In one embodiment, the antibody according to the present invention is a full antibody, preferably IgG, and more preferably IgG4 type.

[0022] In a specific embodiment, the antibody according to the present invention is 1F8, 2B2, 2F5, ch1, ch2, ch16, hu31, hu43, hu44, hu59, hu60 or hu250.

[0023] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention specifically binds to a) IL-17A homodimer and IL-17AF heterodimer, b) blocks the binding between IL-17A and its receptor, and / or c) suppresses the biological activity induced by IL-17A, which is characterized by this.

[0024] In one embodiment, the IL-17A, IL-17AF or IL-17F according to the present invention is selected from one or more of cynomolgus monkeys, mice or humans.

[0025] In one embodiment, the antibody or antigen-binding fragment according to the present invention does not specifically bind to any one or more of a) any one or more of human IL-17F homodimer, IL-17B homodimer, IL-17C homodimer, IL-17D homodimer, IL-17E homodimer, and / or b) any one or more of cynomolgus monkey IL-17F homodimer, mouse IL-17F homodimer.

[0026] On the other hand, the antibody or antigen-binding fragment thereof according to the present invention has a function of suppressing the binding between IL-17A and its receptor, and / or a function of reducing the cell signal transduction and / or biological activity induced by IL-17A.

[0027] In one embodiment, the antibody or antigen-binding protein according to the present invention can suppress the secretion of CXCL1 by epithelial cells induced by IL-17A during in vitro activity evaluation.

[0028] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention can suppress the secretion of CXCL1 in mouse in vivo induced by IL-17A during in vivo activity evaluation.

[0029] In one embodiment, the antibody or antigen-binding protein according to the present invention can suppress the onset of mice and significantly reduce the clinical evaluation score of mouse psoriasis onset and the degree of ear swelling in a psoriasis experimental model induced by imiquimod during in vivo activity evaluation.

[0030] In one embodiment, the isolated antibody or antigen-binding fragment thereof according to the present invention can suppress the swelling of the knee joint in an arthritis experimental model induced by an antigen, such as a cynomolgus monkey AIA-model, during in vivo evaluation.

[0031] The present invention further provides the antibody or its antigen-binding fragment, preferably for use as a drug with hu31, hu43, hu44, hu59, hu60 or hu250, more preferably for use as a drug for treating a pathological disease induced by IL-17A, and / or for use as a drug for treating a pathological disease by suppressing IL-17A signaling.

[0032] In a specific embodiment, the pathological disease induced by IL-17A is, for example, an inflammatory disease or condition such as arthritis, rheumatoid arthritis, psoriasis, ankylosing spondylitis, chronic obstructive pulmonary disease, systemic lupus erythematosus (SLE), lupus nephritis, asthma, multiple sclerosis or cystic fibrosis.

[0033] The present invention provides a method for treating a pathological disease induced by IL-17A, the method including administering an effective amount of the isolated antibody or its antigen-binding fragment according to the present invention so as to reduce the condition, and the hu31, hu43, hu44, hu59, hu60 or hu250 antibody is preferred.

[0034] The present invention further relates to a method for producing the antibody or its antigen-binding fragment of the present invention. The method includes an isolated nucleic acid molecule encoding at least the heavy chain and / or light chain variable region of the antibody or protein of the present invention, or a cloning vector or expression vector containing the nucleic acid, and particularly, in a host cell, for example, recombinant production of the antibody or protein according to the present invention such as hu31, hu43, hu44, hu59, hu60 or hu250.

[0035] The present invention further relates to a host cell containing one or more of the above cloning vectors or expression vectors, and a method for producing a protein containing the antibody or its antigen-binding fragment of the present invention, specifically, for example, a method for producing the hu31, hu43, hu44, hu59, hu60 or hu250 antibody, the method including culturing the host cell, and purifying and recovering the antibody or protein.

[0036] In one embodiment, a protein comprising the isolated antibody or antigen-binding fragment thereof according to the present invention is bound to other active moieties.

[0037] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention may be a monoclonal antibody or antigen-binding fragment thereof, but chimeric antibodies, humanized antibodies or human antibodies or parts thereof are preferred.

[0038] On the other hand, the present invention provides a pharmaceutical composition comprising a protein comprising the antibody or antigen-binding fragment thereof according to any one embodiment of the present invention in combination with one or more pharmaceutically acceptable excipients, diluents or vectors.

[0039] In one embodiment, the pharmaceutical composition contains one or more other active ingredients.

[0040] In a specific embodiment, the pharmaceutical composition is a lyophilized powder. In another specific embodiment, the pharmaceutical composition is a stable liquid formulation containing the antibody or molecule according to the present invention in a therapeutically acceptable amount.

[0041] In one embodiment, the present invention relates to the use of the antibody or antigen-binding fragment thereof in the manufacture of a therapeutic drug for any one pathological condition induced by IL-17A.

[0042] The present invention provides an isolated nucleic acid molecule encoding the above antibody or antigen-binding fragment thereof, an expression vector or recombinant vector containing the above nucleic acid molecule, and a host cell transformed with the above vector.

[0043] The present invention provides a pharmaceutical composition comprising the above antibody or antigen-binding fragment thereof, the above nucleic acid molecule, vector or host cell, and a composition of a pharmaceutically acceptable vector or excipient.

[0044] The present invention provides the use of the above-mentioned antibody or its antigen-binding fragment, nucleic acid molecule, vector or host cell, or the above-mentioned pharmaceutical composition in the manufacture of a therapeutic and / or prophylactic drug for diseases or conditions induced by IL-17A.

[0045] In some embodiments, the above drug is a drug used for the treatment of arthritis, rheumatoid arthritis, psoriasis, ankylosing spondylitis, chronic obstructive pulmonary disease, systemic lupus erythematosus (SLE), lupus nephritis, asthma, multiple sclerosis or cystic fibrosis.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0047] The present invention relates to an antibody that specifically binds to human IL-17A and blocks the biological activities induced by IL-17A. The present invention relates to the full-length IgG-type antibody and its antigen-binding fragment further described in the following specification.

[0048] (Definition) Unless otherwise specified, in the practice of the present invention, conventional techniques in molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, immunology, etc. are employed, which are within the technical scope of this field.

[0049] To make the present invention easier to understand, technical terms are defined as follows. Unless otherwise clearly defined in other parts of this specification, the technical terms used in this specification have the meanings commonly understood by those skilled in the art of the present invention. For definitions and terms in this field, those skilled in the art refer to Current Protocols in Molecular Biology (Ausubel). The abbreviations of amino acid residues are the standard three-letter and / or one-letter codes representing one of the 20 commonly used L-amino acids in this field.

[0050] Unless otherwise clearly explained separately in this specification, the singular expressions used in this specification (including claims) include multiple meanings.

[0051] IL-17 or IL-17A refers to interleukin-17. Unless otherwise specified, the said IL-17 usually refers to human IL-17A.

[0052] IL-17A expressed in vivo is an N-terminal signal peptide consisting of 23 amino acids, with the NCBI accession number NP_443104.1, and mature IL-17A is produced by cleavage. In a specific embodiment, the IL-17A according to the present disclosure is mature IL-17A, which does not contain the N-terminal signal peptide, but has an amino acid sequence of SEQ ID NO:66 and a nucleotide sequence of SEQ ID NO:67.

[0053] IL-17F expressed in vivo is an N-terminal signal peptide consisting of 30 amino acids, with the NCBI accession number NP_443104.1, and mature IL-17F is produced by cleavage. In a specific embodiment, the IL-17F according to this specification is mature IL-17F, which does not contain the N-terminal signal peptide, but has an amino acid sequence of SEQ ID NO:68.

[0054] As understood by those skilled in the art, the IL-17AF according to this specification is a heterodimer of the IL-17A subunit and the IL-17F subunit.

[0055] The term "immune response" refers to, for example, the actions of lymphocytes, antigen-presenting cells, phagocytes, granulocytes, and the above-mentioned cells or soluble macromolecules produced from the liver (including antibodies, cytokines, and complements), by which invading pathogens, cells or tissues infected with pathogens, cancer cells, or in the case of autoimmunity or pathological inflammation, normal human cells or tissues are selectively damaged, destroyed, or removed from the human body.

[0056] The term "signaling pathway" or "signaling activity" generally refers to, for example, a biochemical causal relationship due to the interaction between proteins such as the binding of a growth factor and a receptor, through which a signal is transmitted from one part of a cell to another part of the cell. Generally, the transmission involves specific phosphorylation of one or more tyrosine, serine, or threonine residues in one or more types of proteins in a series of reactions that trigger signal transduction. Usually, since the second-to-last process involves nuclear events, changes in gene expression occur.

[0057] For the antibody or antigen-binding fragment thereof of the present invention, the terms "activity" or "physiological activity", or the terms "biological property" or "biological characteristic" can be used interchangeably with each other, and include, but are not limited to, epitope / antigen affinity and specificity, the ability to neutralize or antagonize IL-17A activity in vivo or in vitro, IC50, the in vivo stability of the antibody, and the immunogenic properties of the antibody. Other biological properties or characteristics of antibodies that can be evaluated well-known in the art include, for example, cross-reactivity (i.e., usually cross-reactivity with non-human homologs of the target peptide, or other proteins or tissues), and the ability to maintain a high expression level of a protein in mammalian cells. The above-mentioned properties or characteristics are observed, measured, or evaluated using well-known techniques in the art, which include, but are not limited to, ion resonance analysis such as ELISA, FACS, or BIACORE, neutralization measurement in an unrestricted in vivo or in vitro, receptor binding, production and / or secretion of cytokines or growth factors, signal transduction, immunohistochemistry of tissue fragments from different origins (human, primate, or any other origin).

[0058] "Antibody" means any form of antibody having the desired biological activity. Thus, when used in the broadest sense, it specifically includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, and camelized single-domain antibodies.

[0059] "Isolated antibody" refers to the purified state of the binding compound, and in such cases, it means that the molecule is substantially free of other biological molecules, such as nucleic acids, proteins, lipids, sugars, cell debris, and other substances such as growth media. The term "isolated" does not mean that such substances are completely absent or that water, buffer, or salt is absent, as long as they are not present in an amount that clearly interferes with the experimental or therapeutic use of the binding compound herein.

[0060] "Monoclonal antibody" is an antibody obtained from a substantially homogeneous group of antibodies, i.e., each antibody comprising this group is the same, except for minor variations that may occur naturally. Monoclonal antibodies have high specificity and are directed against a single antigen epitope. In contrast, a typical (polyclonal) antibody preparation usually contains a large number of antibodies (or antibodies specific for different epitopes) against different epitopes. The modifier "monoclonal" indicates the characteristic of an antibody obtained from a substantially homogeneous group of antibodies and should not be construed as requiring the antibody to be produced through any particular method.

[0061] When present in nature, a "full-length antibody" is an immunoglobulin molecule containing four peptide chains, with two heavy (H) chains (about 50-70 kDa in the full-length case) and two light (L) chains (about 25 kDa in the full-length case) linked to each other by disulfide bonds. Each heavy chain consists of a heavy-chain variable region (abbreviated as VH herein) and a heavy-chain constant region (abbreviated as CH herein). The heavy-chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain consists of a light-chain variable region (abbreviated as VL herein) and a light-chain constant region. The light-chain constant region consists of one domain, CL. The VH region and the VL region are further subdivided into regions called complementarity-determining regions (CDRs) with high variability and framework regions (FRs) conserved therebetween. Each VH region or VL region consists of three CDRs and four FRs arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino-terminal to the carboxyl-terminal. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors (including each cell of the immune system (e.g., effector cells) and the first component of the typical complement system (Clq)).

[0062] An "antigen-binding fragment" of an antibody (the "parent antibody") includes fragments or derivatives of the antibody, but typically includes at least one fragment of the antigen-binding region or variable region of the parent antibody (e.g., one or more CDRs) and retains at least one binding specificity of the parent antibody. Examples of antibody-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules such as sc-Fv, nanobodies formed from antibody fragments, and multispecific antibodies. When antigen-binding activity is expressed in molar concentration, the binding fragment or derivative typically retains at least 10% of its antigen-binding activity. It is preferred that the binding fragment or derivative retains at least 20%, 50%, 70%, 80%, 90%, 95%, 100% or higher affinity of the antigen-binding affinity of the parent antibody. The antigen-binding fragment of an antibody is expected to include conservative or non-conservative amino acid substitutions (referred to as "conservative variants" or "functionally conservative variants" of the antibody) that do not significantly alter its biological activity. The term "binding compound" refers to both the antibody and its binding fragments.

[0063] A "single-chain Fv" or "scFv" antibody refers to an antibody fragment containing the VH and VL domains of an antibody, and these domains are present in a single polypeptide chain. The Fv polypeptide generally further includes a polypeptide linker between the VH and VL domains, whereby the scFv can form the desired structure for antigen binding.

[0064] A "domain antibody" is an immunofunctional immunoglobulin fragment containing only the heavy-chain variable region or the light-chain variable region. In some cases, two or more VH regions are covalently linked with a peptide linker to form a bivalent domain antibody. The two VH regions of the bivalent domain antibody can target the same or different antigens.

[0065] A "bivalent antibody" contains two antigen-binding sites. In some cases, the two binding sites have the same antigen specificity. However, the bivalent antibody may be bispecific.

[0066] The term "bispecific antibody" refers to a small antibody fragment having two antigen-binding sites, said fragment comprising a heavy-chain variable domain (VH) linked to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL or VL-VH). By using a linker that is short enough to prevent pairing between the two domains of the same chain, the domain pairs with the complementary domain of another chain to produce two antigen-binding sites.

[0067] The term "chimeric antibody" refers to an antibody having the variable domain of a first antibody and the constant domain of a second antibody, wherein the first antibody and the second antibody are derived from different species. Generally, the variable domain is an antibody obtained from an experimental animal such as a rodent ("parent antibody"), and the constant domain sequence is obtained from a human antibody, whereby the resulting chimeric antibody is less likely to induce an adverse immune response in a human subject compared to the parent rodent antibody.

[0068] The term "humanized antibody" refers to a form of antibody containing sequences derived from human and non-human (e.g., mouse, rat) antibodies. Generally, a humanized antibody contains substantially all, at least one, and usually two variable domains, wherein all or substantially all of the hypervariable regions correspond to the hypervariable regions of a non-human immunoglobulin, and all or substantially all of the framework (FR) regions are the framework regions of a human immunoglobulin sequence. A humanized antibody optionally contains at least a portion of a human immunoglobulin constant region (Fc).

[0069] The term "fully human antibody" refers to an antibody containing only human immunoglobulin protein sequences. For example, when produced by a mouse, mouse cell, or hybridoma derived from a mouse cell, a fully human antibody may contain mouse glycans. Similarly, the term "mouse antibody" refers to an antibody containing only mouse immunoglobulin sequences. Alternatively, when produced by a rat, rat cell, or hybridoma derived from a rat cell, a fully human antibody may contain rat glycans. Similarly, the term "rat antibody" refers to an antibody containing only rat immunoglobulin sequences.

[0070] An "isotype" is an antibody class provided by a heavy chain constant region gene (e.g., IgG such as IgM, IgE, IgG1 or IgG4). The isotype further includes a modified form of one of these classes, for example, a modification was produced that alters the Fc function, such as enhancing or reducing effector side functions or binding to Fc receptors.

[0071] The terms "nucleic acid" or "polynucleotide" refer to polymers of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and their single-stranded or double-stranded forms. Unless otherwise specified, the terms include nucleic acids containing analogs of known natural nucleotides that have binding properties similar to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. (See U.S. Patent No. 8,278,036 owned by Kariko et al. These disclose mRNA molecules in which uridine is replaced by pseudouridine, methods for synthesizing said mRNA molecules, and methods for use in delivering therapeutic proteins in vivo). For example, methods for modified mRNA may be used, such as those disclosed by U.S. Patent No. 8,278,036 owned by Kariko et al. and Application W02013 / 090186A1 owned by Moderna. Unless otherwise specified, a particular nucleic acid sequence further implicitly includes its conserved and modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, complementary sequences, and specifically designated sequences. Specifically, degenerate codon substitutions are achieved by generating sequences in which the third position of one or more of its selected (or all) codons is replaced by a mixed base and / or deoxyinosine residue (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0072] A "construct" is any recombinant polynucleotide molecule (e.g., plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, bacteriophage, linear or circular single-stranded or double-stranded DNA or RNA polynucleotide molecule), derived from any source, capable of being integrated with the genome or replicating autonomously, and configured to be linked (i.e., operably linked) to one or more polynucleotide molecules in a manner that allows for functional manipulation. A recombinant construct typically contains a polynucleotide of the invention operably linked to transcriptional initiation regulatory sequences that induce transcription of the polynucleotide in a host cell. Both specific and non-specific (i.e., endogenous) promoters are used to induce expression of the nucleic acids of the invention.

[0073] A "vector" is any recombinant polynucleotide construct that may be utilized for the purpose of transformation (i.e., introducing heterologous DNA into a host cell). One type of vector is a "plasmid", which is a circular double-stranded DNA loop to which other DNA segments may be ligated. Another type of vector is a viral vector to which other DNA segments may be ligated to the viral genome. Some vectors are capable of autonomous replication in the introduced host cell (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). When introduced into a host cell, other vectors (e.g., non-episomal mammalian vectors) integrate into the host cell's genome and replicate with the host genome. Also, some vectors are capable of inducing expression of genes to which they are operably linked. These vectors are referred to herein as "expression vectors".

[0074] As used herein, the term "expression vector" is a nucleic acid molecule capable of replicating and expressing a target gene when transformed, transfected or transduced into a host cell. An expression vector contains one or more phenotypic selection markers and an origin of replication to allow for vector maintenance and, if necessary, amplification in the host.

[0075] As used herein, the term "IL-17A antagonist" or "IL-17A blocking molecule" refers to an antibody or its antigen-binding protein that reduces or neutralizes IL-17A activity by suppressing the signal transduction activity of IL-17A induced by IL-17R. It is manifested, for example, in human cell measurements such as the measurement of CXCL1 production by IL-17A in human cells. These measurements are described in more detail in the following examples.

[0076] Unless otherwise clearly defined from the context, "activation", "stimulation" and "treatment" used with respect to a cell or receptor, for example ligand activation, stimulation or treatment used with respect to a cell or receptor, have the same definition. "Ligand" includes natural ligands and synthetic ligands, such as cytokines, cytokine variants, analogs, mutant proteins, and binding compounds derived from antibodies. "Ligand" also includes small molecules, such as peptide mimics of cytokines and peptide mimics of antibodies. "Activation" refers to the activation of a cell that is regulated by internal mechanisms and external or environmental factors. A "response", for example, of a cell, tissue, organ or organism, includes a change in biochemical or physiological behavior (e.g., concentration, density, adhesion or migration within a biological compartment, gene expression rate or differentiation state), where the change is related to activation, stimulation or treatment, or to internal mechanisms such as gene programming.

[0077] As used herein, the term "treatment" or "therapy" for any disease or medical condition means, in one embodiment, the improvement of the disease or medical condition (i.e., the reduction, arrest or decrease of at least one of the development of the disease or its clinical symptoms). In another embodiment, "treatment" or "therapy" refers to the reduction or improvement of at least one physical parameter, including physical parameters not recognizable by the patient. In another embodiment, "treatment" or "therapy" refers to preparing the disease or medical condition in the body (e.g., stabilization of recognizable symptoms), physiologically (e.g., stabilization of physical parameters), or both. Unless otherwise clearly described herein, methods for evaluating the treatment and / or prevention of diseases are generally known in the art.

[0078] The term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrate animals, both mammalian and non-mammalian, such as, for example, non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. As used herein, the term "cyno" or "cynomolgus monkey" refers to cynomolgus monkeys.

[0079] The terms "therapeutically effective amount", "therapeutically effective dose" and "effective amount" refer to an amount that, when the IL-17A antibody of the present invention or an antigen-binding fragment thereof is administered alone or in combination with other therapeutic agents to cells, tissues or a subject, effectively prevents or ameliorates the symptoms of one or more diseases or medical conditions or the development of such diseases or medical conditions. The therapeutically effective dose refers to, for example, an amount that treats, cures, prevents or ameliorates a related medical condition, or an amount that improves the speed of treatment, cure, prevention or amelioration of these medical conditions, i.e., an amount of the antibody or its antigen-binding fragment sufficient for symptom improvement. When the active ingredient is administered alone to an individual, the therapeutically effective dose is only the amount of that ingredient. When administered in combination, regardless of whether it is a combination administration, sequential administration or simultaneous administration, the therapeutically effective dose is the total amount of the active ingredients that produce a therapeutic effect. The effective amount of a therapeutic agent improves at least 10% of the diagnostic criteria or parameters, but usually at least 20%, preferably at least about 30%, more preferably at least 40%, and most preferably at least 50%.

[0080] (Production of antibodies) The antibodies of the present invention may be produced by any suitable method for producing antibodies. Any suitable form of human IL-17A may be used as an immunogen (antigen) for producing antibodies. By way of non-limiting example, any human IL-17A isotype or fragment thereof may be used as an immunogen. Examples include, but are not limited to, the native mature human IL-17A (amino acid sequence SEQ ID No: 66) described herein.

[0081] In a preferred embodiment, hybridoma cells for producing a mouse-derived monoclonal anti-human IL-17A antibody may be produced by methods well known in the art. These methods include, but are not limited to, the hybridoma technology first studied by Kohler et al. (1975) (Nature 256:495-497). Depending on the standard form, it is preferable to isolate mouse pancreatic cells and fuse them with a mouse myeloma cell line by PEG or electrofusion. Then, from the obtained hybridomas, hybridomas for producing antigen-specific antibodies may be selected. For example, on the premise of 50% PEG, a single cell suspension of pancreatic lymphocytes derived from an immunized mouse may be fused with 1 / 6 the number of mouse myeloma cells SP20 (ATCC). The cells are inoculated into a flat-bottom microtiter plate at about 2×10 5 cells / mL and then incubated for 2 weeks in a complete synthetic medium containing 20% fetal bovine serum and a selective medium of 1×HAT (Sigma; HAT is added 24 hours after fusion). After 2 weeks, the cells are cultured in a medium using HT as a substitute for HAT. Thereafter, each well is selected for the anti-human IL-17A monoclonal IgG antibody by ELISA. When a large growth of hybridomas is discovered, usually, the medium is observed after 10-14 days. The hybridomas secreting the antibody are re-inoculated and re-selected, and if they are still positive for human IgG, subcloning is performed at least twice for the anti-human IL-17A monoclonal antibody hybridomas by limiting dilution. Thereafter, in vitro culture of stable subclones is performed to produce a small amount of characterization antibodies in tissue culture medium.

[0082] In a preferred embodiment, the monoclonal hybridoma cells obtained in the present invention are 1F8, 2F5, 2B2, and the antibodies secreted by them specifically bind to IL-17A with high specificity, block the binding of IL-17A and IL-17RA, and suppress the biological activities induced by IL-17A, such as CXCL1 secretion.

[0083] In a preferred embodiment, the present invention measured the DNA sequences of the immunoglobulin variable regions expressed in the candidate hybridoma cells 1F8, 2F5, and 2B2 by a method based on nested primer PCR. Hybridoma cell 1F8 has two types of antibody light chain genes and one type of antibody heavy chain gene, while 2F5 has two types of antibody heavy chain genes and one type of antibody light chain gene. Therefore, the antibodies secreted by 1F8 and 2F5 contain two types of mixed complete antibodies. In this specification, the antibody secreted by 1F8 is referred to as 1F8-1 and 1F8-2, and the antibody secreted by 2F5 is referred to as 2F5-1 and 2F5-2.

[0084]

Table 1

[0085] When antibodies derived from rodents (e.g., mice) are used as therapeutic drugs in vivo, they cause unnecessary antibody immunogenicity, and repeated use generates an immune response against therapeutic antibodies in the human body. These immune responses result in at least the loss of therapeutic effect and, in the worst case, potentially fatal anaphylaxis. One method to reduce the immunogenicity of rodent antibodies involves the production of chimeric antibodies, in which case the mouse variable region is fused with the human constant region (Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84:3439-43). However, the retention of the complete rodent variable region in chimeric antibodies still causes harmful immunogenicity in patients. Transplanting the complementarity-determining region (CDR) loops of the rodent variable domain into the human framework (i.e., humanization) is further used to minimize the reduction of rodent sequences (Jones et al. (1986) Nature 321:522; Verhoeyen et al. (1988) Science 239:1534).

[0086] In some embodiments, the chimeric or humanized antibodies of the present invention are produced based on the sequences of the produced mouse monoclonal hybridoma antibodies. DNA encoding the heavy and light chain immunoglobulins is obtained from the target mouse hybridoma, and engineering improvements are made using standard molecular biology techniques to include non-mouse (e.g., human) immunoglobulin sequences.

[0087] In some embodiments, the chimeric antibodies according to the present invention effectively link the immunoglobulin heavy and light chain variable regions derived from hybridomas with human IgG constant regions using methods known in the art (see, for example, U.S. Patent No. 4,816,567 owned by Cabilly, etc.) to obtain chimeric heavy chains and chimeric light chains. Human IgG is selected from any subtype of, for example, IgG1, IgG2, IgG3, IgG4, with IgG4 being preferred.

[0088] In a specific embodiment, the chimeric antibody of the present invention is obtained by transfecting an expression cell by "mixing and mapping" of an expression plasmid of one type of chimeric light chain and one type of chimeric heavy chain. The binding of the antibody obtained by these "mixing and mapping" to IL-17A is measured by the above binding measurement and other usual binding measurements (e.g., ELISA). Suitable ch1, ch2, ch4, ch7, ch16 have optimal binding and blocking activities, and the amino acid sequences of their variable regions are shown in Table 2.

[0089] [Table 2]

[0090] The precise amino acid sequence boundaries of the CDRs of the variable regions of the antibodies according to the present invention are determined in any one of a number of well-known embodiments, including the Kabat scheme as described by Kabat et al. (1991), Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD (the "Kabat" numbering scheme), and the IMGT scheme as described by Lefranc M.-P. et al. (1999 Nucleic Acids Research, 27, 209-212). In some embodiments, specific definition schemes and amino acid sequences of the CDRs of the preferred murine antibody variable regions of the present invention are shown in Table 3.

[0091]

Table 3

[0092] In some embodiments, the humanized antibodies according to the present invention may insert mouse CDR regions into human germline framework regions using known methods in the art. See U.S. Patent No. 5,225,539 to Winter et al. and U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762; 6,180,370 to Queen et al. Briefly, the applicant searches the human immunoglobulin gene database of the NCBI (http: / / www.ncbi.nlm.nih.gov / igblast / ) site for human germline IgG genes having high homology with the cDNA sequence of the mouse antibody variable region, and humanization is achieved by CDR grafting selected in principle. However, even after exchanging CDR loops, antibodies having binding properties identical to those of the starting antibody cannot be uniformly produced. In humanized antibodies, it is often necessary to change framework residues (FR) (residues involved in supporting CDR loops) in order to maintain the antigen-binding affinity. Kabat et al. (1991) J. Immunol. 147:1709. Briefly, the process of humanization modification includes the following steps. A. Compare the gene sequences of each candidate antibody with the gene sequences of human embryonic antibodies to find sequences with high homology. B. Analyze and examine HLA-DR affinity to select human embryonic framework sequences with low affinity. C. Utilize computer simulation technology to analyze the variable region and the framework amino acid sequences around it by molecular docking to examine their spatial binding modes. By calculating electrostatic forces, van der Waals forces, hydrophilicity / hydrophobicity, and entropy values, analyze the important single amino acids in the gene sequences of each candidate antibody that act with IL-17A to maintain the spatial framework, graft it onto the selected human embryonic gene framework, and based on this, mark the amino acid sites in the framework region that must be retained, and synthesize humanized antibodies.

[0093] In some embodiments, the preferred humanized antibodies obtained in the present invention are hu31, hu43, hu44, hu59, hu60, hu250. The variable regions of the humanized antibodies hu31, hu43, hu44, hu59, hu60, hu250 and their corresponding CDR amino acid sequences are shown in Table 4.

[0094] [Table 4]

[0095] The light / heavy chain amino acid and nucleotide sequences of the humanized antibodies hu31, hu43, hu44, hu59, hu60, hu250 are shown in Table 5.

[0096] [Table 5]

[0097] Other antibodies of the present invention have undergone amino acid deletion, insertion or substitution mutations, but have at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 98%, 99% or 100% identity with the above antibodies (especially the CDR regions described in the above sequences). In some embodiments, the antibody of the present invention is a mutant of any one of hu31, hu43, hu44, hu59, hu60, hu250, the mutant contains a mutant amino acid sequence, and in the mutant amino acid sequence, when compared with the CDR region described in the above sequence, the amino acid deletion, insertion or substitution mutations in the CDR region do not exceed 1, 2, 3, 4 or more than 5.

[0098] Other nucleic acids encoding the antibodies of the present invention have undergone nucleotide deletion, insertion or substitution mutations, but include nucleic acids having at least 60, 70, 80, 90, 95 or 100% identity with the CDR corresponding coding regions described in the above sequences.

[0099] (Antibody expression) In some embodiments, the present invention relates to one or more expression vectors or host cells of the expression vectors, and a method for producing the antibody or antigen-binding fragment thereof of the present invention, wherein the method includes culturing the host cells, and purifying and recovering the antibody or antigen-binding fragment.

[0100] For example, the antibodies of the present invention are produced in host expression cells using a combination of well-known recombinant DNA techniques and gene transfection methods in this field (e.g., Morrison, S. 1985, Science 229:1202). For example, to express an antibody or an antibody fragment thereof, DNA encoding the light chain and heavy chain of the coding portion or full length is obtained by standard molecular biology or biochemical techniques (e.g., DNA chemical synthesis, PCR amplification, or cDNA cloning of a hybridoma using the target antibody to be expressed), and the DNA is inserted into an expression vector such that the gene is effectively linked to transcription and translation control sequences. As used herein, the term "effectively linked" means that the antibody gene is linked to the vector such that the transcription and translation control sequences in the vector perform the intended functions of transcription and translation regulation of the antibody gene. An expression vector and expression regulatory sequences that can coexist with the expression host cell used are selected. The antibody light chain gene and the antibody heavy chain gene may be inserted into different vectors, or more generally, the two genes may be inserted into the same expression vector. The antibody gene is inserted into the expression vector by standard methods (e.g., ligating the antibody gene fragment to a complementary restriction site in the vector, or ligating to blunt ends if no restriction site is present). Using the light and heavy chain variable regions of the antibodies described herein, full-length antibody genes of any antibody isotype are generated, and by inserting these into an expression vector encoding the heavy chain constant region and light chain constant region of the desired isotype, the VH segment and the CH segment in the vector are effectively linked, and the VL segment and the CL segment in the vector are effectively linked. And / or the recombinant expression vector may optionally encode a signal peptide (also called a leader sequence), which leads to the secretion of the antibody chain from the host cell. The antibody chain gene may be cloned into the vector such that the amino-terminal end of the signal peptide and the antibody chain gene are linked in the same reading frame. The signal peptide may be either an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide derived from a non-immunoglobulin).

[0101] Mammalian host cells for expressing the recombinant antibodies of the present invention include a large number of immortalized cell lines obtained from the American Type Culture Collection (ATCC). In particular, it includes Chinese hamster ovary (CHO) cells, NSO, SP2 / O cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells, A549 cells, 293T cells and a large number of other cell lines. Mammalian host cells include cells of humans, mice, rats, dogs, monkeys, pigs, goats, cows, horses, hamsters. By measuring which cell line has a high expression level, a particularly preferred cell line is selected.

[0102] When introducing a recombinant expression vector encoding a heavy chain or an antigen-binding fragment or a fragment thereof, a light chain and / or an antigen-binding fragment thereof into a mammalian host cell, the antibody is expressed in the host cell, or more preferably the antibody is produced by secreting the antibody into the growth medium of the host cell. The host cell is cultured for a sufficient period of time. The antibody is recovered from the medium by standard protein purification methods.

[0103] Antibodies expressed in different cell lines or expressed in genetically engineered animals may exhibit different glycosylations. However, despite the glycosylation of the antibody, all antibodies encoded by the nucleic acid molecules provided herein or comprising the amino acid sequences provided herein are components of the present invention. Similarly, in some embodiments, since the sugar structure of the non-rockweed glycosylated antibody is a common component of natural human serum IgG, it usually has a more potent effect than its rockweed glycosylated counterpart in vitro and in vivo and is not likely to be immunogenic. Therefore, the non-rockweed glycosylated antibody has an advantage.

[0104] (Pharmaceutical Use) The antibodies of the present invention or antigen-binding fragments thereof have diagnostic and therapeutic uses in vitro and in vivo. Preferably, the humanized antibodies hu31, hu43, hu44, hu59, hu60 or hu250 antibodies are applied to the treatment of diseases related to IL-17A.

[0105] On the one hand, when evaluating the in vivo activity, the isolated antibody or its antigen-binding fragment according to the present invention can suppress the onset of mice and significantly reduce the clinical evaluation score of mouse psoriasis onset and the degree of ear swelling in a psoriasis experimental model induced by imiquimod.

[0106] In a specific embodiment, in a psoriasis experimental model induced by imiquimod, the humanized antibodies hu31 and hu44 can significantly suppress the onset of mice and significantly reduce the clinical evaluation score of mouse psoriasis onset and the degree of ear swelling.

[0107] On the other hand, when evaluating in vivo, the isolated antibody or its antigen-binding fragment according to the present invention can suppress the swelling of the knee joint in an arthritis experimental model induced by an antigen, such as the cynomolgus monkey AIA-model.

[0108] In a specific embodiment, in the cynomolgus monkey AIA-model, the humanized antibody hu31 significantly suppresses the increasing tendency of the cynomolgus monkey arthritis clinical evaluation score.

[0109] On the other hand, the present invention provides a method for treating a pathological disease induced by IL-17A, and the method includes administering an isolated antibody or its antigen-binding fragment according to the present invention in an effective amount so as to reduce the disease condition, and the antibodies hu31, hu43, hu44, hu59, hu60 or hu250 are particularly preferred.

[0110] In one embodiment, a protein containing the isolated antibody or its antigen-binding fragment according to the present invention is bound to other active moieties.

[0111] In one embodiment, the protein containing the isolated antibody or its antigen-binding fragment according to the present invention may be a monoclonal antibody or its antigen-binding fragment, but chimeric antibodies, humanized antibodies or human antibodies or a part thereof are preferred.

[0112] On the one hand, the present invention provides a pharmaceutical composition comprising a protein comprising an antibody or an antigen-binding fragment thereof according to an embodiment of the present invention in combination with one or more pharmaceutical excipients, diluents or vectors.

[0113] In an embodiment, the pharmaceutical composition comprises one or more other active ingredients. In a specific embodiment, the pharmaceutical composition is a lyophilized powder. In another specific embodiment, the pharmaceutical composition is a stable liquid formulation comprising the antibody or molecule according to the present invention in a therapeutically acceptable amount.

[0114] Specifically, the present invention provides a method for treating a disease associated with IL-17A and / or an autoimmune disease and an inflammatory disease. In some embodiments, the method comprises administering to a subject in need thereof an isolated antibody or an antigen-binding fragment thereof according to the present invention. The present invention further provides a method for reducing or suppressing a signal transduction response induced by IL-17A or IL-17AF in target cells or tissues by contacting cells with a composition comprising the antibody of the present invention in a therapeutically effective dose.

[0115] In the present invention, the terms "disease induced by IL-17A" or "disease associated with IL-17A" include all diseases and medical conditions in which IL-17A or IL-17AF acts (whether directly or indirectly) in a disease or medical condition, including the cause, development, progression, persistence or pathology of the disease or medical condition. Thus, these terms include conditions associated with or characterized by abnormal IL-17A or IL-17AF levels, and / or diseases or medical conditions that are treated by reducing or suppressing the activity induced by IL-17A / AF (e.g., CXCL1) in target cells or tissues. These diseases or medical conditions include inflammatory conditions and autoimmune diseases such as, for example, arthritis, rheumatoid arthritis, ankylosing spondylitis, multiple sclerosis or psoriasis. These diseases further include anaphylaxis and allergic conditions, hypersensitivity, chronic obstructive pulmonary disease, cystic fibrosis and organ or tissue transplant rejection.

[0116] As used in the present invention, "suppression", "treatment" or "therapy" includes delaying the development of symptoms associated with a medical condition and / or reducing the severity of these symptoms of the medical condition. These terms include improvement of symptoms that are not currently controllable or are harmful, prevention of other symptoms, and improvement or prevention of the underlying cause of these symptoms. Thus, these terms mean providing a beneficial result to a vertebrate subject in which the medical condition, disease or symptoms have already appeared, or to a vertebrate subject in which these medical conditions, diseases or symptoms may appear.

[0117] As used herein, the terms "therapeutically effective amount", "therapeutically effective dose", and "effective amount" refer to an amount that, when the IL-17 binding compound of the present invention is administered to a cell, tissue, or subject alone or in combination with other therapeutic agents, effectively prevents or ameliorates the symptoms of one or more diseases or medical conditions or the development of the disease or medical condition. A therapeutically effective dose refers to an amount of the binding compound sufficient to lead to symptom improvement, for example, an amount that treats, cures, prevents, or ameliorates a related medical condition, or an amount that improves the speed of treatment, cure, prevention, or amelioration of these medical conditions. When the active ingredient is administered alone to an individual, the therapeutically effective dose is only the amount of that ingredient. When administered in combination, the therapeutically effective dose is the total amount of the active ingredients that produce a therapeutic effect, regardless of whether the combination is administered, sequentially, or simultaneously. The effective amount of a therapeutic agent improves at least 10% of the diagnostic criteria or parameters, although at least 20% is typical, at least about 30% is preferred, at least 40% is more preferred, and at least 50% is most preferred.

[0118] (Rheumatoid arthritis (RA)) RA is a progressive systemic disease characterized by inflammation of synovial joints and affects approximately 0.5% of the world's population. See Emery (2006) BMJ 332:152-155. Joint inflammation causes deformity, pain, stiffness, and swelling, and ultimately irreversible joint degeneration. Affected joints include the knees, elbows, neck, and hand and foot joints. Common treatments include using NSAIDs to relieve symptoms and then administering disease-modifying antirheumatic drugs (DMRDs) such as gold, penicillamine, sulfasalazine, and methotrexate to reduce the disease. In recent years, there have been advances in treatment with TNF-α inhibitors such as monoclonal antibodies like Remicade, Humira, Golimumab, and receptor fusion proteins such as etanercept. Treatment with these TNF-α inhibitors significantly reduces the structural damage caused by the disease.

[0119] The anti-IL-17A antibody of the present invention can be applied to the treatment of RA in a subject in need of such treatment. The anti-IL-17A antibody of the present invention can be applied, for example, in combination with other treatments for RA by methotrexate, azathioprine, cyclophosphamide, mycophenolate ethyl, NSAID or TNF-α inhibitor, etc.

[0120] (Psoriasis) The skin is an important screen between the in-vivo environment and the external environment, preventing contact with potential harmful antigens. When an antigen / pathogen invades, T cells, polymorphonuclear cells, macrophages, etc. in the skin contact area infiltrate locally and cause an inflammatory reaction to eliminate the antigen (see, for example, Williams and Kupper, (1996) Life Sci., 58: 1485-1507). Usually, this inflammatory reaction caused by pathogens is strictly monitored, but it stops simultaneously when the pathogens are eliminated. If there is no appropriate control of external stimuli, this inflammatory reaction may occur and skin inflammation may appear. The present invention provides methods for the treatment and diagnosis of skin inflammation. Skin inflammation (resulting from the infiltration of the above cells and cytokines secreted from these cells) includes, for example, several inflammatory diseases such as cicatricial pemphigoid, scleroderma, hidradenitis suppurativa, toxic epidermal necrolysis, acne, osteitis, graft-versus-host disease (GVHD), pyroderma gangrenosum, Behcet's Syndrome, etc. (see, for example, Williams and Griffiths, (2002) Clin. Exp. Dermatol., 27: 585-590). The most common skin inflammation is psoriasis.

[0121] Psoriasis is characterized by rapid proliferation of keratinocytes induced by T cells and infiltration of inflammatory cells. The disease includes several clinically distinct phenotypes that clearly overlap, such as chronic plaque lesions, exanthems, and pustular lesions (see, for example, Gudjonsson et al., (2004) Clin Exp. Immunol. 135: 1-8). Approximately 10% of psoriasis patients develop arthritis. The disease has a complex and strong genetic component, with 60% concordance in monozygotic twins.

[0122] Typical psoriatic lesions are erythematous patches with distinct boundaries covered by thick gray scales. Compared with normal skin, the inflammation and rapid proliferation of psoriatic tissue are related to different histological, antigenic, and cytokine profiles. Cytokines associated with psoriasis are TNF-α, IL-19, IL-18, IL-15, IL-12, IL-7, IFN-γ, IL-17A, and IL-23 (see Gudjonsson et al., supra).

[0123] The anti-IL-17A antibody of the present invention, used alone or in combination with other agents, can also be applied to the prevention, treatment, diagnosis, and prediction of psoriasis flares.

[0124] To produce a pharmaceutical composition or a sterile composition of the IL-17A antibody of the present invention, the antibody is mixed with a pharmaceutically acceptable vector or excipient. See, for example, Remington's Pharmaceutical Sciences and U. S. Pharmacopeia: National Formulary, Mack Publishing Company, Easton, PA (1984).

[0125] By mixing with an acceptable vector, excipient, or stabilizer, therapeutic and diagnostic agents in dosage forms such as, for example, lyophilized powders, pastes, aqueous solutions, or suspensions are produced. In one embodiment, the IL-17A antibody of the present invention is diluted to an appropriate concentration with a sodium acetate solution (pH 5-6), and NaCl or sucrose is added so that the osmotic pressure can be utilized. Other substances (for example, polysorbate 20 or polysorbate 80) may be added to improve stability.

[0126] The mode of administration is due to several factors including the serum or tissue turnover rate of the therapeutic antibody, the level of symptoms, the immunogenicity of the therapeutic antibody, and the accessibility of target cells in the biological matrix. In a preferred mode of administration, delivering sufficient therapeutic antibody improves the condition of the target disease while minimizing adverse side effects. Thus, the amount of biologic to be delivered is determined in part by the specific therapeutic antibody and the severity of the medical condition being treated. Guidelines for selecting the appropriate dose of a therapeutic antibody can be obtained. For example, a clinician can determine the appropriate dose using known or putative parameters or factors in the art that affect treatment. In short, the dose is increased in small increments from a dose slightly less than the optimal dose until the effect or optimal effect required for any minus side effects is obtained. Important diagnostic methods include, for example, methods for diagnosing symptoms of inflammation or the levels of inflammatory cytokines produced. Suitable biologics available are derived from the same species as the animal being targeted for treatment so as to minimize the inflammatory, autoimmune, or proliferative response to the reagent. For example, in the case of human subjects, chimeric, humanized, and fully human antibodies are preferred.

[0127] The present invention includes all combinations of the specific embodiments described. Further embodiments and the full scope of applicability of the present invention will become apparent from the specific description provided below. However, while the preferred embodiments of the present invention have been specifically described and indicated by specific examples, each modification and correction within the spirit and scope of the present invention will become apparent to those skilled in the art from these specific descriptions, and these descriptions and examples are provided in the form of illustration only. For all purposes, all disclosures, patents, and applications cited herein, including the preamble, are incorporated herein by reference in their entirety.

Examples

[0128] <Example 1. Recombinant Protein Human IL-17A-mFc> Plasmid HG12047-G encoding the full-length human IL-17A cDNA sequence (NCBI accession number: NP_002181.1) was purchased from Sino Biological Inc., and the human IL-17A mature fragment (amino acids 24-155 of NCBI accession number NP_002181.1, with the amino acid sequence SEQ ID NO: 66 and the nucleotide sequence SEQ ID NO: 67) was amplified by conventional PCR technology. The amplified fragment was digested with BSPQI and then cloned into a self-constructed eukaryotic expression plasmid system (MXT1-Fc, containing the Fc region of the mouse IgG heavy chain) to produce the recombinant fusion protein expression plasmid IL-17A-mFc. The plasmid evaluated as accurate was transfected into the expression cell 293F by conventional techniques, and the human IL-17A-mFc recombinant protein was obtained by expression and purification. Figure 1 shows the SDS-PAGE electrophoresis diagram of the human IL-17A-mFc recombinant protein.

[0129] <Example 2. Construction of a 293F stable expression cell line expressing human IL-17RA> Plasmid HG10895-G containing the cDNA sequence encoding human full-length IL-17RA was purchased from Sino Biological Inc., and the DNA sequence (SEQ ID NO: No:69) encoding full-length human IL-17RA was amplified by ordinary PCR. The amplified fragment was cloned into a eukaryotic expression plasmid system (HXP) self-constructed by ordinary cloning techniques, which contains a puromycin selection system. The constructed IL-17RA recombinant expression plasmid was transfected into 293F (ATCC) cells. After 24 h of transfection, selection was performed with puromycin (2 μg / ml) until a 293FIL-17RA stable expression cell line bank was formed. For example, monoclonal cells were isolated by ordinary methods such as the limiting dilution method, plated in a 96-well plate at 0.8 cells / well, and after 15 days, IL-17RA-293F monoclonal cells were selected and passaged to form a 293FIL-17RA stable expression cell line. All clones were analyzed and selected by FACS, etc., and top-class expression clones were selected and used for FACS binding assays or functional assays to select hybridoma monoclonal antibodies.

[0130] <Example 3. Binding of Recombinant Protein IL-17A-mFc to 293FIL-17RA Stable Expression Cell Line> The binding specificity between recombinant protein IL-17A-mFc and IL-17RA in 293F cells was detected by FACS experiments. Briefly, cells (293FIL-17RA stable expression cell line) were prepared into a cell suspension of 1×10 6 / ml, and 20 μl / well (actually 2×10 4Cells were seeded at a density of [[number of cells per well]] and the cell suspension was mixed with recombinant protein IL-17A-mFc (3 μg / ml, 20 μl / well; experimental group) or 1% BSA (20 μl / well; negative control group) and incubated at 37°C for 30 minutes. After eluting three times with FACS buffer, anti-mouse IgG (1:200) was added and incubated at room temperature for 30 minutes. After eluting three times with FACS buffer, detection was performed by flow cytometry and the mean fluorescence intensity values (MFI) of each group were compared. As shown in Figure 2, recombinant protein IL-17A-mFc can specifically bind to IL-17RA in 293F cells.

[0131] <Example 4. Production and Selection of Hybridoma Antibodies> Hybridoma antibodies were produced using standard molecular biology techniques. Briefly, natural human IL-17A protein purchased from HumanZyme was used as an antigen and mixed with an equal amount of immunoadjuvant. Five 6-week-old female FVB mice were taken and immunized. After the first immunization, boost immunizations were performed once a week for a total of 7 immunizations. After the last boost immunization, mice with a high anti-IL-17A antibody titration concentration in the serum were selected and a cell fusion experiment was performed. Using standard hybridoma techniques, pancreatic cells were isolated and fused with mouse myeloma cell line SP2 / 0 cells (ATCC). The fused cells were resuspended in RPMI-1640 complete synthetic medium containing HAT and plated in wells with a peritoneal cell feeder layer.

[0132] First, based on the desired antibody / antigen binding characteristics (e.g., binding affinity for IL-17A, ability to block the binding of IL-17A to its receptor, species cross-reactivity, and ability to block the biological effects induced by IL-17A in in vitro measurements), the supernatants secreted from monoclonal hybridomas were evaluated. The supernatant antibodies secreted from hybridomas 1F8, 2B2, 2F5, 2F2, 2H1, and 2H5 were used for further characterization.

[0133] <Example 5. Blocking effect of hybridoma antibody on IL-17A bioactivity in vivo> According to a large number of studies, since IL-17A promotes the expression and release of cytokine CXCL1 in vivo, the change in CXCL1 expression in mouse serum can be quantitatively detected by ELISA to determine the effect of the hybridoma antibody on the bioactivity in mouse in vivo induced by IL-17A. Briefly, 40 10-week-old female Balb / c mice were selected and divided into 8 groups of 5 mice each. Four days before administration, serum was collected and the CXCL1 expression level was detected as the reference value. On the day of administration, candidate hybridoma antibody, physiological saline (control), or reference antibody mAb317 (commercial anti-IL-17A antibody, purchased from R&D) drugs were administered at a dose of 1 mg / kg by intramuscular injection. One hour after administration, natural human IL-17A (HumanZyme) was administered at a dose of 150 μg / kg by subcutaneous injection. Two hours after the injection of human IL-17A, serum was collected, the CXCL1 concentration in the blood was detected, and after comparison with the reference value, the fold change in CXCL1 concentration (mean ± standard error (mean ± SEM)) before and after administration of each group was calculated. The comparative analysis between each antibody group and the control group was performed by Student's-t test, and a significant difference was considered when P<0.05, *P<0.05, **P<0.01, ***P<0.001.

[0134] As shown in Figure 3, both the hybridoma antibody obtained in Example 4 and the commercial antibody mAb317 can significantly suppress the CXCL1 expression in mouse in vivo induced by IL-17A.

[0135] <Example 6. Acquisition of candidate antibody variable region sequences> The DNA sequences of the antibody variable regions expressed in hybridomas 1F8, 2B2, and 2F5 were measured by a method based on nested primer PCR. Briefly, hybridoma cell lines 1F8, 2B2, and 2F5 were each expanded in culture, the cells were collected by centrifugation at 1000 rpm, and total RNA was extracted using Trizol. Using this as a template, first-strand cDNA was synthesized. Then, using the first-strand cDNA as a subsequent template, the corresponding variable region sequences were amplified by PCR. The PCR primers used were based on the Ig-primer group. After the PCR products were recovered and purified and subjected to sequencing analysis, the candidate hybridoma antibody heavy and light chain variable region sequences were obtained. Hybridoma cell 1F8 has two types of antibody light chain variable region gene sequences and one type of antibody heavy chain variable region gene sequence, and 2F5 has two types of antibody heavy chain variable region gene sequences and one type of antibody light chain variable region gene sequence. Therefore, the antibodies secreted by 1F8 and 2F5 contain two types of mixed complete antibodies, and the antibodies secreted by 1F8 are called 1F8-1 and 1F8-2, and the antibodies secreted by 2F5 are called 2F5-1 and 2F5-2.

[0136] The amino acid sequences of the heavy chain variable region and light chain variable region of the antibodies expressed in 1F8, 2F5, and 2B2 are shown in Table 1 (specific description in the specification).

[0137] <Example 7. Construction of Chimeric Antibody Recombinant Expression Vector> The human IgG4 heavy chain constant region Fc fragment and the light chain κ constant region were cloned from human blood cells (Peking Union Medical College Hospital), ligated to the pCDNA3.1 plasmid and modified. The above heavy chain and light chain variable region sequence fragments were synthesized by Genscript. After the heavy chain was digested with Bspq I and the light chain was digested with Bspq I, they were ligated to the corresponding modified pCDNA3.1 plasmid, and the expression plasmids of IgG4 chimeric heavy chain (ch-HC) or light chain (ch-LC) were determined by sequencing. The expression plasmids of the above different chimeric heavy chains and light chains were mixed and paired, transfected into expression cells, and 16 kinds of chimeric antibodies numbered from ch1 to ch16 (see Table 6) were obtained. All subsequent experimental materials were obtained by extracting after transfecting this plasmid into expression cells.

[0138]

Table 6

[0139] <Example 8. Binding Specificity between Chimeric Antibody and Human IL-17A> The binding specificity between the chimeric antibody and human IL-17A was detected by the ordinary ELISA detection method. That is, 0.5 μg / ml of human IL-17A-mFc was coated on a 96-well ELISA plate and incubated at a constant temperature of 37 °C for 60-90 minutes. Next, the solution in the well was discarded, washed 3 times with the washing buffer, and a PBS solution containing 2% BSA was added and blocked for 60 minutes. After washing 3 times with the washing buffer, different concentrations of chimeric antibody diluents were added and incubated at 37 °C for 60 minutes. Next, after washing 3 times with the washing buffer, biotin-anti-IgG4 diluted 1:10000 times was added and incubated at 37 °C for 1 hour. Next, after washing 3 times with the washing buffer, HRP-Strep diluted 1:10000 times with the washing buffer was added and incubated at room temperature for 1 hour. Next, after washing 3 times with the washing buffer, 100 μl of TMB substrate solution was added to develop color and reacted at room temperature for 30 minutes. Next, the reaction was terminated with 100 μl of 2M hydrochloric acid solution and the absorbance was read at 450 nm.

[0140] As shown in Figure 4, the binding of chimeric antibodies ch1, ch2, ch4, ch7, and ch16 to human IL-17A has high specificity, and the EC50 values are 6.62 ng / mL, 5.17 ng / mL, 88.48 ng / mL, 39.96 ng / mL, and 15.42 ng / mL, respectively.

[0141] <Example 9. Blocking effect of chimeric antibodies on the binding of human IL-17A and IL-17RA> Using flow cytometry (FACS) with cell-based competitiveness, the blocking effect of chimeric antibodies on the binding of IL-17A and IL-17RA in cells was measured and detected. Briefly, different concentrations of chimeric antibody dilutions (starting from 10 μg / ml, titrated 3-fold) were mixed with the human IL-17A-mFC (3 μg / ml) obtained in Example 1 that had been pre-labeled with biotin and incubated at room temperature for 30 minutes. Then, the mixture and the cell suspension (293FIL-17RA stable expression cell line obtained in Example 2, 1.5×10 5 cells / well) were incubated at 37°C for 15 minutes. Next, after eluting three times with PBS, 5 μg / ml of anti-mouse IgG was added and incubated at room temperature for 30 minutes. After eluting three times with PBS, the inhibitory effect of chimeric antibodies on the binding of IL-17A and IL-17RA on the surface of 293F cells was detected by flow cytometry.

[0142] As shown in Figure 5, chimeric antibodies ch1, ch2, ch7, and ch16 showed an inhibitory effect on the binding of human IL-17 and IL-17RA on the surface of 293F cells. According to the experimental results, ch1 and ch16 were selected and continued to be humanized.

[0143] <Example 10. Humanization of antibodies> Regarding the humanization of antibodies, first, a human germline IgG gene with high homology to the cDNA sequence of the mouse antibody variable region was searched from the human immunoglobulin gene database of the NCBI (http: / / www.ncbi.nlm.nih.gov / igblast / ) website. Furthermore, the amino acid sequences and their exact boundaries of the variable region CDRs were defined according to the Kabat numbering system or the IMGT numbering system. In principle, human IGVH and IGVk with high homology to the mouse antibody variable region were selected as humanization templates, and humanization was carried out by CDR grafting. Briefly, the process of humanization modification includes the following steps. A. Compare the gene sequences of each candidate antibody with the gene sequences of human embryonic antibodies to find sequences with high homology. B. Analyze and examine the HLA-DR affinity, and select a human embryonic framework sequence with low affinity. C. Utilize computer simulation technology to analyze the variable region and the framework amino acid sequences around it by molecular docking, and examine their spatial binding modes. By calculating electrostatic forces, van der Waals forces, hydrophilicity / hydrophobicity, and entropy values, analyze the important single amino acids in the gene sequences of each candidate antibody that act with IL-17A to maintain the spatial framework, graft it onto the selected human embryonic gene framework, and based on this, mark the amino acid sites in the framework region that must be retained, and synthesize humanized antibodies.

[0144] Refer to Table 3 in the specific description part of the present disclosure content for the mouse antibody variable region CDR definition scheme and its amino acid sequence.

[0145] According to the results selected by the foregoing experiments, the chimeric antibodies ch1 and ch16 were selected and continued to be humanized. After tentatively selecting a series of antibody / antigen binding characteristics (for example, the binding affinity for IL-17A, the ability to block the binding of IL-17A to its receptor), the humanized antibodies hu31, hu43, hu44, hu59, hu60, and hu250 were selected and continued to be verified in the follow-up.

[0146] For the variable regions and CDR amino acid sequences of the humanized antibodies hu31, hu43, hu44, hu59, hu60, and hu250, refer to Table 4 in the specific description section of the present disclosure.

[0147] <Example 11. Binding Specificity between Humanized Antibody and Human IL-17A> The binding specificity between the humanized antibody and human IL-17A was detected by the ordinary ELISA detection method. For the experimental method and steps, refer to Example 8. As shown in Figure 6, the humanized antibodies hu31, hu43, hu44, hu59, hu60, and hu250 specifically bound to IL-17A. The EC50 values were 8.13 ng / mL, 8.64 ng / mL, 6.76 ng / mL, 6.10 ng / mL, 5.78 ng / mL, and 6.35 ng / mL, respectively.

[0148] <Example 12. Blocking Effect of Humanized Antibody on the Binding between Human IL-17A and IL-17RA> Based on cell-based competitive flow cytometry (FACS), the blocking effect of the chimeric antibody on the binding between IL-17A and IL-17RA in cells was measured and detected. For the experimental method and steps, refer to Example 9. As shown in Figure 7, the humanized antibodies hu31, hu43, hu44, hu59, hu60, and hu250 significantly inhibited the specific binding between IL-17A and IL-17RA in cells. The IC50 values were 867.6 ng / mL, 780.8 ng / mL, 828.5 ng / mL, 467.4 ng / mL, 482.8 ng / mL, and 577.8 ng / mL, respectively.

[0149] <Example 13. Antagonism of Humanized Antibody against CXCL1 Expression in Epithelial Cells Induced by IL-17A> IL-17A stimulates the expression and release of the cytokine CXCL1 secreted from multiple types of epithelial cells and other cells. By quantitatively detecting the change in the CXCL1 expression level in the cell supernatant by ELISA, the effect of the humanized antibody on the biological activity in cells induced by IL-17A can be determined.

[0150] HT-29 cells (epithelial cells of human colorectal adenocarcinoma, ATCC) were maintained in culture / measurement medium in flasks treated with tissue culture using standard techniques. The cells were grown in tissue culture flasks such that they reached 50 - 80% confluence on the day of measurement. On the day of measurement, the cells were washed with PBS, and the cells were detached from the culture flasks using trypsin + EDTA to produce a cell suspension. Dilutions of humanized antibodies hu31, hu59, hu60, hu250 or a reference antibody (Secukinumab, Novartis) (starting concentration 55 μg / ml, diluted in a 3-fold concentration gradient) were taken, mixed with human IL-17A (1 μg / ml), plated in 96-well plates, and incubated for 1 h. 100 μl (2×10 4 cells) of an HT-29 cell (ATCC, epithelial cells of human colorectal adenocarcinoma) suspension were added to each well, and the cells were cultured at 37 °C and 7% CO2 for 48 h. The cells were centrifuged at 500×g for 5 min, and the culture supernatant was transferred to a new 96-well plate, and the expression of CXCL1 was detected using an ELISA reagent kit.

[0151] As shown in Figure 8, compared with the reference antibody Secukinumab, the humanized antibodies hu31, hu59, hu60 and hu250 have a stronger antagonistic effect on the IL-17A-induced stimulation of CXCL1 release from epithelial cells.

[0152] <Example 14. Antagonism of humanized antibodies against IL-17A-induced CXCL1 expression in mice> Similar to Example 5, by detecting the change in mouse serum CXCL1 level, the effect of a humanized antibody with bioactivity in vivo induced by IL-17A was determined. Briefly, 40 10-week-old female Balb / c mice were selected and divided into 8 groups of 5 mice each. Four days before administration, serum was collected and the CXCL1 expression level was detected as the reference value. On the day of administration, candidate antibodies (humanized antibodies hu31, hu43, hu44, hu60, and hu250) and the control group IgG4 isotype (hlgG) were each administered at 1 mg / kg by intramuscular injection. One hour after administration, human IL-17A was administered at a dose of 150 μg / kg by subcutaneous injection. Two hours after the injection of human IL-17A, serum was collected, the CXCL1 concentration in the blood was detected, and after comparison with the reference value, the fold change in CXCL1 concentration (mean ± standard error (mean ± SEM)) before and after administration of each group was calculated. The comparative analysis between the candidate antibodies and the control group IgG4 isotype was performed by Student's-t test, and a significant difference was considered at P<0.05, with *P<0.05, **P<0.01, ***P<0.001.

[0153] As shown in Figure 9, compared with the control group IgG4 isotype, the candidate humanized antibodies hu31, hu43, hu44, hu60, and hu250 have a stronger antagonistic effect against the stimulation of IL-17A-induced CXCL1 release in mouse in vivo.

[0154] <Example 15. Study on the therapeutic effect of improvement by humanized antibodies in a mouse psoriasis model induced by imiquimod> Imiquimod was applied to the skin behind the mouse auricle to induce the pathological characteristics of psoriasis, that is, hyperplasia of keratinocytes, aggregation of inflammatory cells, hyperplasia of blood vessels in the dermal papillary layer, etc., to construct a psoriasis mouse model. The therapeutic effect of the drug on psoriasis mice was judged using clinical evaluation scores, ear swelling degree, etc. as indicators.

[0155] 15.1 Experimental method Forty-eight 6- to 8-week-old female C57BL / 6 mice (purchased from the Model Animal Research Institute of Nanjing University, animal certificate number 201605578) were taken, their backs were depilated, and except for the sham operation group, they were sensitized 2 days later. Two days before sensitization, they were randomly divided into 5 groups (8 mice per group). Group I was the sham operation group, Group II was the PBS group, Group III was the KLH control group (isotype IgG), KLH was administered, Group IV was the hu31 group, Group V was the hu43 administration group, and Group VI was the hu44 administration group. The dosage for each group was 50 mg / kg. The above-mentioned each group was administered the drug once by intraperitoneal injection on day 0 and day 3 of grouping. On the day of sensitization (day 1), approximately 62.5 mg of imiquimod cream (Aldara, 5%, 3M Health Care Limited) was applied to the skin of the right ear and back of the mice in Groups II-VI, and it was applied continuously for 4 days.

[0156] 15.2 Evaluation method From the day of sensitization, the thickness of the right ear of the mice was measured daily with a spiral micrometer. Using the thickness of the right ear on day 1 as a control, the swelling thickness value of the mouse ear was calculated. And the body weight of the mice was measured daily, and the skin scale, induration, and erythema conditions were observed and evaluated by scores. A 4-rank score evaluation method of 0 point (no disease), 1 point (mild), 2 point (moderate), 3 point (severe), and 4 point (very severe) was adopted. The results are shown as mean ± standard error (mean±SEM). First, one-way analysis of variance (ANOVA) was used. After differences appeared, the comparison between two groups was performed by Student's -t test, and a P<0.05 was considered a significant difference.

[0157] As shown in Figure 10-1, by administering the humanized antibody of the present invention, symptoms such as skin scale, induration, redness, and swelling of the mouse psoriasis model induced by imiquimod can be significantly suppressed, that is, the score evaluation value is small.

[0158] As shown in Figure 10-2, imiquimod cream was applied to the right ear of the mice from the day of sensitization, and severe swelling appeared in the right ear and the ear thickness increased. However, the humanized antibody of the present invention can significantly improve the swelling condition of the ear.

[0159] In the mouse psoriasis model induced by imiquimod, the humanized antibody of the present invention can significantly suppress the onset of the mouse, and the phenotypes are the reduction of the mouse clinical evaluation score and the swelling of the ear.

[0160] <Example 16. Study on the therapeutic effect of improvement of type II collagen-induced cynomolgus monkey arthritis by humanized antibody> Arthritis induced by type II collagen is an animal model widely used in rheumatoid arthritis (RA) research. It has the same histopathological characteristics as human RA, and is characterized by inflammation of small joints and progressive erosion of cartilage and bone. Since human / humanized biopolymers contain antibodies and always have better cross-reactivity with antigens in cynomolgus monkeys in vivo, the cynomolgus monkey arthritis model is an effective system for detecting the anti-rheumatic effect of the humanized antibody IL-17A according to the present invention. This experiment evaluates the drug effect of the candidate antibody in the cynomolgus monkey rheumatoid arthritis model.

[0161] 16.1 Experimental method Type II bovine collagen (CII, Sichuan University) was dissolved in acetic acid (Lot No. 10000218; Guoyao, Shanghai, China), placed in a refrigerator at 4°C, and stirred overnight. Then, the collagen was emulsified with an equal volume of complete Freund's adjuvant (Lot No.: F5881, Sigma-Aldrich, USA), with the final concentration of the collagen emulsion being 2 mg / ml. On day 0, the animals were anesthetized with ZOLETIL (1.5 - 5 mg / kg, i.m.), and the collagen emulsion was inoculated at multiple sites on the back and the base of the tail. When performing the immunization, anesthesia was maintained with 1.5% - 5% isoflurane as needed. Three weeks later (day 21), the collagen was injected again in the same manner as the first time. This experiment was divided into four groups. G1 was the normal animal group without induction of arthritis, G2 was the solvent control group, G3 was the antibody hu31 administration group, and G4 was the antibody hu59 administration group. When animals with a clinical evaluation score reaching 5% of the maximum clinical evaluation score (192×5% ≈ 10) appeared, they were assigned to each experimental group starting from those reaching the specified score, and this was repeated until all animals meeting the conditions were sequentially assigned to each group. Administration was started after assignment to the group, at a dose of 7.5 mg / kg once a week, and infusion was continued for 30 minutes using an infusion pump for 5 consecutive weeks.

[0162] 16.2 Evaluation method Body weight measurement: One day before the immunization, the body weight of the animals was measured, and then the body weight was measured once a week until the end of the experiment. Arthritis assessment score: The degree of inflammation of the joints of the monkey's limbs was scored on the 0th and 21st days, and then scored once a week until the end of the experiment 21 days later (if the onset was early, the time for weekly scoring of arthritis was advanced accordingly). The criteria for scoring are shown in Table 2. The following 15 joints of each limb were scored. 5 metacarpophalangeal joints (MCP), 4 proximal interphalangeal joints (PIP), 5 distal interphalangeal joints (DIP), and 1 wrist or ankle joint. And the onset degree of the knee / elbow joints of the four limbs was evaluated. The total assessment score of each joint is the arthritis assessment score of this animal, and the maximum score is 192 (16×3×4). Arthritis assessment score criteria: 0 points, normal; 1 point, mild arthritis, mild onset but clearly distinguishable; 2 points, moderate swelling; 3 points, severe arthritis, severe swelling or obvious joint deformation.

[0163] Experimental data are presented as mean ± standard error (mean ± S.E.M). Statistical analysis was performed on the differences in each parameter among the solvent control group, reference drug group, and test drug group, and a statistically significant difference was considered when p < 0.05 (One-way ANOVA / Dunnett).

[0164] As shown in Figure 11-1, the body weight of normal cynomolgus monkeys (G1) was stable. For cynomolgus monkeys after arthritis induction, the average body weight of the monkeys in the solvent-treated group (G2) continued to decrease, while the decreasing trend was suppressed by the test antibodies hu31 and hu59. Therefore, under the experimental conditions, hu31 and hu59 have a certain improvement effect on weight loss caused by arthritis (**P < 0.01, ****P < 0.0001, compared with "G2: solvent group"; One-way ANOVA / Dunnett).

[0165] As shown in Figure 11-2, after the animals were grouped, the clinical arthritis evaluation score of the normal control animals remained at 0, the arthritis evaluation score of the animals in the model-solvent group (G2) gradually increased, and the increasing trend of the clinical arthritis evaluation score was significantly inhibited by the test antibodies hu31 and hu59. Therefore, the test antibodies hu31 and hu59 have an inhibitory effect on the progressive development of arthritis symptoms, and the increasing trend of the clinical arthritis evaluation score in cynomolgus monkeys was significantly inhibited by the test antibody hu31 (***P<0.001, #P<0.05, G2: compared with the solvent group; One-way ANOVA / Dunnett).

[0166] <Example 17. Effect of Humanized Antibodies on Mouse Joint Swelling Induced by NIH3T3-IL-17 Cells> 1. Animals: C57BL / 6, female, 6-8 weeks old, Beijing Vital River Laboratory Animal Technology Co., Ltd. 2. Cells: NIH3T3 cells, NIH3T3 cells expressing human IL-17. 3. Grouping and dosing schedule: NIH3T3 group; NIH3T3-IL-17 + control IgG antibody group (30 mg / kg); NIH3T3-IL-17 + test antibody high-dose administration group (antibody hu31, 3 mg / kg); NIH3T3-IL-17 + test antibody medium-dose administration group (antibody hu31, 10 mg / kg); NIH3T3-IL-17 + test antibody low-dose administration group (antibody hu31, 30 mg / kg); NIH3T3-IL-17 + positive drug administration group (cosentyx, 10 mg / kg).

[0167] 4. Modeling and administration: NIH3T3-IL-17 cells and NIH3T3 control cells (2.5×10 5 cells / mouse, injection volume per mouse 25 μL) were injected into the joint cavity of the right ankle joint of each group of mice, respectively. One day before model selection, antibodies hu31 (3, 10, 30 mg / kg) and cosentyx (10 mg / kg) were administered by intraperitoneal injection. Although it was involved in the administration, the effect of the hu31 injection solution of the antibody on the mouse arthritis model was considered once every three days. 5. Detection: The thickness of the mouse ankle joint was measured with calipers, and the degree of swelling was calculated.

[0168] 6. Experimental results: As shown in Figure 12, NIH3T3 cells stably expressing hIL-17 were injected into the mouse ankle joint cavity, and severe swelling of the mouse ankle joint was observed the next day.

[0169] The swelling inhibition rate was calculated from the thickness of the mouse ankle joint. The calculation formula is: inhibition rate (%) = (ankle joint thickness of NIH3T3-IL-17 group - ankle joint thickness of administration group) / (ankle joint thickness of NIH3-IL-17 group - ankle joint thickness of NIH3T3 group) × 100. According to the results, the inhibitory effect of each dose drug group on mouse ankle joint swelling could be observed from the day after administration until the end of the test, and the inhibitory efficiency was maximized on the 6th day after administration. On the 10th day, the inhibition rates of each group of antibody hu31 (3, 10, 30 mg / kg) were 49.4%, 65.9%, and 74.1% respectively. The inhibition rate of cosentyx (10 mg / kg) for swelling was 67.1%. According to the results of calculating the average inhibition rate of each group for different days, the average inhibition rates of each group of 3, 10, and 30 mg / kg were 45.2%, 57.0%, and 73.9% respectively. The inhibition rate of cosentyx (10 mg / kg) for swelling was 60.4%. According to the experimental results, the antibody hu31 can dose-dependently inhibit mouse ankle joint swelling induced by IL-17, and the effect of 10 mg / kg is equivalent to that of the positive control drug cosentyx (10 mg / kg). The effect of 30 mg / kg is superior to that of the positive control drug cosentyx (10 mg / kg).

[0170] <Example 18. Effect of humanized antibody on mouse air pouch inflammation induced by NIH3T3-IL-17 cells> 1. Animals: C57BL / 6, male, 6-8 weeks old, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. 2. Cells: NIH3T3 cells, NIH3T3 cells expressing human IL-17. 3. Reagents: Gr1-FITC antibody, Biolegend 4. Grouping and dosing regimen: NIH3T3 cell group NIH3T3-IL-17 cell group + control IgG antibody group (30 mg / kg); NIH3T3-IL-17 cell group + test antibody high-dose administration group (antibody hu31, 30 mg / kg); NIH3T3-IL-17 cell group + test antibody medium-dose administration group (antibody hu31, 10 mg / kg); NIH3T3-IL-17 cell group + test antibody low-dose administration group (antibody hu31, 3 mg / kg); Route of administration: Intraperitoneal injection.

[0171] 5. Modeling: Air sac: 2.5 ml of air was injected into the back of the mice on day 0 and day 3, respectively. Cells were injected into the air sac from day 5. The number of cells injected per mouse was 2×10 5 cells / 500 μl PBS. There were 8 mice per group.

[0172] 6. Detection: The total number of cells in the washing solution was calculated from the migration of leukocytes into the air sac, and the ratio of Gr1 + cells was measured by flow cytometry to calculate the number of neutrophils. Number of neutrophils = total number of cells × ratio of Gr1 + cells

[0173] 7. Experimental results As shown in Figure 13, NIH-3T3 cells stably expressing hIL-17A were injected into the air sac on the back of the mice. From the total number of infiltrated cells, the number of infiltrated leukocytes in the air sac of the NIH3T3-IL-17 cell group was significantly increased compared with the NIH3T3 cell group, and Grl +The ratio and number of cells also increased, but the model selection was successful. On the day of modeling, the antibody hu31 (doses of 3, 10, and 30 mg / kg, respectively) was administered by intraperitoneal injection and was involved in the administration. The total number of infiltrated cells and Gr1 + The inhibition rate is calculated from the number of cells. The calculation formula is: inhibition rate (%) = (number of cells in the NIH3T3-IL-17-IgG group - number of cells in the administration group) / (number of cells in the NIH3T3-IL-17-IgG group - number of cells in the NIH3T3 group) × 100. According to the results, the inhibition rates of the total number of infiltrated cells in each group of antibody hu31 (3, 10, 30 mg / kg) are 50.0%, 56.7%, and 78.3%, respectively. Gr1 + According to the results of calculating the inhibition rate of the number of cells, the average inhibition rate of Gr1 + cells in each group of antibody hu31 (3, 10, 30 mg / kg) are 59.1%, 54.5%, and 81.8%, respectively, indicating that antibody hu31 can dose-dependently inhibit the infiltration of total mouse cells and inflammatory cells induced by IL-17.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to IL-17A, comprising a heavy chain variable region (VH) containing HCDR1, HCDR2, and HCDR3, and a light chain variable region (VL) containing LCDR1, LCDR2, and LCDR3, wherein the amino acid sequences of said HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and the amino acid sequences of said LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, respectively, and the sequences of SEQ ID NO: 1, 2, 3, 13, 14, and 15 are according to the IMGT numbering system; or wherein the amino acid sequences of said HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively, and the amino acid sequences of said LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, respectively, and the sequences of SEQ ID NO: 10, 11, 12, 22, 23, and 24 are according to the Kabat numbering system; or wherein the amino acid sequences of said HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NO: 60, SEQ ID NO: 61, and SEQ ID NO: 62, respectively, and the amino acid sequences of said LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO: 63, SEQ ID NO: 64, and SEQ ID NO: 65, respectively, and the sequences of SEQ ID NO: 60, 61, 62, 63, 64, and 65 are according to the IMGT numbering system, an antibody or antigen-binding fragment thereof, characterized in that.

2. comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein said VH contains the amino acid sequence shown in SEQ ID NO: 25 and said VL contains the amino acid sequence shown in SEQ ID NO: 29; or The VH contains the amino acid sequence shown in SEQ ID NO: 28, and the VL contains the amino acid sequence shown in SEQ ID NO: 32; or The VH contains the amino acid sequence shown in SEQ ID NO: 33 or 35, and the VL contains the amino acid sequence shown in SEQ ID NO: 34; or The VH contains the amino acid sequence shown in SEQ ID NO: 35, and the VL contains the amino acid sequence shown in SEQ ID NO: 36; or The VH contains the amino acid sequence shown in SEQ ID NO: 37, and the VL contains the amino acid sequence shown in SEQ ID NO: 38 or 39; or The VH contains the amino acid sequence shown in SEQ ID NO: 40, and the VL contains the amino acid sequence shown in SEQ ID NO:

41. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that the antibody or antigen-binding fragment thereof is a mouse antibody, a chimeric antibody, or a humanized antibody.

4. The antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that the antibody is a complete antibody, and the antigen-binding fragment is selected from a single-chain antibody, a Fab antibody, a Fab' antibody, a (Fab')2 antibody, and a bispecific (multispecific) antibody.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, characterized in that the antibody or antigen-binding fragment thereof is of any IgG subtype.

6. The antibody or antigen-binding fragment thereof according to claim 5, characterized in that the antibody or antigen-binding fragment thereof is IgG1, IgG2, IgG3 or IgG4.

7. It contains a light chain (LC) and a heavy chain (HC), The amino acid sequence of the light chain is shown in SEQ ID NO: 43, and the amino acid sequence of the heavy chain is shown in SEQ ID NO: 42 or 44; or, The amino acid sequence of the light chain is shown in SEQ ID NO: 45, and the amino acid sequence of the heavy chain is shown in SEQ ID NO: 44; or, The amino acid sequence of the light chain is shown in SEQ ID NO: 47 or 48, and the amino acid sequence of the heavy chain is shown in SEQ ID NO: 46; or, The amino acid sequence of the light chain is shown in SEQ ID NO: 50, and the amino acid sequence of the heavy chain is shown in SEQ ID NO: 49, The antibody or antigen-binding fragment thereof according to claim 1, characterized in that.

8. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7.

9. The nucleotide sequences of the heavy and light chains of the isolated nucleic acid molecule are selected from the group consisting of SEQ ID NO: 51 and 52, SEQ ID NO: 53 and 52, SEQ ID NO: 53 and 54, SEQ ID NO: 55 and 56, SEQ ID NO: 55 and 57, and SEQ ID NO: 58 and 59, the isolated nucleic acid molecule according to claim 8.

10. An expression vector containing the isolated nucleic acid molecule according to claim 8 or 9.

11. The expression vector according to claim 10, wherein the expression vector is a recombinant vector.

12. A host cell transformed with the expression vector according to claim 10 or 11.

13. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, the isolated nucleic acid molecule according to claim 8 or 9, the expression vector according to claim 10 or 11, or the host cell according to claim 12, and a pharmaceutically acceptable vector or excipient.

14. Use of an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 7, an isolated nucleic acid molecule according to claim 8 or 9, an expression vector according to claim 10 or 11, a host cell according to claim 12, or a pharmaceutical composition according to claim 13, in the manufacture of a medicament for the treatment and / or prophylaxis of a disease or disorder induced by IL-17A.

15. Use according to claim 14, characterized in that the medicament is a medicament used for the treatment of arthritis, rheumatoid arthritis, psoriasis, ankylosing spondylitis, chronic obstructive pulmonary disease, systemic lupus erythematosus (SLE), lupus nephritis, asthma, multiple sclerosis or cystic fibrosis.

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