CN113735975A

CN113735975A - anti-IL-11R antibody and application thereof

Info

Publication number: CN113735975A
Application number: CN202111045651.9A
Authority: CN
Inventors: 周加滔; 陈苍沙; 万云凤; 曹生田; 徐愉林; 李文佳
Original assignee: Sunshine Lake Pharma Co Ltd
Current assignee: Guangdong HEC Pharmaceutical
Priority date: 2021-09-07
Filing date: 2021-09-07
Publication date: 2021-12-03
Anticipated expiration: 2041-09-07
Also published as: CN113735975B

Abstract

The invention relates to an anti-IL-11R antibody and application thereof, wherein the antibody has higher neutralization activity on IL-11R. Under the action of the anti-IL-11R antibody, the fibrosis of lung fibroblast HFL-1 can be effectively inhibited, and the anti-IL-11R antibody has good thermal stability and good clinical application prospect in the aspect of treating fibrosis-related diseases.

Description

anti-IL-11R antibody and application thereof

Technical Field

The invention relates to the technical field of biology, and particularly relates to an anti-IL-11R antibody and application thereof.

Background

Interleukin 11(IL-11) is a cytokine secreted by bone marrow stromal cells, and the human IL-11 gene is located in chromosome 19, the q13.3-q13.4 region. IL-11 is a member of the IL-6 cytokine family, which also includes IL-27, IL-31, Leukemia Inhibitory Factor (LIF), oncostatin M (OSM), ciliary neurotrophic factor (CNTF), and the like. IL-6 family cytokines induce signal transduction via the common signal transduction receptor beta subunit gp130 and a specific receptor alpha subunit. For IL-11, binding of this cytokine to its specific receptor IL-11Ra induces gp130 homodimerization and acts on effector cells to promote hematopoietic progenitor cell differentiation maturation, maintain epithelial cell homeostasis, stimulate the liver to produce polar phase reaction proteins with anti-inflammatory effects such as ferritin, C-reactive protein, etc., mainly by activating the JAK1/STAT3 (tyrosine protein kinase pathway) pathway. With the progress of research, the extensive cellular biological activities of IL-11 are still further discovered.

Fibrosis (fibrosis) can occur in many organs, and the major pathological changes are fibrous connective tissue increase in organ tissues, parenchymal cell reduction, and continued progression can lead to structural destruction and hypofunction of organs, or even failure, seriously threatening human health and life. Diseases characterized by excessive fibrosis include, but are not limited to: systemic sclerosis, scleroderma, hypertrophic cardiomyopathy, Dilated Cardiomyopathy (DCM), atrial fibrillation, ventricular fibrillation, myocarditis, liver cirrhosis, nephropathy, eye diseases, asthma, cystic fibrosis, arthritis, idiopathic pulmonary fibrosis and the like. Despite the great impact on human health, therapeutic and diagnostic methods for fibrosis remain an unmet medical need. The patent document EP 3428188A 1 discloses a new function of IL-11 for the treatment of pulmonary fibrosis, before which TGF-beta1 was considered as a key factor mainly inducing fibrosis (Leask, A.and Abraham, D.J. (2004), TGF-beta signaling and the fibrous stress, the FASEB Journal,18: 816-. There are several companies that are developing globally for drugs that inhibit TGF-beta1 and other targets.

However, few drugs for treating fibrosis are available on the market, for example, only two drugs of pirfenidone and nintedanib are currently available on the market for treating pulmonary fibrosis diseases, and the curative effects of the two drugs are not ideal. There is a great need in the art to develop new drugs to address this dilemma.

Disclosure of Invention

The invention aims to provide a high-affinity anti-IL-11R antibody and application thereof, wherein the antibody has high neutralizing activity on IL-11R and can be used for treating fibrosis-related diseases.

To this end, in a first aspect, the present invention provides an anti-IL-11R antibody comprising a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 and a light chain variable region comprising LCDR1, LCDR2 and LCDR 3;

wherein the amino acid sequences of the HCDR1, HCDR2 and HCDR3 are selected from the group consisting of:

HCDR1：GFTFSSYT(SEQ ID NO：28)，HCDR2：ISSGGSYT(SEQ ID NO：29)，HCDR3：AREDYDGFAY(SEQ ID NO：30)；

alternatively, HCDR 1: GFTFSDFY (SEQ ID NO: 31), HCDR 2: SRDNTKDYTT (SEQ ID NO: 32), HCDR 3: ARAHYYGFGAMDY (SEQ ID NO: 33);

alternatively, HCDR 1: GFTFSSSA (SEQ ID NO: 34), HCDR 2: VSSGGTYT (SEQ ID NO: 35), HCDR 3: ARHSPDDGYFVDY (SEQ ID NO: 36);

alternatively, HCDR 1: GFIFSDYG (SEQ ID NO: 37), HCDR 2: ISNLAYSF (SEQ ID NO: 38), HCDR 3: VRADEGLGY (SEQ ID NO: 39);

alternatively, HCDR 1: GYSFTGYT (SEQ ID NO: 40), HCDR 2: INPDNGGI (SEQ ID NO: 41), HCDR 3: AINYYGLDY (SEQ ID NO: 42);

alternatively, HCDR 1: GFNIKNYY (SEQ ID NO: 43), HCDR 2: VDPENGNT (SEQ ID NO: 44), HCDR 3: VLYRYGFAY (SEQ ID NO: 45);

alternatively, HCDR 1: GYAFTNYL (SEQ ID NO: 46), HCDR 2: ISPDNGNT (SEQ ID NO: 47), HCDR 3: ARNRYGIDS (SEQ ID NO: 48);

alternatively, HCDR 1: GYTFRNYG (SEQ ID NO: 49), HCDR 2: INTFTGEP (SEQ ID NO: 50), HCDR 3: AREELRSGHYGFAC (SEQ ID NO: 51);

alternatively, HCDR 1: GFTLSSYT (SEQ ID NO: 52), HCDR 2: ISSGGSYI (SEQ ID NO: 53), HCDR 3: TRDLGNDDFTYYFDS (SEQ ID NO: 54);

alternatively, HCDR 1: GFTFSDFY (SEQ ID NO: 31), HCDR 2: SRDKAKDYTT (SEQ ID NO: 55), HCDR 3: ARVHYYGFGAMDY (SEQ ID NO: 56);

alternatively, HCDR 1: GFTFSYYA (SEQ ID NO: 57), HCDR 2: ISSSGRYT (SEQ ID NO: 58), HCDR 3: ARTDGYYPDY (SEQ ID NO: 59);

alternatively, HCDR 1: GYAFTNYL (SEQ ID NO: 46), HCDR 2: ISPDNGNT (SEQ ID NO: 47), HCDR 3: ARNRYGIDY (SEQ ID NO: 60);

wherein the amino acid sequences of the LCDR1, LCDR2 and LCDR3 are selected from the group consisting of:

LCDR1：QSLVFSNGNTY(SEQ ID NO：61)，LCDR2：KVS(SEQ ID NO：62)，LCDR3：SQMTHVPYT(SEQ ID NO：63)；

alternatively, LCDR 1: QSVDYYGDSY (SEQ ID NO: 64), LCDR 2: AAS (SEQ ID NO: 65), LCDR 3: QQSNEDPWT (SEQ ID NO: 66);

alternatively, LCDR 1: QNIVHSNGNTY (SEQ ID NO: 67), LCDR 2: KVS (SEQ ID NO: 62), LCDR 3: FQGSHVPPT (SEQ ID NO: 68);

alternatively, LCDR 1: QTLVDSNVNNY (SEQ ID NO: 69), LCDR 2: KVS (SEQ ID NO: 62), LCDR 3: SQSTHVPWT (SEQ ID NO: 70);

alternatively, LCDR 1: QSIVHSTGVTY (SEQ ID NO: 71), LCDR 2: RVS (SEQ ID NO: 72), LCDR 3: FQGSHVPVT (SEQ ID NO: 73);

alternatively, LCDR 1: TGAVTTSNY (SEQ ID NO: 74), LCDR 2: GTN (SEQ ID NO: 75), LCDR 3: VLWHSNHWV (SEQ ID NO: 76);

alternatively, LCDR 1: KSLLHSNGITY (SEQ ID NO: 77), LCDR 2: QMS (SEQ ID NO: 78), LCDR 3: AQNLELPHT (SEQ ID NO: 79);

alternatively, LCDR 1: QSIVFSNGITY (SEQ ID NO: 80), LCDR 2: KVS (SEQ ID NO: 62), LCDR 3: FQGSHVPPT (SEQ ID NO: 68);

alternatively, LCDR 1: QDINNY (SEQ ID NO: 81), LCDR 2: YTS (SEQ ID NO: 82), LCDR 3: QQGKTFPT (SEQ ID NO: 83);

alternatively, LCDR 1: QSVDYDGDSY (SEQ ID NO: 84), LCDR 2: AAS (SEQ ID NO: 65), LCDR 3: QQSNEDPWT (SEQ ID NO: 66);

alternatively, LCDR 1: QSIVHSNGNTY (SEQ ID NO: 85), LCDR 2: KVS (SEQ ID NO: 62), LCDR 3: FQGSHVPPT (SEQ ID NO: 68);

alternatively, LCDR 1: KSLLHNNGITY (SEQ ID NO: 86), LCDR 2: QMS (SEQ ID NO: 78), LCDR 3: AQNLELPHT (SEQ ID NO: 79);

alternatively, LCDR 1: QSVDYYGDNY (SEQ ID NO: 87), LCDR 2: AAS (SEQ ID NO: 65), LCDR 3: QQSNEDPWT (SEQ ID NO: 66).

According to the anti-IL-11R antibody provided by the invention, the amino acid sequences of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are analyzed according to Kabat standards.

In some embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are selected from the group consisting of:

HCDR1：GFTFSSYT(SEQ ID NO：28)，HCDR2：ISSGGSYT(SEQ ID NO：29)，HCDR3：AREDYDGFAY(SEQ ID NO：30)，LCDR1：QSLVFSNGNTY(SEQ ID NO：61)，LCDR2：KVS(SEQ ID NO：62)，LCDR3：SQMTHVPYT(SEQ ID NO：63)；

alternatively, HCDR 1: GFTFSDFY (SEQ ID NO: 31), HCDR 2: SRDNTKDYTT (SEQ ID NO: 32), HCDR 3: ARAHYYGFGAMDY (SEQ ID NO: 33), LCDR 1: QSVDYYGDSY (SEQ ID NO: 64), LCDR 2: AAS (SEQ ID NO: 65), LCDR 3: QQSNEDPWT (SEQ ID NO: 66);

alternatively, HCDR 1: GFTFSSSA (SEQ ID NO: 34), HCDR 2: VSSGGTYT (SEQ ID NO: 35), HCDR 3: ARHSPDDGYFVDY (SEQ ID NO: 36), LCDR 1: QNIVHSNGNTY (SEQ ID NO: 67), LCDR 2: KVS (SEQ ID NO: 62), LCDR 3: FQGSHVPPT (SEQ ID NO: 68);

alternatively, HCDR 1: GFIFSDYG (SEQ ID NO: 37), HCDR 2: ISNLAYSF (SEQ ID NO: 38), HCDR 3: VRADEGLGY (SEQ ID NO: 39), LCDR 1: QTLVDSNVNNY (SEQ ID NO: 69), LCDR 2: KVS (SEQ ID NO: 62), LCDR 3: SQSTHVPWT (SEQ ID NO: 70);

alternatively, HCDR 1: GYSFTGYT (SEQ ID NO: 40), HCDR 2: INPDNGGI (SEQ ID NO: 41), HCDR 3: AINYYGLDY (SEQ ID NO: 42), LCDR 1: QSIVHSTGVTY (SEQ ID NO: 71), LCDR 2: RVS (SEQ ID NO: 72), LCDR 3: FQGSHVPVT (SEQ ID NO: 73);

alternatively, HCDR 1: GFNIKNYY (SEQ ID NO: 43), HCDR 2: VDPENGNT (SEQ ID NO: 44), HCDR 3: VLYRYGFAY (SEQ ID NO: 45), LCDR 1: TGAVTTSNY (SEQ ID NO: 74), LCDR 2: GTN (SEQ ID NO: 75), LCDR 3: VLWHSNHWV (SEQ ID NO: 76);

alternatively, HCDR 1: GYAFTNYL (SEQ ID NO: 46), HCDR 2: ISPDNGNT (SEQ ID NO: 47), HCDR 3: ARNRYGIDS (SEQ ID NO: 48), LCDR 1: KSLLHSNGITY (SEQ ID NO: 77), LCDR 2: QMS (SEQ ID NO: 78), LCDR 3: AQNLELPHT (SEQ ID NO: 79);

alternatively, HCDR 1: GYTFRNYG (SEQ ID NO: 49), HCDR 2: INTFTGEP (SEQ ID NO: 50), HCDR 3: AREELRSGHYGFAC (SEQ ID NO: 51), LCDR 1: QSIVFSNGITY (SEQ ID NO: 80), LCDR 2: KVS (SEQ ID NO: 62), LCDR 3: FQGSHVPPT (SEQ ID NO: 68);

alternatively, HCDR 1: GFTLSSYT (SEQ ID NO: 52), HCDR 2: ISSGGSYI (SEQ ID NO: 53), HCDR 3: TRDLGNDDFTYYFDS (SEQ ID NO: 54), LCDR 1: QDINNY (SEQ ID NO: 81), LCDR 2: YTS (SEQ ID NO: 82), LCDR 3: QQGKTFPT (SEQ ID NO: 83);

alternatively, HCDR 1: GFTFSDFY (SEQ ID NO: 31), HCDR 2: SRDKAKDYTT (SEQ ID NO: 55), HCDR 3: ARVHYYGFGAMDY (SEQ ID NO: 56), LCDR 1: QSVDYDGDSY (SEQ ID NO: 84), LCDR 2: AAS (SEQ ID NO: 65), LCDR 3: QQSNEDPWT (SEQ ID NO: 66);

alternatively, HCDR 1: GFTFSYYA (SEQ ID NO: 57), HCDR 2: ISSSGRYT (SEQ ID NO: 58), HCDR 3: ARTDGYYPDY (SEQ ID NO: 59), LCDR 1: QSIVHSNGNTY (SEQ ID NO: 85), LCDR 2: KVS (SEQ ID NO: 62), LCDR 3: FQGSHVPPT (SEQ ID NO: 68);

alternatively, HCDR 1: GYAFTNYL (SEQ ID NO: 46), HCDR 2: ISPDNGNT (SEQ ID NO: 47), HCDR 3: ARNRYGIDY (SEQ ID NO: 60), LCDR 1: KSLLHNNGITY (SEQ ID NO: 86), LCDR 2: QMS (SEQ ID NO: 78), LCDR 3: AQNLELPHT (SEQ ID NO: 79);

alternatively, HCDR 1: GFTFSDFY (SEQ ID NO: 31), HCDR 2: SRDNTKDYTT (SEQ ID NO: 32), HCDR 3: ARAHYYGFGAMDY (SEQ ID NO: 33), LCDR 1: QSVDYYGDNY (SEQ ID NO: 87), LCDR 2: AAS (SEQ ID NO: 65), LCDR 3: QQSNEDPWT (SEQ ID NO: 66).

In some embodiments, the amino acid sequence of the heavy chain variable region is as set forth in any one of the following groups: SEQ ID NO: 1-13.

In some embodiments, the light chain variable region has the amino acid sequence set forth in any one of the following groups: SEQ ID NO: 14-26.

In one embodiment, the amino acid sequence of the heavy chain variable region is SEQ ID NO: 1, the amino acid sequence of the light chain variable region is ID NO: 14.

in one embodiment, the amino acid sequence of the heavy chain variable region is SEQ ID NO: 2, the amino acid sequence of the light chain variable region is ID NO: 15.

in one embodiment, the amino acid sequence of the heavy chain variable region is SEQ ID NO: 3, the amino acid sequence of the light chain variable region is ID NO: 16.

in one embodiment, the amino acid sequence of the heavy chain variable region is SEQ ID NO: 4, the amino acid sequence of the light chain variable region is ID NO: 17.

in one embodiment, the amino acid sequence of the heavy chain variable region is SEQ ID NO: 5, the amino acid sequence of the light chain variable region is ID NO: 18.

in one embodiment, the amino acid sequence of the heavy chain variable region is SEQ ID NO: 6, the amino acid sequence of the light chain variable region is ID NO: 19.

in one embodiment, the amino acid sequence of the heavy chain variable region is SEQ ID NO: 7, the amino acid sequence of the light chain variable region is ID NO: 20.

in one embodiment, the amino acid sequence of the heavy chain variable region is SEQ ID NO: 8, the amino acid sequence of the light chain variable region is ID NO: 21.

in one embodiment, the amino acid sequence of the heavy chain variable region is SEQ ID NO: 9, the amino acid sequence of the light chain variable region is ID NO: 22.

in one embodiment, the amino acid sequence of the heavy chain variable region is SEQ ID NO: 10, the amino acid sequence of the light chain variable region is ID NO: 23.

in one embodiment, the amino acid sequence of the heavy chain variable region is SEQ ID NO: 11, the amino acid sequence of the light chain variable region is ID NO: 24.

in one embodiment, the amino acid sequence of the heavy chain variable region is SEQ ID NO: 12, the amino acid sequence of the light chain variable region is ID NO: 25.

in one embodiment, the amino acid sequence of the heavy chain variable region is SEQ ID NO: 13, the amino acid sequence of the light chain variable region is ID NO: 26.

in some embodiments, the anti-IL-11R antibody is a full-length antibody, a Fab fragment, F (ab)₂Fragments, two-chain Fv fragments or single-chain Fv fragments

In some embodiments, the anti-IL-11R antibody is a monoclonal antibody.

In some embodiments, the anti-IL-11R antibody is of the IgG1, IgG2a, IgG2b, or IgG3 subtype.

In some embodiments, the anti-IL-11R antibody further comprises a heavy chain constant region selected from the IgG1, IgG2, IgG3, or IgG4 subtype.

In certain embodiments, the heavy chain constant region is of the IgG1 subtype.

In one embodiment, the heavy chain constant region comprises the amino acid sequence as set forth in SEQ ID NO: 88, or a pharmaceutically acceptable salt thereof.

In some embodiments, the anti-IL-11R antibody further comprises a light chain constant region selected from a Kappa or Lambda subtype.

In certain embodiments, the light chain constant region is of the Kappa subtype.

In one embodiment, the light chain constant region comprises the amino acid sequence set forth as SEQ ID NO: 89.

In a second aspect of the invention, there is provided a nucleic acid molecule encoding an anti-IL-11R antibody according to the invention.

In a third aspect of the invention, there is provided a vector comprising a nucleic acid molecule according to the invention.

In a fourth aspect of the invention, there is provided a host cell comprising a nucleic acid molecule according to the invention, or comprising a vector according to the invention.

In a fifth aspect of the invention, there is provided a pharmaceutical composition comprising an anti-IL-11R antibody of the invention and a pharmaceutically acceptable carrier; wherein the anti-IL-11R antibody of the invention is present in a therapeutically effective amount.

In a sixth aspect of the present invention, there is provided a method for producing an anti-IL-11R antibody according to the present invention, comprising the steps of: allowing the host cell according to the fourth aspect of the present invention to express the anti-IL-11R antibody according to the present invention, and isolating and purifying to obtain the anti-IL-11R antibody.

In a seventh aspect of the invention, there is provided the use of an anti-IL-11R antibody as described herein in the manufacture of a medicament for the prevention, alleviation or treatment of a disease or condition having a fibrotic disorder.

In some embodiments, the fibrotic disorder is a symptom of fibrotic hyperplasia in a tissue or organ of the subject.

In some embodiments, the fibrotic disorder is a fibrotic disorder selected from liver, biliary, lung, kidney, bladder, heart, blood vessel, eye, skin, pancreas, gastrointestinal, bone marrow, penis, breast, or muscle.

In some embodiments, the disease with fibrotic disorders includes silicosis, asbestosis, coallung, tuberculosis, viral pneumonia, pneumocystis infection, secondary lung disease, idiopathic interstitial pneumonia (including idiopathic pulmonary fibrosis, etc.), bronchiolitis obliterans complicated with organizing pneumonia, pulmonary lymphangioleiomyomatosis, systemic lupus erythematosus, rheumatoid arthritis, progressive systemic sclerosis, polymyositis, dermatomyositis, mixed connective tissue disease, diffuse alveolar hemorrhage syndrome, pulmonary alveolar proteinosis, eosinophilic pneumonia, pulmonary vasculitis, lymphocytic interstitial pneumonia, ischemic heart disease, hypertensive heart disease, viral myocarditis, hemochromatosis cardiomyopathy, amyloidosis, glycogen-accumulating cardiomyopathy, diabetic cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, myocardial infarction, pulmonary inflammation, systemic lupus erythematosus, chronic inflammatory bowel disease, chronic inflammatory, Liver cirrhosis (including viral cirrhosis, schistosomiasis cirrhosis, alcoholic cirrhosis, biliary cirrhosis, metabolic cirrhosis, toxic cirrhosis, dystrophic cirrhosis, cardiogenic cirrhosis, etc.), acute pancreatitis, pancreatic duct obstruction, glomerulonephritis, pyelonephritis, renal calculus, hyperuricemia, hypercalciuria, spleen fibroproliferative disease, retinal fibroplasia, myelofibrosis, etc.

Compared with the prior art, the technical scheme of the invention has the following advantages: the invention provides an anti-IL-11R antibody, which has higher neutralizing activity on IL-11R through binding activity detection and competitive blocking activity detection. Under the action of the anti-IL-11R antibody provided by the invention, the fibrosis of lung fibroblast HFL-1 can be effectively inhibited, and the anti-IL-11R antibody has good thermal stability and good clinical application prospect in the aspect of treating fibrosis related diseases.

Drawings

Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The drawings are only for purposes of illustrating the preferred embodiments and are not to be construed as limiting the invention. In the drawings:

FIG. 1: the result of measuring the binding activity of the anti-IL-11R antibody to IL-11R;

FIG. 2: results of detection of competitive blocking activity of anti-IL-11R antibody;

FIG. 3: western Blot detection result of the expression ACTA of the TGF-beta induced HFL-1 cells under the action of anti-IL-11R antibody;

FIG. 4: western Blot detection results of the expression of ACTA by the TGF-beta induced HFL-1 cells under the action of anti-IL-11R antibodies with the concentrations of 5 mu g/mL and 25 mu g/mL;

FIG. 5: a graph showing the results of measuring the binding affinity of the anti-IL-11R antibody to IL-11R;

FIG. 6: the results of the thermostability assay for anti-IL-11R antibodies are shown.

Detailed Description

Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.

The examples herein employ, unless otherwise indicated, molecular biology, microbiology, cell biology, biochemistry, and immunology techniques which are conventional in the art.

Unless otherwise indicated, terms used in the present application have meanings commonly understood by those skilled in the art.

The term "antibody" as used herein refers to a polypeptide capable of being attached to an immunoglobulin molecule via at least one variable region located in the immunoglobulin moleculeThe antigen recognition site in (a) specifically binds to an immunoglobulin molecule of a target. "antibody" as used herein includes not only intact (i.e., full-length) antibodies, but also antigen-binding fragments thereof (e.g., Fab, F (ab)₂Fv), variants thereof, fusion proteins comprising an antibody portion, humanized antibodies, chimeric antibodies, diabodies, linear antibodies, single chain antibodies, multispecific antibodies (e.g., bispecific antibodies), and any other modified configuration of an immunoglobulin molecule comprising an antigen recognition site of a desired specificity, including glycosylated variants of an antibody, amino acid sequence variants of an antibody, and covalently modified antibodies.

Typically, a complete or full-length antibody comprises two heavy chains and two light chains. Each heavy chain contains a heavy chain variable region (VH) and a heavy chain constant region (CH). Each light chain contains a light chain variable region (VL) and a light chain constant region (CL). Full-length antibodies can be of any class, such as IgD, IgE, IgG, IgA, or IgM (or subclasses thereof), but the antibodies need not belong to any particular class. Depending on the antibody amino acid sequence of the constant domain of the heavy chain, immunoglobulins can be assigned to different classes. Generally, there are five main classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these classes can be further classified into subclasses, such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA 2.

As used herein, the term "antigen-binding portion" refers to a portion or region of an intact antibody molecule that is responsible for binding an antigen. The antigen-binding portion may comprise a heavy chain variable region (VH), a light chain variable region (VL), or both. It is well known to those skilled in the art that each of VH and VL typically contains three complementarity determining regions CDR1, CDR2, and CDR3, which are the regions of the variable region that have the greatest impact on the affinity and specificity of an antibody. For a given antibody variable region amino acid sequence, the variable region amino acid sequence of the middle CDR amino acid sequence can be obtained by analysis in a manner known in the art.

As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide may be inserted. When a vector allows the expression of a protein encoded by a polynucleotide inserted therein, the vector is referred to as an expression vector. The vector may be used to express the carried genetic material element in a host cell by transformation, transduction, or transfection into the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, phages, cosmids, artificial chromosomes such as Yeast Artificial Chromosomes (YACs), Bacterial Artificial Chromosomes (BACs) or P1-derived artificial chromosomes (PACs); bacteriophages such as lambda bacteriophage or M13 bacteriophage and animal viruses. Animal viruses that may be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, papovaviruses (e.g., SV 40). The vector may contain a number of elements for controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may comprise an origin of replication.

As used herein, "host cell" refers to a cell into which an exogenous nucleic acid has been introduced, including progeny of such a cell. Host cells include the initially transformed cells and progeny derived therefrom (irrespective of the number of passages). Progeny may not be identical to the parent cell in nucleic acid content, but may contain mutations. Included herein are mutant progeny that have the same function or biological activity as screened or selected in the originally transformed cell. Host cells are any type of cell system that can be used to produce the fusion proteins of the invention. Host cells include cultured mammalian cells such as CHO cells, BHK cells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER. C6 cells or hybridoma cells, yeast cells, bacterial cells such as E.coli, insect cells and plant cells, etc., and also include cells contained in transgenic animals, transgenic plants or cultured plants or animal tissues.

Herein, a "pharmaceutical composition" refers to a formulation in a form that allows the biological activity of the active ingredient contained therein to be effective, and that is free of other ingredients that have unacceptable toxicity to a subject that will receive administration of the pharmaceutical composition.

As used herein, a "therapeutically effective amount" refers to an amount effective to achieve a desired therapeutic or prophylactic result, including, for example, eliminating, reducing, delaying, minimizing, or preventing an adverse effect of a disease.

Herein, "pharmaceutically acceptable carrier" refers to an ingredient in the pharmaceutical composition other than the active ingredient that is not toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

Herein, "treatment" refers to an attempt to alter the natural course of disease in the treated individual, and may be a clinical intervention performed for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, slowing the rate of disease progression, ameliorating or eliminating the disease state, and regression or improved prognosis.

Herein, an "individual" or "subject" is a mammal. Mammals include, but are not limited to, primates (e.g., human and non-human primates such as monkeys) or other mammals (e.g., cows, sheep, cats, dogs, horses, rabbits, and rodents such as mice and rats). In particular, the individual or subject is a human.

The conventional single or three letter code for natural amino acids is used herein:

in some embodiments of the invention, the amino acid sequence of the heavy chain variable region of an anti-IL-11R antibody is provided as comprising the amino acid sequence of SEQ ID NO: 1-13, see table 1; the amino acid sequence of the light chain variable region that provides the humanized anti-PDL 1 antibody includes SEQ ID NO: 14-26, see table 2.

TABLE 1 heavy chain variable region of anti-IL-11R antibody

TABLE 2 light chain variable regions of anti-IL-11R antibodies

In some embodiments of the invention, anti-IL-11R antibodies are provided as shown in table 3, with the numbering (naming) and shorthand numbering of the antibodies shown in table 3 being used further below.

TABLE 3 anti-IL-11R antibody variable region sequences

EXAMPLE 1 preparation of antigen

Designing a fusion protein which sequentially comprises a signal peptide, a human IL-11RA protein extracellular domain and human IgG1 Fc from an amino terminal to a carboxyl terminal, wherein the amino acid sequence of the signal peptide is SEQ ID NO: 27, the amino acid sequence of the extracellular domain of the human IL-11RA protein is SEQ ID NO: bits 22-367 of 27. Constructing the nucleic acid sequence encoding the fusion protein on a pcDNA3.4 vector to obtain an expression vector pcDNA3.4-IL 11R-Fc; pcDNA3.4-IL11R-Fc was transfected into mammalian cells CHO for expression for 14 days using the transient transfer method (Expifactamine CHO Transfection Kit, Thermo Fisher; cat # A29129); cell supernatants were harvested after expression was complete and purified using Protein A purification column (GE Healthcare, Cat: 17040201); obtaining recombinant protein antigen IL-11R-Fc for immunizing animals after ultrafiltration.

Example 2 animal immunization

Female Balb/c mice (purchased from Swake Lirioda, Hunan) were collected, and the recombinant protein antigen IL-11R-Fc prepared in example 1 was emulsified with an immunological adjuvant sufficiently and then immunized with the procedures shown in Table 4.

TABLE 4 immune animal scheduling table

A small amount of mouse orbital blood is harvested on the 35 th day, and the antibody titer is detected by adopting a conventional ELISA method; the spleen was removed from the mice sacrificed at day 41, and isolated mouse spleen cells were obtained and used in example 3.

Example 3 antibody secreting cell fusion and screening

The mouse spleen cells isolated in example 2 were fused with an immortalized mouse myeloma cell SP2/0, and the resulting fused cells were plated in a 96-well plate and subjected to pressure-screening and static culture for about 14 days. The culture medium in the 96-well plate is prepared according to the following formula: 1 XHAT medium supplement (Gibco, H0137-10VL), 10% Hybridoma Feeder addition factor (Boolong, CM-2001), 1 XL-Glutamine 200mM (Gibco, 25030-. After the fused cells grow out of the clone groups, cell supernatants are taken for antigen binding ELISA screening. The fused cell mass capable of binding to IL-11R-his obtained by ILISA screening was transferred to a 24-well plate and cultured for 3 days, and then the supernatant was again subjected to ELISA affinity assay. Cells with both ELISA assays bound to antigen were transferred to 6-well plates for culture. After 3 days of culture, taking the supernatant, performing flow cytometry to determine the competitive binding of the antigen and the antibody, and selecting a cell strain with high secretion specificity for amplification culture and cryopreservation. Screening to obtain 17 positive cell strains with high affinity and high neutralization activity, wherein the numbers are respectively as follows: 55A3, 58B10, 59E4, 1A5, 1A11, 7B12, 8A6, 23D7, 28D7, 29F10, 32A3, 36F2, 23A10, 30G2, 10C1, 15H11, 18B4, for subcloning in example 4.

EXAMPLE 4 subcloning of Positive cell lines

The 17 positive cell lines obtained in example 3 were subcloned. Blowing and uniformly mixing the cells in the positive holes, and counting the number of the living cells by a counting plate; each Hybridoma fused cell mass was plated in 4 96-well plates at a density of 1 cell/well in RPMI Medium 1640 Medium + 10% FBS + 10% Hybridoma Feeder plus factor + (1X) L-Glutamine 200mM + (1X) HT Supplement (Gibco, 11067030) + (1X) penicilimin-Streptomycin. After 14 days of culture, the supernatant of the subclone cell culture fluid was subjected to ELISA detection and flow competitive assay detection, and the detection results are shown in Table 5, thereby finally obtaining monoclonal antibodies derived from single antibody secreting cells.

TABLE 5 detection results of ELISA affinity assay and flow competition assay

Wherein N/A indicates that after two rounds of subcloning of hybridoma cells, subclones with significant affinity for IL-11R have not been obtained.

EXAMPLE 5 sequencing of monoclonal antibodies

This example sequenced antibodies expressed by the subcloned cells obtained in example 4, and the specific steps included: take 1X 10⁶～5×10⁶The total RNA was extracted from each cell using the Takara MiniBEST Universal RNA Extraction Kit, and the cDNA was obtained by reverse transcription using the PrimeScript RT Reagent Kit (Takara). The above steps were carried out according to the instructions provided by the manufacturer. Gene amplification primers for heavy chain variable region (VH) and light chain variable region (VL) of antibody based on Ig-Primer Sets (Novagen, cat # 69831-3) was designed and synthesized by Huada Gene. Amplifying genes of a heavy chain variable region and a light chain variable region from a first chain of cDNA, cloning the genes into a pMD18-T vector to obtain expression vectors of the heavy chain variable region and the light chain variable region, transforming the expression vectors into escherichia coli DH5 alpha, and selecting a single clone to obtain gene sequences of the heavy chain variable region (VH) and the light chain variable region (VL) by sequencing of Huada Gene company. The sequences of the sequenced antibodies are shown in Table 3.

Example 6 antibody subtype identification

The monoclonal hybridoma cell strain corresponding to the antibody obtained by screening in example 4 was injected into Balb/C mice for ascites antibody production, and then protein G was used for purification. Antibody subtype analysis was performed using the purified antibody, and the antibody subtype detection method was performed using a kit (KMIM-2), and the results are shown in Table 6.

TABLE 6 antibody subtype identification ELISA results

Example 7 full Length expression of antibodies

The variable region sequences of the 13-strain antibodies obtained in example 6 were constructed and expressed as full-length (IgG) antibodies, respectively. Wherein, the antibody heavy chain constant region is IgG1 subtype, and the amino acid sequence thereof is shown as SEQ ID NO: 88; the antibody light chain constant region is of Kappa subtype, and the amino acid sequence of the antibody light chain constant region is shown as SEQ ID NO: 89 is shown. The sequence synthesis of the gene was requested to Huada Gene Co. After the full-length gene of the antibody is obtained, transient transfection and expression are carried out through CHO cells after vector construction.

Example 8 in vitro binding Activity assay

This example detects the binding activity of the antibody provided by the present invention to IL-11R by flow cytometry, and comprises the following steps: providing a cell line membrane-IL-11R CHO stably expressing IL-11R, taking the antibody prepared in example 6, diluting each antibody in a gradient manner, and mixing with 1 × 10⁵After incubation for 1h for membrane-IL-11R CHO, wash 3 times with PBS, and add flowThe secondary antibody, Goat pAb to Ms IgG (FITC) (Abcam, ab6785), was detected on a flow cytometer. The graph of the detection results is shown in FIG. 1, in which the horizontal axis represents the log of the concentration of the antibody and the vertical axis represents the fluorescence intensity value (MFI), and each abbreviated symbol corresponds to the subclone number in Table 6. The experimental results show that the binding activity of each antibody provided by the invention and IL-11R is different; as the concentration of the antibody provided by the invention increases, the signal value generated by the binding of the antibody and IL-11R is stronger.

Example 9 in vitro blocking Activity assay

This example evaluates whether the antibodies provided by the present invention are capable of blocking the binding of IL-11 to IL-11R, comprising the steps of: providing a cell strain membrane-IL-11R CHO for stably expressing IL-11R and providing a recombinant protein antigen IL-11R-Fc; the antibodies prepared in example 6 were diluted in a gradient and incubated with the recombinant protein antigen IL-11R-Fc for 1h, followed by incubation with 1X 10⁵After incubation of membrane-IL-11R CHO at room temperature for 1h, the cells were washed 3 times with PBS, and a flow-type secondary antibody, Goat pAb to Human IgG (FITC) (Abcam, Ab97224), was added and detected on a flow cytometer. The graph of the detection results is shown in FIG. 2, in which the horizontal axis represents the concentration (log value) of the antibody and the vertical axis represents the percentage of the competitive blocking activity, and a smaller value of the percentage indicates a stronger competitive blocking activity of the antibody to be detected, and each abbreviated symbol corresponds to the subclone number in Table 6.

Example 10 functional assays for inhibition of transformation of pulmonary fibroblasts into fibrosis of HFL-1

HFL-1 cells act as lung fibroblasts and, when induced by profibrotic factors such as TGF-beta1, express IL-11 cytokines in large amounts, eventually transforming into fibrosis. During the transformation of HFL-1 cells into fibrotic cells, the ACTA2 marker protein was continuously accumulated.

In this example, HFL-1 lung fibroblasts were treated at 1.2X 10⁵One/well is laid in a 24-well plate and starved with serum-free medium overnight; after overnight incubation, appropriate amounts of TGF-beta1 cytokine were added, set as experimental: adding 7 antibodies to be tested (antibodies No. 2, No. 6, No. 7, No. 8, No. 9, No. 10, No. 13 prepared in example 6) with 25 μ g/ml working concentration, and adding 25 μ g/ml concentrated solutionIgG control and NC control; after 24h of co-incubation, cells were harvested for lysis. The cell lysate is detected and analyzed by a general Western Blot method, the detection result is shown in figure 3, and according to the detection result, the ACTA2 protein expression level of the HFL-1 cell is obviously reduced under the action of the anti-IL-11R antibody with the abbreviation number of 6#, which indicates that the anti-IL-11R antibody provided by the invention has the function of obviously inhibiting the conversion of the HFL-1 cell to fibrosis.

This example further tests the fibrosis-inhibiting effect of the anti-IL-11R antibody, abbreviated as 6#, at working concentrations of 5. mu.g/ml and 25. mu.g/ml, using the same experimental procedures as those described above except for the working concentrations of the antibody, and the results are shown in FIG. 4. according to the results, the anti-IL-11R antibody provided by the present invention has significant function of inhibiting the transformation of HFL-1 cells into fibrosis at both concentrations of 5. mu.g/ml and 25. mu.g/ml.

EXAMPLE 11 determination of antigen binding affinity

This example uses the biofilm interference technique (BLI) to determine the binding affinity of the antibody of the present invention to an antigen, and comprises the following steps: IL-11R-His protein was provided and diluted to 5 concentration gradients (625nM, 312.5nM, 156.2nM, 78.1nM, 39.1 nM); carrying out biotinylation labeling on the anti-IL-11R antibody with the abbreviation number 6 #; the SA sensor was used to capture biotinylated anti-IL-11R antibody, gradient concentrations of IL-11R-His protein as analyte, binding time 200s, dissociation time 1000s, buffer 20mM PBS, 0.15M NaCl, 0.1% BSA, 0.05% Tween 20, pH7.4. The affinity constant (KD) of the anti-IL-11R antibody with the abbreviation number 6# to IL-11R was obtained by analysis with BLI kinetics/affinity software, and the specific results are shown in Table 7 and FIG. 5.

TABLE 7 affinity assay results for anti-IL-11R antibody and IL-11R

KD(M)	kon(1/Ms)	kdis(1/s)	Full R²
				4.24E-09	1.83E+04	7.76E-05	0.998472

Example 12 thermal stability test

In this example, the assay for determining the thermal stability of the anti-IL-11R antibody provided by the present invention was performed by using the MicroCalTM VP-Capillary DSC system, which comprises the following steps: the anti-IL-11R antibody, abbreviated as # 6, was diluted to 0.4mg/ml with PBS buffer under the following assay conditions: the heating rate is 90 ℃/h, and the temperature range is 25-110 ℃. The results of the measurements are shown in Table 8 and FIG. 6.

TABLE 8 Tm values of anti-IL-11R antibodies

Thalf	DH	TmOnset	Tm1	Tm2
					5.62	315000	56.75	68.52	75.2

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.

Sequence listing

<110> Guangdong Dongyuang pharmaceutical Co., Ltd

<120> anti-IL-11R antibody and application thereof

<160> 89

<170> PatentIn version 3.5

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

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

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Thr Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Asp Trp Val

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Ala Thr Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val

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Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

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

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Ala Arg Glu Asp Tyr Asp Gly Phe Ala Tyr Trp Gly Gln Gly Thr Leu

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

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Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Ser Asp Phe

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Tyr Met Glu Trp Val Arg Gln Pro Pro Gly Lys Arg Leu Glu Trp Ile

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

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

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Leu Tyr Leu Gln Met Asn Ala Leu Arg Pro Glu Asp Thr Ala Ile Phe

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Tyr Cys Ala Arg Ala His Tyr Tyr Gly Phe Gly Ala Met Asp Tyr Trp

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Gly Gln Gly Thr Ser Val Thr Val Ser Ser

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

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Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Ser

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Ala Leu Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Asp Trp Val

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Ala Thr Val Ser Ser Gly Gly Thr Tyr Thr Tyr Phe Pro Asp Ser Met

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Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

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

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Ala Arg His Ser Pro Asp Asp Gly Tyr Phe Val Asp Tyr Trp Gly Gln

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Gly Thr Leu Val Thr Val Ser Ala

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

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Ser Arg Lys Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser Asp Tyr

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Gly Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val

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Ala Phe Ile Ser Asn Leu Ala Tyr Ser Phe Tyr Tyr Ala Asp Thr Val

50 55 60

Thr Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

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

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Val Arg Ala Asp Glu Gly Leu Gly Tyr Trp Gly Gln Gly Thr Leu Val

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Thr Val Ser Ala

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

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Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr

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Thr Met Asn Trp Val Arg Gln Ser His Gly Lys Asn Leu Glu Trp Ile

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Gly Leu Ile Asn Pro Asp Asn Gly Gly Ile Arg Tyr Asn Gln Lys Phe

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

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

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Ala Ile Asn Tyr Tyr Gly Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu

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

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Leu Val Lys Leu Ser Cys Asn Ala Ser Gly Phe Asn Ile Lys Asn Tyr

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Tyr Ile His Trp Val Arg Arg Arg Pro Glu Gln Gly Leu Glu Trp Ile

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

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Gln Asp Lys Ala Ser Met Thr Ala Asp Thr Ser Ser Asn Thr Ala Asp

65 70 75 80

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

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Val Leu Tyr Arg Tyr Gly Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val

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

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Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr

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Leu Ile Glu Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Val

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Gly Ile Ile Ser Pro Asp Asn Gly Asn Thr Asn Tyr Asn Glu Lys Phe

50 55 60

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

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Phe Cys

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Ala Arg Asn Arg Tyr Gly Ile Asp Ser Trp Gly Gln Gly Thr Thr Leu

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

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Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Arg Asn Tyr

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Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met

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Gly Trp Ile Asn Thr Phe Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe

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Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys

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Gln Gly Thr Leu Val Thr Val Ser Ala

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Asp Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

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

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Thr Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val

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

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Lys Gly Arg Ser Thr Ile Ser Arg Asp Asp Ala Asn Asn Thr Leu Tyr

65 70 75 80

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

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Thr Arg Asp Leu Gly Asn Asp Asp Phe Thr Tyr Tyr Phe Asp Ser Trp

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Gly Gln Gly Thr Thr Leu Thr Val

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Ser Asp Phe

20 25 30

Tyr Met Glu Trp Val Arg Gln Pro Pro Gly Lys Arg Leu Glu Trp Ile

35 40 45

Ala Ala Ser Arg Asp Lys Ala Lys Asp Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

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

65 70 75 80

Leu Tyr Leu Gln Met Asn Ala Leu Arg Ala Glu Asp Thr Ala Ile Tyr

85 90 95

Tyr Cys Ala Arg Val His Tyr Tyr Gly Phe Gly Ala Met Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Ser Val Thr Val Ser His

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Glu Val Met Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

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

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Ala Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val

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

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Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr

65 70 75 80

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

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Ala Arg Thr Asp Gly Tyr Tyr Pro Asp Tyr Trp Gly Gln Gly Thr Thr

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Leu Thr Val Ser Gln

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

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Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr

20 25 30

Leu Ile Glu Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

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Gly Val Ile Ser Pro Asp Asn Gly Asn Thr Asn Tyr Asn Glu Lys Phe

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

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Met Gln Leu Ser Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Phe Cys

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Ala Arg Asn Arg Tyr Gly Ile Asp Tyr Trp Gly Gln Gly Thr Thr Leu

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Thr Val Ser His

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

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Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Ser Asp Phe

20 25 30

Tyr Met Glu Trp Val Arg Gln Pro Pro Gly Lys Arg Leu Glu Trp Ile

35 40 45

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

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

65 70 75 80

Leu Tyr Leu Gln Met Asn Ala Leu Arg Pro Glu Asp Thr Ala Ile Phe

85 90 95

Tyr Cys Ala Arg Ala His Tyr Tyr Gly Phe Gly Ala Met Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Ser Val Thr Val Ser Ser

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

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Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val Phe Ser

20 25 30

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

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Thr Arg Phe Ser Gly Val Pro

50 55 60

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

65 70 75 80

Ser Gly Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Met

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

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Val Ser Ala

115

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

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Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Tyr

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Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

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Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala

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Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

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Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Asn

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Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Lys Ser Asn

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

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Asn Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Asn Ile Val His Ser

20 25 30

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

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

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

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly

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

100 105 110

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

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Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Thr Leu Val Asp Ser

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Asn Val Asn Asn Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

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

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser

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Thr His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

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

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser

20 25 30

Thr Gly Val Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

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

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Leu Tyr Tyr Cys Phe Gln Gly

85 90 95

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

100 105 110

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

1 5 10 15

Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser

20 25 30

Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Asp His Leu Phe Thr Ser

35 40 45

Leu Ile Gly Gly Thr Asn Asn Arg Thr Pro Gly Val Pro Ala Arg Phe

50 55 60

Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala

65 70 75 80

Gln Thr Glu Asp Glu Ala Ile Tyr Phe Cys Val Leu Trp His Ser Asn

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His Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

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

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Thr Ser Val Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser

20 25 30

Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

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

50 55 60

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

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn

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

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

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val Phe Ser

20 25 30

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

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

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

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Leu Tyr Tyr Cys Phe Gln Gly

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

100 105 110

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Glu Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Asn Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Phe Leu Thr Ile Thr Ser Leu Glu Gln

65 70 75 80

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

85 90 95

Phe Gly Gly Gly Ser Lys Leu Lys Ser Asn

100 105

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

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Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp

20 25 30

Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Val Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Ile Thr Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Asn

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Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

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<213> Artificial Sequence (Artificial Sequence)

<400> 24

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

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser

20 25 30

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

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

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

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Lys Ser Asn

100 105 110

<210> 25

<211> 112

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 25

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

1 5 10 15

Thr Ser Val Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Asn

20 25 30

Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

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

50 55 60

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

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn

85 90 95

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

100 105 110

<210> 26

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 26

Glu Phe Asp Gly Ser Ala Ser Leu His Ala Cys Arg Ser Thr Ala Thr

1 5 10 15

Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Tyr Gly Asp Asn Tyr

20 25 30

Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His Pro Val Glu Glu

65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Asn Glu Asp Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 27

<211> 367

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 27

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

1 5 10 15

Val Gln Cys Ser Ser Pro Cys Pro Gln Ala Trp Gly Pro Pro Gly Val

20 25 30

Gln Tyr Gly Gln Pro Gly Arg Ser Val Lys Leu Cys Cys Pro Gly Val

35 40 45

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

50 55 60

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

65 70 75 80

Ala Asp Ser Thr Asp Glu Gly Thr Tyr Ile Cys Gln Thr Leu Asp Gly

85 90 95

Ala Leu Gly Gly Thr Val Thr Leu Gln Leu Gly Tyr Pro Pro Ala Arg

100 105 110

Pro Val Val Ser Cys Gln Ala Ala Asp Tyr Glu Asn Phe Ser Cys Thr

115 120 125

Trp Ser Pro Ser Gln Ile Ser Gly Leu Pro Thr Arg Tyr Leu Thr Ser

130 135 140

Tyr Arg Lys Lys Thr Val Leu Gly Ala Asp Ser Gln Arg Arg Ser Pro

145 150 155 160

Ser Thr Gly Pro Trp Pro Cys Pro Gln Asp Pro Leu Gly Ala Ala Arg

165 170 175

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

180 185 190

Thr Glu Val Asn Pro Leu Gly Ala Ser Thr Arg Leu Leu Asp Val Ser

195 200 205

Leu Gln Ser Ile Leu Arg Pro Asp Pro Pro Gln Gly Leu Arg Val Glu

210 215 220

Ser Val Pro Gly Tyr Pro Arg Arg Leu Arg Ala Ser Trp Thr Tyr Pro

225 230 235 240

Ala Ser Trp Pro Cys Gln Pro His Phe Leu Leu Lys Phe Arg Leu Gln

245 250 255

Tyr Arg Pro Ala Gln His Pro Ala Trp Ser Thr Val Glu Pro Ala Gly

260 265 270

Leu Glu Glu Val Ile Thr Asp Ala Val Ala Gly Leu Pro His Ala Val

275 280 285

Arg Val Ser Ala Arg Asp Phe Leu Asp Ala Gly Thr Trp Ser Thr Trp

290 295 300

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

305 310 315 320

Ile Pro Ala Trp Gly Gln Leu His Thr Gln Pro Glu Val Glu Pro Gln

325 330 335

Val Asp Ser Pro Ala Pro Pro Arg Pro Ser Leu Gln Pro His Pro Arg

340 345 350

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

355 360 365

<210> 28

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 28

Gly Phe Thr Phe Ser Ser Tyr Thr

1 5

<210> 29

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 29

Ile Ser Ser Gly Gly Ser Tyr Thr

1 5

<210> 30

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 30

Ala Arg Glu Asp Tyr Asp Gly Phe Ala Tyr

1 5 10

<210> 31

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 31

Gly Phe Thr Phe Ser Asp Phe Tyr

1 5

<210> 32

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 32

Ser Arg Asp Asn Thr Lys Asp Tyr Thr Thr

1 5 10

<210> 33

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 33

Ala Arg Ala His Tyr Tyr Gly Phe Gly Ala Met Asp Tyr

1 5 10

<210> 34

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 34

Gly Phe Thr Phe Ser Ser Ser Ala

1 5

<210> 35

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 35

Val Ser Ser Gly Gly Thr Tyr Thr

1 5

<210> 36

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 36

Ala Arg His Ser Pro Asp Asp Gly Tyr Phe Val Asp Tyr

1 5 10

<210> 37

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 37

Gly Phe Ile Phe Ser Asp Tyr Gly

1 5

<210> 38

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 38

Ile Ser Asn Leu Ala Tyr Ser Phe

1 5

<210> 39

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 39

Val Arg Ala Asp Glu Gly Leu Gly Tyr

1 5

<210> 40

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 40

Gly Tyr Ser Phe Thr Gly Tyr Thr

1 5

<210> 41

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 41

Ile Asn Pro Asp Asn Gly Gly Ile

1 5

<210> 42

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 42

Ala Ile Asn Tyr Tyr Gly Leu Asp Tyr

1 5

<210> 43

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 43

Gly Phe Asn Ile Lys Asn Tyr Tyr

1 5

<210> 44

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 44

Val Asp Pro Glu Asn Gly Asn Thr

1 5

<210> 45

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 45

Val Leu Tyr Arg Tyr Gly Phe Ala Tyr

1 5

<210> 46

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 46

Gly Tyr Ala Phe Thr Asn Tyr Leu

1 5

<210> 47

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 47

Ile Ser Pro Asp Asn Gly Asn Thr

1 5

<210> 48

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 48

Ala Arg Asn Arg Tyr Gly Ile Asp Ser

1 5

<210> 49

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 49

Gly Tyr Thr Phe Arg Asn Tyr Gly

1 5

<210> 50

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 50

Ile Asn Thr Phe Thr Gly Glu Pro

1 5

<210> 51

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 51

Ala Arg Glu Glu Leu Arg Ser Gly His Tyr Gly Phe Ala Cys

1 5 10

<210> 52

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 52

Gly Phe Thr Leu Ser Ser Tyr Thr

1 5

<210> 53

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 53

Ile Ser Ser Gly Gly Ser Tyr Ile

1 5

<210> 54

<211> 15

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 54

Thr Arg Asp Leu Gly Asn Asp Asp Phe Thr Tyr Tyr Phe Asp Ser

1 5 10 15

<210> 55

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 55

Ser Arg Asp Lys Ala Lys Asp Tyr Thr Thr

1 5 10

<210> 56

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 56

Ala Arg Val His Tyr Tyr Gly Phe Gly Ala Met Asp Tyr

1 5 10

<210> 57

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 57

Gly Phe Thr Phe Ser Tyr Tyr Ala

1 5

<210> 58

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 58

Ile Ser Ser Ser Gly Arg Tyr Thr

1 5

<210> 59

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 59

Ala Arg Thr Asp Gly Tyr Tyr Pro Asp Tyr

1 5 10

<210> 60

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 60

Ala Arg Asn Arg Tyr Gly Ile Asp Tyr

1 5

<210> 61

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 61

Gln Ser Leu Val Phe Ser Asn Gly Asn Thr Tyr

1 5 10

<210> 62

<211> 3

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 62

Lys Val Ser

1

<210> 63

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 63

Ser Gln Met Thr His Val Pro Tyr Thr

1 5

<210> 64

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 64

Gln Ser Val Asp Tyr Tyr Gly Asp Ser Tyr

1 5 10

<210> 65

<211> 3

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 65

Ala Ala Ser

1

<210> 66

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 66

Gln Gln Ser Asn Glu Asp Pro Trp Thr

1 5

<210> 67

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 67

Gln Asn Ile Val His Ser Asn Gly Asn Thr Tyr

1 5 10

<210> 68

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 68

Phe Gln Gly Ser His Val Pro Pro Thr

1 5

<210> 69

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 69

Gln Thr Leu Val Asp Ser Asn Val Asn Asn Tyr

1 5 10

<210> 70

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 70

Ser Gln Ser Thr His Val Pro Trp Thr

1 5

<210> 71

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 71

Gln Ser Ile Val His Ser Thr Gly Val Thr Tyr

1 5 10

<210> 72

<211> 3

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 72

Arg Val Ser

1

<210> 73

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 73

Phe Gln Gly Ser His Val Pro Val Thr

1 5

<210> 74

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 74

Thr Gly Ala Val Thr Thr Ser Asn Tyr

1 5

<210> 75

<211> 3

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 75

Gly Thr Asn

1

<210> 76

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 76

Val Leu Trp His Ser Asn His Trp Val

1 5

<210> 77

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 77

Lys Ser Leu Leu His Ser Asn Gly Ile Thr Tyr

1 5 10

<210> 78

<211> 3

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 78

Gln Met Ser

1

<210> 79

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 79

Ala Gln Asn Leu Glu Leu Pro His Thr

1 5

<210> 80

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 80

Gln Ser Ile Val Phe Ser Asn Gly Ile Thr Tyr

1 5 10

<210> 81

<211> 6

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 81

Gln Asp Ile Asn Asn Tyr

1 5

<210> 82

<211> 3

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 82

Tyr Thr Ser

1

<210> 83

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 83

Gln Gln Gly Lys Thr Phe Pro Thr

1 5

<210> 84

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 84

Gln Ser Val Asp Tyr Asp Gly Asp Ser Tyr

1 5 10

<210> 85

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 85

Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr

1 5 10

<210> 86

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 86

Lys Ser Leu Leu His Asn Asn Gly Ile Thr Tyr

1 5 10

<210> 87

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 87

Gln Ser Val Asp Tyr Tyr Gly Asp Asn Tyr

1 5 10

<210> 88

<211> 330

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 88

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 89

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 89

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

Claims

1. An anti-IL-11R antibody comprising a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 and a light chain variable region comprising LCDR1, LCDR2, and LCDR 3;

HCDR1：GFTFSDFY(SEQ ID NO：31)，HCDR2：SRDNTKDYTT(SEQ ID NO：32)，HCDR3：ARAHYYGFGAMDY(SEQ ID NO：33)；

HCDR1：GFTFSSSA(SEQ ID NO：34)，HCDR2：VSSGGTYT(SEQ ID NO：35)，HCDR3：ARHSPDDGYFVDY(SEQ ID NO：36)；

HCDR1：GFIFSDYG(SEQ ID NO：37)，HCDR2：ISNLAYSF(SEQ ID NO：38)，HCDR3：VRADEGLGY(SEQ ID NO：39)；

HCDR1：GYSFTGYT(SEQ ID NO：40)，HCDR2：INPDNGGI(SEQ ID NO：41)，HCDR3：AINYYGLDY(SEQ ID NO：42)；

HCDR1：GFNIKNYY(SEQ ID NO：43)，HCDR2：VDPENGNT(SEQ ID NO：44)，HCDR3：VLYRYGFAY(SEQ ID NO：45)；

HCDR1：GYAFTNYL(SEQ ID NO：46)，HCDR2：ISPDNGNT(SEQ ID NO：47)，HCDR3：ARNRYGIDS(SEQ ID NO：48)；

HCDR1：GYTFRNYG(SEQ ID NO：49)，HCDR2：INTFTGEP(SEQ ID NO：50)，HCDR3：AREELRSGHYGFAC(SEQ ID NO：51)；

HCDR1：GFTLSSYT(SEQ ID NO：52)，HCDR2：ISSGGSYI(SEQ ID NO：53)，HCDR3：TRDLGNDDFTYYFDS(SEQ ID NO：54)；

HCDR1：GFTFSDFY(SEQ ID NO：31)，HCDR2：SRDKAKDYTT(SEQ ID NO：55)，HCDR3：ARVHYYGFGAMDY(SEQ ID NO：56)；

HCDR1：GFTFSYYA(SEQ ID NO：57)，HCDR2：ISSSGRYT(SEQ ID NO：58)，HCDR3：ARTDGYYPDY(SEQ ID NO：59)；

HCDR1：GYAFTNYL(SEQ ID NO：46)，HCDR2：ISPDNGNT(SEQ ID NO：47)，HCDR3：ARNRYGIDY(SEQ ID NO：60)；

the amino acid sequences of the LCDR1, the LCDR2 and the LCDR3 are selected from the following groups:

LCDR1：QSVDYYGDSY(SEQ ID NO：64)，LCDR2：AAS(SEQ ID NO：65)，LCDR3：QQSNEDPWT(SEQ ID NO：66)；

LCDR1：QNIVHSNGNTY(SEQ ID NO：67)，LCDR2：KVS(SEQ ID NO：62)，LCDR3：FQGSHVPPT(SEQ ID NO：68)；

LCDR1：QTLVDSNVNNY(SEQ ID NO：69)，LCDR2：KVS(SEQ ID NO：62)，LCDR3：SQSTHVPWT(SEQ ID NO：70)；

LCDR1：QSIVHSTGVTY(SEQ ID NO：71)，LCDR2：RVS(SEQ ID NO：72)，LCDR3：FQGSHVPVT(SEQ ID NO：73)；

LCDR1：TGAVTTSNY(SEQ ID NO：74)，LCDR2：GTN(SEQ ID NO：75)，LCDR3：VLWHSNHWV(SEQ ID NO：76)；

LCDR1：KSLLHSNGITY(SEQ ID NO：77)，LCDR2：QMS(SEQ ID NO：78)，LCDR3：AQNLELPHT(SEQ ID NO：79)；

LCDR1：QSIVFSNGITY(SEQ ID NO：80)，LCDR2：KVS(SEQ ID NO：62)，LCDR3：FQGSHVPPT(SEQ ID NO：68)；

LCDR1：QDINNY(SEQ ID NO：81)，LCDR2：YTS(SEQ ID NO：82)，LCDR3：QQGKTFPT(SEQ ID NO：83)；

LCDR1：QSVDYDGDSY(SEQ ID NO：84)，LCDR2：AAS(SEQ ID NO：65)，LCDR3：QQSNEDPWT(SEQ ID NO：66)；

LCDR1：QSIVHSNGNTY(SEQ ID NO：85)，LCDR2：KVS(SEQ ID NO：62)，LCDR3：FQGSHVPPT(SEQ ID NO：68)；

LCDR1：KSLLHNNGITY(SEQ ID NO：86)，LCDR2：QMS(SEQ ID NO：78)，LCDR3：AQNLELPHT(SEQ ID NO：79)；

LCDR1：QSVDYYGDNY(SEQ ID NO：87)，LCDR2：AAS(SEQ ID NO：65)，LCDR3：QQSNEDPWT(SEQ ID NO：66)。

2. the anti-IL-11R antibody of claim 1, wherein the amino acid sequence of the heavy chain variable region is any one of the group consisting of: SEQ ID NO: 1-13.

3. The anti-IL-11R antibody of claim 1, wherein the light chain variable region has the amino acid sequence set forth in any one of the following groups: SEQ ID NO: 14-26.

4. The anti-IL-11R antibody of claim 1, wherein the amino acid sequences of the heavy chain variable region and the light chain variable region are selected from the group consisting of:

the amino acid sequence of the heavy chain variable region is SEQ ID NO: 1, the amino acid sequence of the light chain variable region is ID NO: 14;

the amino acid sequence of the heavy chain variable region is SEQ ID NO: 2, the amino acid sequence of the light chain variable region is ID NO: 15;

the amino acid sequence of the heavy chain variable region is SEQ ID NO: 3, the amino acid sequence of the light chain variable region is ID NO: 16;

the amino acid sequence of the heavy chain variable region is SEQ ID NO: 4, the amino acid sequence of the light chain variable region is ID NO: 17;

the amino acid sequence of the heavy chain variable region is SEQ ID NO: 5, the amino acid sequence of the light chain variable region is ID NO: 18;

the amino acid sequence of the heavy chain variable region is SEQ ID NO: 6, the amino acid sequence of the light chain variable region is ID NO: 19;

the amino acid sequence of the heavy chain variable region is SEQ ID NO: 7, the amino acid sequence of the light chain variable region is ID NO: 20;

the amino acid sequence of the heavy chain variable region is SEQ ID NO: 8, the amino acid sequence of the light chain variable region is ID NO: 21;

the amino acid sequence of the heavy chain variable region is SEQ ID NO: 9, the amino acid sequence of the light chain variable region is ID NO: 22;

the amino acid sequence of the heavy chain variable region is SEQ ID NO: 10, the amino acid sequence of the light chain variable region is ID NO: 23;

the amino acid sequence of the heavy chain variable region is SEQ ID NO: 11, the amino acid sequence of the light chain variable region is ID NO: 24;

the amino acid sequence of the heavy chain variable region is SEQ ID NO: 12, the amino acid sequence of the light chain variable region is ID NO: 25;

the amino acid sequence of the heavy chain variable region is SEQ ID NO: 13, the amino acid sequence of the light chain variable region is ID NO: 26.

5. the anti-IL-11R antibody of any one of claims 1-4, wherein the anti-IL-11R antibody is a full-length antibody, a Fab fragment, F (ab)₂A fragment, a two-chain Fv fragment, or a single-chain Fv fragment;

preferably, the anti-IL-11R antibody is a monoclonal antibody;

preferably, the anti-IL-11R antibody is of the IgG1, IgG2a, IgG2b or IgG3 subtype;

preferably, the anti-IL-11R antibody further comprises a heavy chain constant region selected from the group consisting of IgG1, IgG2, IgG3, IgG4 subtypes; preferably, the heavy chain constant region comprises the amino acid sequence as set forth in SEQ ID NO: 88;

preferably, the anti-IL-11R antibody further comprises a light chain constant region selected from Kappa or Lambda subtypes; preferably, the light chain constant region comprises the amino acid sequence as set forth in SEQ ID NO: 89.

6. A biomaterial, characterized in that it is any of the following (i), (ii), (iii):

(i) a nucleic acid comprising a nucleotide sequence encoding the anti-IL-11R antibody of any one of claims 1-5;

(ii) (ii) a vector comprising the nucleic acid of (i);

(iii) a host cell comprising a nucleic acid as described in (i) and/or a vector as described in (ii).

7. A pharmaceutical composition comprising the anti-IL-11R antibody of any one of claims 1-5 as an active ingredient; wherein the anti-IL-11R antibody is present in a therapeutically effective dose;

preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

8. A method for preparing an anti-IL-11R antibody according to the present invention comprises the steps of: allowing host cells of one of the biomaterials of claim 6 to express the anti-IL-11R antibody, and isolating and purifying to obtain the anti-IL-11R antibody.

9. Use of an anti-IL-11R antibody of any one of claims 1-5 in the manufacture of a medicament for the prevention, alleviation or treatment of a disease with a fibrotic disorder.

10. The use of claim 9, wherein the fibrotic disorder is a fibrotic disorder selected from the liver, the gallbladder, the lung, the kidney, the bladder, the heart, a blood vessel, the eye, the skin, the pancreas, the gastrointestinal tract, the bone marrow, the penis, the breast, or the muscle;

preferably, the disease with fibrotic disorders includes silicosis, asbestosis, pneumonitis, tuberculosis, viral pneumonia, pneumocystis infection, secondary lung disease, idiopathic interstitial pneumonia, bronchiolitis obliterans with organizing pneumonia, pulmonary lymphangioleiomyomata, systemic lupus erythematosus, rheumatoid arthritis, progressive systemic sclerosis, polymyositis, dermatomyositis, mixed connective tissue disease, diffuse alveolar hemorrhage syndrome, pulmonary alveolar proteinosis, eosinophilic pneumonia, pulmonary vasculitis, lymphocytic interstitial pneumonia, ischemic heart disease, hypertensive heart disease, viral myocarditis, hemochromatosis cardiomyopathy, amyloidosis, glycogen-accumulating cardiomyopathy, diabetic cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, cirrhosis, acute pancreatitis, pancreatic duct obstruction, pulmonary fibrosis, myocardial infarction, pulmonary fibrosis, pulmonary, Glomerulonephritis, pyelonephritis, renal calculus, hyperuricemia, hypercalciuria, spleen fibroplasia disease, retina fibroplasia, and myelofibrosis.