Novel anti-mouse NLRP3 monoclonal antibody and application thereof

Abstract

The invention provides a novel anti-mouse NLRP3 monoclonal antibody and application thereof, and particularly discloses two monoclonal antibodies aiming at NLRP3 protein, nucleic acid sequences of coding antibodies and antibody fragments and preparation methods of the monoclonal antibodies and the nucleic acid sequences. The result of the invention shows that the two monoclonal antibodies (named as 9-B8-3-2-C5 and 9-C9-1) prepared by the invention have a specific recognition effect on the NLRP3 protein.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a novel anti-mouse NLRP3 monoclonal antibody and application thereof. Background Art

[0002] As pattern recognition receptors distributed in the cytoplasm, the NLR family can recognize pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) in the cytoplasm and is an important receptor in the natural immune signaling pathway that resists intracellular pathogen infection. Currently, 23 NLRs have been found in humans and 34 NLRs have been found in mice. NLR consists of three domains. The C-terminal is a leucine-rich repeat sequence (LRR), which is mainly responsible for recognizing and binding specific PAMPs and DAMPs; the middle is the central nucleotide binding / oligomerization domain (NOD) domain, which is a characteristic domain shared by NLR family members, also known as the NACHT domain; the N-terminal is the effector domain, which can be a caspase recruitment domain (CARD), a pyrin domain (PYD) or a baculovirus inhibitor of apoptosis repeat (BIR), etc., responsible for transmitting signals downstream. NLR is divided into four different subfamilies according to its different N-terminal domains: NLRA subfamily, NLRB subfamily, NLRC subfamily and NLRP subfamily.

[0003] NLRP is the largest subfamily in the NLR family. Currently, 14 NLRPs have been discovered. Except for NLRP10, which does not contain an LRR domain, other NLRPs contain PYD, NACHT, and LRR domains. Among the many NLRPs, NLRP3 has been studied in the most detail. NLRP3 is mainly expressed in macrophages and peripheral blood leukocytes. Its N-terminal PYD effector domain can recognize and bind to PAMPs and DAMPs, thereby inducing conformational changes in itself, exposing the NOD domain, and then oligomerizing. It also recruits apoptosis-associated speck-like protein (ASC) adaptor protein through PYD-PYD interaction to form the NLRP3 inflammasome containing NLRP3, ASC, and caspase-1.

[0004] When studying the biological function of NLRP3, it was found that all NLRP3 antibodies currently on the market cannot specifically recognize the natural (non-denatured) NLRP3, such as anti-NLRP3 (D4D8T, Cat#15101, Cell Signaling Technology, synthetic peptide corresponding to the residues around immunogen Ala306), anti-NLRP3 (Cryo2, Cat#AG-20B-0014-C100, Adipogen, immunogen aa1-93), anti-NLRP3 (Rabbit pAb, Cat#A12694, Abclonal, immunogen aa250-350), and anti-NLRP3 (Cat#MAB7578, R&D systems, immunogen aa1-153).

[0005] Therefore, there is an urgent need in this field to screen out hybridoma cell lines that can secrete anti-mouse non-denatured NLRP3 monoclonal antibodies to provide a powerful tool for studying the biological functions of NLRP3. Summary of the invention

[0006] The purpose of the present invention is to provide a hybridoma cell line that can secrete anti-mouse non-denatured NLRP3 monoclonal antibodies, thereby providing a powerful tool for studying the biological function of NLRP3.

[0007] Another object of the present invention is to specifically identify non-denatured NLRP3 protein in immunofluorescence detection.

[0008] The first aspect of the present invention provides a heavy chain variable region of an antibody, wherein the heavy chain variable region comprises the following three complementarity determining regions CDR:

[0009] CDR1 shown in SEQ ID NO: 1,

[0010] CDR2 shown in SEQ ID NO:3, and

[0011] CDR3 shown in SEQ ID NO:5; or

[0012] CDR1 shown in SEQ ID NO:2,

[0013] CDR2 shown in SEQ ID NO:4, and

[0014] The sequence of CDR3 is as shown in NDY.

[0015] In another preferred embodiment, any one of the above amino acid sequences also includes a derivative sequence that is optionally added, deleted, modified and / or substituted with at least one (such as 1-3, preferably 1-2, and more preferably 1) amino acid and can retain the specific recognition activity of the NLRP3 protein.

[0016] In another preferred embodiment, the NLRP3 protein is a natural or non-denatured NLRP3 protein.

[0017] In another preferred example, the heavy chain variable region further includes a human FR region or a mouse FR region.

[0018] In another preferred example, the heavy chain variable region has an amino acid sequence shown in any one of SEQ ID NOs: 6-7.

[0019] The second aspect of the present invention provides a heavy chain of an antibody, wherein the heavy chain has the heavy chain variable region as described in the first aspect of the present invention.

[0020] In another preferred embodiment, the heavy chain of the antibody further includes a heavy chain constant region.

[0021] In another preferred embodiment, the heavy chain constant region is of human, mouse or rabbit origin.

[0022] The third aspect of the present invention provides a light chain variable region of an antibody, wherein the light chain variable region comprises the following three complementarity determining regions CDR:

[0023] CDR1' shown in SEQ ID NO:8,

[0024] CDR2' shown in SEQ ID NO:10, and

[0025] CDR3' shown in SEQ ID NO:11; or

[0026] CDR1' shown in SEQ ID NO:9,

[0027] CDR2' shown in SEQ ID NO:10, and

[0028] CDR3′ shown in SEQ ID NO:12

[0029] In another preferred embodiment, any one of the above amino acid sequences also includes a derivative sequence that is optionally added, deleted, modified and / or substituted with at least one (such as 1-3, preferably 1-2, and more preferably 1) amino acid and can retain the specific recognition activity of the NLRP3 protein.

[0030] In another preferred example, the light chain variable region further includes a human FR region or a mouse FR region.

[0031] In another preferred example, the light chain variable region has an amino acid sequence shown in any one of SEQ ID NOs: 13-14.

[0032] The fourth aspect of the present invention provides a light chain of an antibody, wherein the light chain has the light chain variable region as described in the third aspect of the present invention.

[0033] In another preferred embodiment, the light chain of the antibody further includes a light chain constant region.

[0034] In another preferred embodiment, the light chain constant region is of human, mouse or rabbit origin.

[0035] The fifth aspect of the present invention provides an antibody having:

[0036] (1) the heavy chain variable region as described in the first aspect of the present invention; and / or

[0037] (2) The light chain variable region as described in the third aspect of the present invention.

[0038] In another preferred embodiment, the antibody has: the heavy chain as described in the second aspect of the present invention; and / or the light chain as described in the fourth aspect of the present invention.

[0039] In another preferred embodiment, the antibody is an antibody specific against NLRP3 protein.

[0040] In another preferred embodiment, the antibody is selected from the following group: animal-derived antibodies, chimeric antibodies, humanized antibodies, or a combination thereof.

[0041] In another preferred embodiment, the CDR region of the humanized antibody comprises 1, 2, or 3 amino acid changes.

[0042] In another preferred embodiment, the animal is a non-human mammal, preferably a mouse, a sheep or a rabbit.

[0043] In another preferred embodiment, the antibody is a double-chain antibody or a single-chain antibody.

[0044] In another preferred embodiment, the antibody is a full-length antibody protein or an antigen-binding fragment.

[0045] In another preferred embodiment, the antibody is a bispecific antibody or a multispecific antibody.

[0046] In another preferred embodiment, the antibody is a monoclonal antibody.

[0047] In another preferred embodiment, the antibody is a partially or fully humanized monoclonal antibody.

[0048] In another preferred embodiment, the antibody further comprises a heavy chain constant region and / or a light chain constant region.

[0049] In another preferred embodiment, the heavy chain constant region is of human origin, and / or the light chain constant region is of human origin.

[0050] In another preferred example, the heavy chain variable region of the antibody further includes a human framework region, and / or the light chain variable region of the antibody further includes a human framework region.

[0051] In another preferred example, the heavy chain variable region of the antibody further includes a framework region of mouse origin, and / or the light chain variable region of the antibody further includes a framework region of mouse origin.

[0052] In another preferred embodiment, the heavy chain variable region sequence of the antibody is as shown in any one of SEQ ID NOs: 6-7; and / or

[0053] The light chain variable region sequence of the antibody is shown in any one of SEQ ID NOs: 13-14.

[0054] In another preferred embodiment, the amino acid sequence of the heavy chain variable region has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 6-7.

[0055] In another preferred embodiment, the amino acid sequence of the light chain variable region has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 13-14.

[0056] In another preferred embodiment, the number of added, deleted, modified and / or substituted amino acids does not exceed 40% of the total number of amino acids in the initial amino acid sequence, preferably 20%, more preferably 10%.

[0057] In another preferred embodiment, the at least one amino acid sequence that has been added, deleted, modified and / or substituted has an amino acid sequence with a homology of at least 80%.

[0058] In another preferred embodiment, the derivative sequence after addition, deletion, modification and / or substitution of at least one amino acid has the activity of binding to NLRP3 protein.

[0059] In another preferred example, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:6, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:13.

[0060] In another preferred example, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:7, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:14.

[0061] In another preferred embodiment, the antibody is of IgG type.

[0062] In another preferred embodiment, the antibody is in the form of a drug conjugate.

[0063] The sixth aspect of the present invention provides a recombinant protein, wherein the recombinant protein has:

[0064] (i) the heavy chain variable region of the first aspect of the invention, the heavy chain of the second aspect of the invention, the light chain variable region of the third aspect of the invention, the light chain of the fourth aspect of the invention, or the antibody of the fifth aspect of the invention; and

[0065] (ii) optionally a tag sequence to facilitate expression and / or purification.

[0066] In another preferred embodiment, the tag sequence includes a 6His tag.

[0067] In another preferred embodiment, the recombinant protein (or polypeptide) includes a fusion protein.

[0068] In another preferred embodiment, the recombinant protein is a monomer, a dimer, or a polymer.

[0069] In another preferred embodiment, the recombinant protein specifically binds to NLRP3 protein.

[0070] The seventh aspect of the present invention provides a CAR construct, wherein the antigen binding region of the CAR construct is a scFv that specifically binds to the NLRP3 protein, and the scFv has the heavy chain variable region as described in the first aspect of the present invention and the light chain variable region as described in the third aspect of the present invention.

[0071] The eighth aspect of the present invention provides a recombinant immune cell, wherein the immune cell expresses an exogenous CAR construct as described in the seventh aspect of the present invention; or the immune cell expresses or exposes the antibody described in the fifth aspect of the present invention outside the cell membrane.

[0072] In another preferred embodiment, the immune cells are selected from the following group: NK cells and T cells.

[0073] In another preferred embodiment, the immune cells are from humans or non-human mammals (such as mice).

[0074] The ninth aspect of the present invention provides an antibody-drug conjugate, wherein the antibody-drug conjugate comprises:

[0075] (a) an antibody portion, the antibody portion being selected from the group consisting of a heavy chain variable region as described in the first aspect of the invention, a heavy chain as described in the second aspect of the invention, a light chain variable region as described in the third aspect of the invention, a light chain as described in the fourth aspect of the invention, or an antibody as described in the fifth aspect of the invention, or a combination thereof; and

[0076] (b) a coupling portion coupled to the antibody portion, wherein the coupling portion is selected from the group consisting of a detectable marker, a drug, a toxin, a cytokine, a radionuclide, an enzyme, a gold nanoparticle / nanorod, a nanomagnetic particle, a viral coat protein or a VLP, or a combination thereof.

[0077] In another preferred embodiment, the antibody portion and the coupling portion are coupled via a chemical bond or a linker.

[0078] In another preferred embodiment, the radioactive nuclides include:

[0079] (i) a diagnostic isotope selected from the group consisting of Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, or a combination thereof; and / or

[0080] (ii) therapeutic isotopes, wherein the therapeutic isotopes are selected from the group consisting of Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd-103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133Yb-169, Yb-177, or a combination thereof.

[0081] In another preferred embodiment, the coupling moiety is a drug or a toxin.

[0082] In another preferred embodiment, the drug is a cytotoxic drug.

[0083] In another preferred embodiment, the cytotoxic drug is selected from the following group: anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, or a combination thereof.

[0084] Examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors. Typical cytotoxic drugs include, for example, auristatins, camptothecins, duocarmycins, etoposides, maytansines and maytansinoids (e.g., DM1 and DM4), taxanes, benzodiazepines or benzodiazepine containing drugs (e.g., pyrrolo[1,4]benzodiazepines (PBDs), indolinobenzodiazepines and oxazolidinobenzodiazepines), vinca alkaloids, or combinations thereof.

[0085] In another preferred embodiment, the toxin is selected from the following group:

[0086] auristatins (e.g., auristatin E, auristatin F, MMAE and MMAF), chlortetracycline, maytansinoids, ricin, ricin A-chain, combretastatin, duocarmycin, dolastatin, adriamycin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracin dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotonin, calicheamicin, saponin, officinalis) inhibitors, glucocorticoids, or a combination thereof.

[0087] In another preferred embodiment, the coupling moiety is a detectable label.

[0088] In another preferred embodiment, the conjugate is selected from: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computer tomography) contrast agents, or enzymes capable of producing detectable products, radionuclides, biotoxins, cytokines (such as IL-2), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorods, viral particles, liposomes, nanomagnetic particles, prodrug-activating enzymes (such as DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), and chemotherapeutic agents (such as cisplatin).

[0089] In another preferred embodiment, the immunoconjugate contains: multivalent (eg, bivalent) (a).

[0090] In another preferred embodiment, the multivalency refers to the presence of multiple repeats of (a) in the amino acid sequence of the immunoconjugate.

[0091] The tenth aspect of the present invention provides a use of an active ingredient, wherein the active ingredient is selected from the following group: the heavy chain variable region as described in the first aspect of the present invention, the heavy chain as described in the second aspect of the present invention, the light chain variable region as described in the third aspect of the present invention, the light chain as described in the fourth aspect of the present invention, or the antibody as described in the fifth aspect of the present invention, the recombinant protein as described in the sixth aspect of the present invention, or a combination thereof, wherein the active ingredient is used to prepare a drug for preventing and / or treating inflammatory and autoimmune diseases.

[0092] In another preferred embodiment, the inflammatory and autoimmune diseases are selected from the group consisting of gout, Alzheimer's disease, type 2 diabetes, atherosclerosis, cryopyrin-associated periodic syndrome (CAPS), non-alcoholic hepatitis (NASH), inflammatory bowel disease, or a combination thereof.

[0093] In another preferred embodiment, the antibody is in the form of an antibody-drug conjugate (ADC).

[0094] The eleventh aspect of the present invention provides a pharmaceutical composition, wherein the pharmaceutical composition comprises:

[0095] (i) an active ingredient, wherein the active ingredient is selected from the group consisting of the heavy chain variable region as described in the first aspect of the present invention, the heavy chain as described in the second aspect of the present invention, the light chain variable region as described in the third aspect of the present invention, the light chain as described in the fourth aspect of the present invention, or the antibody as described in the fifth aspect of the present invention, the recombinant protein as described in the sixth aspect of the present invention, the immune cell as described in the eighth aspect of the present invention, the antibody-drug conjugate as described in the ninth aspect of the present invention, or a combination thereof; and

[0096] (ii) a pharmaceutically acceptable carrier.

[0097] In another preferred embodiment, the pharmaceutical composition is a liquid preparation.

[0098] In another preferred embodiment, the pharmaceutical composition is an injection.

[0099] In another preferred example, the pharmaceutical composition comprises 0.01 to 99.99% of the heavy chain variable region as described in the first aspect of the present invention, the heavy chain as described in the second aspect of the present invention, the light chain variable region as described in the third aspect of the present invention, the light chain as described in the fourth aspect of the present invention, or the antibody as described in the fifth aspect of the present invention, the recombinant protein as described in the sixth aspect of the present invention, the immune cell as described in the eighth aspect of the present invention, the antibody-drug conjugate as described in the ninth aspect of the present invention, or a combination thereof and 0.01 to 99.99% of a pharmaceutical carrier, wherein the percentage is the mass percentage of the pharmaceutical composition.

[0100] In another preferred embodiment, the pharmaceutical composition is used to prevent and / or treat inflammatory and autoimmune diseases.

[0101] The twelfth aspect of the present invention provides a polynucleotide, wherein the polynucleotide encodes a polypeptide selected from the following group:

[0102] (1) the heavy chain variable region as described in the first aspect of the present invention, the heavy chain as described in the second aspect of the present invention, the light chain variable region as described in the third aspect of the present invention, the light chain as described in the fourth aspect of the present invention, or the antibody as described in the fifth aspect of the present invention; or

[0103] (2) The recombinant protein according to the sixth aspect of the present invention;

[0104] (3) The CAR construct as described in the seventh aspect of the present invention.

[0105] The thirteenth aspect of the present invention provides a vector, wherein the vector contains the polynucleotide as described in the twelfth aspect of the present invention.

[0106] In another preferred embodiment, the vector includes: bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vectors.

[0107] The fourteenth aspect of the present invention provides a genetically engineered host cell, wherein the host cell contains the vector as described in the thirteenth aspect of the present invention or the polynucleotide as described in the twelfth aspect of the present invention is integrated into its genome.

[0108] A fifteenth aspect of the present invention provides a method for detecting NLRP3 protein or a fragment thereof in a sample in vitro, the method comprising the steps of:

[0109] (1) contacting a sample with the antibody described in the fifth aspect of the present invention;

[0110] (2) Detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of NLRP3 protein or its fragment in the sample.

[0111] In another preferred embodiment, the detection includes diagnostic or non-diagnostic.

[0112] The sixteenth aspect of the present invention provides a detection plate, characterized in that the detection plate comprises: a substrate (support plate) and a test strip, and the test strip contains the antibody as described in the fifth aspect of the present invention or the antibody-drug conjugate as described in the ninth aspect of the present invention.

[0113] A seventeenth aspect of the present invention provides a kit, comprising:

[0114] (1) a first container, wherein the first container contains the antibody according to the fifth aspect of the present invention; and / or

[0115] (2) a second container, wherein the second container contains a secondary antibody against the antibody according to the fifth aspect of the present invention;

[0116] Alternatively, the kit contains the detection plate as described in the sixteenth aspect of the present invention.

[0117] An eighteenth aspect of the present invention provides a method for preparing a recombinant polypeptide, the method comprising:

[0118] (a) culturing the host cell according to the fourteenth aspect of the present invention under conditions suitable for expression;

[0119] (b) isolating a recombinant polypeptide from the culture, wherein the recombinant polypeptide is the antibody according to the fifth aspect of the present invention or the recombinant protein according to the sixth aspect of the present invention.

[0120] The nineteenth aspect of the present invention provides a pharmaceutical combination comprising:

[0121] (i) a first active ingredient, wherein the first active ingredient is selected from the group consisting of the heavy chain variable region as described in the first aspect of the present invention, the heavy chain as described in the second aspect of the present invention, the light chain variable region as described in the third aspect of the present invention, the light chain as described in the fourth aspect of the present invention, or the antibody as described in the fifth aspect of the present invention, the recombinant protein as described in the sixth aspect of the present invention, the immune cell as described in the eighth aspect of the present invention, the antibody-drug conjugate as described in the ninth aspect of the present invention, or a combination thereof;

[0122] (ii) a second active ingredient, which includes other drugs for preventing and / or treating inflammatory and autoimmune diseases.

[0123] The twentieth aspect of the present invention provides a method for preventing and / or treating inflammatory and autoimmune diseases, characterized in that the method comprises: administering to a subject in need thereof an antibody as described in the fifth aspect of the present invention, a recombinant protein as described in the sixth aspect of the present invention, a CAR construct as described in the seventh aspect of the present invention, an immune cell as described in the eighth aspect of the present invention, an antibody-drug conjugate as described in the ninth aspect of the present invention, a pharmaceutical composition as described in the eleventh aspect of the present invention, a drug combination as described in the nineteenth aspect of the present invention, or a combination thereof.

[0124] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0125] Figure 1 The NLR gene family structure is shown.

[0126] Figure 2 The components of the NLRP3 inflammasome complex are shown.

[0127] Figure 3 The expression of NLRP3 protein in cells was detected using a commercially available NLRP3 antibody.

[0128] Figure 4 The construction of the recombinant plasmid Ms-N3-throhFc is shown.

[0129] Figure 5 The expression of recombinant plasmid Ms-N3-thro-hFc in HEK293F cells is shown. Note: M: protein marker, 1: pcDNA4.0, 2: Ms-N3-throhFc.

[0130] Figure 6 The purification of Ms-N3 protein using Protein A affinity column is shown. Elution peak of Ms-N3 protein purified by Protein A affinity chromatography column. Note: The X-axis represents the volume (unit: mL) passed through the system, the Y-axis represents the UV value (unit: mAU), and the arrow represents the Ms-N3 protein, whose UV absorption peak maximum is 900 mAU.

[0131] Figure 7 Western blot identification and Coomassie brilliant blue staining results of Ms-N3 protein are shown.

[0132] (A and B) Western blot results of Ms-N3 protein purification. Note: M: protein marker, 1: sample solution, 2, 3: flow-through, 4: purified Ms-N3 protein. (C) SDS-PAGE gel staining results of Ms-N3 protein purification. Note: M: protein marker, 1: sample solution, 2, 3: flow-through, 4: purified Ms-N3 protein.

[0133] Figure 8 The immune effect detection of serum of mice immunized with Ms-N3 protein is shown.

[0134] Fig. 9 Demonstration of the specificity of monoclonal antibody recognition of NLRP3 by immunocytofluorescence assay

[0135] (A) The specificity of monoclonal antibody 9-B8-3-2-C5 in recognizing NLRP3 was detected by immunocytofluorescence assay. (B) The specificity of monoclonal antibody 9-C9-1 in recognizing NLRP3 was detected by immunocytofluorescence assay.

[0136] Note: Scale bar: 50 μm.

[0137] Fig.10 Shows subtype identification of monoclonal antibodies

[0138] (A) Identification of subtypes of monoclonal antibody 9-B8-3-2-C5. (B) Identification of subtypes of monoclonal antibody 9-C9-1.

[0139] Fig.11 The results of PCR amplification of the heavy and light chain variable region genes of the monoclonal antibodies are shown. DETAILED DESCRIPTION

[0140] The inventors, through extensive and in-depth research and a large number of screenings, unexpectedly developed for the first time a class of antibodies with specific recognition activity for NLRP3 protein, and highly specific chimeric antigen receptor immune cells based on the antibodies. Specifically, the present invention unexpectedly obtained monoclonal antibodies with extremely excellent affinity and specificity for recognizing NLRP3 protein (especially natural or non-denatured NLRP3 protein). The results of the present invention show that the two monoclonal antibodies (named 9-B8-3-2-C5 and 9-C9-1) prepared by the present invention have specific recognition effects on NLRP3 protein. On this basis, the present invention was completed.

[0141] the term

[0142] In order to more easily understand the present disclosure, some terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms should have the meaning given below. Other definitions are set forth throughout the application.

[0143] The term "about" can refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.

[0144] As used herein, a "chimeric antigen receptor (CAR)" is a fusion protein comprising an extracellular domain capable of binding to an antigen, a transmembrane domain derived from a different polypeptide than the extracellular domain, and at least one intracellular domain. "Chimeric Antigen Receptor (CAR)" is also known as a "chimeric receptor", "T-body" or "chimeric immune receptor (CIR)". The "extracellular domain capable of binding to an antigen" refers to any oligopeptide or polypeptide capable of binding to a certain antigen. "Intracellular domain" refers to any oligopeptide or polypeptide known as a domain that transmits signals to activate or inhibit intracellular biological processes.

[0145] As used herein, "domain" refers to a region of a polypeptide that is independent of other regions and folds into a specific structure.

[0146] As used herein, "single-chain variable region fragment (ScFv)" refers to a single-chain polypeptide derived from an antibody that retains the ability to bind to an antigen. Examples of ScFv include antibody polypeptides formed by recombinant DNA technology, and wherein the Fv regions of immunoglobulin heavy chain (H chain) and light chain (L chain) fragments are connected via spacer sequences. Various methods for modifying ScFv are known to those skilled in the art.

[0147] As used herein, the term "treatment" refers to administering an internal or external therapeutic agent, including any anti-NLRP3 protein antibody and composition thereof, to a patient who has one or more disease symptoms, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, the therapeutic agent is administered to the patient in an amount (therapeutically effective amount) that is effective in alleviating one or more disease symptoms.

[0148] As used herein, the term "optional" or "optionally" means that the event or situation described subsequently may occur but need not occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable region of a specific sequence may have but need not have, and may be 1, 2 or 3.

[0149] "Sequence identity" of the present invention means the degree of identity between two nucleic acids or two amino acid sequences when optimally aligned and compared with mutations such as appropriate substitutions, insertions or deletions. The sequence identity between the sequences described in the present invention and the sequences with which they are identical can be at least 85%, 90% or 95%, preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%.

[0150] As used herein, Mouse-NLRP3 is abbreviated as Ms-N3.

[0151] NLRP3 protein

[0152] As pattern recognition receptors distributed in the cytoplasm, the NLR family can recognize pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) in the cytoplasm and is an important receptor in the natural immune signaling pathway that resists intracellular pathogen infection. Currently, 23 NLRs have been found in humans and 34 NLRs have been found in mice. NLR is composed of three domains. The C-terminal is a leucine-rich repeat sequence (LRR), which is mainly responsible for recognizing and binding specific PAMPs and DAMPs; the middle is the central nucleotide binding / oligomerization domain (NOD) domain, which is a characteristic domain shared by NLR family members, also known as the NACHT domain; the N-terminal is the effector domain, which can be a caspase recruitment domain (CARD), a pyrin domain (PYD) or a baculovirus inhibitor of apoptosis repeat (BIR), etc., responsible for transmitting signals downstream. NLR is divided into four different subfamilies according to its different N-terminal domains: NLRA subfamily, NLRB subfamily, NLRC subfamily and NLRP subfamily (such as Figure 1 shown).

[0153] NLRP is the largest subfamily in the NLR family. Currently, 14 NLRPs have been discovered. Except for NLRP10, which does not contain an LRR domain, other NLRPs contain PYD, NACHT, and LRR domains. Among the many NLRPs, NLRP3 has been studied in the most detail. NLRP3 is mainly expressed in macrophages and peripheral blood leukocytes. Its N-terminal PYD effector domain can recognize and bind to PAMPs and DAMPs, thereby inducing conformational changes in itself, exposing the NOD domain, and then oligomerizing. It also recruits apoptosis-associated speck-like protein (ASC) adaptor proteins through PYD-PYD interactions to form NLRP3 inflammasomes containing NLRP3, ASC, and caspase-1 (such as Figure 2 shown).

[0154] The innate immune system prevents infectious or non-infectious damage by releasing pro-inflammatory cytokines. NLRP3, as a general sensor, plays an important role in the host's defense against bacterial, fungal and viral infections. An increasing number of studies have shown that excessive activation of NLRP3 inflammasomes is closely related to a variety of inflammatory diseases, including inflammatory bowel disease, autoimmune diseases, gout, Alzheimer's disease, obesity, asthma, etc.

[0155] Antibody

[0156] As used herein, the term "antibody" or "immunoglobulin" is a heterotetrameric glycoprotein of about 150,000 daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.

[0157] As used herein, the term "variable" means that some parts of the variable region in an antibody are different in sequence, which forms the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three fragments called complementary determining regions (CDRs) or hypervariable regions in the variable regions of light and heavy chains. The more conservative part of the variable region is called the framework region (FR). The variable regions of natural heavy and light chains each contain four FR regions, which are roughly in a β-folded configuration, connected by three CDRs forming a connecting loop, and can form a partially folded structure in some cases. The CDRs in each chain are closely together through the FR region and together with the CDRs of the other chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Volume I, 647-669 pages (1991)). The constant region does not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in the antibody's antibody-dependent cytotoxicity.

[0158] The "light chains" of vertebrate antibodies (immunoglobulins) can be classified into one of two distinct classes (called kappa and lambda) based on the amino acid sequence of their constant regions. Immunoglobulins can be divided into different classes based on the amino acid sequence of their heavy chain constant regions. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant regions corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known in the art.

[0159] As used herein, the term "monoclonal antibody (mAb)" refers to an antibody obtained from a substantially homogeneous population, i.e., the individual antibodies contained in the population are identical, except for a few naturally occurring mutations that may be present. Monoclonal antibodies are highly specific for a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (which typically have different antibodies directed against different determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they are synthesized by hybridoma culture and are not contaminated by other immunoglobulins. The modifier "monoclonal" indicates the characteristic of the antibody, which is obtained from a substantially homogeneous antibody population, and should not be construed as requiring any special method to produce the antibody.

[0160] The present invention also includes monoclonal antibodies having the corresponding amino acid sequence of the NLRP3 protein monoclonal antibody, monoclonal antibodies having the variable region chain of the NLRP3 protein monoclonal antibody, and other proteins or protein conjugates and fusion expression products having these chains. Specifically, the present invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a light chain and a heavy chain containing a hypervariable region (complementarity determining region, CDR), as long as the hypervariable region is identical to the hypervariable region of the light chain and heavy chain of the present invention or at least 90% homology, preferably at least 95% homology.

[0161] As known to those skilled in the art, immunoconjugates and fusion expression products include: drugs, toxins, cytokines, radionuclides, enzymes and other diagnostic or therapeutic molecules combined with the NLRP3 protein anti-monoclonal antibody or its fragment to form a conjugate. The present invention also includes a cell surface marker or antigen combined with the anti-NLRP3 protein monoclonal antibody or its fragment.

[0162] The term "antigen-binding fragment of an antibody" (or simply "antibody fragment") refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that fragments of a full-length antibody can be used to perform the antigen-binding function of an antibody. Examples of binding fragments encompassed by the term "antigen-binding fragment of an antibody" include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab') 2 Fragment, a bivalent fragment comprising two Fab fragments connected by a disulfide bridge on the hinge region; (iii) an Fd fragment consisting of a VH and CH1 domain; (iv) an Fv fragment consisting of the VH and VL domains of a single arm of an antibody. The Fv antibody contains the variable region of the antibody heavy chain and the variable region of the light chain, but no constant region, and is the smallest antibody fragment with all antigen binding sites. Generally, the Fv antibody also contains a polypeptide linker between the VH and VL domains, and is capable of forming the structure required for antigen binding.

[0163] The present invention includes not only complete monoclonal antibodies, but also antibody fragments having immunological activity, such as Fab or (Fab') 2 Fragment; Antibody heavy chain; Antibody light chain.

[0164] The term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive or non-consecutive amino acids in a unique spatial conformation.

[0165] The terms "specific binding", "selective binding", "selectively binds" and "specifically binds" refer to the binding of an antibody to a predetermined epitope on an antigen. -7 M, for example, less than about 10 -8 M, 10 -9 M or l0 -10 The term "antigenic determinant" as used herein refers to a discrete three-dimensional site on an antigen that is recognized by the antibody or antigen-binding fragment of the present invention.

[0166] The present invention includes not only complete antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of the antibodies.

[0167] In the present invention, antibodies include murine, chimeric, humanized or fully human antibodies prepared by techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, including human and non-human parts, can be prepared using DNA recombinant techniques well known in the art. The term "murine antibody" in the present invention refers to a monoclonal antibody against NLRP3 protein prepared according to the knowledge and skills in the art. The term "chimeric antibody" is an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can reduce the immune response induced by the murine antibody. The term "humanized antibody", also known as CDR-grafted antibody, refers to an antibody produced by transplanting the CDR sequence of a mouse into the variable region framework of a human antibody, that is, different types of human germline antibody framework sequences. Humanized antibodies can overcome the heterologous response induced by chimeric antibodies due to carrying a large amount of murine protein components. Such framework sequences can be obtained from public DNA databases or published references including germline antibody gene sequences. In order to avoid a decrease in activity caused by a decrease in immunogenicity, the human antibody variable region framework sequence may be subjected to minimal reverse mutation or back mutation to maintain activity.

[0168] In the present invention, the antibodies may be monospecific, bispecific, trispecific, or more multispecific.

[0169] As used herein, the term "heavy chain variable region" is used interchangeably with "VH".

[0170] As used herein, the term "variable region" and "complementarity determining region (CDR)" are used interchangeably.

[0171] The term "CDR" refers to one of the six hypervariable regions within the variable domain of an antibody that primarily contribute to antigen binding. One of the most commonly used definitions of the six CDRs is provided by Kabat EA et al. (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242).

[0172] In a preferred embodiment of the present invention, the heavy chain of the antibody comprises the above-mentioned heavy chain variable region and a heavy chain constant region, and the heavy chain constant region may be of murine or human origin.

[0173] As used herein, the term "light chain variable region" is used interchangeably with "VL."

[0174] In a preferred embodiment of the present invention, the heavy chain variable region and light chain variable region of the antibody respectively include three complementary determining regions CDR (amino acid sequence and nucleotide sequence) as shown in Table A1 and Table A2 below:

[0175] Table A1

[0176]

[0177] Table A2

[0178]

[0179] In another preferred example, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.: 6-7.

[0180] In another preferred example, the amino acid sequence of the light chain variable region is shown in SEQ ID NO.: 13-14.

[0181] In a preferred embodiment of the present invention, the light chain of the antibody comprises the above-mentioned light chain variable region and a light chain constant region, and the light chain constant region may be of murine or human origin.

[0182] The function of the antibody of the present invention is determined by the specific gene sequence of the light chain and heavy chain variable region genes of the antibody, which can specifically bind to the NLRP3 protein and prevent and / or treat inflammation and autoimmune diseases. Using the antibody variable region gene or complementary determining region (CDR) gene, different forms of genetically engineered antibodies can be transformed and produced in any expression system using prokaryotic and eukaryotic cells.

[0183] In the present invention, the terms "antibodies of the present invention", "proteins of the present invention", or "polypeptides of the present invention" are used interchangeably and refer to antibodies that specifically bind to NLRP3 proteins, such as proteins or polypeptides having heavy chain variable regions (such as the amino acid sequences shown in SEQ ID NOs.: 6-7) and / or light chain variable regions (such as the amino acid sequences shown in SEQ ID NOs.: 13-14). They may or may not contain the initiating methionine.

[0184] In another preferred embodiment, the antibody is a mouse or human or human-mouse chimeric monoclonal antibody against NLRP3 protein, and its heavy chain constant region and / or light chain constant region can be a humanized heavy chain constant region or light chain constant region. More preferably, the humanized heavy chain constant region or light chain constant region is a heavy chain constant region or light chain constant region of human IgG1, IgG2, etc.

[0185] Generally, the antigen binding properties of an antibody can be described by three specific regions located in the variable regions of the heavy and light chains, called variable regions (CDRs). This segment is divided into four framework regions (FRs). The amino acid sequences of the four FRs are relatively conservative and do not directly participate in the binding reaction. These CDRs form a ring structure, and the β-folds formed by the FRs in between are close to each other in spatial structure. The CDRs on the heavy chain and the CDRs on the corresponding light chains constitute the antigen binding site of the antibody. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR region.

[0186] The variable regions of the heavy and / or light chains of the antibodies of the present invention are particularly interesting because at least part of them are involved in binding to antigens. Therefore, the present invention includes molecules having monoclonal antibody light and heavy chain variable regions with CDRs, as long as their CDRs have more than 90% (preferably more than 95%, and most preferably more than 98%) homology with the CDRs identified herein. The present invention includes not only complete monoclonal antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of the antibodies.

[0187] As used herein, the terms "fragment", "derivative" and "analog" refer to polypeptides that substantially retain the same biological function or activity as the antibodies of the present invention. The polypeptide fragments, derivatives or analogs of the present invention may be (i) polypeptides in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) polypeptides having a substitution group in one or more amino acid residues, or (iii) polypeptides formed by fusion of a mature polypeptide with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iv) polypeptides formed by fusion of an additional amino acid sequence to the polypeptide sequence (such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, or a fusion protein formed with a 6His tag). According to the teachings herein, these fragments, derivatives and analogs are within the scope known to those skilled in the art.

[0188] The antibody of the present invention refers to a polypeptide having specific NLRP3 protein binding activity and including the above-mentioned CDR region. The term also includes variant forms of polypeptides containing the above-mentioned CDR region and having the same function as the antibody of the present invention. These variant forms include (but are not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, and optimally 1-10) amino acid deletions, insertions and / or substitutions, and addition of one or several (usually within 20, preferably within 10, and more preferably within 5) amino acids at the C-terminus and / or N-terminus. For example, in the art, when amino acids with similar or similar properties are substituted, the function of the protein is generally not changed. For another example, adding one or several amino acids to the C-terminus and / or N-terminus generally does not change the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the present invention.

[0189] Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the antibody of the present invention under high or low stringency conditions, and polypeptides or proteins obtained using antiserum against the antibody of the present invention.

[0190] The present invention also provides other polypeptides, such as fusion proteins comprising human antibodies or fragments thereof. In addition to almost full-length polypeptides, the present invention also includes fragments of antibodies of the present invention. Typically, the fragment has at least about 50 consecutive amino acids of the antibody of the present invention, preferably at least about 60 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids.

[0191] In the present invention, "conservative variants of the antibodies of the present invention" refer to polypeptides formed by replacing at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids with amino acids having similar or similar properties compared to the amino acid sequence of the antibodies of the present invention. These conservative variant polypeptides are preferably generated by amino acid substitution according to Table B.

[0192] Table B

[0193] Initial residue Representative replacement Preferred substitutions Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala Leu

[0194] The present invention also provides a polynucleotide molecule encoding the above-mentioned antibody or its fragment or its fusion protein. The polynucleotide of the present invention can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand. The coding region sequence encoding the mature polypeptide can be the same as the coding region sequence shown in SEQ ID NO.: 15-29 or it can be a degenerate variant. As used herein, "degenerate variant" in the present invention refers to a nucleic acid sequence that encodes an amino acid sequence identical to the polypeptide of the present invention, but is different from the coding region sequence shown in SEQ ID NO.: 15-29.

[0195] The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence encoding only a mature polypeptide; a coding sequence of a mature polypeptide and various additional coding sequences; a coding sequence of a mature polypeptide (and optional additional coding sequences) and non-coding sequences.

[0196] The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or may include additional coding and / or non-coding sequences.

[0197] The present invention also relates to polynucleotides that hybridize with the above sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that can hybridize with the polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refer to: (1) hybridization and elution at relatively low ionic strength and relatively high temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) addition of denaturing agents during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, preferably at least 95%. In addition, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO.: 1-14.

[0198] The full-length nucleotide sequence of the antibody of the present invention or its fragment can usually be obtained by PCR amplification, recombination or artificial synthesis. A feasible method is to synthesize the relevant sequence by artificial synthesis, especially when the fragment length is short. Usually, a fragment with a very long sequence can be obtained by synthesizing multiple small fragments first and then connecting them. In addition, the coding sequence of the heavy chain and the expression tag (such as 6His) can be fused together to form a fusion protein.

[0199] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the proliferated host cells by conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in the present invention include biomolecules in isolated form.

[0200] At present, the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention by chemical synthesis.

[0201] The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0202] Host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells such as CHO, COS7, 293 cells, etc.

[0203] Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with CaCl 2 Another method is to use MgCl 2 If necessary, transformation can also be performed by electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0204] The obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used in the culture can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cells. After the host cells grow to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

[0205] The recombinant polypeptide in the above method can be expressed in the cell, on the cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be separated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.

[0206] The antibodies of the invention may be used alone or in combination or conjugated to a detectable marker (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or any combination of these.

[0207] Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing a detectable product.

[0208] Therapeutic agents that may be conjugated include, but are not limited to, insulin, IL-2, interferon, calcitonin, GHRH peptide, intestinal peptide analogs, albumin, antibody fragments, cytokines, and hormones.

[0209] In addition, therapeutic agents that can be combined or coupled with the antibodies of the present invention include but are not limited to: 1. radionuclides; 2. biological toxins; 3. cytokines such as IL-2; 4. gold nanoparticles / nanorods; 5. viral particles; 6. liposomes; 7. nanomagnetic particles; 8. prodrug activating enzymes; 10. chemotherapeutic agents (e.g., cisplatin) or any form of nanoparticles, etc.

[0210] The present invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment or its fusion protein, and a pharmaceutically acceptable carrier. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 6-8, although the pH value may vary depending on the properties of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): oral, respiratory, intratumoral, intraperitoneal, intravenous, or topical administration.

[0211] The pharmaceutical composition of the present invention can be used to directly bind to the NLRP3 protein, and thus can be used to prolong the half-life of the drug. In addition, other therapeutic agents can also be used simultaneously.

[0212] The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the above-mentioned monoclonal antibody (or its conjugate) of the present invention and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 microgram / kg body weight to about 10 mg / kg body weight per day. In addition, the polypeptide of the present invention can also be used with other therapeutic agents.

[0213] When using a pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to a mammal, wherein the safe and effective amount is usually at least about 10 micrograms / kg body weight, and in most cases does not exceed about 8 milligrams / kg body weight, preferably the dosage is about 10 micrograms / kg body weight to about 1 milligram / kg body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health status, which are all within the skill range of skilled physicians.

[0214] Carrier

[0215] The nucleic acid sequence encoding the desired molecule can be obtained using recombinant methods known in the art, such as, for example, by screening a library from a cell expressing the gene, by obtaining the gene from a known vector comprising the gene, or by directly isolating from cells and tissues comprising the gene using standard techniques. Alternatively, the gene of interest can be produced synthetically.

[0216] The present invention also provides vectors into which the expression cassette of the present invention is inserted. Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer because they allow long-term, stable integration of transgenes and their proliferation in daughter cells. Lentiviral vectors have advantages over vectors derived from oncogenic retroviruses such as murine leukemia viruses because they can transduce non-proliferating cells, such as hepatocytes. They also have the advantage of low immunogenicity.

[0217] In brief summary, the expression cassette or nucleic acid sequence of the present invention is usually operably connected to a promoter and incorporated into an expression vector. The vector is suitable for replication and integration into eukaryotic cells. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters that can be used to regulate the expression of the desired nucleic acid sequence.

[0218] The expression constructs of the present invention can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods of gene delivery are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, 5,589,466, which are incorporated herein by reference in their entirety. In another embodiment, the present invention provides a gene therapy vector.

[0219] The nucleic acid can be cloned into many types of vectors. For example, the nucleic acid can be cloned into such vectors, which include but are not limited to plasmids, phagemids, phage derivatives, animal viruses and cosmids. Specific vectors of interest include expression vectors, replication vectors, probe generation vectors and sequencing vectors.

[0220] Further, expression vector can be provided to cell in the form of viral vector. Viral vector technology is well known in the art and is described in, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses and slow viruses. Generally, suitable vectors are included in at least one organism that works on the origin of replication, promoter sequence, convenient restriction enzyme sites and one or more selectable markers (for example, WO01 / 96584; WO01 / 29058; and U.S. Patent number 6,326,193).

[0221] Many virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to a subject's cells in vivo or in vitro. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In one embodiment, a lentiviral vector is used.

[0222] Additional promoter elements, such as enhancers, can regulate the frequency of transcription initiation. Typically, these are located in the 30-110 bp region upstream of the start site, although recently it has been shown that many promoters also contain functional elements downstream of the start site. The spacing between promoter elements is often flexible so that when an element is inverted or moved relative to another, the promoter function is maintained. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it is shown that individual elements can work cooperatively or independently to start transcription.

[0223] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence that can drive any polynucleotide sequence operably connected thereto to express at a high level. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr (Epstein-Barr) virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, such as but not limited to actin promoter, myosin promoter, heme promoter, and creatine kinase promoter. Further, the present invention should not be limited to the application of constitutive promoters. Inducible promoters are also considered to be part of the present invention. The use of an inducible promoter provides a molecular switch that can turn on the expression of a polynucleotide sequence operably linked to an inducible promoter when such expression is desired, or turn off expression when expression is undesirable. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0224] In order to evaluate the expression of CAR polypeptides or parts thereof, the expression vector introduced into the cell may also include any one or both of a selectable marker gene or a reporter gene to facilitate identification and selection of expressing cells from a cell population seeking to be transfected or infected by a viral vector. In other aspects, selectable markers may be carried on a single DNA segment and used for co-transfection procedures. Both selectable markers and reporter genes may be flanked by appropriate regulatory sequences so that they can be expressed in host cells. Useful selectable markers include, for example, antibiotic resistance genes, such as neo and the like.

[0225] Reporter gene is used to identify the cells of potential transfection and to evaluate the functionality of regulatory sequences. Generally, reporter gene is the following gene: it is not present in or expressed by a receptor organism or tissue, and it encodes a polypeptide whose expression is clearly indicated by some easily detectable properties such as enzymatic activity. After DNA has been introduced into the receptor cell, the expression of the reporter gene is measured at the appropriate time. Suitable reporter gene can include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase or green fluorescent protein (for example, Ui-Tei et al., 2000FEBS Letters479:79-82). Suitable expression systems are well known and can be prepared using known techniques or commercially available. Generally, the construct with a minimum of 5 flanking regions showing the highest level of reporter gene expression is identified as a promoter. Such a promoter region can be connected to a reporter gene and used to evaluate the ability of a reagent to regulate promoter-driven transcription.

[0226] Methods for introducing genes into cells and expressing genes into cells are known in the art. In the context of expression vectors, vectors can be easily introduced into host cells, such as mammalian, bacterial, yeast or insect cells, by any method known in the art. For example, expression vectors can be transferred into host cells by physical, chemical or biological means.

[0227] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0228] Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used methods for inserting genes into mammalian cells such as human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, etc. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0229] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles and liposomes. An exemplary colloidal system used as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0230] In the case of using a non-viral delivery system, an exemplary delivery vehicle is a liposome. Consider using lipid preparations to introduce nucleic acid into a host cell (in vitro, ex vivo or in vivo). On the other hand, the nucleic acid can be associated with a lipid. Nucleic acids associated with lipids can be encapsulated in the aqueous interior of the liposome, dispersed in the lipid bilayer of the liposome, attached to the liposome through a connecting molecule associated with both the liposome and the oligonucleotide, trapped in the liposome, compounded with the liposome, dispersed in a solution comprising lipids, mixed with lipids, combined with lipids, included in lipids as a suspension, included in micelles or compounded with micelles, or otherwise associated with lipids. The lipids, lipid / DNA or lipid / expression vectors associated with the composition are not limited to any specific structure in the solution. For example, they can be present in a bilayer structure, as micelles or have a "collapsed (collapsed)" structure. They can also be simply dispersed in a solution, possibly forming aggregates of size or shape inhomogeneity. Lipid is a fatty substance, which can be a naturally occurring or synthetic lipid. For example, lipids include fat droplets that occur naturally in the cytoplasm as well as compounds that contain long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0231] In a preferred embodiment of the present invention, the vector is a plasmid, such as a conventional mammalian expression plasmid.

[0232] preparation

[0233] The present invention provides a composition containing the heavy chain variable region described in the first aspect of the present invention, the heavy chain described in the second aspect of the present invention, the light chain variable region described in the third aspect of the present invention, the light chain described in the fourth aspect of the present invention, the antibody described in the fifth aspect, the recombinant protein described in the sixth aspect of the present invention, the CAR construct described in the seventh aspect of the present invention, the immune cell described in the eighth aspect of the present invention, or the antibody-drug conjugate described in the ninth aspect of the present invention, and a pharmaceutically acceptable carrier, diluent or excipient. In one embodiment, the preparation is a liquid preparation. Preferably, the preparation is an injection.

[0234] In one embodiment, the formulation may include a buffer such as neutral buffered saline, sulfate buffered saline, etc.; a carbohydrate such as glucose, mannose, sucrose or dextran, mannitol; a protein; a polypeptide or an amino acid such as glycine; an antioxidant; a chelating agent such as EDTA or glutathione; an adjuvant (e.g., aluminum hydroxide); and a preservative. The formulation of the present invention is preferably formulated for intravenous administration.

[0235] Test Uses and Kits

[0236] The antibodies of the present invention can be used in detection applications, for example, for detecting a sample to provide diagnostic information.

[0237] In the present invention, the sample (specimen) used includes cells, tissue samples and biopsy specimens. The term "biopsy" used in the present invention should include all types of biopsies known to those skilled in the art. Therefore, the biopsy used in the present invention can include, for example, tissue samples prepared by endoscopic methods or puncture or needle biopsy of an organ.

[0238] Samples used in the present invention include fixed or preserved cell or tissue samples.

[0239] The present invention also provides a kit containing the antibody (or fragment thereof) of the present invention. In a preferred embodiment of the present invention, the kit further comprises a container, instructions for use, a buffer, etc. In a preferred embodiment, the antibody of the present invention can be fixed to a detection plate.

[0240] The main advantages of the present invention include:

[0241] (1) The present invention develops a class of antibodies that have specific recognition activity for NLRP3 protein or a class of antibodies that can specifically recognize NLRP3 protein.

[0242] (2) The present invention unexpectedly obtains monoclonal antibodies with extremely excellent affinity and specificity for recognizing NLRP3 protein (especially native or non-denatured NLRP3 protein). The results of the present invention show that the two monoclonal antibodies prepared by the present invention (named 9-B8-3-2-C5 and 9-C9-1) have specific recognition effects on NLRP3 protein.

[0243] (3) The present invention prepares monoclonal antibodies against NLRP3, which provides an important basis for future related experimental research. In this study, the recombinant plasmid Ms-N3-throhFc was constructed, and the Ms-N3 protein was purified by Protein A affinity chromatography, and then immunized against Nlrp3 - / -Mice. The hybridoma cell lines 9-B8-3-2-C5 and 9-C9-1 that secrete anti-mouse NLRP3 monoclonal antibodies were successfully screened. Experiments have shown that the antibodies produced by the hybridoma cells can be used in immunofluorescence experiments and specifically recognize mouse NLRP3 protein, providing a powerful tool for further exploration of the biological function of NLRP3.

[0244] (4) The present invention uses these NLRP3 antibodies—Cryo2, pAb (A12694) and mAb7578, and uses immunofluorescence to detect the expression of these antibodies in WT and Nlrp3 - / - The results showed that these antibodies could not specifically recognize NLRP3 protein ( Figure 3 ). Therefore, this study hopes to screen hybridoma cell lines that can secrete anti-mouse non-denatured NLRP3 monoclonal antibodies to provide a powerful tool for studying the biological function of NLRP3.

[0245] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0246] Unless otherwise specified, the materials and reagents used in the examples of the present invention are commercially available products.

[0247] Example 1 Construction, expression and purification of Ms-N3-throFc recombinant plasmid

[0248] 1. Construction of recombinant plasmid Ms-N3-throhFc

[0249] The third exon gene (Ms-N3) of Mus musculus-Nlrp3 (Gene ID: 216799) was amplified using pcDNA4.0 v5-msNLRP3 plasmid as template and ms-F1 and ms-R1 as specific primers. The PCR product was analyzed by 1% agarose gel electrophoresis, and the amplified fragment was approximately 1755 bp, which was consistent with the expected size ( Figure 4 A). Then the target gene and pcDNA4.0 empty plasmid were double-digested with restriction endonucleases NheⅠ-HF and SacⅡ ( Figure 4 B), T4 DNA enzyme ligation and transformation into competent Escherichia coli DH5α, and positive clone 1 was screened after sequence alignment.

[0250] Since the target gene of mouse contains a BamHⅠ-HF restriction site, we first extract the plasmid from positive clone 1 as a template, use ms-F2 and ms-R2 as specific primers, and perform point mutation on the BamHⅠ restriction site in the middle of the target sequence (a synonymous mutation at 899bp, aga becomes agg), thereby removing the BamHⅠ-HF restriction site and screening positive clone 2. Then, use positive clone 2 as a template, use ms-F3 and ms-R2 as specific primers for PCR amplification, and double-digest the PCR amplification product and p36-G3-hFc plasmid with restriction endonucleases BamHⅠ-HF and NotⅠ-HF, respectively ( Figure 4 C), after ligation and transformation, a single colony was picked and shaken, and the sequence was compared to find the successful positive clone 3. Finally, the plasmid extracted from positive clone 3 was used as a template, and ms-F4 and ms-R4 were used as specific primers for point mutation (insertion of bovine thrombin cleavage site), and positive clone 4 was screened, which was the final recombinant plasmid Ms-N3-throhFc.

[0251] We used the above method to construct a recombinant plasmid Ms-N3-throhFc with a bovine thrombin cleavage sequence and a human-Fc fragment, which contained the Ms-N3 sequence (1755 bp), a bovine thrombin cleavage site sequence (30 bp) and a human-Fc fragment (696 bp). The total target gene size was approximately 2481 bp. Figure 4 D is the identification result of the recombinant plasmid Ms-N3-throhFc after the plasmid was successfully constructed and double-digested with BamHI-HF and NotⅠ-HF. After digestion, a band of the target gene was found at around 1750 bp.

[0252] Table 2-1. Primers

[0253]

[0254] Example 2 Expression, preparation and further purification of Ms-N3-throFc recombinant protein

[0255] 1. Expression and identification of recombinant plasmid Ms-N3-throFc in HEK293F cells

[0256] We transfected the recombinant plasmid Ms-N3-throFc and pcDNA4.0 empty plasmid into HEK293F cells to identify their expression. 48 hours after transfection, 0.5-1mL of cell suspension was taken, centrifuged and resuspended in PBS to wash away the culture medium, and the cells were treated with 1× Loading Buffer. The expression of the recombinant protein was detected by Western Blot. The results are as follows: Figure 5As shown, the Ms-N3-throFc recombinant protein was correctly expressed, and its molecular weight was about 93.9 kDa, which was consistent with the expectation.

[0257] 2. Protein A affinity column purification of Ms-N3 protein

[0258] Since suspension cell culture has more advantages and the expression of recombinant plasmids in HEK293F cells has been identified, we chose to expand the culture and further purify in HEK293F cells. The recombinant plasmid Ms-N3-throFc was transformed into HEK293F cells, and the culture medium was added in appropriate amounts every day to maintain the logarithmic growth state of the cells. After 72 hours of plasmid transfection, the cells were harvested by centrifugation and stored at -80°C.

[0259] After processing the cells collected multiple times (about 2g), the protein was purified using a Protein A affinity column. The sample was loaded at a rate of 0.2mL / min, and the target protein specifically bound to the affinity column. Then the column was removed and bovine thrombin was added to digest for 12 hours at 4°C. The column was reinstalled and eluted with equilibrium buffer at a rate of 0.1mL / min until the ultraviolet absorption value displayed by AKTA reached the baseline. About 2mL of the eluate containing the target protein was collected. Finally, the hFc protein was eluted with elution buffer. Figure 6 It is the elution peak of Ms-N3 protein purified by Protein A, and the maximum value of the UV absorption peak is 900mAU.

[0260] 3. Western Blot Identification and Coomassie Brilliant Blue Staining Results of Ms-N3 Protein

[0261] We used Western Blot analysis and SDS-PAGE gel Coomassie blue staining to identify the samples collected during the purification of Ms-N3 protein. Figure 7 As shown in A and B, the Ms-N3-throhFc protein in the sample solution before purification can be recognized by both anti-human IgG (Fc specific) antibody and anti-NLRP3 antibody at about 94kDa; after the sample solution is purified, due to the separation of Ms-N3 protein from hFc protein, Ms-N3 appears at 69kDa and can only be recognized by anti-NLRP3 antibody. The same sample was identified by SDS-PAGE gel Coomassie brilliant blue staining, and the results showed that we obtained a higher purity Ms-N3 protein through Protein A affinity chromatography column ( Figure 7 C). The purified two protein samples were concentrated by ultrafiltration and then filtered with a 0.22 μm filter. After being packaged, they were stored in a -80°C refrigerator for subsequent mouse immunization experiments.

[0262] Example 4. Preparation and identification of Ms-N3 mouse monoclonal antibody

[0263] 1. Detection of immune effects in Ms-N3 mice

[0264] Purified Ms-N3 protein was used to immunize Nlrp3- / - mice. In order to detect the immune effect of mice, we cut the tail and collected blood on the 7th day after the third immunization to obtain mouse serum. Indirect ELISA was used with Ms-N3 protein as substrate to detect the antibody titer in mouse serum. Figure 8 The results showed that the serum antibody titers of immunized mice met the requirements of subsequent experiments.

[0265] 2. Preliminary screening of positive hybridoma cells

[0266] After the serum titer of the immunized mice in the Ms-N3 group was tested, the mice with good immune effect were selected for the last immunization before fusion, that is, ms-4 and ms-2 were selected for immunization. After cell fusion, the hybridoma cells were cultured with HAT selection medium, and after 10-14 days, the positive clones were preliminarily screened by indirect ELISA method. The positive clones screened out in each group are as follows: ms-4 group: 1-D8-1, 2-H3, 3-B2-1, 3-C2-1, 3-C2-2, 3-C2-3, 5-D9-1, 7-E1, 7-E7, 7-G6-1; ms-2 group: 1-B9-1, 1-B9-2, 1-B9-3, 1-B9-4, 2-D3-3, 2-F8-2, 4-D8-3, 6-G10-2, 7-G8-2, 8-D4-1, 9-B7-1, 9-C9-1, 9-E2-1, 9-B8-1, 9-B8-3.

[0267] 3. Detection of the specificity of monoclonal antibodies for NLRP3 by immunocytofluorescence assay

[0268] On the seventh day, WT and Nlrp3- / - mouse BMDM cells were stimulated with LPS and ATP solution, and the immunocytofluorescence method was used to detect whether the antibodies secreted by the 9-B8-3-2-C5 and 9-C9-1 hybridoma cell lines could specifically recognize activated NLRP3. After the cell slides were treated with immunofluorescence, the results were photographed using a confocal microscope. Fig. 9 As shown in A and B. Monoclonal antibodies 9-B8-3-2-C5 and 9-C9-1 can specifically recognize NLRP3 in WT BMDM cells compared with Nlrp3- / - BMDM cells.

[0269] 4. Monoclonal Antibody Subtype Identification

[0270] The screened monoclonal antibodies 9-B8-3-2-C5 and 9-C9-1 that can specifically recognize mouse NLRP3 were identified using a mouse monoclonal antibody subtype identification kit. The results are as follows Fig.10As shown in A and B, the heavy chain subtype of the detected monoclonal antibodies was IgM, and the light chain subtype was Kappa.

[0271] 5. PCR amplification of heavy and light chain variable region genes of monoclonal antibodies

[0272] The two hybridoma cells were lysed with Trizol and RNA was extracted. The cDNA was synthesized according to the method of the reverse transcription kit of Promega. The primers of the heavy chain variable region (VH) and the light chain variable region (VL) were VH-F: 5'-AGGTGCAGCTGCAGGAGTCTGG-3', VH-R: 5'-TGAGGAGACGGTGACCGTGGTCCCTTGGCCCC-3', VL-F: 5'GACATTGAGCTCACCCAGTCTCCA-3', VL-R: 5'-GGGAAGATGGATCCAGTTGGTGCAGCAGC-3'. After amplifying the target gene according to the method of the PCR amplification kit of Takara, 1% agarose gel electrophoresis analysis showed that the amplified fragment was about 300 bp ( Fig.11 ). After the PCR product was recovered from the gel, the following results were obtained by sequencing:

[0273] The sequence of VH of 9-B8-3-2-C5 is:

[0274] GTCGGCTATGCGAGCTGGGGCTTCAGTGAGCTGTCCTGCAAGGCTTCTGGCTACACCTTCACAAGCTATGGTATAAGCTGGGTGAAGCAGAGAACTGGACAGGGCCTTGAGTGGATTGGAGAGATTTATCCTAGAAGTGGTAATACTTACTACAATGAGAAGTTCAAGGGCAAG GCCACACTGACTGCAGACAAATCCTCCAGCACAGCGTACATGGAGCTCCGCAGCCTGACATCTGAGGACTCTGCGGTCTATTTCTGTGCAAGACGGTACTATGATTACGACGAGGGGCAGGCCTGGTTTGCTTACTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAA(SEQID NO.26);

[0275] Its amino acid sequence is:

[0276] VGYASWGFSELSCKASGYTFT SYGIS WVKQRTGQGLEWIG EIYPRSGNTYYNEKFKGKATLTADKSSSTAYMELRSLTSEDSAVYFCA RRYYDYDEGQAWFAY WGQGTTVTVSS(SEQ ID NO.6)

[0277] The sequence of the VL of 9-B8-3-2-C5 is:

[0278] ATTGTTCTGCATCTCAGGGGAGAGGTCACCATAACCTGCAGTGCCAGCTCAAGTGTAAGTTACATGCACTGGTTCCAGCAGAAGCCAGGCACTTCTCCCAAACTCTGGATTTATAGCACATCCAACCTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGATCTGGGACCTCTTACTCTCTCACAATCAGCCGAATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAAAGGAGTAGTTACCCACCCACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGGGCTGATGCTGCACCAACTGGATCCATCTTCCCA(SEQ ID NO.27);

[0279] Its amino acid sequence is:

[0280] IVLHLRGEVTITC SASSSVSYMH WFQQKPGTSPKLWIY STSNLAS GVPARFSGSGSGTSYSLTISRMEAEDAATYYC QQRSSYPPT FGGGTKLEIKRADAAPTGSIFP(SEQ ID NO.13).

[0281] The sequence of the VH of 9-C9-1 is:

[0282] AGTCTATATCTGGGGCTCAGTGAGCTGTCCTGCAAGGCTTCTGGCTACACCTTTACTAGCTACTGGATGCACTGGGTAAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGGATACATTAATCCTAGCAGTGGTTATACTAAGTACAATCAGAAGTTCAAGGACAAGGCCACATTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAGCTGAGCAGCCTGACATATGAGGACTCTGCAGTCTATTACTGTGCAAGGAATGACTACTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA(SEQ ID NO.28);

[0283] Its amino acid sequence is:

[0284] SLYLGLSELSCKASGYTFT SYW WVKQRPGQGLEWIG YINPSSGYTKYNQKFKD KATLTADKSSSTAYMQLSSLTYEDSAVYYCAR NDY WGQGTTVTVSS(SEQ ID NO.7).

[0285] The sequence of the VL of 9-C9-1:

[0286] GGGTCTTGTTCTGCATCTCAGGGGAAGGTCACCATGACCTGCAGGGCCAGCTCAAGTGTAAGTTCCAGTTACTTGCACTGGTACCAGCAGAAGTCAGGTGCCTCCCCCAAACTCTGGATTTATAGCACATCCAACTTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGTGTGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTACAGTGGTTACCCACTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGGGCTGATGCTGCACCAACTGGATCCATCTTC(SEQ ID NO.29);

[0287] Its amino acid sequence is:

[0288] GSCSASQGKVTMTC RASSSVSSSYLH WYQQKSGASPKLWIY STSNLAS GVPARFSGSGSGTSYSLTISSVEAEDAATYYCQQYSGYPLTFGAGTKLELKR ADAAPTGSIF(SEQ ID NO.14)

[0289] Note: The underlined parts in the above VH and VL sequences are CDR1, CDR2, and CDR3, respectively.

[0290] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A heavy chain variable region of an antibody, characterized in that The heavy chain variable region includes the following three complementarity determining regions CDR: CDR1 shown in SEQ ID NO: 1, CDR2 shown in SEQ ID NO:3, and CDR3 shown in SEQ ID NO:5; or CDR1 shown in SEQ ID NO:2, CDR2 shown in SEQ ID NO:4, and The sequence of CDR3 is as shown in NDY.

2. A heavy chain of an antibody, characterized in that The heavy chain has the heavy chain variable region as described in claim 1.

3. A light chain variable region of an antibody, characterized in that: The light chain variable region includes the following three complementarity determining regions CDR: CDR1' shown in SEQ ID NO:8, CDR2' shown in SEQ ID NO:10, and CDR3' shown in SEQ ID NO:11; or CDR1' shown in SEQ ID NO:9, CDR2' shown in SEQ ID NO:10, and CDR3' shown in SEQ ID NO:

12.

4. A light chain of an antibody, characterized in that The light chain has the light chain variable region as described in claim 3.

5. An antibody, characterized in that The antibody has: (1) the heavy chain variable region of claim 1; and / or (2) The light chain variable region according to claim 3.

6. A recombinant protein, characterized in that The recombinant protein has: (i) the heavy chain variable region of claim 1, the heavy chain of claim 2, the light chain variable region of claim 3, the light chain of claim 4, or the antibody of claim 5; and (ii) optionally a tag sequence to facilitate expression and / or purification.

7. A CAR construct, characterized in that: The antigen binding region of the CAR construct is a scFv that specifically binds to the NLRP3 protein, and the scFv has the heavy chain variable region as described in claim 1 and the light chain variable region as described in claim 3.

8. A recombinant immune cell, characterized in that The immune cell expresses the exogenous CAR construct of claim 7; or the immune cell expresses or exposes the antibody of claim 5 outside the cell membrane.

9. An antibody-drug conjugate, characterized in that: The antibody-drug conjugate contains: (a) an antibody portion, the antibody portion being selected from the group consisting of: The heavy chain variable region of claim 1, the heavy chain of claim 2, the light chain variable region of claim 3, the light chain of claim 4, or the antibody of claim 5, or a combination thereof; and (b) a coupling portion coupled to the antibody portion, wherein the coupling portion is selected from the group consisting of a detectable marker, a drug, a toxin, a cytokine, a radionuclide, an enzyme, a gold nanoparticle / nanorod, a nanomagnetic particle, a viral coat protein or a VLP, or a combination thereof.

10. A use of an active ingredient, characterized in that: The active ingredient is selected from the following group: the heavy chain variable region of claim 1, the heavy chain of claim 2, the light chain variable region of claim 3, the light chain of claim 4, or the antibody of claim 5, the recombinant protein of claim 6, or a combination thereof, and the active ingredient is used to prepare a drug for preventing and / or treating inflammatory and autoimmune diseases.

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