WO2019061962A1 - Anti-interleukin-17a antibody, preparation method therefor, and application thereof - Google Patents

Abstract

Provided is an anti-interleukin-17A (IL-17A) antibody, a preparation method therefor and an application thereof. Specifically, said antibody is an anti-IL-17A monoclonal antibody. Said antibody can be combined in a highly specific way with an IL-17A antigen, has high affinity and low immunogenicity, and is used for preparing drugs for preventing or treating diseases related to IL-17A, such as any kind of inflammations or autoimmune diseases.

Description

Anti-interleukin 17A antibody, preparation method and application thereof Technical field

The present invention relates to the field of medicine, and in particular to an antibody targeted to interleukin 17A (also referred to as IL-17), a preparation method thereof and use thereof.

Background technique

To date, six members of the IL-17 family have been identified: IL-17A (IL-17), IL-17B, IL-17C, IL-17D, IL-17E (IL-25), IL-17F. These interleukin-17 cytokines can bind to the corresponding receptors and mediate different inflammatory responses.

IL-17A was originally found to be secreted by activated CD4 ⁺ T cells. This type of characteristic T cell subset that secretes IL-17A is called Th17 cell. In addition to Th17 cells, cytotoxic CD8 ⁺ T cells (Tc17), γδT cells, natural killer T cells (NKT-17), and B cells can also express IL-17A under specific conditions. Innate immune cells, including monocytes, neutrophils, natural killer cells, and lymphoid tissue-like (Lti-like) cells can also produce IL-17A. Recently, in a study on trypanosome infection, it was found that B cells can also produce IL-17A. Some non-immune cells, such as intestinal pancreatic cells and intestinal epithelial cells, can also produce IL-17A under stress. Since Th17 cells are the most widely distributed in the body and have a wide range of effects in the inflammatory response, it is generally considered to be the main source of IL-17A. The congenital cells that produce IL-17A mainly participate in the host's anti-infective immune response as the early defense cells of the body.

IL-17A is a homodimer that is linked by a disulfide bond from two chains of 155 amino acids with a molecular weight of 35 kDa. The structure of IL-17 consists of a signal peptide (AA) consisting of 23 amino acids and a region of 123 amino acid chains.

The receptor for IL-17 binding to the type I cell surface is called IL-17R, and there are at least three of them: IL-17RA, IL-17RB and IL-17RC. IL-17A and IL-17F bind to IL-17RA and IL-17RC receptor complexes in the form of homodimers or heterodimers to transduce signals and participate in autoimmune diseases, multiple inflammatory responses, and The host is resistant to an immune response. IL-17C binds to IL-17RA and IL-17RE receptor complexes to activate downstream signaling and promotes anti-infective immunity, autoimmune diseases and inflammatory responses. IL-17B was found to bind IL-17RB, but its downstream signal remains unclear. IL-17RB also forms a receptor complex with IL-17RA to mediate IL-17E causing a type II immune response. Il-17E has also been reported to promote apoptosis in tumor cells. The receptor and downstream signals of IL-17D and the ligand and downstream signals of the orphan receptor IL-17RD are still unclear.

IL-17A primarily induces signal activation of non-hematopoietic-derived cells including epithelial cells and stromal cells. IL-17A induces the expression of a variety of inflammatory factors and chemokines to promote the recruitment of a variety of immune cells, thereby promoting autoimmune diseases. Studies have found that IL-17A and IL-17F, and their major secretory T cell subsets, Th17 cells also play important roles in a variety of autoimmune diseases, including autoimmune diseases such as rheumatoid arthritis (rheumatoid). Arthritis, RA) and multiple sclerosis (MS), as well as inflammatory boweldise (IBD), psoriasis, systemic lupus erythematosus (SLE), and type 1 diabetes ( Type1diabetes, T1D).

IL-17A and IL-17F exert their functions of promoting inflammatory response mainly by inducing target cells to express various inflammatory factors and chemokines. IL-17A binds to the cell surface receptor IL-17RA and recruits IL-17RC to form a heterodimer that mediates downstream signaling pathways. IL-17 binds to its receptor and activates TRAF6 (TNF-receptor associated factor 6). IL-17 shares the same transcriptional pathway as IL-1 and TNF, which activates NF-kB and three MAP (mitogen-activated protein) enzymes, including ERK1, ERK2, JNK, and p38. These pathways are found in both synovial and chondrocytes.

Therefore, in view of the role and function of IL-17A in various related diseases, there remains a need in the art to develop improved anti-IL-17A-specific antibodies suitable for treating patients.

Summary of the invention

It is an object of the present invention to provide an antibody against IL-17A, a process for its preparation and use.

In a first aspect of the invention, there is provided a heavy chain variable region of an antibody, the heavy chain variable region comprising the following three complementarity determining region CDRs:

CDR1 shown in SEQ ID NO: 7,

CDR2 shown in SEQ ID NO: 8, and

CDR3 shown in SEQ ID NO:9.

In another preferred embodiment, the amino acid sequence of any one of the above amino acid sequences further comprises at least one of optionally adding, deleting, modifying and/or substituting (eg, 1-3, preferably 1-2, more preferably 1) amino acid and a derivative sequence capable of retaining IL-17A binding affinity.

In another preferred embodiment, the heavy chain variable region further comprises a FR region of a human source or a FR region of a murine source.

In another preferred embodiment, the heavy chain variable region has the amino acid sequence set forth in SEQ ID NO: 1.

In another preferred embodiment, the heavy chain variable region has the amino acid sequence set forth in SEQ ID NO: 5.

In a second aspect of the invention, there is provided a heavy chain of an antibody having a heavy chain variable region according to the first aspect of the invention.

In another preferred embodiment, the heavy chain of the antibody further comprises a heavy chain constant region.

In another preferred embodiment, the heavy chain constant region is of human, murine or rabbit origin.

In a third aspect of the invention, there is provided a light chain variable region of an antibody, the light chain variable region comprising the following three complementarity determining region CDRs:

CDR1' shown in SEQ ID NO: 10,

The amino acid sequence is CDR2' of GAT, and

CDR3' shown in SEQ ID NO:11.

In another preferred embodiment, the amino acid sequence of any one of the above amino acid sequences further comprises at least one of optionally adding, deleting, modifying and/or substituting (eg, 1-3, preferably 1-2, more preferably 1) amino acid and a derivative sequence capable of retaining IL-17A binding affinity.

In another preferred embodiment, the light chain variable region further comprises a human FR region or a murine FR region.

In another preferred embodiment, the light chain variable region has the amino acid sequence set forth in SEQ ID NO: 2.

In another preferred embodiment, the light chain variable region has the amino acid sequence set forth in SEQ ID NO: 6.

In a fourth aspect of the invention, there is provided a light chain of an antibody having a light chain variable region according to the third aspect of the invention.

In another preferred embodiment, the light chain of the antibody further comprises a light chain constant region.

In another preferred embodiment, the light chain constant region is of human, murine or rabbit origin.

In a fifth aspect of the invention, an antibody is provided, the antibody having:

(1) a heavy chain variable region according to the first aspect of the invention; and/or

(2) a light chain variable region according to the third aspect of the invention;

Alternatively, the antibody has: a heavy chain according to the second aspect of the invention; and/or a light chain according to the fourth aspect of the invention.

In another preferred embodiment, the antibody for human IL-17A protein EC (preferably wild type) binding force ₅₀ 5-50ng / ml.

In another preferred embodiment, the antibody for human IL-17A protein EC (preferably wild type) binding force ₅₀ 12.6ng / ml.

In another preferred embodiment, the antibody is selected from the group consisting of an animal-derived antibody, a chimeric antibody, a humanized antibody, or a combination thereof.

In another preferred embodiment, the antibody is a diabody, or a single chain antibody.

In another preferred embodiment, the antibody is a monoclonal antibody.

In another preferred embodiment, the antibody is a partially or fully humanized monoclonal antibody.

In another preferred embodiment, the heavy chain variable region sequence of the antibody is as set forth in SEQ ID NO.: 1 or 5; and/or

The light chain variable region sequence of the antibody is set forth in SEQ ID NO.: 2 or 6.

In another preferred embodiment, the heavy chain variable region sequence of the antibody is set forth in SEQ ID NO.: 1; and the light chain variable region sequence of the antibody is set forth in SEQ ID NO.: 2.

In another preferred embodiment, the heavy chain variable region sequence of the antibody is set forth in SEQ ID NO.: 5; and the light chain variable region sequence of the antibody is set forth in SEQ ID NO.: 6.

In another preferred embodiment, the antibody is in the form of a drug conjugate.

In a sixth aspect of the invention, a recombinant protein is provided, the recombinant protein having:

(i) a heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, a light chain variable region according to the third aspect of the invention, a fourth invention according to the invention The light chain of the aspect, or the antibody of the fifth aspect of the invention;

(ii) an optional tag sequence that facilitates expression and/or purification.

In another preferred embodiment, the tag sequence comprises a 6His tag.

In another preferred embodiment, the recombinant protein (or polypeptide) comprises a fusion protein.

In another preferred embodiment, the recombinant protein is a monomer, a dimer, or a multimer.

In a seventh aspect of the invention, a CAR construct is provided, wherein the scFV segment of the monoclonal antibody antigen-binding region of the CAR construct is a binding region that specifically binds to IL-17A, and the scFv has a The heavy chain variable region of the first aspect of the invention and the light chain variable region of the third aspect of the invention.

In an eighth aspect of the invention, a recombinant immune cell is provided, the immune cell expressing an exogenous CAR construct according to the seventh aspect of the invention.

In another preferred embodiment, the immune cells are selected from the group consisting of NK cells, T cells.

In another preferred embodiment, the immune cells are from a human or non-human mammal (e.g., a mouse).

In a ninth aspect of the invention, there is provided an antibody drug conjugate comprising:

(a) an antibody portion selected from the group consisting of a heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, according to the third aspect of the invention Light chain variable region, a light chain according to the fourth aspect of the invention, or an antibody of the fifth aspect of the invention, or a combination thereof;

(b) a coupling moiety coupled to the antibody moiety, the coupling moiety being selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.

In another preferred embodiment, the antibody moiety is coupled to the coupling moiety via a chemical bond or linker.

According to a tenth aspect of the invention, there is provided a use of an active ingredient selected from the group consisting of the heavy chain variable region according to the first aspect of the invention, the weight according to the second aspect of the invention a light chain variable region according to the third aspect of the invention, a light chain according to the fourth aspect of the invention, or an antibody according to the fifth aspect of the invention, the recombinant according to the sixth aspect of the invention a protein, an immune cell according to the eighth aspect of the invention, the antibody drug conjugate according to the ninth aspect of the invention, or a combination thereof, wherein the active ingredient is used for (a) preparing a detection reagent or kit; / or (b) preparation of a medicament for preventing and/or treating an IL-17A-related disease.

In another preferred embodiment, the active ingredient is used to prevent and/or treat an IL-17A related disease.

In another preferred embodiment, the IL-17A-associated disease is selected from the group consisting of inflammation, autoimmune disease, or a combination thereof; preferably an autoimmune disease.

In another preferred embodiment, the disease is selected from the group consisting of psoriasis, psoriatic arthritis, ankylosing spondylitis, multiple sclerosis, inflammatory arthritis, or a combination thereof, preferably inflammatory arthritis.

In another preferred embodiment, the inflammatory arthritis is selected from the group consisting of osteoarthritis, rheumatoid arthritis, rheumatoid arthritis, or a combination thereof, preferably rheumatoid arthritis.

In another preferred embodiment, the antibody is in the form of a drug conjugate (ADC).

In another preferred embodiment, the detection reagent or kit is for diagnosing an IL-17A related disease.

In another preferred embodiment, the detection reagent or kit is for detecting IL-17A protein in a sample.

In another preferred embodiment, the detection reagent is a test piece.

In an eleventh aspect of the invention, there is provided a pharmaceutical composition comprising:

(i) an active ingredient, the active ingredient being selected from the group consisting of a heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, according to the third aspect of the invention a light chain variable region, a light chain according to the fourth aspect of the invention, or an antibody according to the fifth aspect of the invention, the recombinant protein according to the sixth aspect of the invention, according to the eighth aspect of the invention Immunocyte, an antibody drug conjugate according to the ninth aspect of the invention, or a combination thereof;

(ii) a pharmaceutically acceptable carrier.

In another preferred embodiment, the pharmaceutical composition is a liquid formulation.

In another preferred embodiment, the pharmaceutical composition is an injection.

In a twelfth aspect of the invention, a polynucleotide is provided, the polynucleotide encoding a polypeptide selected from the group consisting of:

(1) The heavy chain variable region according to the first aspect of the invention, the heavy chain according to the second aspect of the invention, the light chain variable region according to the third aspect of the invention, the fourth aspect of the invention The light chain of the aspect, or the antibody of the fifth aspect of the invention; or

(2) a recombinant protein according to the sixth aspect of the invention;

(3) A CAR construct according to the seventh aspect of the invention.

In a thirteenth aspect of the invention, the invention provides a vector comprising the polynucleotide of the twelfth aspect of the invention.

In another preferred embodiment, the vector comprises: a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, a mammalian cell virus such as an adenovirus, a retrovirus, or other vector.

According to a fourteenth aspect of the present invention, a genetically engineered host cell comprising the vector or genome according to the thirteenth aspect of the present invention, which is integrated with the twelfth aspect of the present invention, is provided Polynucleotide.

In a fifteenth aspect of the invention, there is provided a method of detecting IL-17A protein in a sample (including a diagnostic or non-diagnostic) in vitro, the method comprising the steps of:

(1) contacting the sample with an antibody according to the fifth aspect of the invention in vitro;

(2) Detecting whether an antigen-antibody complex is formed, wherein the formation of a complex means that IL-17A protein is present in the sample.

According to a sixteenth aspect of the invention, there is provided a test panel comprising: a substrate (support plate) and a test strip, the test strip comprising the antibody according to the fifth aspect of the invention or The immunoconjugate of the ninth aspect of the invention.

In a seventeenth aspect of the invention, a kit is provided, the kit comprising:

(1) a first container comprising the antibody of the fifth aspect of the invention; and/or

(2) a second container comprising a secondary antibody against the antibody of the fifth aspect of the invention;

Alternatively, the kit contains the test plate according to the sixteenth aspect of the invention.

According to an eighteenth aspect of the invention, a method for preparing a recombinant polypeptide, the method comprising:

(a) cultivating a host cell according to the fourteenth aspect of the invention under conditions suitable for expression;

(b) isolating the recombinant polypeptide from the culture, the recombinant polypeptide according to the fifth aspect of the invention or the recombinant protein according to the sixth aspect of the invention.

A nineteenth aspect of the invention provides a method of an IL-17A-associated disease, the method comprising: administering to a subject in need thereof an antibody according to the fifth aspect of the invention, antibody-drug coupling of the antibody Or a CAR-T cell expressing the antibody, or a combination thereof.

It is to be understood that within the scope of the present invention, the various technical features of the present invention and the various technical features specifically described hereinafter (as in the embodiments) may be combined with each other to form a new or preferred technical solution. Due to space limitations, we will not repeat them here.

Detailed ways

The present inventors have unexpectedly obtained an anti-IL-17A monoclonal antibody having extremely excellent affinity and specificity, and a humanized antibody obtained based on the antibody, through extensive and intensive research. Antibodies of the invention bind with high specificity can be IL-17A antigen, which has high affinity (ELISA assay which EC ₅₀ of about 15.4ng / ml), and significantly inhibit the binding of IL-17A and IL-17 receptor, and There are no visible side effects to the mammal itself. The present invention has been completed on this basis.

the term

As used herein, the term "conjugate" refers to a soluble receptor or fragment thereof or analog thereof, or an antibody or fragment thereof, or an analog thereof, that is capable of binding to a target. The "IL-17A conjugate" as used herein refers to an antibody or a fragment thereof or an analog thereof which specifically recognizes IL-17A and binds to IL-17A.

As used herein, the terms "administering" and "treating" mean that the exogenous drug, therapeutic, diagnostic, or composition is applied to an animal, human, subject, cell, tissue, organ, or biological fluid. "Administration" and "treatment" can refer to treatment, pharmacokinetics, diagnostics, research, and experimental methods. The treatment of the cells includes contact of the reagents with the cells, contact of the reagents with the fluid, and contact of the fluid with the cells. "Administration" and "treatment" also mean in vitro and ex vivo treatment by reagents, diagnostics, binding compositions or by another cell. "Treatment" when applied to a human, animal or research subject, refers to therapeutic treatment, prophylactic or preventive measures, research and diagnosis; includes IL-17A conjugates with humans or animals, subjects, cells, tissues, Contact with physiological compartments or physiological fluids.

As used herein, the term "treating" refers to the administration of a therapeutic agent for internal or external use to a patient, comprising any of the IL-17A conjugates of the invention, and compositions thereof, which have one or more symptoms of the disease, and are known Therapeutic agents have a therapeutic effect on these symptoms. Typically, the patient is administered in an amount (a therapeutically effective amount) of a therapeutic agent effective to alleviate the symptoms of one or more diseases.

As used herein, the term "optional" or "optionally" means that the subsequently described event or circumstance may occur but does not necessarily have to occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable region of a particular sequence may have, but is not required to be, one, two or three.

antibody

As used herein, the term "antibody" refers to an immunoglobulin that is a tetrapeptide chain structure formed by the joining of two identical heavy chains and two identical light chains by interchain disulfide bonds. The amino acid composition and sequence of the immunoglobulin heavy chain constant region are different, so the antigenicity is also different. Accordingly, immunoglobulins can be classified into five classes, or different types of immunoglobulins, namely, IgM, IgD, IgG, IgA, and IgE, and the heavy chain constant regions corresponding to different classes of immunoglobulins are respectively referred to as α. , δ, ε, γ, and μ. IgG stands for the most important class of immunoglobulins and can be divided into four subclasses due to differences in chemical structure and biological function: IgG1, IgG2, IgG3 and IgG4. Light chains are divided into kappa or lambda chains by the constant region. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.

The sequence of about 110 amino acids near the N-terminus of the antibody heavy and light chains varies greatly, being the variable region (V region); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region (C region). The variable region includes three hypervariable regions (HVR) and four relatively conserved FR regions (FR). The amino acid sequences of the four FRs are relatively conservative and are not directly involved in the binding reaction. The three hypervariable regions determine the specificity of the antibody, also known as the complementarity determining region (CDR). Each of the light chain variable region (LCVR) and the heavy chain variable region (HCVR) consists of three CDR regions and four FR regions, and the order from the amino terminus to the carboxy terminus is FR1, CDR1, FR2, CDR2. FR3, CDR3, FR4. The three CDR regions of the light chain, i.e., the light chain hypervariable region (LCDR), refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain, the heavy chain hypervariable region (HCDR), refer to HCDR1, HCDR2, and HCDR3. The CDR amino acid residues of the LCVR and HCVR regions of the antibodies or antigen-binding fragments of the invention conform to known Kabat numbering rules (LCDR1-3, HCDR2-3) in number and position, or in accordance with the kabat and chothia numbering rules (HCDR1) ). The four FR regions in the native heavy and light chain variable regions are generally in a beta-sheet configuration, joined by three CDRs forming a linker, and in some cases may form a partial beta sheet structure. The CDRs in each chain are closely joined together by the FR region and together with the CDRs of the other chain form the antigen binding site of the antibody. The amino acid sequence of the same type of antibody can be compared to determine which amino acids constitute the FR or CDR regions. The constant regions are not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, such as antibody-dependent cytotoxicity of the participating antibodies.

As used herein, the term "antigen-binding fragment" refers to a Fab fragment having an antigen-binding activity, a Fab' fragment, a F(ab')2 fragment, or a single Fv fragment. The Fv antibody contains the antibody heavy chain variable region, the light chain variable region, but no constant region, and has the smallest antibody fragment of the entire antigen binding site. In general, Fv antibodies also comprise a polypeptide linker between the VH and VL domains and are capable of forming the desired structure for antigen binding.

As used herein, the term "antigenic determinant" refers to a three-dimensional spatial site that is discrete on an antigen and that is recognized by an antibody or antigen-binding fragment of the invention.

The present invention encompasses not only intact antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies with other sequences. Accordingly, the invention also includes fragments, derivatives and analogs of the antibodies.

In the present invention, antibodies include murine, chimeric, humanized or fully human antibodies prepared by techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, including human and non-human portions, can be prepared using recombinant DNA techniques well known in the art.

As used herein, the term "monoclonal antibody" refers to an antibody that is secreted from a clone of a single cell source. Monoclonal antibodies are highly specific and target a single epitope. The cell may be a eukaryotic, prokaryotic or phage clonal cell line.

As used herein, the term "chimeric antibody" is obtained by splicing a V region gene of a murine antibody with a C region gene of a human antibody into a chimeric gene, and then inserting the vector into an antibody molecule expressed by the host cell. It not only retains the high specificity and affinity of the parental mouse antibody, but also enables its human Fc segment to effectively mediate biological effects.

The term "humanized antibody", as used herein, is a variable region engineered form of a murine antibody of the invention having a CDR region derived from (or substantially derived from) a non-human antibody, preferably a mouse monoclonal antibody. And FR regions and constant regions derived substantially from human antibody sequences; the CDR region sequences of the murine antibodies are grafted onto different types of human germline antibody framework sequences. Because CDR sequences are responsible for most of the antibody-antigen interactions, recombinant antibodies that mimic the properties of a particular naturally occurring antibody can be expressed by constructing an expression vector.

In the present invention, the antibody may be monospecific, bispecific, trispecific, or more multiple specificity.

In the present invention, the antibody of the present invention further includes a conservative variant thereof, which means that there are up to 10, preferably up to 8, more preferably up to 5, optimally compared to the amino acid sequence of the antibody of the present invention. Up to 3 amino acids are replaced by amino acids of similar or similar nature to form a polypeptide. These conservative variant polypeptides are preferably produced by amino acid substitution according to Table A.

Table A

Initial residue	Representative substitution	Preferred substitution
Ala(A)	Val; Leu; Ile	Val
Arg(R)	Lys; Gln; Asn	Lys
Asn(N)	Gln;His;Lys;Arg	Gln
Asp(D)	Glu	Glu
Cys(C)	Ser	Ser
Gln(Q)	Asn	Asn
Glu(E)	Asp	Asp
Gly(G)	Pro; Ala	Ala
His(H)	Asn; Gln; Lys; Arg	Arg
Ile(I)	Leu;Val;Met;Ala;Phe	Leu
Leu(L)	Ile;Val;Met;Ala;Phe	Ile
Lys(K)	Arg; Gln; Asn	Arg
Met(M)	Leu;Phe;Ile	Leu
Phe(F)	Leu;Val;Ile;Ala;Tyr	Leu
Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
Thr(T)	Ser	Ser
Trp(W)	Tyr;Phe	Tyr
Tyr(Y)	Trp;Phe;Thr;Ser	Phe
Val(V)	Ile; Leu; Met; Phe; Ala	Leu

anti-IL-17A antibody

As used herein, the term "IL-17A" generally refers to native or recombinant human IL-17A, as well as non-human homologs of human IL-17A. Unless otherwise indicated, the molar concentration of IL-17A was calculated using the molecular weight of the homodimer of IL-17A (eg, 30 KDa for human IL-17A).

As used herein, the term "human IL-17A (huIL-17A)" refers to the mature form of human IL-17A protein accession numbers NP-002180 and AAT22064 (ie residues 24-155), as well as its natural variants and polymorphisms. Sex.

The present invention provides a highly specific and high affinity antibody against IL-17A comprising a heavy chain and a light chain, the heavy chain comprising a heavy chain variable region (VH) amino acid sequence, the light chain comprising a light chain Variable region (VL) amino acid sequence.

Preferably, the respective CDRs of the heavy chain variable region (VH) amino acid sequence and the light chain variable region (VL) amino acid sequence are selected from the group consisting of:

A1) SEQ ID No.: 7;

A2) SEQ ID No.: 8;

A3) SEQ ID No.: 9;

A4) SEQ ID No.: 10;

A5) GAT;

A6) SEQ ID No.: 11;

A7) at least one (eg, 1-5, 1-3, preferably 1-2, and more preferably 1) amino acid of any one of the above amino acid sequences is added, deleted, modified, and/or substituted. A sequence with IL-17A binding affinity.

In another preferred embodiment, the sequence formed by adding, deleting, modifying and/or substituting at least one amino acid sequence preferably has a homology of at least 80%, preferably at least 85%, more preferably at least 90. %, optimally at least 95% of the amino acid sequence.

The antibody of the present invention may be a double-stranded or single-chain antibody, and may be selected from an animal-derived antibody, a chimeric antibody, a humanized antibody, more preferably a humanized antibody, a human-animal chimeric antibody, and more preferably a whole human. Sourced antibodies.

The antibody derivative of the present invention may be a single chain antibody, and/or an antibody fragment such as Fab, Fab', (Fab') ₂ or other known antibody derivatives in the field, and IgA, IgD, IgE. Any one or more of IgG and IgM antibodies or antibodies of other subtypes.

Wherein the animal is preferably a mammal, such as a mouse.

The antibody of the invention may be a murine antibody, chimeric antibody, humanized antibody, CDR grafted and/or modified antibody that targets human IL-17A.

In a preferred embodiment of the invention, any one or more of the above SEQ ID No.: 7, 8 and 9 or which have been added, deleted, modified and/or substituted for at least one amino acid have IL- The 17A binding affinity sequence is located in the CDR region of the heavy chain variable region (VH).

In a preferred embodiment of the invention, any one or more of the above SEQ ID No.: 10, amino acid sequence: GAT and SEQ ID No.: 11, or they are added, deleted, modified and/or A sequence having an IL-17A binding affinity that replaces at least one amino acid, is located in the CDR region of the light chain variable region (VL).

In a more preferred embodiment of the invention, the VH CDR1, CDR2, CDR3 are each independently selected from any one or more of SEQ ID No.: 7, 8, and 9, or they are added, deleted, modified And/or a sequence having at least one amino acid having IL-17A binding affinity; VL CDR1, CDR2, CDR3 are each independently selected from any one of SEQ ID No.: 10, amino acid sequence: GAT and SEQ ID No.: Or a sequence of several sequences, or a sequence having IL-17A binding affinity for addition, deletion, modification and/or substitution of at least one amino acid.

In the above aspect of the invention, the number of amino acids added, deleted, modified and/or substituted is preferably not more than 40%, more preferably not more than 35%, more preferably 1-33% of the total amino acid number of the initial amino acid sequence. More preferably, it is 5-30%, more preferably 10-25%, and still more preferably 15-20%.

In the present invention, the number of amino acids added, deleted, modified and/or substituted is usually 1, 2, 3, 4 or 5, preferably 1-3, more preferably 1-2. The best is 1.

Antibody preparation

Any method suitable for the production of monoclonal antibodies can be used to produce the anti-IL-17A antibodies of the invention. For example, an animal can be immunized with a linked or naturally occurring IL-17A homodimer or a fragment thereof. Suitable immunization methods can be used, including adjuvants, immunostimulants, repeated booster immunizations, and one or more routes can be used.

Any suitable form of IL-17 can be used as an immunogen (antigen) for the production of a non-human antibody specific for IL-17A, and the biological activity of the antibody is screened. The stimulating immunogen can be a full length mature human IL-17A, including a native homodimer, or a peptide containing a single/multiple epitope. The immunogen can be used alone or in combination with one or more immunogenic enhancers known in the art. The immunogen can be purified from natural sources or produced in genetically modified cells. The DNA encoding the immunogen can be genomic or non-genomic (e.g., cDNA) in its source. The DNA encoding the immunogen can be expressed using a suitable genetic vector including, but not limited to, an adenoviral vector, an adeno-associated viral vector, a baculovirus vector, a material, and a non-viral vector.

An exemplary method of producing an anti-human IL-17A antibody of the invention is described in Example 1.

The humanized antibody can be selected from any class of immunoglobulins, including IgM, IgD, IgG, IgA, and IgE. In the present invention, the antibody is an IgG antibody, and an IgG1 subtype is used. Optimization of the necessary constant domain sequences is readily achieved by screening antibodies using the biological assays described in the Examples below to produce the desired biological activity.

Likewise, any type of light chain can be used in the compounds and methods herein. In particular, kappa, lambda chains or variants thereof are useful in the compounds and methods of the invention.

An exemplary method of humanizing an anti-human IL-17A antibody of the invention is described in Example 5.

The sequence of the DNA molecule of the antibody or fragment thereof of the present invention can be obtained by a conventional technique such as PCR amplification or genomic library screening. In addition, the coding sequences of the light and heavy chains can also be fused together to form a single chain antibody.

Once the relevant sequences are obtained, the recombinant sequence can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it to a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.

In addition, synthetic sequences can be used to synthesize related sequences, especially when the fragment length is short. Usually, a long sequence of fragments can be obtained by first synthesizing a plurality of small fragments and then connecting them. The DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.

The invention also relates to vectors comprising the appropriate DNA sequences described above, as well as appropriate promoters or control sequences. These vectors can be used to transform appropriate host cells to enable them to express proteins.

The host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Preferred animal cells include, but are not limited to, CHO-S, CHO-K1, HEK-293 cells.

The step of transforming a host cell with recombinant DNA as described in the present invention can be carried out by techniques well known in the art. The obtained transformant can be cultured by a conventional method, and the transformant expresses the polypeptide encoded by the gene of the present invention. Depending on the host cell used, it is cultured under appropriate conditions using a conventional medium.

Typically, the resulting host cells are cultured under conditions suitable for expression of the antibody of the invention. Then using conventional immunoglobulin purification steps, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography, etc. The antibodies of the present invention are purified by conventional separation and purification means well known to those skilled in the art.

The resulting monoclonal antibodies can be identified by conventional means. For example, the binding specificity of a monoclonal antibody can be determined by immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

application

The invention provides the use of an antibody of the invention, for example, for the preparation of a diagnostic preparation, or for the preparation of a medicament for the prevention and/or treatment of a disease associated with IL-17A. The IL-17A-related diseases include inflammatory diseases, autoimmune diseases, and the like, including but not limited to psoriasis, psoriatic arthritis, ankylosing spondylitis, multiple sclerosis, inflammatory bowel disease (such as Crohn Disease, ulcerative colitis, etc.), osteoarthritis, rheumatoid arthritis (RA), rheumatoid arthritis or osteoporosis, inflammatory fibrosis (eg scleroderma, pulmonary fibrosis and sclerosis), Asthma (including allergic asthma), allergies, and cancer.

Pharmaceutical composition

The invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition comprising the above antibody or active fragment thereof or a fusion protein thereof or an ADC thereof or a corresponding CAR-T cell, and a pharmaceutically acceptable carrier. Generally, these materials can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium wherein the pH is usually from about 5 to about 8, preferably from about 6 to about 8, although the pH may be The nature of the formulation and the condition to be treated vary. The formulated pharmaceutical compositions can be administered by conventional routes including, but not limited to, intratumoral, intraperitoneal, intravenous, or topical administration.

The antibody of the present invention may also be a cell therapy for expression of a nucleotide sequence in a cell, for example, the antibody is used for chimeric antigen receptor T cell immunotherapy (CAR-T) and the like.

The pharmaceutical composition of the present invention can be directly used for binding to an IL-17A protein molecule, and thus can be used for the prevention and treatment of diseases associated with IL-17A. In addition, other therapeutic agents can be used simultaneously.

The pharmaceutical composition of the present invention contains a safe and effective amount (e.g., 0.001 to 99% by weight, preferably 0.01 to 90% by weight, more preferably 0.1 to 80% by weight) of the above-mentioned monoclonal antibody (or a conjugate thereof) of the present invention and pharmacy An acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should be matched to the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably prepared under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example from about 1 microgram per kilogram body weight to about 5 milligrams per kilogram body weight per day. In addition, the polypeptides of the invention may also be used with other therapeutic agents.

When a pharmaceutical composition is used, a safe and effective amount of the pharmaceutical composition is administered to the mammal, wherein the safe and effective amount is usually at least about 10 micrograms per kilogram of body weight, and in most cases no more than about 50 milligrams per kilogram of body weight. Preferably, the dose is from about 10 micrograms per kilogram of body weight to about 20 milligrams per kilogram of body weight. Of course, specific doses should also consider factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled physician.

Detection use and kit

The antibodies of the invention can be used in detection applications, for example for detecting samples, to provide diagnostic information.

In the present invention, the sample (sample) used includes cells, tissue samples, and biopsy specimens. The term "biopsy" as used herein shall include all types of biopsies known to those skilled in the art. Thus, the biopsy used in the present invention may include a tissue sample prepared, for example, by an endoscopic method or a puncture or needle biopsy of an organ.

Samples used in the present invention include fixed or preserved cell or tissue samples.

The invention also provides a kit comprising an antibody (or a fragment thereof) of the invention, and in a preferred embodiment of the invention, the kit further comprises a container, instructions for use, a buffer, and the like. In a preferred embodiment, the antibody of the invention may be immobilized on a test plate.

The main advantages of the invention

(a) The antibody of the present invention has excellent biological activity and specificity, and has high binding ability (EC ₅₀ can be as high as about 10-15 ng/ml by ELISA). In addition, IL-17A has a good binding affinity and can be used as an antibody targeting IL-17A.

(b) The humanized antibody of the present invention not only has better affinity with IL-17A but also has lower immunogenicity than the murine antibody.

(c) The antibody of the present invention can significantly inhibit the binding of IL-17A to the IL-17 receptor without any visible side effects to the mammal itself.

The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are not intended to limit the scope of the invention. Experimental methods in which the specific conditions are not indicated in the following examples are generally carried out according to the conditions described in conventional conditions, for example, Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturing conditions. The conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight and parts by weight.

Example 1 Mouse monoclonal antibody against human IL-17A - preparation method of original antibody

1.1 Preparation of hybridoma cells producing murine monoclonal antibodies

Firstly, human IL-17A protein was used as an antigen. After emulsification with adjuvant, BALB/c mice were subjected to multi-subcutaneous immunization, and the serum titer of the immunized mice was monitored. After the requirement, the mouse spleen cells and myeloma were taken (Sp2/ 0) Hybridomas polyclonal cells obtained by fusion of cells and screening by HAT.

1.2 Indirect ELISA - screening method for hybridoma cells

High specific binding polyclonal was screened by ELISA, monoclonal culture was performed, and highly specific monoclonal cells were screened by ELISA; IL-6 release assay was performed by HT1080 cells, and cell function was screened. The effect of the monoclonal cell line, and then the affinity and half-life were analyzed by the Biacore method, and finally the monoclonal cells expressing IL-17A were obtained.

experiment material:

Recombinant human IL-17A, Sino Biological, 12047-HNAS

experiment method:

Human IL-17A was prepared into 1μg/ml coating solution with CBS, 50μL/well was added to the enzyme standard plate, and coated at 2～8°C for more than 12 hours. The residual liquid of the plate was discarded, and 3% milk was added, 200 μL per well, room temperature. Closed for 1 hour. One well of not less than 200 μL of PBST was added to each well, and the hybridoma supernatant was diluted to 100 μg/ml, and 10 gradients were further diluted 10 times, and 100 μL/well was added to the plate. Incubate for 1 hour at room temperature, add not less than 200 μL of PBST per well, wash 4 times, and add 25,000-fold diluted HRP-conjugated goat anti-mouse IgG Fc (purchased from Jackson) with 3% milk-PBST, 100 μL/well . After incubating for 1 hour at room temperature, not less than 200 μL of PBST was added to each well, washed 6 times, and patted dry. Add TMB coloring solution to 100 μL per well. After reacting for 5 minutes at room temperature, the reaction was terminated by adding 2 M H ₂ SO ₄ , 50 μL/well. The enzyme-labeled plate on which the reaction was stopped was placed on a microplate reader, and the absorbance OD450 value was read at a wavelength of 450 nm.

test results:

Table 1. Comparison of hybridomas to human IL-17a binding activity

sample name	EC 50 (ng/mL)
1B1/7A2	310.9
1B1/7C8	214.6
7D6/5G2	173
7D6/5H8	194.4
7D6/6B11	210.1
7D6/6G11	226.4
4F9/4H1	42.41

14F10/9A1	34.42
14F10/9F6	32.79
1B1/8E1	268.9
1B1/8E5	448.2

As can be seen from Table 1, among the plurality of selected antibodies, the murine antibody produced by the hybridoma 14F10/9F6 has a higher binding activity to human IL-17a.

Example 2 Anti-IL-17A Antibody V-gene Sequence Cloning

The DNA sequence encoding the variable region of the mouse antibody expressed by hybridoma 14F10/9F6 was determined based on the 5' RACE technique. Briefly, gene-specific cDNAs for heavy and light chains were prepared using the SMART 5' RACE Synthesis Kit (TAKARA, No. 634859) according to the manufacturer's instructions. The PCR product was analyzed by agarose gel electrophoresis. The variable region size of both the heavy and light chains is approximately 500 base pairs. The amplified amplified PCR product obtained by the reaction was cloned into the vector pEASY-Blunt Simple plasmid (Beijing Full Gold, No. CB111-02) and transformed into Stellar E. coli competent cells (TAKARA, No. 636763). )in. Clones were screened by colony PCR with universal M13 forward or reverse primers, and 2-3 clones were selected from each reaction for DNA sequencing analysis. Each sequencing reaction result of each clone was analyzed using the Expasy-translate tool (http://web.expasy.org/translate/). The sequencing results showed that the sequence of the anti-IL17A antibody V region expressed by 14F10/9F6 was as follows:

IL17-HC5 SEQ ID NO:1

IL17-LC5 SEQ ID NO: 2

Wherein underlined are CDR1', CDR2', CDR3' (SEQ ID NO.: 10, amino acid sequence: GAT and SEQ ID NO.: 11).

Table 2: Murine anti-IL-17A antibody CDR sequences

Example 3 Construction and Expression of Chimeric Antibodies

3.1 Preparation of chimeric antibodies

Chimeric heavy and light chains were constructed by ligating PCR cloned mouse 14F10/9F6 VH and VL region cDNAs to human IgGl and k constant regions, respectively. The 5' and 3' ends of the mouse cDNA sequence were modified with PCR primers designed to add appropriate leader sequences to each strand and to increase restriction sites that enable cloning into the existing recombinant antibody expression vector pHB-Fc. . The pHB-Fc plasmid vector was prepared as follows: the pcDNA/HA-FLAG (Accession#: FJ524378) vector was used as the starting plasmid, and the restriction region EcoRI was followed by the constant region sequence of human IgG1 or k, endonuclease HindIII. The human cytomegalovirus (HCMV) promoter sequence (Accession#: X17403) was added to the front of the ampicillin tolerance gene, and the Chinese hamster glutamine synthetase gene was added in front of the HCMV promoter (Accession#: X03495). .

The host cell used for protein expression was CHO-K1 cells (Cat# CCL-61) purchased from ATCC. The cells were acclimated into CHO-K1 cells that were cultured in suspension in serum-free medium (EX-CELLTM 302) after a series of domestication steps. Using the cells, the constructed light and heavy chain recombinant expression plasmids were transferred into cells by electroporation. Put in the incubator for 3-5 days. The antibody concentration from the CHO-K1 transfection supernatant was measured by indirect ELISA. This shows that transfected CHO-K1 cells secrete approximately 30 mg/L of chimeric antibody.

Novartis humanized anti-IL-17 antibody (Novartis mAb) as a positive control was cloned according to the humanized sequence provided in US 7,807,155 B2 (AIN 457) and transiently transfected for expression.

3.2 Determination of chimeric antibodies

experiment material:

Recombinant human IL-17A, Sino Biological, 12047-HNAS

experiment method:

The method was the same as in Example 1. The chimeric antibody was used in place of the hybridoma supernatant, and the HRP-conjugated rabbit anti-human IgG Fc antibody (Luoyang Qiongtong Experimental Material Center) was used instead of HRP-conjugated goat anti-mouse IgG Fc. Human IL-17A binding activity.

test results:

Table 3. Comparison of chimeric antibodies against human IL-17A binding activity

sample	EC 50 (ng/ml)
Positive control (Novartis mAb)	35.11
Negative control (PBS)	-
Chimeric antibody	29.98

The results demonstrated that the chimeric antibody has stronger binding to human IL-17A than the murine antibody and the positive control.

Example 4 Immunological cross-reactivity of different species of chimeric antibodies

In this example, the antigen-antibody binding ability of the anti-IL-17A antibody to IL-17A of different species was determined by the ELSIA method.

experiment material:

Recombinant human IL-17A, Sino Biological, 12047-HNAS

Recombinant mouse IL-17A, Sino Biological, PBV10159R-010

Recombinant marmoset IL-17A, Sino Biological, 90287-CNAE-100

experiment method:

The method was the same as in Example 1. The chimeric antibody was used in place of the hybridoma supernatant, and the HRP-conjugated rabbit anti-human IgG Fc antibody (Luoyang Qiongtong Experimental Material Center) was used instead of HRP-conjugated goat anti-mouse IgG Fc. Human IL-17A, mouse IL-17A and simian IL-17A binding activity.

test results:

Table 4. Binding antibody to mouse IL-17A binding results

sample	EC 50 (ng/ml)
Positive control (Novartis mAb)	-
Negative control (PBS)	-
Chimeric antibody	-

Table 5. Binding antibody to human IL-17A binding results

sample	EC 50 (ng/ml)
Positive control (Novartis mAb)	35.11
Negative control (PBS)	-
Chimeric antibody	29.98

Table 6. Binding antibody to simian IL-17A binding results

sample	EC 50 (ng/ml)
Positive control (Novartis mAb)	34.1
Negative control (PBS)	-
Chimeric antibody	26.39

As can be seen from Tables 4-6, the chimeric antibody of the present invention binds to simian IL-17A in addition to human IL-17A, but does not bind to mouse IL-17A, and the chimeric antibody and The affinity of human and simian IL-17A was better than that of the positive control.

Example 5 Preparation of Humanized Antibody

The humanization of antibodies was carried out by the following method. Briefly, the variable chain sequences of antibodies are compared to the available sequences in the NCBI protein database, and by identification and analysis, the human framework on which the CDR-grafted heavy and light chains are suitably constructed is finally determined.

Based on subsequent testing and screening, one preferred FR region is the humanized FR region derived from hu-VH (SEQ ID NO: 3) and the light chain is hu-VL (SEQ ID NO: 4):

hu-VH SEQ ID NO:3

hu-VL SEQ ID NO: 4

During the modification, according to the amino acid residues conserved in the FR region of the human antibody and the important amino acid residues in the FR region of the antibody, a design site was designed, and the variable region of the antibody was subjected to humanized mutation design, and the PCR technique was used to amplify and construct the human. The point mutation antibody expression plasmid was derived. The humanized point mutant antibody expression plasmid was separately expressed by CHO-K1 (ATC C, NO. CCL-61) cells, and purified to obtain a humanized antibody protein. A humanized antibody with excellent performance was obtained by ELISA, receptor binding inhibition assay, Biacore and cell activity assay.

The VH and VL sequences of the humanized antibody are shown in SEQ ID NO.: 5 and 6, respectively:

IL17-HC5-1D4 SEQ ID NO: 5

IL17-LC5-1C5 SEQ ID NO:6

The experimental results show that the humanized antibody has better affinity and specificity than the murine antibody, see Example 6.

Example 6 Determination of Humanized Antibody

experiment material:

Recombinant human IL-17A, Sino Biological, 12047-HNAS

experiment method:

The method was the same as in Example 1. The hybridoma supernatant was replaced with a humanized antibody, and the HRP-conjugated rabbit anti-human IgG Fc antibody (Luoyang Qiongtong Experimental Material Center) was used instead of HRP-conjugated goat anti-mouse IgG Fc. Human IL-17A binding activity.

test results:

Table 7. Comparison of human IL-17A binding activity by humanized antibodies

sample	EC 50 (ng/ml)
Chimeric antibody	29.98

Negative control (PBS)	-
Humanized antibody	12.6

The results showed that, after humanization, the inventors unexpectedly obtained a humanized antibody which not only did not decrease in the binding activity of human IL-17A, but further improved. Murine antibody EC ₅₀ values of the humanized antibody is about 3 times. It was also found that the antibody of the present invention has a lower EC ₅₀ value and a stronger binding activity to human IL-17A than the positive control antibody Novartis mAb.

Example 7 Affinity Detection of Humanized Monoclonal Antibodies

In this example, the antigen-antibody binding kinetics and affinity were determined using the BIACORE method.

experiment material:

Recombinant human IL-17A, Sino Biological, 12047-HNAS

Amino Coupling Kit, GE, BR-1000-50

HBS-EP (10X), GE, BR-1006-69

Human Antibody Captrue Kit, GE, BR-1008-39

experiment method:

A human antibody capture antibody (Human Antibody Capture Antiboy) and an anti-human capture-CM5 chip (Anti-Human Capture-CM5 chip) were immobilized on an S-series sensor chip (Sereis S Sensor Chip CM5) using an amino coupling kit. The mixture was equilibrated at room temperature for 20 to 30 minutes, and the chip was loaded into the instrument. Dilute the antigen with equilibration buffer, dilute the antigen with a 10 ml initial dilution of 5 concentration gradients, and set 2 zero concentrations (ie equilibration buffer) and one replicate concentration (typically the lowest concentration repeat). The antibody sample was diluted to the experimental working concentration with an equilibration buffer and sealed at 2 to 8 °C. After the sample analysis is completed, the corresponding analysis program is used to analyze the data, confirming that there is no obvious reference binding, selecting the kinetics, 1:1 binding model (Kinetics, 1:1 binding modle), fitting analysis, and obtaining the power of the sample. Learning parameters.

test results:

Table 8. Affinity test results of humanized antibody and human IL-17a

sample	Ka (1/Ms)	Kd (1/s)	KD(M)
Positive control (Novartis mAb)	1.46E+06	1.61E-04	1.11E-10
Chimeric antibody	1.04E+06	1.01E-04	9.70E-11
Humanized antibody	9.56E+06	1.81E-04	1.90E-11

The affinity constant (KD(M)) of human IL-17A showed that the affinity of the humanized antibody of the present invention was comparable to that of the chimeric antibody, and both were nearly an order of magnitude higher than the positive control antibody, and had a stronger affinity.

In addition, the present inventors also measured the biological activity, antigen binding specificity, and in vivo efficacy test of humanized antibody cells, and found that the humanized antibody of the present invention has good biological activity and specificity, and can be significantly improved. Symptoms of IL-17A related diseases.

All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the the In addition, it should be understood that various modifications and changes may be made by those skilled in the art in the form of the present invention.

Claims (10)

1. A heavy chain variable region of an antibody, characterized in that said heavy chain variable region comprises the following three complementarity determining region CDRs:

   CDR1 shown in SEQ ID NO: 7,

   CDR2 shown in SEQ ID NO: 8, and

   CDR3 shown in SEQ ID NO:9.
2. A heavy chain of an antibody, characterized in that the heavy chain has the heavy chain variable region of claim 1.
3. A light chain variable region of an antibody, wherein the light chain variable region comprises the following three complementarity determining region CDRs:

   CDR1' shown in SEQ ID NO: 10,

   The amino acid sequence is CDR2' of GAT, and

   CDR3' shown in SEQ ID NO:11.
4. A light chain of an antibody, characterized in that the light chain has the light chain variable region of claim 3.
5. An antibody characterized in that the antibody has:

   (1) The heavy chain variable region of claim 1; and/or

   (2) The light chain variable region of claim 3;

   Alternatively, the antibody has: the heavy chain of claim 2; and/or the light chain of claim 4.
6. The antibody according to claim 5, wherein the antibody is selected from the group consisting of an animal-derived antibody, a chimeric antibody, a humanized antibody, or a combination thereof.
7. The antibody according to claim 5, wherein the heavy chain variable region sequence of the antibody is as shown in SEQ ID NO.: 1 or 5; and/or

   The light chain variable region sequence of the antibody is set forth in SEQ ID NO.: 2 or 6.
8. A recombinant protein characterized in that the recombinant protein has:

   (i) a heavy chain variable region according to claim 1, a heavy chain according to claim 2, a light chain variable region according to claim 3, a light chain according to claim 4, or The antibody of claim 5;

   (ii) an optional tag sequence that facilitates expression and/or purification.
9. A CAR construct, characterized in that the scFV segment of the monoclonal antibody antigen-binding region of the CAR construct is a binding region that specifically binds to IL-17A, and the scFv has the composition of claim 1. A heavy chain variable region and a light chain variable region according to claim 3.
10. Use of an active ingredient selected from the group consisting of the heavy chain variable region of claim 1, the heavy chain of claim 2, the light chain of claim 3 A light chain according to claim 4, an antibody according to claim 5, a recombinant protein according to claim 8, or a combination thereof, wherein the active ingredient is used for (a) preparation detection An agent or kit; and/or (b) a medicament for the prevention and/or treatment of a disease associated with IL-17A.