CN106928354B

CN106928354B - anti-I L-1 β monoclonal antibody and application thereof

Info

Publication number: CN106928354B
Application number: CN201710276999.6A
Authority: CN
Inventors: 冯晓; 王涛; 金磊; 秦锁富; 陈宇珩
Original assignee: GeneScience Pharmaceuticals Co Ltd
Current assignee: GeneScience Pharmaceuticals Co Ltd
Priority date: 2017-04-25
Filing date: 2017-04-25
Publication date: 2020-07-17
Anticipated expiration: 2037-04-25
Also published as: CN106928354A

Abstract

The invention relates to the technical field of antibody medicaments, and discloses a fully human anti-human I L-1 β monoclonal antibody prepared by screening by using a phage antibody library technology and using a genetic engineering method, a DNA molecule encoding the monoclonal antibody, a DNA molecule expression vector containing the encoding monoclonal antibody, a host cell and application thereof.A monoclonal antibody against I β 1-1 β 0 can block the combination of human I β 3-1 β 2 and human I β 5-1R, and further can reduce the initial induction fever, expansion lymphocyte response and stimulation of acute phase response of optional I L-l β by combining I L-1 β 4 and blocking I L-1 β 6 and a receptor signal path thereof.A monoclonal antibody against I L-1 β can be used for detecting the expression of I L-1 β and simultaneously used for preventing and treating the cryopyrin-related periodic syndrome.

Description

anti-I L-1 β monoclonal antibody and application thereof

Technical Field

The invention relates to the technical field of antibody medicines, in particular to a fully human anti-I L-1 β monoclonal antibody and application thereof.

Background

Interleukin 1 β (I L-1 β) is an important cytokine and polypeptide regulator produced mainly by mononuclear macrophages and plays a regulatory role in cellular immune activation I L-1 β, contains 153 amino acids, has a theoretical molecular weight of 17.5 KD.I L-1 β, does not contain disulfide bonds, but two cysteine (Cys) residues are necessary for the activity of I L-1 β.

I L-1 β has strong biological activity, including (1) mediating inflammatory reaction, wherein I L-1 β not only can cause inflammatory reaction, but also can induce the expression of cyclooxygenase 2 (COX-2), iNOS, I L-6 and other inflammatory factors, thereby further activating stroma cells and immunocytes to generate more I L-1 β to participate in inflammatory reaction, (2) immunoregulation which synergistically stimulates T cells and induces a plurality of cells to generate other lymphokines, (3) participating in the formation of cachexia and having the effect of negative nitrogen balance, and can stimulate skeletal muscle decomposition protein, (4) inducing acute phase protein to participate in acute phase reaction, (5) inducing fibroblast proliferation and the like.

I L-1 β can bind to I L-1 RA receptor on effector cell and rapidly cause downstream signal transduction, and induces inflammatory response of organism in different cell types, such as monocyte, macrophage, epithelial cell, vascular endothelial cell, chondrocyte and fibroblast, and I L-1 β is considered as a proinflammatory factor in organism, and its production brings more harm to organism, and abnormal expression of I L-1 β is also a typical characteristic of some diseases, and its abnormal expression may cause pathological changes and destruction of organism normal tissue.

The normal immune system of the human body is in a state of equilibrium in which proinflammatory and anti-inflammatory cells and molecules are regulated to promote normal immune defenses in the body without destroying the body's tissues. Once this regulatory balance is broken, nonspecific stimulation and activation can lead to the generation and release of large numbers of potent destructive immune and inflammatory molecules. Overproduction of pro-inflammatory cytokines or cytokine production in the wrong biological context is closely related to morbidity and mortality in a number of diseases.

I L-l β is involved in a variety of biological pathways and is a potent molecule that can act by triggering as few as one or two receptors.I L-l β 0, as a signaling molecule, can act at very low concentrations, even on the micromolar scale.I L-l β 1 initially induces fever, expands lymphocytes and stimulates acute phase responses to be recognized.I L-l β can induce inflammatory responses upon infection.I L-1 β is an inflammatory cytokine that is widely involved in various pathological injury processes such as human tissue destruction, edema formation, etc. it promotes the production of β defensin-4, but in general, destruction greater than defense, e.g., juvenile systemic idiopathic arthritis (sJIA) is an autoinflammatory disease mediated by interleukin-1 β.

Canakinumab (canakinumab) is a whole-human mab that rapidly and selectively blocks I L-1 β, approved by the FDA at 6 months of 2009, and is used to treat children and adults with cryopyrin-associated periodic syndrome (CAPS), including and limited to familial cold-type autoinflammatory syndrome (FCAS) and Muckle-Wells syndrome (MWS), canakinumab reduces gout attacks by 48% to 75% compared to colchicine, effectively relieves pain compared to triamcinolone acetonide, and has a gout attack rate that is 94%, but has a higher incidence of adverse reactions.

Disclosure of Invention

The invention aims to provide a fully human anti-I L-1 β monoclonal antibody, a nucleotide molecule for coding the monoclonal antibody, a vector, a host cell and application thereof, wherein the sequence of the antibody gene variable region related to the invention can construct a full-length antibody molecule which can be used as a medicine for clinically treating indications caused by abnormal expression of I L-1 β.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides an anti-I L-1 β monoclonal antibody, which has a heavy chain variable region and a light chain variable region:

the amino acid sequence of the heavy chain variable region is shown as any one of SEQ ID No. 1-6; or by substitution, deletion or addition of one or more amino acids; or an amino acid sequence which has at least 80 percent of homology with the amino acid sequence shown in the formula (I) and has the same or similar functions;

and/or

(II) the variable region of the light chain has an amino acid sequence as shown in any one of SEQ ID Nos. 7-12; by substitution, deletion or addition of one or more amino acids; or an amino acid sequence which has at least 80% homology with the amino acid sequence shown in the (II) and has the same or similar functions.

Preferably, the plurality of amino acids that are substituted, deleted or added is 2,3,4,5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32.

Preferably, the anti-I L-1 β monoclonal antibody has any one of the following groups of heavy chain variable regions and light chain variable regions:

the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 1, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 10;

(ii) the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 11;

(iii) the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 11;

(iv) the amino acid sequence of the heavy chain variable region is represented by SEQ ID NO:2, and the amino acid sequence of the light chain variable region is represented by SEQ ID NO: 12;

(V) the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 3, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 12;

(VI) the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 4, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 11.

Preferably, the heavy chain type of the anti-I L-1 β monoclonal antibody is IgG1, IgG2, IgG3 or Ig4, and more preferably IgG 4.

Preferably, the light chain of the anti-I L-1 β monoclonal antibody of the present invention is of the kappa or lambda type, more preferably lambda.

The monoclonal antibody of the present invention includes not only the variable region but also the constant region. Preferably, the constant region is any one of human IgG1, IgG2, IgG3 or IgG 4.

The invention also provides a DNA molecule for encoding the anti-I L-1 β monoclonal antibody.

Preferably, the DNA molecule has

(I) the nucleotide sequence of the heavy chain variable region as set forth in any one of SEQ ID NOS 13-18; or a sequence which differs from the nucleotide sequence of (I) by virtue of the degeneracy of the genetic code;

and/or

(II) the nucleotide sequence of the light chain variable region as shown in any one of SEQ ID Nos. 7-12; or a sequence which differs from the nucleotide sequence of (II) by virtue of the degeneracy of the genetic code.

The invention also provides an expression vector which comprises a DNA molecule for coding the anti-I L-1 β monoclonal antibody.

The invention also provides a host cell which is transformed or transfected by the expression vector and is a prokaryotic cell or a eukaryotic cell.

The invention also provides an antigen with an amino acid sequence shown as SEQ ID NO. 25 or SEQ ID NO. 26.

The invention also provides a conjugate comprising the anti-I L-1 β monoclonal antibody covalently linked to a chemical marker or a biomarker.

Preferably, the chemical label is an isotopic label, an immunotoxin label and/or a chemical drug label; the biomarker is a biotin label, an avidin label, or an enzyme label.

The invention also provides a conjugate formed by coupling the anti-I L-1 β monoclonal antibody and/or the conjugate with a solid medium or a semi-solid medium.

The invention also provides a pharmaceutical composition, which comprises the anti-human I L-1 β monoclonal antibody and/or the conjugate.

The invention also provides application of the anti-I L-1 β monoclonal antibody, the conjugate and/or the conjugate in preparation of products for detecting expression of I L-1 β.

Further, the invention also provides a kit comprising the anti-I L-1 β monoclonal antibody, the conjugate and/or the conjugate.

The invention also provides application of the anti-I L-1 β monoclonal antibody, the conjugate and/or the conjugate in preparation of a preparation for blocking the combination of I L-1 β and I L-1 RA.

The invention also provides application of the anti-I L-1 β monoclonal antibody, the conjugate and/or the conjugate in preparation of a medicament for preventing or treating the cold imidacloprid-related periodic syndrome.

Preferably, the cryopyrin-associated periodic syndrome is familial cryoprecipital autoinflammatory syndrome and muckle-viri syndrome, tumor necrosis factor receptor-associated periodic fever syndrome, hyper immunoglobulin D syndrome/mevalonate kinase deficiency and/or familial mediterranean fever.

The invention discloses a fully human anti-human I L-1 β monoclonal antibody which is screened by using a phage antibody library technology and prepared by using a genetic engineering method, and discloses a DNA molecule encoding the monoclonal antibody, a DNA molecule expression vector containing the encoding monoclonal antibody, a host cell and application thereof, wherein the anti-I β -1 β 1 monoclonal antibody is used as a novel anti-I β -1 β antibody, can bind to human I β -1 β, monkey I β -1 β and mouse I β -1 β, has excellent affinity and specificity, can block the binding of human I β -1 β 3 and human I β -1R in a dose-dependent manner, can bind to I β 0-1 β 5 in blood and block the binding of the human I β -1R in a dose-dependent manner, can effectively inhibit the fever generated by the stimulation of I β -1 β, can further increase the lymphocyte reaction and the acute reaction of the cell surface human I β, can be used for preventing the fever-induced by the immune deficiency syndrome of the immune globulin I β -1-warm syndrome, the immune deficiency syndrome, the immune globulin I-1-warm syndrome and the immune deficiency syndrome, the immune globulin I β and the immune globulin receptor-cold-cycle syndrome, and the immune deficiency syndrome.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 SDS-PAGE electrophoresis detection of purified human I L-1 β -linker peptide-mIgG 1Fc and positive antibodies, wherein A is SDS-PAGE electrophoresis detection of purified human I L-1 β -linker peptide-mIgG 1Fc, lane M protein molecular weight marker, lane 1 human I L-1 β -linker peptide-mIgG 1Fc (non-reduced), lane 2 human I L-1 β -linker peptide-mIgG 1Fc (reduced), B is SDS-PAGE electrophoresis detection of positive antibodies (kanamycin), lane M protein molecular weight marker, lane 1 kanamycin (non-reduced), lane 2 kanamycin (reduced);

FIG. 2is a map of a phagemid vector pCANTAB5E-SF vector;

FIG. 3is a photograph of human PBMC for total RNA electrophoretic detection, in lane M DNA marker D L2000, lane 1 total RNA;

FIG. 4 is an electrophoresis test chart of PCR amplification products of human VH and V L, wherein, Lane M is DNA marker D L2000, Lanes 1-4 are PCR amplification products of human VH, Lane 5-8 are PCR amplification products of human V L;

FIG. 5 shows SDS-PAGE electrophoresis to detect purified 6 candidate antibodies; lane M protein molecular weight markers; lane 1:048Ab-1 purified antibody (reduced); lane 2:048Ab-2 purified antibody (reduced); lane 3:048Ab-3 purified antibody (reduced); lane 4:048Ab-4 purified antibody (reduced); lane 5:048Ab-5 purified antibody (reduced); lane 6: 048Ab-6 purification of antibody (reduction); lane 7: 048Ab-1 purified antibody (non-reduced); lane 8: 048Ab-2 purified antibody (non-reduced); lane 9: 048Ab-3 purified antibody (non-reduced); lane 10: 048Ab-4 purified antibody (non-reduced); lane 11: 048Ab-5 purified antibody (non-reduced); lane 12: 048Ab-6 purified antibody (non-reduced);

FIG. 6 shows the detection of the cell activity of preferably 6 candidate antibodies; IC of 6 candidate antibodies 048Ab-1, 048Ab-2, 048Ab-3, 048Ab-4, 048Ab-5 and 048Ab-6₅₀2200pM, 2103pM, 1254pM,1106pM,7343pM, 2597pM respectively;

FIG. 7 shows that the preferred antibody 048Ab-4 is used for in vivo drug effect detection in animals, 048Ab-4 can significantly inhibit mI L-6 expression induced by I L-1 β in mice, and the expression level is reduced from 1411 +/-428.3 pg/m L to 34.13 +/-9.93 pg/m L.

Detailed Description

The invention has been described in terms of preferred embodiments, and it will be apparent to those skilled in the art that the techniques of the invention can be practiced and applied by modifying or appropriately combining the methods described herein without departing from the spirit, or scope of the invention.

An "antibody" refers to a protein composed of one or more polypeptides that specifically bind to an antigen. One form of antibody constitutes the basic building block of an antibody. This form is a tetramer, which is composed of two identical pairs of antibody chains, each pair having a light chain and a heavy chain. In each pair of antibody chains, the variable regions of the light and heavy chains are joined together and are responsible for binding to antigen, while the constant regions are responsible for the effector functions of the antibody.

The currently known antibody types include kappa and lambda light chains, and α, gamma (IgG1, IgG2, IgG3, IgG4), and mu heavy chains or their other type equivalents.A full-length immunoglobulin "light chain" (about 25kDa or about 214 amino acids) comprises a variable region formed by about 110 amino acids at the NH 2-terminus, and a kappa or lambda constant region at the COOH-terminus.

"antibody" includes any isotype of antibody or immunoglobulin, or antibody fragments that retain specific binding to an antigen, including but not limited to Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single chain antibodies, and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein. The antibody may be labeled and detected, for example, by a radioisotope, an enzyme capable of producing a detectable substance, a fluorescent protein, biotin, or the like. The antibodies can also be bound to a solid support, including but not limited to polystyrene plates or beads, and the like.

The invention screens the humanized antibody aiming at I L-1 β by a phage display technology, and obtains a complete antibody by a genetic engineering method, thereby providing a medicament for treating related diseases caused by abnormal expression of I L-1 β.

The specific preparation and related detection method of the anti-I L-1 β monoclonal antibody comprises the following steps of constructing an I L-1 β fully human antibody library, including optimization and modification of phage display carriers, separation of PBMC cells, RNA extraction, cDNA reverse transcription, amplification and enzyme digestion, connection, electrotransformation and the like of antibody genes, screening antibodies, including antigen preparation, phage display screening, E L ISA screening and the like, constructing a full-length antibody, expressing, purifying, measuring affinity, measuring cell activity and the like.

and/or

In some embodiments of the invention, the plurality of amino acids that are substituted, deleted, or added is 2,3,4,5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32.

In some embodiments of the invention, the anti-I L-1 β monoclonal antibody has:

(1) a heavy chain variable region having an amino acid sequence of SEQ ID NO. 1,2,3,4,5,6 and a light chain variable region having an amino acid sequence of SEQ ID NO. 7; or

(2) A heavy chain variable region having an amino acid sequence of SEQ ID NO. 1,2,3,4,5,6 and a light chain variable region having an amino acid sequence of SEQ ID NO. 8; or

(3) A heavy chain variable region having an amino acid sequence of SEQ ID NO. 1,2,3,4,5,6 and a light chain variable region having an amino acid sequence of SEQ ID NO. 9; or

(4) A heavy chain variable region having an amino acid sequence of SEQ ID NO. 1,2,3,4,5,6 and a light chain variable region having an amino acid sequence of SEQ ID NO. 10; or

(5) A heavy chain variable region having an amino acid sequence of SEQ ID NO. 1,2,3,4,5,6 and a light chain variable region having an amino acid sequence of SEQ ID NO. 11; or

(6) The heavy chain variable region having the amino acid sequence of

SEQ ID NO

1,2,3,4,5,6 and the light chain variable region having the amino acid sequence of SEQ ID NO 12.

Specifically, the amino acid sequence of the heavy chain variable region of the monoclonal antibody against I L-1 β is shown as SEQ NO. 1, and the amino acid sequence of the light chain variable region is shown as SEQ NO. 7.

Or, the amino acid sequence of the heavy chain variable region of the monoclonal antibody of anti-I L-1 β is shown as SEQ NO. 2, and the amino acid sequence of the light chain variable region is shown as SEQ NO. 7.

Or, the amino acid sequence of the heavy chain variable region of the monoclonal antibody of anti-I L-1 β is shown as SEQ NO. 3, and the amino acid sequence of the light chain variable region is shown as SEQ NO. 7.

Or, the amino acid sequence of the heavy chain variable region of the monoclonal antibody of anti-I L-1 β is shown as SEQ NO. 4, and the amino acid sequence of the light chain variable region is shown as SEQ NO. 7.

Or, the amino acid sequence of the heavy chain variable region of the monoclonal antibody of anti-I L-1 β is shown as SEQ NO. 5, and the amino acid sequence of the light chain variable region is shown as SEQ NO. 7.

Or, the amino acid sequence of the heavy chain variable region of the monoclonal antibody of anti-I L-1 β is shown as SEQ NO. 6, and the amino acid sequence of the light chain variable region is shown as SEQ NO. 7.

Specifically, the amino acid sequence of the heavy chain variable region of the monoclonal antibody against I L-1 β is shown as SEQ NO. 1, and the amino acid sequence of the light chain variable region is shown as SEQ NO. 8.

Or, the amino acid sequence of the heavy chain variable region of the monoclonal antibody of anti I L-1 β is shown as SEQ NO. 2, and the amino acid sequence of the light chain variable region is shown as SEQ NO. 8.

Or, the amino acid sequence of the heavy chain variable region of the monoclonal antibody of anti I L-1 β is shown as SEQ NO. 3, and the amino acid sequence of the light chain variable region is shown as SEQ NO. 8.

Or, the amino acid sequence of the heavy chain variable region of the monoclonal antibody of anti I L-1 β is shown as SEQ NO. 4, and the amino acid sequence of the light chain variable region is shown as SEQ NO. 8.

Or, the amino acid sequence of the heavy chain variable region of the monoclonal antibody of anti-I L-1 β is shown as SEQ NO. 5, and the amino acid sequence of the light chain variable region is shown as SEQ NO. 8.

Or, the amino acid sequence of the heavy chain variable region of the monoclonal antibody of anti-I L-1 β is shown as SEQ NO. 6, and the amino acid sequence of the light chain variable region is shown as SEQ NO. 8.

Or, the amino acid sequence of the heavy chain variable region of the monoclonal antibody of anti I L-1 β is shown as SEQ NO 1, and the amino acid sequence of the light chain variable region is shown as SEQ NO 9.

Or, the amino acid sequence of the heavy chain variable region of the monoclonal antibody of anti I L-1 β is shown as SEQ NO. 2, and the amino acid sequence of the light chain variable region is shown as SEQ NO. 9.

Or, the amino acid sequence of the heavy chain variable region of the monoclonal antibody of anti I L-1 β is shown as SEQ NO. 3, and the amino acid sequence of the light chain variable region is shown as SEQ NO. 9.

Or, the amino acid sequence of the heavy chain variable region of the monoclonal antibody of anti-I L-1 β is shown as SEQ NO. 4, and the amino acid sequence of the light chain variable region is shown as SEQ NO. 9.

Or, the amino acid sequence of the heavy chain variable region of the monoclonal antibody of anti-I L-1 β is shown as SEQ NO. 5, and the amino acid sequence of the light chain variable region is shown as SEQ NO. 9.

Or, the amino acid sequence of the heavy chain variable region of the monoclonal antibody of anti-I L-1 β is shown as SEQ NO. 6, and the amino acid sequence of the light chain variable region is shown as SEQ NO. 9.

Or, the amino acid sequence of the heavy chain variable region of the monoclonal antibody of anti I L-1 β is shown as SEQ NO 1, and the amino acid sequence of the light chain variable region is shown as SEQ NO 10.

Or, the amino acid sequence of the heavy chain variable region of the monoclonal antibody of anti I L-1 β is shown as SEQ NO. 2, and the amino acid sequence of the light chain variable region is shown as SEQ NO. 10.

Or, the amino acid sequence of the heavy chain variable region of the monoclonal antibody of anti I L-1 β is shown as SEQ NO 3, and the amino acid sequence of the light chain variable region is shown as SEQ NO 10.

Or, the amino acid sequence of the heavy chain variable region of the monoclonal antibody of anti-I L-1 β is shown as SEQ NO. 4, and the amino acid sequence of the light chain variable region is shown as SEQ NO. 10.

Or, the amino acid sequence of the heavy chain variable region of the monoclonal antibody of anti-I L-1 β is shown as SEQ NO. 5, and the amino acid sequence of the light chain variable region is shown as SEQ NO. 10.

Or, the amino acid sequence of the heavy chain variable region of the monoclonal antibody of anti-I L-1 β is shown as SEQ NO. 6, and the amino acid sequence of the light chain variable region is shown as SEQ NO. 10.

Or, the amino acid sequence of the heavy chain variable region of the monoclonal antibody of anti-I L-1 β is shown as SEQ NO. 7, and the amino acid sequence of the light chain variable region is shown as SEQ NO. 10.

Or, the amino acid sequence of the heavy chain variable region of the monoclonal antibody of anti I L-1 β is shown as SEQ NO 1, and the amino acid sequence of the light chain variable region is shown as SEQ NO 11.

Or, the amino acid sequence of the heavy chain variable region of the monoclonal antibody of anti-I L-1 β is shown as SEQ NO. 2, and the amino acid sequence of the light chain variable region is shown as SEQ NO. 11.

Or, the amino acid sequence of the heavy chain variable region of the monoclonal antibody of anti-I L-1 β is shown as SEQ NO 3, and the amino acid sequence of the light chain variable region is shown as SEQ NO 11.

Or, the amino acid sequence of the heavy chain variable region of the monoclonal antibody of anti-I L-1 β is shown as SEQ NO. 4, and the amino acid sequence of the light chain variable region is shown as SEQ NO. 11.

Or, the amino acid sequence of the heavy chain variable region of the monoclonal antibody of anti-I L-1 β is shown as SEQ NO. 5, and the amino acid sequence of the light chain variable region is shown as SEQ NO. 11.

Or, the amino acid sequence of the heavy chain variable region of the monoclonal antibody of anti-I L-1 β is shown as SEQ NO. 6, and the amino acid sequence of the light chain variable region is shown as SEQ NO. 11.

Or, the amino acid sequence of the heavy chain variable region of the monoclonal antibody of anti-I L-1 β is shown as SEQ NO. 7, and the amino acid sequence of the light chain variable region is shown as SEQ NO. 11.

Or, the amino acid sequence of the heavy chain variable region of the monoclonal antibody of anti I L-1 β is shown as SEQ NO 1, and the amino acid sequence of the light chain variable region is shown as SEQ NO 12.

Or, the amino acid sequence of the heavy chain variable region of the monoclonal antibody of anti-I L-1 β is shown as SEQ NO. 2, and the amino acid sequence of the light chain variable region is shown as SEQ NO. 12.

Or, the amino acid sequence of the heavy chain variable region of the monoclonal antibody of anti-I L-1 β is shown as SEQ NO. 3, and the amino acid sequence of the light chain variable region is shown as SEQ NO. 12.

Or, the amino acid sequence of the heavy chain variable region of the monoclonal antibody of anti-I L-1 β is shown as SEQ NO. 4, and the amino acid sequence of the light chain variable region is shown as SEQ NO. 12.

Or, the amino acid sequence of the heavy chain variable region of the monoclonal antibody of anti-I L-1 β is shown as SEQ NO. 5, and the amino acid sequence of the light chain variable region is shown as SEQ NO. 12.

Or, the amino acid sequence of the heavy chain variable region of the monoclonal antibody of anti-I L-1 β is shown as SEQ NO. 6, and the amino acid sequence of the light chain variable region is shown as SEQ NO. 12.

Or, the amino acid sequence of the heavy chain variable region of the monoclonal antibody of anti-I L-1 β is shown as SEQ NO. 7, and the amino acid sequence of the light chain variable region is shown as SEQ NO. 12.

The anti-I L-1 β monoclonal antibodies provided herein are capable of binding to human I L-1 β and monkey I L-1 β in certain embodiments, the affinity between the antibody and its target is characterized by Ka, Kd (dissociation constant), KD (equilibrium dissociation constant), and the anti-I L-1 β monoclonal antibodies provided herein have a KD value of no greater than 1.76 nM.

After the anti-I L-1 β monoclonal antibody provided by the invention can neutralize I L-1 β, the expression of I L-6 of human lung fibrocyte MRC-5 containing an I L-1 receptor is reduced by induction of I L-1 β, and the IC50 value of the inhibition is not higher than 7343 pM.

The anti-I L-1 β monoclonal antibody provided by the invention can also inhibit the expression of I L-6 in a human I β 0-1 β (R & D system) activated mouse, can obviously inhibit the expression of mI L-6 after 75ng of I L-1 β is injected into each mouse, and the expression quantity of mI L-6 is reduced to 34.13 +/-9.93 pg/m L from 1411 +/-428.3 pg/m L.

6 anti-I β 0-1 β 2 monoclonal antibodies in the anti-I L-1 β monoclonal antibodies provided by the invention have good affinity with human I β 1-1 β 4 and monkey I β 3-1 β, can block the combination of I L-1 β and I L-1R, and reduce the response of optional I L-l β to induced fever, amplified lymphocytes and stimulate acute phase response initially, 6 anti-I L-1 β monoclonal antibodies are respectively named as 048Ab-1, 048Ab-2, 048Ab-3, 048Ab-4, 048Ab-5 and 048Ab-6, and particularly have any one of the following groups of heavy chain variable regions and light chain variable regions:

the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO:1, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO:10 (H1L 4, named 048 Ab-1);

(ii) the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:11 (H1L 5, designated as 048 Ab-2);

(iii) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:2, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:11 (H2L 5, designated 048 Ab-3);

(iv) the amino acid sequence of the heavy chain variable region is represented by SEQ ID NO:2, and the amino acid sequence of the light chain variable region is represented by SEQ ID NO:12 (H2L 6, designated 048 Ab-4);

(V) the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO:3, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO:12 (H3L 6, named 048 Ab-5);

(VI) the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO:4, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO:11 (H4L 5, named 048 Ab-6).

Wherein, the amino acid sequence of the heavy chain variable region is shown as SEQ NO:2, the anti-I L-1 β monoclonal antibody 048Ab-4(H2 β 16) shown as SEQ NO:12 of the amino acid sequence of the light chain variable region has the best effect of blocking the combination of I β 2-1 β 0 and I β 4-1R, after I β 5-1 β 3is neutralized, the expression of I L-6 induced by I L-1 β of human lung fiber cell MRC-5 containing I β 6-1 receptor is reduced, the IC50 value of the inhibition is as low as 1106Pm, the expression of mI L-6 induced by human I L-1 β can be obviously inhibited in mice, and the expression amount is reduced to 34.13 +/-9.93/m L from 1411 +/-428.3/m L.

The heavy chain type of the anti-I L-1 β monoclonal antibody is preferably IgG1, IgG2, IgG3 or IgG4, and more preferably IgG 4.

The light chain type of the anti-I L-1 β monoclonal antibody is preferably kappa or lambda, more preferably lambda.

The monoclonal antibodies of the invention are fully human.

Preferably, the DNA molecule has

and/or

Wherein, the chemical label includes but is not limited to isotope, immunotoxin, chemical drug.

Preferably, the immunotoxin is aflatoxin, diphtheria toxin, pseudomonas aeruginosa exotoxin, ricin, abrin, mistletoe agglutinin, calycosin, PAP, nystatin, gelonin or luffa toxin.

The biomarkers include, but are not limited to, biotin, avidin, or enzyme markers.

Preferably, the enzyme is labeled horseradish peroxidase or alkaline phosphatase.

Preferably, the solid medium or non-solid medium is selected from colloidal gold, polystyrene plates or beads.

The invention also provides a pharmaceutical composition comprising the anti-human VEGFR2 monoclonal antibody and/or the conjugate.

In some embodiments, the kit further comprises a coating buffer, a washing solution, a blocking solution, and/or a color developing solution.

In some embodiments, the coating buffer is a carbonate buffer.

In some embodiments, the wash solution includes at least one of PBS, Tween, sodium chloride, potassium chloride, disodium hydrogen phosphate, dipotassium hydrogen phosphate.

In some embodiments, the blocking solution comprises at least one of PBS and BSA.

In some embodiments, the color developing solution comprises at least one of a TMB solution, a substrate buffer, and a stop solution.

Wherein, in some embodiments, the substrate buffer comprises citric acid and disodium hydrogen phosphate and the stop solution is an aqueous hydrogen peroxide solution.

Further, in some embodiments, the kit further comprises PBS, goat anti-mouse IgG Fc, and a TITC secondary antibody.

The preparation for blocking the combination of I L-1 β and I L-1 RA and the medicine for preventing or treating the periodic syndrome related to the coldness imidacloprid can be injection or injection powder, and the technicians in the field can add the anti-I L-1 β monoclonal antibody, the conjugate and/or the conjugate into various pharmaceutically acceptable common auxiliary materials required for preparing the injection or the injection powder according to the prior art to prepare the injection or the injection powder.

In order to further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Unless otherwise specified, the reagents and apparatuses used in the examples of the present invention are commercially available products and commercially available.

EXAMPLE 1 preparation of antigenic proteins

1. Antigen and positive antibody gene synthesis and expression vector construction:

the amino acid sequence design of the fusion human I L-1 β mature protein and the connecting peptide-mIgG 1Fc is shown as SEQ ID NO. 25 the amino acid sequence design of the fusion human I L-1 β mature protein and the connecting peptide-6 his amino acid sequence is shown as SEQ ID NO. 26.

The amino acid sequence corresponding to the human I L-1 β mature protein fusion protein (I L-1 β -connecting peptide-mIgG 1Fc or I L-1 β -connecting peptide-6 his) designed above is subjected to codon artificial optimization, the codon optimized sequence is shown as SEQ ID NO:27 or 28, a HindIII enzyme cutting site and a Kozak sequence GCCGCCACC are added to the 5 'end, a termination codon TAG and an EcoR I enzyme cutting site are added to the 3' end, Suzhou Hongsu company is entrusted to synthesize optimized DNA, and the optimized DNA is cloned into a pUC57simple (provided by Suzhou Honghu company) vector to obtain human pUC57simple-I L-1 β -connecting peptide-mIgG 1Fc and pUC57simple-I L-1 β -connecting peptide-6 his plasmids.

Carrying out enzyme digestion (Hind III and EcoR I) on human plasmids pUC57simple-I L-1 β -connecting peptide-mIgG 1Fc, pUC57simple-I β 0-1 β 1-connecting peptide-6 his and a vector pGS003, carrying out electrophoretic recovery on the obtained fusion gene fragments I L-1 β -connecting peptide-mIgG 1Fc and I L-1 β -connecting peptide-6 his, carrying out connection reaction and recombination with a pGS003 vector to obtain expression plasmids pGS 003-human I L-1 β -connecting peptide-mIgG 1Fc and pGS 003-human I L-1 β -connecting peptide-6 his.

Positive antibody gene synthesis and expression vector construction:

the pAb antibody sequence is shown as PABH shown in SEQ ID NO:29 and PAB L shown in SEQ ID NO: 30.

The amino acid sequence corresponding to the antibody sequence is subjected to codon artificial optimization, a Hind III restriction site and a Kozak sequence GCCGCCACC are added at the 5 'end, a termination codon TAG and an EcoR I restriction site are added at the 3' end, Kinshire corporation is entrusted to synthesize optimized DNA, the optimized DNA is cloned into a pUC57simple (provided by Kinshire corporation) vector to obtain pUC57simple-PCABH and pUC57simple-PCAB L plasmids, the pUC57simple-PCABH and pUC57simple-PCAB L are subjected to restriction enzyme (Hind III and EcoR I), gene fragments PCABH and PCAB L obtained by electrophoretic recovery are subjected to ligation reaction with a pGS003 vector to be recombined to obtain pGS003-PCABH and pGS003-PCAB L.

2. Instantaneous expression

pGS003-PCABH, pGS003-PCAB L, pGS 003-human I L-1 β -linker-mIgG 1Fc and pGS 003-human I L-1 β -linker-6 his were transiently expressed.

Using FreeStyle^TM293F cells were transiently expressed in Freestyle Medium 24 hours prior to transfection, in 125ml Erlenmeyer flasks were seeded with 0.6 × 10⁶293F cells per ml, 5% CO at 37 ℃₂Shaking the culture in an incubator at 130 rpm. When in transfection, 30 mu l of 293fectin is firstly added into 1ml of OPTIMEM, and after being fully and uniformly mixed, the mixture is incubated for 5 minutes at room temperature; at the same time, the recombinant vector and 293fectin are added with 1. mu.g plasmid according to the ratio of 1:1, namely 1. mu.l 293fectin is added, and the total DNA amount is taken as 30. mu.g, and is dissolved in 1ml optiMEM. Then fully mixing the DNA diluent and the 293fectin diluent with the total volume of 2ml, incubating for 15 minutes at room temperature, then adding all the mixture into a cell culture hole, uniformly mixing, and carrying out 5% CO treatment at 37 DEG C₂Shaking at 130rpm in incubator for 7 days. And (4) centrifuging the culture solution at a high speed, and performing vacuum filtration by using a microporous filter membrane.

3. Protein purification

According to the operation method provided by the manufacturer, a Protein A column (Protein purification liquid chromatography system/AKTAPurifier 100, GE) is adopted for purification, purified human I L-1 β -mIgG1Fc fusion Protein is obtained, AKTA is washed cleanly by ultrapure water, and 1ml of rProtianA Fast Flow prepacked column is connected to AKTA, cleaning is carried out, 5 column volumes are cleaned by 1M HAC, and the balance is 5 column volumes balanced by 20mM PB 0.15M NaCl (pH 7.0).

The cell expression supernatant sample is centrifuged at 1000rpm × 5min, the supernatant is centrifuged at 8000rpm × 30min, 20ml of the centrifuged supernatant is applied at a flow rate of 0.2ml/min, the equilibrium is 20mM PB 0.15M NaCl (pH 7.0) for 5 column volumes, the wash at 0.2ml/min is 20mM PB 1M NaCl (pH 7.0) for 5 column volumes, and the equilibrium is 20mM PB 0.15M NaCl (pH 7.0) for 5 column volumes.

And (3) elution: 20mM sodium citrate buffer (pH 3.0) at a flow rate of 0.2 ml/min. The collection started when UV280 to 100mAU and stopped when it dropped to 100 mAU. The pH of the sample is adjusted to 6-8 by 1M Tris, and SDS-PAGE electrophoresis detects the result as shown in figure 1.

Example 2: construction of natural humanized single-chain antibody phage display library

1. Construction of phagemid vectors

Selecting pCANTAB5E as a phage display vector, carrying out vector modification according to cloning and phage display requirements, wherein the modification result is shown in figure 2. SfiI-NcoI-XhoI + L inker + NheI-NotI sequence (SEQ ID NO:31) is subjected to gene synthesis, then enzyme digestion is carried out by SfiI and NotI, and the vector is subjected to ligation reaction and recombination with pCANTAB5E to construct a modified vector pCANTAB 5E-SF.

2. PBMC isolation and mRNA extraction

Fresh peripheral blood was aseptically collected from healthy volunteers, and lymphocytes were separated from the blood using a lymphocyte separation medium (GE) and used by Invitrogen corporation

Extraction of 100 × 10 from reagent (15596-⁶Total RNA of individual cells, the results are shown in FIG. 3.

3. Antibody library primer design, synthesis and RT-PCR

Primers for amplifying the heavy and light chains of human antibodies and correct restriction enzyme sites were designed based on the antibody gene sequence information published by the Kabat, V-base2 and IMGT websites, primers were synthesized by suzhou hong biotech limited, purified by PAGE, and the primer sequences are shown in tables 1 and 2:

TABLE 1 primers for amplifying heavy chain variable regions

R＝A/G，Y＝C/T，M＝A/C，K＝G/T，S＝C/G，W＝A/T，H＝A/C/T，B＝C/G/T，V＝A/C/G，D＝A/G/T

TABLE 2 primers for amplifying light chain variable regions

Primer name	Sequence (5 '→ 3')
		h-k_F1	ggtggcggtggtagtgctagcGAWAYWGTGATGACMCAGYMTC
h-k_F2	ggtggcggtggtagtgctagcGAAGTTATGTTGATGCAGTCTCT
		h-k_F3	ggtggcggtggtagtgctagcGAWGTTGTGMTGACAYRGTC
h-k_F4	ggtggcggtggtagtgctagcRHCATCYRGATGACCCAGTCTSCA
		h-k_F5	ggtggcggtggtagtgctagcRACATCCAGATGAATTCAGTCTCC
h-k_F6	ggtggcggtggtagtgctagcGAMATYGTRHTGACMCAGTCTCCA
		h-k_F7	ggtggcggtggtagtgctagcRATWTCCAGATGACYCAGTCTCCA
h-k_F8	ggtggcggtggtagtgctagcGATRTTGTGATGACTSAGYCTCCA
		h-k_F9	ggtggcggtggtagtgctagcGAAATWGTGWTGAYGCAGTCTCCA
h-k_F10	ggtggcggtggtagtgctagcGAAACGACACTCACGCAGTCTCCA
		h-k_F11	ggtggcggtggtagtgctagcGAMATTSTGYTGACHCAGWCTCCA
h-k_F12	ggtggcggtggtagtgctagcGABAYTGTGATSRCCCAGACTCCA
		h-k_F13	ggtggcggtggtagtgctagcRMCATCCAGDTGAYBCAGYCTCC
h-k_F14	ggtggcggtggtagtgctagcGATGCTGYGAWGACCCAACCTCCA
		h-k_R1	ATGAGTTTTTGTTCTGCGGCCGCTTTGATHTCCASYTTGGTCCCH
h-k_R2	ATGAGTTTTTGTTCTGCGGCCGCTTTAATCTCCAGTCGTGTCCCT
		h-λ_F1	ggtggcggtggtagtgctagcCAGYCTGYKCTGACYCAGV
h-λ_F2	ggtggcggtggtagtgctagcCAGGCAGGGCWGACTCAGCMMC
		h-λ_F3	ggtggcggtggtagtgctagcCAGCYTGTGCTGACTCARTCRYCC
h-λ_F4	ggtggcggtggtagtgctagcCACGTTATACTGACTCAACCGCCC
		h-λ_F5	ggtggcggtggtagtgctagcCRGCCYGTGCTGACTCARCYGCC
h-λ_F6	ggtggcggtggtagtgctagcCTGSCTGTGCTRACTCAGGCCCC
		h-λ_F7	ggtggcggtggtagtgctagcCAGBCTGTGCTGACTCAGCCR
h-λ_F8	ggtggcggtggtagtgctagcCAGTCTGTSBTGACGCAGCCGCC
		h-λ_F9	ggtggcggtggtagtgctagcCAGDCTGTGRTGACYCAGGARCC
h-λ_F10	ggtggcggtggtagtgctagcCARTCTGCCCTGAYTCAGCCTS
		h-λ_R1	ATGAGTTTTTGTTCTGCGGCCGCKAGGACGGTSACCTTGGTSCCAST
h-λ_R2	ATGAGTTTTTGTTCTGCGGCCGCTAGGACGGTCAGCTCSGTCCCCTC
		h-λ_R3	ATGAGTTTTTGTTCTGCGGCCGCKAGGRCGGTCAGCTKGGTSCCTCC
h-λ_R4	ATGAGTTTTTGTTCTGCGGCCGCTAAAATGATCAGCTGGGTTCCTCC

PrimeScript by Takara corporation was used^TMII OligodT primer in first chain cDNA synthesis kit (6210A), reverse transcription was performed to synthesize first chain cDNA using total RNA extracted from PBMC as template, and the heavy chain variable region VH and light chain variable region V L of antibody gene were amplified by PCR technique using the primers in Table 1 and Table 2 above under the condition of 95 deg.C 5min, and the amplification was performed 25 times by denaturation at 95 deg.C for 30S, annealing at 56 deg.C for 30S, extension at 72 deg.C for 1min, and final cycle extension at 72 deg.C for 3min, and electrophoresis detection was performed after completion of amplification, as shown in FIG. 4.

4. Construction of human VH and V L Single chain antibody libraries

V L was first cloned into pCANTAB5E-SF vector by NheI and NotI, and then VH was cloned into the vector of the previous step by NcoI and XhoI.

The pCANTAB5E-SF vector and the amplified and purified PCR product of the human light chain variable region were digested with NheI and NotI. Connecting the enzyme-digested 4.4. mu.g vector with 1. mu.g fragment, purifying the connection product, and electrotransfering to 10 pieces of 200. mu.l TG1 electrotransferAdding SOC into the cells (2.5kV, 2cm electric shock cup), culturing at 37 deg.C for 1h, taking a small amount of resuscitating bacteria, diluting, coating on 10 phi 15cm plates, culturing overnight, and collecting the solution 2.02 × 10⁸And (3) carrying out single cloning, scraping and washing a phi 15cm flat plate by using 50ml of 2YT to obtain a light chain antibody library, taking part of bacterial liquid to prepare small quality-improved particles, adding glycerol with the final concentration of 30% (V/V) into the residual library bacterial liquid, and storing at-80 ℃.

The light chain antibody library plasmid and the amplified and purified PCR product of the human heavy chain variable region are cut by NcoI and XhoI, 5.8 mu g of cut carrier is connected with 1.2 mu g of fragment, the connection product is purified and then transferred to 20 200 mu lTG1 electrotransfer competent cells (2.5kV, 2cm specification electric shock cup), SOC is added, the cells are cultured for 1h at 37 ℃, part of resuscitation bacteria are taken to be diluted and coated on a flat plate for calculating the library capacity, the rest bacteria liquid is coated on 20 phi 15cm flat plates for overnight culture, and the library capacity is calculated to be 5.31 × 10 on the next day⁸And (3) carrying out single cloning, scraping and washing a phi 15cm flat plate by using 50ml of 2YT to obtain humanized VH and V L single-chain antibody libraries, preparing small quality-improved particles by taking part of bacterial liquid, adding glycerol with the final concentration of 30% (V/V) into the residual library bacterial liquid, and storing at-80 ℃.

Example 3: phage display and screening of humanized antibody libraries

1. Phage display and panning of antibody libraries

The human VH and V L single-chain antibody library bacteria with 100 times library capacity are taken to inoculate 880ml of 2YT-AG culture medium (containing 100 mu g/ml ampicillin and 2% glucose), cultured at 37 ℃ and 200rpm until OD600 is 0.5-0.6, added with helper phage with 100 times cell density, infected for 1.5h, centrifuged to collect the bacteria, and then 400ml of 2YT-AK culture medium (containing 100 mu g/ml ampicillin and 75 mu g/ml kanamycin) is used for resuspension of the cells, and cultured at 30 ℃ and 200rpm overnight.

Centrifuging the culture of the previous step at 4 deg.C for 20min at 10000g, collecting supernatant, adding 1/4 volume of PEG/NaCl, mixing, standing on ice for 1h, centrifuging at 4 deg.C for 25min at 12000g, discarding supernatant, placing the centrifuge tube on flat paper, removing liquid, re-suspending the phage precipitate with 2ml precooled 1 × PBS, centrifuging at 4 deg.C for 10min at 12000g, transferring supernatant to new 15ml centrifuge tube, and adding 3% BSA to obtain final productObtaining initial phage in the first round, coating immune tube with I L-1 β -His as antigen, blocking with 2% M-PBS, and adding 10¹³The first round of initial phage was antibody antigen binding, PBST was used to wash away unbound phage, 1ml of Glycine-HCl (pH2.2) was used to elute phage, the eluted phage was re-infected with TG1, amplification of the eluted product was performed, and PEG/NaCl precipitation was used to purify phage for the next round of screening. And 4 rounds of enrichment screening of the phage library are carried out, the antigen amount is sequentially reduced, the washing strength is sequentially enhanced, and the titer of each round of elution products is determined.

2. Induced expression of monoclonal and E L ISA screening

Performing limited dilution and plate coating on the bacterial liquid subjected to the 1 st to 4 th rounds of elutriation, and culturing overnight; selecting the monoclonal to be cultured in a 96-hole deep-hole plate which is subpackaged with 0.5 ml/hole of 2YT-AG culture medium for overnight; the overnight culture was then transferred to a 96-well deep-well plate containing 0.5 ml/well of 2YT-AG medium at a ratio of 1:10, cultured until OD600 became 0.5-0.6, and the cells were collected by centrifugation at 3000g, resuspended in 2YT-AI medium (containing 100. mu.g/ml ampicillin and 1mM IPTG), induced overnight at 30 ℃, and the supernatant was transferred by centrifugation the next day to a clean 96-well deep-well plate and added with 3% BSA at final concentration to obtain a monoclonal phage sample.

Coating a 96-well enzyme label plate by using I L-1 β -His as an antigen, sealing, adding 50 mu l of monoclonal phage sample into each well, incubating for 1h at 25 ℃, then adding 200 mu l of PBST into each well, oscillating for 5-10S, discarding the solution, repeating for 3-5 times, then adding 50 mu l of anti-M13-HRP antibody PBS diluent into each well, incubating for 1h at 25 ℃, then adding 200 mu l of PBST into each well, oscillating for 5-10S, discarding the solution, repeating for 5 times, adding 50 mu l of TMB color developing solution into each well, developing for 3-10 min (the specific color developing time is determined according to the color developing speed), and then adding 50 mu l of 1M H into each well₂SO₄Terminating color development, measuring OD450 value by using enzyme-labeling instrument, selecting 860E L ISA positive samples according to the E L ISA data of the monoclonal phage, carrying out competitive E L ISA screening, diluting the positive control antibody to 200 mu g/ml, mixing 50 mu l of diluent with 50 mu l of monoclonal phage sample, carrying out E L ISA of the above steps, and displaying that 18 monoclonal antibodies and the positive control antibody existEpitope competition, selecting these clones for sequencing; and (3) taking the overnight culture bacterial liquid in the 2YT-AG culture medium of the 96-well deep-well plate, and performing sequencing analysis to finally obtain the amino acid sequence of the variable region of the unique monoclonal antibody, which is shown as SEQ ID NO 1-12, and the nucleotide sequence is shown as SEQ ID NO 13-24.

Example 4: full Length antibody preparation

1. Construction of full-Length antibody transient transfection expression vector

pGS003-hIgG4CH and pGS003-hIg lambda C L were selected as expression vectors for constructing heavy and light chains of full-length antibodies against human I L-1 β, respectively, wherein hIgG4CH and hIg lambda C L were previously constructed into pGS003 vectors, primers were designed based on the gene sequences of VH and V L and the multiple cloning sites in the vectors, after PCR amplification, 6 VH and 6V L antibody genes were cloned into pGS003-hIgG4CH and pGS003-hIg lambda C L, respectively, using an in vitro recombination method (Suzhou Xunli, iMulli multi-fragment recombinant cloning kit), as identified in Table 3. after the correct insertion of the antibody genes by sequencing, the recombinant expression vector was transformed into E.coli TOP10F ', a single colony was picked up and inoculated into ampicillin-containing 100. mu.g/ml of ampicillin L B medium, incubated at 37 ℃ for 16 hours, using Zymo Research's extraction kit, finally plasmid concentration was determined by light shaking at 280 OD260, and OD 9.8. plasmid purity was measured by OD 9.

TABLE 3 List of heavy and light chain transient transfection expression vectors

Heavy chain expression vector name	Heavy chain variable region sequence	Light chain expression vector name	Light chain variable region sequence
				H1	SEQ NO:1	L1	SEQ NO:7
H2	SEQ NO:2	L2	SEQ NO:8
				H3	SEQ NO:3	L3	SEQ NO:9
H4	SEQ NO:4	L4	SEQ NO:10
				H5	SEQ NO:5	L5	SEQ NO:11
H6	SEQ NO:6	L6	SEQ NO:12

2. Transfection, expression and detection in mammalian cells 293E

After the 6 heavy chain expression vectors and the 6 light chain expression vectors are combined in pairs (36 combinations in total), transient transfection expression evaluation of a 2ml293E system is carried out, and the expression quantity and the detection value of E β 0ISA of the antibody combined with I L-1 β are detected, and the results are shown in Table 4. according to the expression quantity and the detection value of E β 2ISA of the antibody combined with I β 1-1 β, relatively low EC50 value and relatively high E L ISARmax value are selected, and 6 full-length antibodies of H1L 4, H1L 5, H2L 5, H2L 6, H3L 6 and H4L 5 are preferably selected and named as 048Ab-1, 048Ab-2, 048Ab-3, 048Ab-4, 048Ab-5 and 048 Ab-6.

Table 46 × 6 expression amount, EC50 value and E L ISA Rmax value of transient transfection expression of combined full-length antibody minor line

Transient transfection expression of 6 preferred antibodies using 293E in Freestyle Medium 24 hours prior to transfection, 0.5 × 10 was seeded in 1L cell culture flasks⁶Cell/ml 293E cells 300ml, 5% CO at 37 ℃₂Shaking and culturing at 120rpm in an incubator. When in transfection, 300 mu l of 293fectin is firstly added into 5.7ml of OPTIMEM, and after fully and uniformly mixing, the mixture is incubated for 2 minutes at room temperature; at the same time, 150. mu.g of each of the heavy and light chain expression plasmids was diluted to 6ml using OptiMEM. Mixing the diluted transfection reagent and plasmid, incubating at room temperature for 15 min, adding the mixture into cells, mixing, and adding 5% CO at 37 deg.C₂Shaking at 120rpm in incubator for 7 days.

3. Purification and detection of antibodies

The cell culture broth was centrifuged at 2000g for 20min, the supernatant was collected, and the amount of antibody expression in the supernatant was determined by Octet, see Table 5.

TABLE 56 detection of expression levels of 300ml of preferred antibodies transiently transfected

Name of antibody	Heavy chain sequence	Light chain sequence	Transient transfection expression amount (mg/L)
				048Ab-1	SEQ NO:1	SEQ NO:10	115.9
048Ab-2	SEQ NO:1	SEQ NO:11	186.8
				048Ab-3	SEQ NO:2	SEQ NO:11	171.2
048Ab-4	SEQ NO:2	SEQ NO:12	232.7
				048Ab-5	SEQ NO:3	SEQ NO:12	184.6
048Ab-6	SEQ NO:4	SEQ NO:11	38.5

The supernatant was filtered through a 0.22 micron filter, then passed through a MabSelect Sure affinity chromatography column (GE), 20mM citric acid-sodium citrate, pH3.0, eluted, pH adjusted to neutral with 1M Tris base, and adjusted to an isotonic solution by addition of 10 xPBS. The purified protein was detected by SDS-PAGE using 4-20% gradient gels (Kinsley), and the results are shown in lanes 7-12 of FIG. 5. And (3) taking the purified antibody, analyzing the purified product by SDS-PAGE under a reducing condition, and identifying the property and purity of the antibody, wherein the heavy chain is 50KD and the light chain is 25KD under the reducing condition, and the result is shown in lanes 1-6 of figure 5.

Example 5: preferred antibody affinity assays

The affinity of the anti-I L-1 β full-length antibody was measured using a Biacore T200 instrument by coupling human antibodies to a CM5 biosensing chip (GE Healthcare) using a human antibody capture kit from GEhealthcare, capturing candidate antibodies or positive controls with anti-human Fc antibodies on the chip, flowing different concentrations of human I L-1 β at a flow rate of 30. mu.l/min over the candidate antibodies on the chip, binding human I L-1 β to the candidate antibodies, at a binding time of 120S and a dissociation time of 300S, and performing kinetic fitting using BIAanalysis software (GE Healthcare) to obtain affinity constant results as shown in Table 6 below, which indicate that 6 preferred antibodies all have good binding activity to human I L-1 β.

TABLE 6 determination of affinity of candidate antibodies to human I L-1 β

Name of antibody	Ka(1/Ms)	Kd(1/s)	KD(M)
				048Ab-1	5.93E+04	7.66E-05	1.29E-09
048Ab-2	5.75E+04	6.04E-05	1.05E-09
				048Ab-3	5.68E+04	5.92E-05	1.04E-09
048Ab-4	4.84E+04	7.15E-05	1.48E-09
				048Ab-5	5.51E+04	9.67E-05	1.76E-09
048Ab-6	4.90E+04	6.00E-05	1.23E-09
				Positive control (Kanacumab)	2.23E+04	1.02E-04	4.56E-09

The positive control is canakinumab against I L-1 β.

Example 6: preferred antibody cell Activity assays

Human lung fibroblast MRC-5 contains I L-1 receptor, is recognized and activated by I L-1L 1, induces expression of I L0-6, after I L-1 β antibody neutralizes I L-1 β, expression of I L-6 is reduced, so that activity of I L-1 β antibody is evaluated by detecting expression level of I L-6, 5000MRC-5 cells are inoculated into each hole of a 96-well plate, and 5% CO is performed at 37 DEG C₂After overnight incubation, 4nM I L-1 β (R)&D System) 24h, I L-6E L ISA kit (R)&D system) test the level of supernatant I L-6 the I L-6 protein amounts of control and stimulated groups were 47.16 + -18.91 and 10571 + -174 pg/m L0, respectively, treatment with 50nM I L1-1L 2 antibody almost completely inhibited I L-1 β from inducing I L-6 expression, I L-6 expression < 1% after neutralization of I L-1 β antibody (FIG. 6A). for the preferred 6 candidate antibodies, 10 gradients were diluted 3-fold using 50nM to 0.0025nM to generate a dose response curve from which the I L-1 β antibody activity IC was calculated₅₀(FIG. 6B), the results show the IC of 6 candidate antibodies₅₀2200pM, 2103pM, 1254pM,1106pM,7343pM and 2597pM respectively show good neutralization activity, and 048Ab-4 is selected according to the cell activity result to carry out activity detection in animals.

Example 7 in vivo detection of I L-1 β antibody Activity in mice:

human I L-1 β (R & D system) can activate the expression of I β -6 in mice, I β -1 β antibody neutralizes I β -1 β to reduce the expression of mI β -6, the expression of mI β -6 changes the activity of I638-1 6865 antibody, 8-week-old BA β B/C female mice with the weight of 18-20g are selected, 7 mice in each group are selected, 75ng I L-1L is injected into each mouse in each experimental group, then 75 mu g I L1-1L antibody is injected into tail vein immediately, the eyeball blood is extracted after 2h, serum is separated, the optimal 048-4 in the serum is selected according to the result of cell activity, the in vivo activity of the animals is tested by injecting PGI L-72 Ab kit (R & D system ISA), and the expression of mI L-6 in the serum is tested by the method that the optimal 048-4 Ab 6 is selected according to the result of cell activity, the expression of mI L-L is tested by the mI L-L antibody, and the expression of the mI L-L is shown by the biological map 13 + -L/L.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

SEQUENCE LISTING

<110> Changchun golden race pharmaceutical Limited liability company

<120> anti-I L-1 β monoclonal antibody and application thereof

<130>MP1705733

<160>31

<170>PatentIn version 3.3

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

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

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

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35 40 45

Gly Ser Ile Arg Gly Lys Ala Tyr Gly Gly Ser Ser Glu Tyr Ala Ala

50 55 60

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

65 70 75 80

Thr Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Asn Arg Glu Val Glu Tyr Cys Lys Ser Pro Glu Asn Tyr Cys

100 105 110

Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210>5

<211>128

<212>PRT

<213> Artificial sequence

<400>5

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

1 5 10 15

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

20 25 30

Ala Met Asn Trp Phe Arg Gln Ala Pro Gly Met Gly Leu Glu Trp Val

35 40 45

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

50 55 60

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

65 70 75 80

Ala Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

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

100 105 110

Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210>6

<211>128

<212>PRT

<213> Artificial sequence

<400>6

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Gly Ser Gly Phe Thr Phe Gly Glu Tyr

20 25 30

Ala Ile Asn Trp Phe Arg Gln Ala Pro Gly Met Gly Leu Glu Trp Val

35 40 45

Gly Ser Ile Arg Gly Lys Ala Tyr Gly Gly Ser Ser Glu Tyr Ala Ala

50 55 60

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

65 7075 80

Ala Tyr Leu Arg Met Asn Gly Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Asn Arg Glu Val Glu Tyr Cys Lys Ser Pro Glu Asn Tyr Cys

100 105 110

Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210>7

<211>106

<212>PRT

<213> Artificial sequence

<400>7

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

1 5 10 15

Thr Ala Ser Ile Thr Cys Ser Gly Asp Arg Met Gly Asp Lys Phe Ala

20 25 30

Gly Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr

35 40 45

Gln Asp Thr Lys Arg Pro Thr Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

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

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys His Ala Trp Asp AsnSer Ser Val Ile

85 90 95

Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210>8

<211>106

<212>PRT

<213> Artificial sequence

<400>8

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

1 5 10 15

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

20 25 30

Ser Trp Tyr Arg Gln Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr

35 40 45

Gln Asp Ile Met Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

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

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys His Ala Trp Asp Asn Asn Ser Val Ile

85 90 95

Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210>9

<211>106

<212>PRT

<213> Artificial sequence

<400>9

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

1 5 10 15

Thr Ala Ser Ile Thr Cys Ser Gly Asp Arg Met Gly Asp Lys Tyr Ala

20 25 30

Gly Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr

35 40 45

Gln Asp Ile Lys Arg Pro Thr Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

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

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys His Ala Trp Asp Ser Asn Ser Val Val

85 90 95

Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210>10

<211>106

<212>PRT

<213> Artificial sequence

<400>10

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

1 510 15

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

20 25 30

Gly Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr

35 40 45

Gln Asp Ile Lys Arg Pro Thr Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

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

65 70 75 80

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

85 90 95

Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210>11

<211>106

<212>PRT

<213> Artificial sequence

<400>11

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

1 5 10 15

Thr Ala Ser Ile Thr Cys Ser Gly Asp Arg Met Gly Asp Lys Phe Ala

20 25 30

Gly Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr

35 40 45

Gln Asp Ile Lys Arg Pro Thr Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

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

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys His Ala Trp Asp Ser Asn Ser Val Ile

85 90 95

Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210>12

<211>106

<212>PRT

<213> Artificial sequence

<400>12

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

1 5 10 15

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

20 25 30

Cys Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr

35 40 45

Gln Asp Thr Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln AlaMet

65 70 75 80

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

85 90 95

Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210>13

<211>384

<212>DNA

<213> Artificial sequence

<400>13

gaggtgcagc tggtggagtc tgggggaggc ttggtaaggc cagggcggtc cctgagactc 60

tcctgtacag gttctggatt caccttaggt gattatacca tgaactggtt ccgccaggct 120

ccagggatgg ggctggagtg ggtaggtttg atcagaggca aagcctatgg tgggagcagc 180

gaatacgccg cgtctgtgaa agatagattc accatctcaa gagatgattc caaaagcatc 240

gcctatctgc aaatgaacag cctgaaaacc gaggacacag ccgtgtatta ctgtaataga 300

gaggtagagt attgcaaatc gtcgcaaaac tactgctacg gtatggacgt ctggggccaa 360

gggaccacgg tcaccgtctc ctcc 384

<210>14

<211>384

<212>DNA

<213> Artificial sequence

<400>14

gaggtgcagc tggtggagtc tgggggaggc ttggtaaagc cagggcggtc cctgagactc 60

tcctgtacag gttctggatt cacctttggt gattatgctt tgaactggtt ccgccaggct 120

ccagggatgg ggctggagtg ggtaggtttc attagaggca aagcctatgg tgggacaaca 180

gaatacgccg cgtctgtgaa aggcagattc accatctcaa gagatgattc caaaagcatc 240

gcctatctgc aaatgaacag cctgaaaacc gaggacacag ccgtgtatta ctgtaataga 300

gaggtagagt attgcaggtc gtccgagaac tactgctacg gtatggacgt ctggggccaa 360

gggaccacgg tcaccgtctc ctcc 384

<210>15

<211>384

<212>DNA

<213> Artificial sequence

<400>15

gaggtgcagc tggtggagtc tgggggaggc ttggtaaagc cagggcggtc cctgagactc 60

tcctgtacag gttctggatt caccttaggt gattatgcca tgaactggtt ccgccaggct 120

ccagggatgg ggctggagtg ggtaggttcg agcagaggca aagcctatgg tgggaccagc 180

gaatacgccg cgtctgcgaa aggtagattc accatctcaa gagatgattc caaaagcatc 240

gcctatctgc aaatgaacag cctgaaaacc gaggacacag ccgtgtatta ctgtaatagg 300

gaggtagagt tttgcagatc gccggaaaac tactgctacg gtatggacgt ctggggccaa 360

gggaccacgg tcaccgtctc ctcc 384

<210>16

<211>384

<212>DNA

<213> Artificial sequence

<400>16

gaggtgcagc tggtggagtc tgggggaggc ttggtaaagc cagggcggtc cctgagactc 60

tcctgtacag gttctggatt caccttaggt gattatgcca tgaactggtt ccgccaggct 120

ccagggatgg ggctggagtg ggtaggttcg atcagaggca aggcctatgg tgggagcagc 180

gaatacgccg cgtctgcgat aggtagattc accatctcaa gagatgattc caaaagcatc 240

acctatctgc aaatgaacag cctgaaaacc gaggacacag ccgtgtatta ctgtaataga 300

gaggtagagt attgcaaatc gccggaaaac tactgctacg gtatggacgt ctggggccaa 360

gggaccacgg tcaccgtctc ctcc 384

<210>17

<211>384

<212>DNA

<213> Artificial sequence

<400>17

gaggtgcagc tggtggagtc tgggggaggc ttggtaaagc cagggcggtc cctgagactc 60

tcctgtacag gttctggatt cacctttggt gattatgcca tgaactggtt ccgccaggct 120

ccagggatgg ggctggagtg ggtaggttcc accagaggca aagcctatgg tgggagcagc 180

gtatacgccg cgtctgcgaa aggtagattc accatctcaa gagatgattc caaaagcatc 240

gcctatctgc aaatgaacag cctgaaaacc gaggacacag ccgtgtatta ctgtaataga 300

gaggtagagt attgcaaatc gccgcaaaac tactgctacg gtatggacgt ctggggccaa 360

gggaccacgg tcaccgtctc ctcc 384

<210>18

<211>384

<212>DNA

<213> Artificial sequence

<400>18

gaggtgcagc tggtggagtc tgggggaggc ttggtaaagc cagggcggtc cctgagactc 60

tcctgtacaggttctggatt cacctttggt gaatatgcca taaactggtt ccgccaggct 120

ccagggatgg ggctggagtg ggtaggttcc atcagaggca aagcctatgg tgggagcagc 180

gaatacgccg cgtctgcgaa aggtagattc accatctcaa gagatgattc caaaaggatc 240

gcctatctgc gaatgaacgg cctgaaaacc gaagacacag ccgtgtatta ctgtaataga 300

gaggtagagt attgcaaatc gccggaaaac tactgctacg gtatggacgt ctggggccaa 360

gggaccacgg tcaccgtctc ctcc 384

<210>19

<211>318

<212>DNA

<213> Artificial sequence

<400>19

tcctatgagt tgactcagcc accctcagtg tccgtgtccc caggacagac agccagcatc 60

acctgctctg gagatagaat gggggataaa tttgctggct ggtatcagca gaagccaggc 120

cagtcccctg tgctggtcat ctatcaagat accaagcggc ccaccgggat ccctgagcga 180

ttctctggct ccaactctgg gaacacagcc actctgacca tcagcgggac ccaggctatg 240

gatgaggctg actattactg tcacgcgtgg gacaacagca gcgtgatttt cggcggaggg 300

accaagctga ccgtccta 318

<210>20

<211>318

<212>DNA

<213> Artificial sequence

<400>20

tcctatgagt tgactcagcc accctcagtg tccgtgtccc caggacagac agccagcatc 60

acctgctctg gagatagact gggggataaa tttgctagct ggtatcggca gaagccaggc 120

cagtcccctg tgctggtcat ctatcaagat atcatgcggc ccagcgggat ccctgagcga 180

ttctctggct ccaactccgg gaacacagcc actctgacca tcagcgggac ccaggctatg 240

gatgaggctg actattactg tcacgcgtgg gacaacaaca gcgtgatttt cggcggaggg 300

accaagctga ccgtccta 318

<210>21

<211>318

<212>DNA

<213> Artificial sequence

<400>21

tcctatgagt tgactcagcc accctcagtg tccgtgtccc caggacagac agccagcatc 60

acctgctctg gagatagaat gggggataaa tatgctggct ggtatcagca gaagccaggc 120

cagtcccctg tgctggtcat ctatcaagat atcaagcggc ccaccgggat ccctgagcga 180

ttctctggct ccaactctgg gaacacagcc actctgacca tcagcgggac ccaggctatg 240

gatgaggctg actattactg tcacgcgtgg gacagcaaca gcgtggtttt cggcggaggg 300

accaagctga ccgtccta 318

<210>22

<211>318

<212>DNA

<213> Artificial sequence

<400>22

tcctatgagt tgactcagcc accctcagtg tccgtgtccc caggacagac agccagcatc 60

acctgctctg gagatagact gggggataaa tttgctggct ggtatcagca gaagccaggc 120

cagtcccctg tgctggtcat ctatcaagat atcaagcggc ccaccgggat ccctgagcga 180

ttctctggct ccaactctgg gaacacagcc actctgacca tcagcgggac ccaggctatg 240

gatgaggctg agtattactg tcacgcgtgg gacagcaaca ccgtgatttt cggcggaggg 300

accaagctga ccgtccta 318

<210>23

<211>318

<212>DNA

<213> Artificial sequence

<400>23

tcctatgagt tgactcagcc accctcagtg tccgtgtccc caggacagac agccagcatc 60

acctgctctg gagatagaat gggggataaa tttgctggct ggtatcagca gaagccaggc 120

cagtcccctg tgctggtcat ctatcaagat atcaagcggc ccaccgggat ccctgagcga 180

ttctctggct ccaactctgg gaacacagcc actctgacca tcagcgggac ccaggctatg 240

gatgaggctg actattactg tcacgcgtgg gacagcaaca gcgtgatttt cggcggaggg 300

accaagctga ccgtccta 318

<210>24

<211>383

<212>DNA

<213> Artificial sequence

<400>24

gaggtgcagc tggtggagtc tgggggaggc ttggtaaagc cagggcggtc cctgagactc 60

tcctgtacag gttctggatt cacctttggt gattatgctt tgaactggtt ccgccaggct 120

ccagggatgg ggctggagtg ggtaggtttc attagaggca aagcctatgg tgggacaaca 180

gaatacgccg cgtctgtgaa aggcagattc accatctcaa gagatgattc caaaagcatc 240

gcctatctgc aaatgaacag cctgaaaacc gaggacacag ccgtgtatta ctgtaataga 300

gaggtagagt attgcaggtc gtccgagaac tactgctacg gtatggacgt ctggggccaa 360

gggaccacgg tcaccgtctc ctc 383

<210>25

<211>404

<212>PRT

<213> Artificial sequence

<400>25

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

1 5 10 15

Val His Ser Ala Pro Val Arg Ser Leu Asn Cys Thr Leu Arg Asp Ser

20 25 30

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

35 40 45

His Leu Gln Gly Gln Asp Met Glu Gln Gln Val Val Phe Ser Met Ser

50 55 60

Phe Val Gln Gly Glu Glu Ser Asn Asp Lys Ile Pro Val Ala Leu Gly

65 70 75 80

Leu Lys Glu Lys Asn Leu Tyr Leu Ser Cys Val Leu Lys Asp Asp Lys

85 90 95

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

100 105 110

Lys Met Glu Lys Arg Phe Val Phe Asn Lys Ile Glu Ile Asn Asn Lys

115 120 125

Leu Glu Phe Glu Ser Ala Gln Phe Pro Asn Trp Tyr Ile Ser Thr Ser

130 135 140

Gln Ala Glu Asn Met Pro Val Phe Leu Gly Gly Thr Lys Gly Gly Gln

145 150 155 160

Asp Ile Thr Asp Phe Thr Met Gln Phe Val Ser Ser Gly Gly Gly Gly

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

Ser Pro Gly Lys

<210>26

<211>183

<212>PRT

<213> Artificial sequence

<400>26

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

1 5 10 15

Val His Ser Ala Pro Val Arg Ser Leu Asn Cys Thr Leu Arg Asp Ser

20 25 30

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

35 40 45

His Leu Gln Gly Gln Asp Met Glu Gln Gln Val Val Phe Ser Met Ser

50 55 60

Phe Val Gln Gly Glu Glu Ser Asn Asp Lys Ile Pro Val Ala Leu Gly

65 70 75 80

Leu Lys Glu Lys Asn Leu Tyr Leu Ser Cys Val Leu Lys Asp Asp Lys

85 90 95

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

100 105 110

Lys Met Glu Lys Arg Phe Val Phe Asn Lys Ile Glu Ile Asn Asn Lys

115 120 125

Leu Glu Phe Glu Ser Ala Gln Phe Pro Asn Trp Tyr Ile Ser Thr Ser

130 135 140

Gln Ala Glu Asn Met Pro Val Phe Leu Gly Gly Thr Lys Gly Gly Gln

145 150 155 160

Asp Ile Thr Asp Phe Thr Met Gln Phe Val Ser Ser Gly Gly Gly Gly

165 170 175

Ser His His His His His His

180

<210>27

<211>1238

<212>DNA

<213> Artificial sequence

<400>27

aagcttaatt gccgccacca tgggatggtc ttgtattatt ctgtttctgg tcgctaccgc 60

tactggcgtc cactctgcac ctgtacgatc actgaactgc acgctccggg actcacagca 120

aaaaagcttg gtgatgtctg gtccatatga actgaaagct ctccacctcc agggacagga 180

tatggagcaa caagtggtgt tctccatgtc ctttgtacaa ggagaagaaa gtaatgacaa 240

aatacctgtg gccttgggcc tcaaggaaaa gaatctgtac ctgtcctgcg tgttgaaaga 300

tgataagccc actctacagc tggagagtgt agatcccaaa aattacccaa agaagaagat 360

ggaaaagcga tttgtcttca acaagataga aatcaataac aagctggaat ttgagtctgc 420

ccagttcccc aactggtaca tcagcacctc tcaagcagaa aacatgcccg tcttcctggg 480

agggaccaaa ggcggccagg atataactga cttcaccatg caatttgtgt cttccggggg 540

tggaggctct gtgcccaggg attgtggttg taagccttgc atatgtacag tcccagaagt 600

atcatctgtc ttcatcttcc ccccaaagcc caaggatgtg ctcaccatta ctctgactcc 660

taaggtcacg tgtgttgtgg tagacatcag caaggatgat cccgaggtcc agttcagctg 720

gtttgtagat gatgtggagg tgcacacagc tcagacgcaa ccccgggagg agcagttcaa 780

cagcactttc cgctcagtca gtgaacttcc catcatgcac caggactggc tcaatggcaa 840

ggagttcaaa tgcagggtca acagtgcagc tttccctgcc cccatcgaga aaaccatctc 900

caaaaccaaa ggcagaccga aggctccaca ggtgtacacc attccacctc ccaaggagca 960

gatggccaag gataaagtca gtctgacctg catgataaca gacttcttcc ctgaagacat 1020

tactgtggag tggcagtgga atgggcagcc agcggagaac tacaagaaca ctcagcccat 1080

catggacaca gatggctctt acttcgtcta cagcaagctc aatgtgcaga agagcaactg 1140

ggaggcagga aatactttca cctgctctgt gttacatgag ggcctgcaca accaccatac 1200

tgagaagagc ctctcccact ctcctggtaa atgaattc 1238

<210>28

<211>575

<212>DNA

<213> Artificial sequence

<400>28

aagcttaatt gccgccacca tgggatggtc ttgtattatt ctgtttctgg tcgctaccgc 60

tactggcgtc cactctgcac ctgtacgatc actgaactgc acgctccggg actcacagca 120

aaaaagcttg gtgatgtctg gtccatatga actgaaagct ctccacctcc agggacagga 180

tatggagcaa caagtggtgt tctccatgtc ctttgtacaa ggagaagaaa gtaatgacaa 240

aatacctgtg gccttgggcc tcaaggaaaa gaatctgtac ctgtcctgcg tgttgaaaga 300

tgataagccc actctacagc tggagagtgt agatcccaaa aattacccaa agaagaagat 360

ggaaaagcga tttgtcttca acaagataga aatcaataac aagctggaat ttgagtctgc 420

ccagttcccc aactggtaca tcagcacctc tcaagcagaa aacatgcccg tcttcctggg 480

agggaccaaa ggcggccagg atataactga cttcaccatg caatttgtgt cttccggggg 540

tggaggctct caccatcacc accatcattg aattc 575

<210>29

<211>118

<212>PRT

<213> Artificial sequence

<400>29

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

1 5 10 15

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

20 25 30

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

35 40 45

Ala Ile Ile Trp Tyr Asp Gly Asp Asn Gln Tyr Tyr Ala Asp Ser Val

50 55 60

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

65 70 75 80

Leu Gln Met Asn Gly Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

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

100 105 110

Leu Val Thr Val Ser Ser

115

<210>30

<211>107

<212>PRT

<213> Artificial sequence

<400>30

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

1 5 10 15

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

20 25 30

Leu His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Gln Ser Phe Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Glu Ala

65 70 75 80

Glu Asp Ala Ala Ala Tyr Tyr Cys His Gln Ser Ser Ser Leu Pro Phe

85 90 95

Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys

100 105

<210>31

<211>120

<212>DNA

<213> Artificial sequence

<400>31

ggcccagccg gccatggcct aaggatccta aaccgtctcg agcggtggtg gcggtagtgg 60

cggtggtggt agcggtggcg gtggtagtgc tagcgacatc ctgcagtgaa aggcggccgc 120

Claims

1. An anti-I L-1 β monoclonal antibody, which has any one of the following groups of heavy chain variable region and light chain variable region:

(ii) the amino acid sequence of the heavy chain variable region is represented by SEQ ID NO:1, and the amino acid sequence of the light chain variable region is represented by SEQ ID NO: 11;

(iii) the amino acid sequence of the heavy chain variable region is represented by SEQ ID NO:2, and the amino acid sequence of the light chain variable region is represented by SEQ ID NO: 11;

2. The anti-I L-1 β monoclonal antibody of claim 1, wherein the heavy chain type is IgG1, IgG2, IgG3 or IgG4 and the light chain type is kappa, lambda.

3. The anti-I L-1 β monoclonal antibody of claim 1 or 2, further comprising a constant region, wherein the constant region is any one of human IgG1, IgG2, IgG3, or IgG 4.

4. A DNA molecule encoding the anti-I L-1 β monoclonal antibody of any one of claims 1-3.

5. The DNA molecule of claim 4, having

and

(II) the nucleotide sequence of the light chain variable region as shown in any one of SEQ ID Nos. 19-24; or a sequence which differs from the nucleotide sequence of (II) by virtue of the degeneracy of the genetic code.

6. An expression vector comprising a DNA molecule encoding the anti-I L-1 β monoclonal antibody of any one of claims 1-3.

7. A host cell transformed or transfected with the expression vector of claim 6, wherein the host cell is a prokaryotic cell or a eukaryotic cell.

8. A conjugate comprising the anti-I L-1 β monoclonal antibody of any one of claims 1-3 covalently linked to a chemical label or a biomarker.

9. The conjugate of claim 8, wherein the chemical label is an isotopic label, an immunotoxin label, and/or a chemical drug label; the biomarker is a biotin label, an avidin label, or an enzyme label.

10. A conjugate formed by coupling the anti-I L-1 β monoclonal antibody of any one of claims 1 to 3 and/or the conjugate of any one of claims 8 to 9 to a solid or semi-solid medium.

11. A pharmaceutical composition comprising an anti-human I L-1 β monoclonal antibody as defined in any one of claims 1 to 3 and/or a conjugate as defined in any one of claims 8 to 9 and/or a conjugate as defined in claim 10.

12. Use of the anti-I L-1 β monoclonal antibody of any one of claims 1 to 3, the conjugate of any one of claims 8 to 9 and/or the conjugate of claim 10 for the preparation of a product for detecting expression of I L-1 β.

13. A kit comprising an anti-I L-1 β monoclonal antibody according to any one of claims 1 to 3, a conjugate according to any one of claims 8 to 9 and/or a conjugate according to claim 10.

14. Use of an anti-I L-1 β monoclonal antibody as claimed in any one of claims 1 to 3, a conjugate as claimed in any one of claims 8 to 9 and/or a conjugate as claimed in claim 10 for the manufacture of a formulation for blocking the binding of I L-1 β to I L-1 RA.

15. Use of the anti-I L-1 β monoclonal antibody of any one of claims 1 to 3, the conjugate of any one of claims 8 to 9 and/or the conjugate of claim 10 for the manufacture of a medicament for the prevention or treatment of a cold imidacloprid-associated periodic syndrome.

16. The use according to claim 15, wherein the cryopyrin-associated periodic syndrome is familial cryopanel autoinflammatory syndrome and muckle-viri syndrome, tumor necrosis factor receptor-associated periodic fever syndrome, hyper-immunoglobulin D syndrome/mevalonate kinase deficiency and/or familial mediterranean fever.