Anti-human interleukin 2 antibody and use thereof

Abstract

The application provides an anti-human interleukin 2 monoclonal antibody, a CBA kit comprising the antibody and a detection method thereof. Specifically, the application provides an antibody against human interleukin 2 and a CBA kit comprising the antibody. The kit provided by the application can complete quantitative detection of human interleukin 2, has high specificity and detection sensitivity, and has a good development and application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological detection. Specifically, the present application relates to an anti-human interleukin 2 antibody and its application. BACKGROUND

[0002] Interleukin-2 (IL-2) is a cytokine that plays an important role in the chemotactic factor family. IL-2 is composed of a bundle structure of four alpha-helix cores and beta fragments, with a molecular weight of 15.5 kD. It is mainly produced by CD4+ T cells stimulated by antigens, but CD8+ T cells, dendritic cells, NKT cells, and mast cells can also produce small amounts of IL-2. IL-2 is a cytokine with a wide range of biological activities. It belongs to a type of lymphocyte growth factor, and has the effect of promoting the proliferation and differentiation of T cells after being stimulated by antigens. Its main principle of action is to strengthen the secretion of natural killer (NK) cells by cytokines. NK cells are stimulated by proliferation signals, and their killing power is effectively improved due to the lack of MHC hindrance. Lymphocyte-activated killer (LAK) cells receive signal induction, B lymphocytes proliferate faster, thereby promoting antibody secretion, and the activity of autophagy cells increases. It has pharmacological effects such as antiviral, immune enhancement, and antitumor in clinical practice.

[0003] Interleukin-2 (IL-2) plays an important role in the regulation of the immune system. Under normal circumstances, the level of IL-2 will change with the body's immune status. The normal range of IL-2 is 5-50 pg / ml. The level of IL-2 can be affected by many factors, including age, gender, body weight, disease status, etc. In some diseases, the level of IL-2 will change abnormally, for example, in some cancers, autoimmune diseases or infectious diseases, the level of IL-2 may increase or decrease. Therefore, for the diagnosis and treatment of specific diseases, doctors need to understand the level and changes of IL-2 in patients. Therefore, it is of great clinical significance to develop a high-sensitivity method for detecting interleukin 2 protein.

[0004] Most of the detection methods on the market are ELISA methods, that is, enzyme-linked immunoassay. Human interleukin-2 (IL-2) ELISA KIT is based on the classic double antibody sandwich ELISA method, which uses HRP conjugated antibody to catalyze TMB to produce color material, and calculates the content of human interleukin-2 in the sample based on the standard curve. The detection principle of this method is: double antibody sandwich method is used to determine the level of human interleukin-2 (IL-2) in the sample. The purified human interleukin-2 (IL-2) antibody is used to coat the microplate to form a solid phase antibody. Human interleukin-2 (IL-2) is added to the coated monoclonal antibody microplate, and then combined with HRP labeled human interleukin-2 (IL-2) antibody to form an antibody-antigen-enzyme labeled antibody complex. After thorough washing, the substrate TMB is added for color development. TMB is converted to blue under the catalysis of HRP enzyme, and is converted to the final yellow under the action of acid. The color depth is positively correlated with the human interleukin-2 (IL-2) in the sample. The absorbance (OD value) is measured at 450 nm wavelength by enzyme labeling instrument, and the concentration of human interleukin-2 (IL-2) in the sample is calculated by standard curve. ELISA method is the most common, but its experimental steps are complex and the throughput is low. Due to the limitation of the bottom area of the enzyme labeled plate, a large amount of coated antibody or antigen is required, which conflicts with the pain points of difficult access to clinical samples, and the sensitivity and linearity still need to be improved. For example, the sensitivity of Human IL-2 ELISA Kit (Cat: KIT11848) of SinoBiological Company is 4.47 pg / ml, and the linear range is 18.75-1200 pg / mL. The linearity and sensitivity of this kit still have a large improvement space.

[0005] Currently, some detection methods on the market use the AlphaLISA method. AlphaLISA technology primarily relies on the interaction between alpha donor and acceptor microbeads. When a biological reaction brings the donor and acceptor microbeads close together, a laser-excited cascade reaction generates a highly amplified signal. Specifically, under 680nm laser irradiation, the photosensitizer on the donor microbeads converts oxygen from the surrounding environment into more reactive singlet oxygen. This singlet oxygen diffuses to the acceptor microbeads, producing a series of chemiluminescent reactions, ultimately transferring energy to europium and emitting a light signal at a wavelength of 615nm. In the absence of specific interactions between biomolecules, singlet oxygen cannot diffuse to the acceptor microbeads, and no signal is generated. Alternatively, an antibody is biotinylated and binds to a streptavidin-coated donor microbead, while the acceptor microbead directly binds to another antibody. In the presence of cytokines, the binding of antibodies to cytokines brings the donor and acceptor microbeads closer. Excitation of the donor microbeads causes singlet oxygen to diffuse to the acceptor microbeads, thereby emitting a light signal at a wavelength of 615nm. The detection signal of AlphaLISA is directly proportional to the amount of cytokines present in the sample. AlphaLISA technology also uses a sandwich method to detect cytokines; this method has high sensitivity and a wide detection range, as seen in Revvity's... The Human Interleukin-2 (IL2) Detection Kit (catalog number AL221C / F) has a sensitivity of 1.8 pg / ml and a linear range of 1.8-100,000 pg / mL. However, this technology requires a multi-functional microplate reader capable of performing AlphaLISA detection, which is expensive. Furthermore, AlphaLISA detection kits are much more expensive than other cytokine detection kits on the market, resulting in high detection costs and making them unsuitable for high-throughput sample detection.

[0006] Currently, some detection methods on the market use the CBA method. CBA (Cytometric Bead Array) is a multi-index flow cytometry protein quantification technology for microsamples. It is a multiplex protein quantification method based on a flow cytometry detection system, capable of simultaneously detecting multiple indicators in a single sample. Based on the CBA principle, corresponding cytokine antibodies are attached to specific fluorescently encoded microspheres. When a specific antigen is present in the sample, these capture antibodies attached to the fluorescently encoded microspheres will bind to it. Finally, by adding phycoerythrin (PE)-labeled specific detection antibodies, a microsphere-capture antibody-antigen-PE-labeled detection antibody complex is formed. Under the phycocyanin (APC) channel of the flow cytometer, the microspheres are recognized and used to determine the antigen; under the PE channel of the flow cytometer, the fluorescence intensity of PE on the microspheres is positively correlated with the concentration of each analyte in the sample. Each calibrator and its corresponding fluorescence signal can be fitted to a concentration-fluorescence intensity standard curve, and the concentration of each antigen can be calculated using the curve equation. For example, BD's HumanIL-2BD. TM The Cytometric Bead Array (CBA) kit has a detection limit of 11.2 pg / mL and a linear range of 10⁻²,500 pg / mL. There is still room for improvement in terms of sensitivity and linearity.

[0007] Other members of the IL-2 family, such as IL-4, IL-7, IL-9, IL-15, and IL-21, have extremely similar characteristics to IL-2, and are prone to false positives when detecting human IL-2 in samples. Therefore, there is a need in the field for a highly specific method for detecting human IL-2.

[0008] To improve the specificity and sensitivity of CBA detection methods, the current industry consensus is to develop corresponding CBA detection kits using monoclonal antibodies against interleukin-2. However, currently available CBA detection kits targeting interleukin-2 generally have low sensitivity and specificity. Furthermore, the mouse monoclonal antibodies used rely on traditional hybridoma methods for development and production, making the preparation process more complex than that of recombinant monoclonal antibodies and resulting in significant batch-to-batch variability. Therefore, detection kits developed using mouse monoclonal antibodies face challenges such as low sensitivity, narrow linear range, and difficulty in controlling batch-to-batch variability.

[0009] Therefore, there is a need in this field to establish a highly sensitive, wide detection range, and highly specific method for detecting IL-2. Summary of the Invention

[0010] The purpose of this invention is to provide a highly sensitive, wide detection range, and highly specific method for detecting IL-2.

[0011] This invention provides an anti-human interleukin-2 antibody and an antibody combination.

[0012] The present application provides a detection kit comprising an anti-human interleukin 2 antibody combination and application thereof.

[0013] In a first aspect of the present application, an antibody against human interleukin 2 protein is provided, the antibody comprising a heavy chain and a light chain, the heavy chain comprising a heavy chain variable region HCVR, and the light chain comprising a light chain variable region LCVR, wherein the heavy chain variable region comprises the following complementarity determining regions CDRs:

[0014] a CDR-H1 with an amino acid sequence as set forth in SEQ ID No: 2;

[0015] a CDR-H2 with an amino acid sequence as set forth in SEQ ID No: 4;

[0016] a CDR-H3 with an amino acid sequence as set forth in SEQ ID No: 6;

[0017] the light chain variable region comprising the following complementarity determining regions CDRs

[0018] a CDR-L1 with an amino acid sequence as set forth in SEQ ID No: 8;

[0019] a CDR-L2 with an amino acid sequence as set forth in SEQ ID No: 10;

[0020] a CDR-L3 with an amino acid sequence as set forth in SEQ ID No: 12.

[0021] In another preferred embodiment, the heavy chain variable region and the light chain variable region further comprise a framework region (FR), respectively.

[0022] In another preferred embodiment, the CDR1, CDR2 and CDR3 of the heavy chain variable region and the light chain variable region are separated by framework regions FR1, FR2, FR3 and FR4, respectively.

[0023] In another preferred embodiment, the HCVR amino acid sequence of the antibody is as set forth in SEQ ID No: 14, or has a sequence identity of > 85%, > 90%, > 95%, > 96%, > 97%, > 98% or > 99% thereto.

[0024] In another preferred embodiment, the LCVR amino acid sequence of the antibody is as set forth in SEQ ID No: 16, or has a sequence identity of > 85%, > 90%, > 95%, > 96%, > 97%, > 98% or > 99% thereto.

[0025] In another preferred embodiment, the HCVR of the antibody has an amino acid sequence as set forth in SEQ ID No: 14, and / or the LCVR has an amino acid sequence as set forth in SEQ ID No: 16.

[0026] In another preferred embodiment, the light chain and / or the heavy chain of the antibody further comprises a constant region.

[0027] In another preferred embodiment, the constant region is a human constant region.

[0028] In another preferred embodiment, the heavy chain of the antibody has an amino acid sequence as set forth in SEQ ID No: 18, or has a sequence identity of >85%, >90%, >95%, >96%, >97%, >98%, or >99% thereto.

[0029] In another preferred embodiment, the light chain of the antibody has an amino acid sequence as set forth in SEQ ID No: 20, or has a sequence identity of >85%, >90%, >95%, >96%, >97%, >98%, or >99% thereto.

[0030] In another preferred embodiment, the heavy chain of the antibody has an amino acid sequence as set forth in SEQ ID No: 18, and / or the light chain has an amino acid sequence as set forth in SEQ ID No: 20.

[0031] In another preferred embodiment, any of the above amino acid sequences further comprises a derivative sequence that optionally has at least one amino acid added, deleted, modified, and / or substituted, and is capable of retaining the binding affinity to human interleukin 2 protein.

[0032] In another preferred embodiment, the number of the added, deleted, modified, and / or substituted amino acids does not exceed 30%, preferably 20%, and more preferably 10%, of the total number of the original amino acids.

[0033] In another preferred embodiment, the antibody is an animal-derived antibody, a chimeric antibody, or a humanized antibody.

[0034] In another preferred embodiment, the antibody is a single-chain antibody, a double-chain antibody, or an antigen-binding fragment.

[0035] In a second aspect of the present application, a fusion protein is provided, the fusion protein comprising:

[0036] (1) the antibody as described in the first aspect of the present application; and

[0037] (2) an optional tag sequence that facilitates expression and / or purification.

[0038] In another preferred embodiment, the tag comprises an Fc tag, a FLAG tag, a 6His tag, or a combination thereof.

[0039] In a third aspect of the present application, a polynucleotide encoding the antibody of the first aspect of the present application or the fusion protein of the second aspect of the present application is provided.

[0040] In a fourth aspect of the present application, a vector containing the polynucleotide of the third aspect of the present application is provided.

[0041] In another preferred embodiment, the expression vector is selected from the group consisting of DNA, RNA, viral vector, plasmid, transposon, other gene transfer system, or a combination thereof. Preferably, the expression vector comprises a viral vector, such as lentivirus, adenovirus, AAV virus, retrovirus, or a combination thereof.

[0042] In a fifth aspect of the present application, a genetically engineered host cell containing the vector of the fourth aspect of the present application or the polynucleotide of the third aspect of the present application integrated into the genome is provided.

[0043] In another preferred embodiment, the host cell comprises a prokaryotic cell or a eukaryotic cell.

[0044] In another preferred embodiment, the host cell is selected from the group consisting of E. coli, yeast cell, mammalian cell.

[0045] In a sixth aspect of the present application, an antibody conjugate is provided, which comprises:

[0046] (a) an antibody moiety, which is the antibody of the first aspect of the present application; and

[0047] (b) a conjugating moiety conjugated to the antibody moiety, which is selected from the group consisting of a detectable label, a drug, or a combination thereof.

[0048] In another preferred embodiment, the antibody moiety is conjugated to the detectable label via a chemical bond or a linker.

[0049] In another preferred embodiment, the detectable label is selected from the group consisting of a fluorescent or luminescent label, biotin, a radioactive label, an MRI (magnetic resonance imaging) or CT (computed tomography) contrast agent, or an enzyme capable of producing a detectable product, gold nanoparticle / nanorod, nanomagnetic particle, or any form of nanoparticle.

[0050] In another preferred embodiment, the detectable label is biotin.

[0051] In another preferred embodiment, the drug is a small molecule drug, a biological factor, or a combination thereof.

[0052] In another preferred embodiment, the drug is a cytotoxic drug (toxin).

[0053] In another preferred embodiment, the cytotoxic drug is selected from the group consisting of an anti-tubulin drug, a DNA minor groove binding agent, a DNA replication inhibitor, an alkylating agent, an antibiotic, a folate antagonist, an antimetabolite drug, a chemotherapeutic sensitizer, a topoisomerase inhibitor, a vinca alkaloid, or a combination thereof.

[0054] In a seventh aspect of the present application, there is provided an antibody combination against human interleukin 2 protein, the antibody combination comprising:

[0055] an antibody S1, a heavy chain variable region of the antibody S1 comprising the following complementarity determining regions (CDRs):

[0056] a CDR-H1 having an amino acid sequence as set forth in SEQ ID No: 1,

[0057] a CDR-H2 having an amino acid sequence as set forth in SEQ ID No: 3,

[0058] a CDR-H3 having an amino acid sequence as set forth in SEQ ID No: 5,

[0059] a light chain variable region of the antibody S1 comprising the following complementarity determining regions (CDRs):

[0060] a CDR-L1 having an amino acid sequence as set forth in SEQ ID No: 7,

[0061] a CDR-L2 having an amino acid sequence as set forth in SEQ ID No: 9,

[0062] a CDR-L3 having an amino acid sequence as set forth in SEQ ID No: 11; and

[0063] an antibody S2, the antibody S2 being the antibody as described in the first aspect of the present application.

[0064] In another preferred embodiment, the heavy chain variable region and the light chain variable region further comprise a framework region (FR), respectively.

[0065] In another preferred embodiment, the CDR1, CDR2 and CDR3 of the heavy chain variable region and the light chain variable region are separated by a framework region FR1, FR2, FR3 and FR4, respectively.

[0066] In another preferred embodiment, the HCVR of the antibody S1 has an amino acid sequence as set forth in SEQ ID No: 13 and / or the LCVR has an amino acid sequence as set forth in SEQ ID No: 15.

[0067] In another preferred embodiment, the HCRV of antibody S2 has an amino acid sequence as set forth in SEQ ID No: 14 and / or the LCVR has an amino acid sequence as set forth in SEQ ID No: 16.

[0068] In another preferred embodiment, the light chain and / or the heavy chain of the antibody further comprises a constant region.

[0069] In another preferred embodiment, the constant region is a human constant region.

[0070] In another preferred embodiment, the heavy chain of antibody S1 has an amino acid sequence as set forth in SEQ ID No: 17 and / or the light chain has an amino acid sequence as set forth in SEQ ID No: 19.

[0071] In another preferred embodiment, the heavy chain of antibody S2 has an amino acid sequence as set forth in SEQ ID No: 18 and / or the light chain has an amino acid sequence as set forth in SEQ ID No: 20.

[0072] In another preferred embodiment, any of the above amino acid sequences further comprises a derivative sequence in which at least one amino acid is optionally added, deleted, modified and / or substituted, and which is capable of retaining the binding affinity to human interleukin 2 protein.

[0073] In another preferred embodiment, the number of the added, deleted, modified and / or substituted amino acids does not exceed 30%, preferably 20%, and more preferably 10% of the total number of amino acids in the original amino acid sequence.

[0074] In another preferred embodiment, the antibody is an animal-derived antibody, a chimeric antibody or a humanized antibody.

[0075] In another preferred embodiment, the antibody is a single-chain antibody, a double-chain antibody, or an antigen-binding fragment.

[0076] In an eighth aspect of the present application, there is provided use of the antibody of the first aspect of the present application, the polynucleotide of the third aspect of the present application, the vector of the fourth aspect of the present application, the host cell of the fifth aspect of the present application, the antibody conjugate of the sixth aspect of the present application, or the antibody combination of the seventh aspect of the present application, for the manufacture of:

[0077] i) a reagent or a kit for detecting human interleukin 2 protein;

[0078] ii) a preparation or a medicament for treating human interleukin 2 related diseases.

[0079] In another preferred embodiment, the human interleukin 2 related diseases are selected from the group consisting of inflammation related diseases, tumors, autoimmune diseases, or a combination thereof.

[0080] In another preferred embodiment, the preparation or medicament is in a liquid dosage form, preferably an injection.

[0081] In another preferred embodiment, the medicament is a cell therapy medicament.

[0082] In a ninth aspect of the present application, there is provided a human interleukin 2 protein detection kit, comprising the antibody according to the first aspect of the present application, the antibody conjugate according to the sixth aspect of the present application, or the antibody combination according to the seventh aspect of the present application.

[0083] In another preferred embodiment, the detection kit is used for a double antibody sandwich method.

[0084] In another preferred embodiment, the detection kit is used for CBA detection, ELISA detection, and / or colloidal gold detection.

[0085] In another preferred embodiment, the kit is a CBA kit.

[0086] In another preferred embodiment, the detection kit comprises the antibody combination according to the sixth aspect of the present application, wherein the antibody S1 is immobilized on a carrier and used for capturing human interleukin 2 protein, and the antibody S2 is conjugated with a detection label.

[0087] In another preferred embodiment, the detection kit comprises microspheres (such as fluorescent microspheres), which are coated with the antibody S1.

[0088] In another preferred embodiment, the detection kit comprises the antibody S2 with biotin.

[0089] In another preferred embodiment, the detection kit further comprises a human interleukin 2 protein standard.

[0090] In another preferred embodiment, the detection kit further comprises a component selected from the group consisting of a fluorescent protein-labeled streptavidin, a lyophilized interleukin 2 standard, a washing solution, a sample diluent, a matrix buffer, or a combination thereof.

[0091] In another preferred embodiment, the fluorescent protein is phycoerythrin.

[0092] In a tenth aspect of the present application, there is provided a method for detecting interleukin 2 in a sample, using the antibody according to the first aspect of the present application, the antibody conjugate according to the sixth aspect of the present application, the antibody combination according to the seventh aspect of the present application, or the detection kit according to the ninth aspect of the present application.

[0093] In another preferred embodiment, the method comprises the following steps:

[0094] 1) immobilizing the antibody S1 on a carrier;

[0095] 2) labelling antibody S2 with a detectable label;

[0096] 3) incubating said antibodies S1, S2 and the sample to be tested, so that antibodies S1, S2 bind to interleukin 2 in the sample;

[0097] 4) washing away the reagents not bound to the carrier, and detecting said detectable label, thereby detecting interleukin 2.

[0098] In another preferred embodiment, the detection is quantitative detection.

[0099] In another preferred embodiment, the carrier is a microsphere, preferably a fluorescent microsphere.

[0100] In another preferred embodiment, the detectable label is biotin.

[0101] In another preferred embodiment, in step 4), the step comprises:

[0102] adding fluorescent protein-labelled avidin and incubating so as to link the biotin on antibody S2, and detecting said fluorescent protein.

[0103] In another preferred embodiment, the avidin is streptavidin.

[0104] In another preferred embodiment, the fluorescent protein is phycoerythrin.

[0105] In another preferred embodiment, the method further comprises the step of detecting a standard of interleukin 2 so as to obtain a standard curve, and calculating the content of interleukin 2 in the sample to be tested according to the standard curve.

[0106] In another preferred embodiment, the interleukin 2 is human interleukin 2.

[0107] In another preferred embodiment, the sample to be tested is selected from the group consisting of cell culture medium, whole blood, serum, plasma, body fluid, or a combination thereof.

[0108] In an eleventh aspect of the present application, there is provided a pharmaceutical composition comprising:

[0109] (i) an antibody as described in the first aspect of the present application, a polynucleotide as described in the third aspect of the present application, a vector as described in the fourth aspect of the present application, a host cell as described in the fifth aspect of the present application, an antibody conjugate as described in the sixth aspect of the present application, or an antibody combination as described in the seventh aspect of the present application, or a combination thereof; and

[0110] (ii) a pharmaceutically acceptable carrier.

[0111] In another preferred embodiment, the pharmaceutical composition is in the form of an injection.

[0112] In another preferred embodiment, the pharmaceutical composition further comprises other drugs for treating inflammation-related diseases, tumors, or autoimmune diseases.

[0113] It should be understood that, within the scope of the present application, the above technical features of the present application and the technical features specifically described hereinafter (e.g., in the examples) can be combined with each other to form new or preferred technical solutions. Due to the limited space, they are not listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0114] The following drawings are used to illustrate specific embodiments of the present application and are not intended to limit the scope of the present application as defined by the claims.

[0115] Figure 1 Results of human IL-2 immunized mice are shown.

[0116] Figure 2 Results of binding of anti-human interleukin monoclonal antibodies 1# and 2# to antigens human IL-4, IL-7, IL-9, IL-15, and IL-21 are shown.

[0117] Figure 3 Graph showing the standard curve for detecting human IL-2 concentration. DETAILED DESCRIPTION

[0118] The present inventors have developed, for the first time, an anti-human interleukin 2 antibody and its use. Specifically, the present application provides an anti-human interleukin 2 monoclonal antibody and a human interleukin 2 CBA detection kit comprising the antibody. The kit of the present application has higher sensitivity and a wider detection range. The kit also has very high specificity, which can avoid the interference of human interleukin 4, human interleukin 7, human interleukin 9, human interleukin 15, and human interleukin 21, making the detection more accurate, and has good development and application prospects. On this basis, the present application is completed.

[0119] Terminology

[0120] For easier understanding of the present application, certain technical and scientific terms are defined in detail below. Unless otherwise clearly defined in this text, all other technical and scientific terms used in this text have the meanings generally understood by those of ordinary skill in the art to which the present application belongs. Before describing the present application, it should be understood that the present application is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary. It should also be understood that the terms used herein are intended only to describe specific embodiments and are not intended to be limiting, and the scope of the present application will be limited only by the appended claims.

[0121] As used herein, the term "about," when used in reference to a numerically recited value, means that the value can vary from the recited value by not more than 1%. For example, as used herein, the expression "about 100" includes all values between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0122] As used herein, the terms "comprising," "including," "containing," and variations thereof, are interchangeable and mean that the composition or process described includes, but is not limited to, whatever follows the term. In other words, these terms do not exclude additional, unrecited elements or method steps.

[0123] As used herein, the term "pharmaceutically acceptable carrier" refers to a material that is not biologically or otherwise undesirable, i.e., the material can be administered to a human and / or animal without excessive adverse side effects or interactions with other pharmaceuticals.

[0124] As used herein, the term "therapeutically effective amount" refers to an amount that produces functional or therapeutic effects for which it is administered, and which is acceptable to the person and / or animal. Those of ordinary skill in the art will understand that the "therapeutically effective amount" can vary depending on the form of the pharmaceutical composition, the route of administration, the excipient used, the severity of the disease, and the combination with other drugs, etc.

[0125] Interleukin 2

[0126] In the present specification, the term "interleukin 2 (IL-2)" is a cytokine that plays an important role in the chemokine family, and IL-2 is a bundled structure consisting of 4 alpha-helix cores and beta fragments, and has a molecular weight of 15.5 kD. The amino acid sequence of IL-2 is shown in SEQ ID NO: 21.

[0127] SEQ ID No: 21:

[0128] MYKMQLLSCIALTLVLVANSAPITSSSTKETEQQMEQLLLDLQLLLNGVNNYENPQLSRMLTFKFYTPKKATEFTHLQCLAEELKNLEEVLGLPQSKNVHLTDTKELISNMNVTLLKLKGSETSYNCEYDDETATITEFLNKWITFCQSIFSTLTIL-2 is a cytokine with a wide range of biological activities, IL-2 belongs to a kind of lymphocyte growth factor, has the effect of promoting T cell proliferation and cell transformation differentiation after being stimulated by antigen, its principle of action is mainly through strengthening the secretion of natural killer (NK) cells, NK cells are stimulated by proliferation signal, at the same time, due to the MHC hindrance, the killing power of NK cells is effectively improved. Lymphokine-activated killer (LAK) cells receive signal induction, B lymphocyte proliferation rate is accelerated, thereby promoting antibody secretion, the activity of autophagy cells is increased, which has pharmacological effects of antiviral, improving immunity, antitumor and the like in clinic.

[0129] The present application provides antibodies, detection kits and detection methods for detecting interleukin 2 (particularly human interleukin 2).

[0130] Antibodies

[0131] In the present application, the term "antibody" should be interpreted in the broadest sense, with various antibody structures, including but not limited to Y-type antibodies, so-called full-length antibodies, antigen-binding portions of Y-type antibodies, and their genetic or chemical modifications. Among them, "antigen-binding portion" refers to one or more portions or fragments of Y-type antibodies, which can retain the ability of the antibody to specifically bind to mouse interleukin 2.

[0132] In the present application, the term "monoclonal antibody" (mAb) includes a highly homogeneous antibody population with substantially the same antigenic determinant. That is, in the antibody population, individual antibodies are essentially the same, except for a small amount of mutations that may occur naturally. Monoclonal antibodies can exhibit a single binding specificity and affinity for a specific epitope on an antigen. Each monoclonal antibody can be directed to the same or substantially the same epitope on an antigen, compared with polyclonal antibodies which usually contain antibodies against different epitopes. The modifier "monoclonal" indicates that the antibody is obtained from a substantially homogeneous antibody population, and cannot be interpreted as requiring production by any particular method. The antibody can be prepared by various methods, including but limited to hybridoma method, recombinant DNA method, phage antibody library and similar methods.

[0133] In the present application, the term "murine antibody" or "anti-human interleukin 2 monoclonal antibody" or similar terms with the modifier "murine" means that the antibody has complementarity determining regions (CDRs) derived from murine immunoglobulin sequences. In one embodiment, the anti-human interleukin 2 murine monoclonal antibody can comprise CDRs and framework regions (FRs) of an antibody derived from rabbit immunoglobulin sequences. In one embodiment, the anti-human interleukin 2 murine antibody or murine monoclonal antibody can comprise CDRs of an antibody derived from murine immunoglobulin sequences. In one embodiment, the anti-human interleukin 2 murine monoclonal antibody can be an antibody whose CDR regions are derived from murine immunoglobulin sequences, and whose FRs are derived from the germline immunoglobulin sequences of another mammal (e.g., mouse or human). The term "anti-human interleukin 2 murine monoclonal antibody" can also include antibodies having amino acid residues encoded by non-rabbit immunoglobulin sequences, e.g., mutations introduced by in vitro random or point-specific mutagenesis, or mutations introduced by in vivo somatic mutation. However, the term "anti-human interleukin 2 murine monoclonal antibody" does not include antibodies whose CDR regions are derived from the germline of another mammal (e.g., human).

[0134] In the present application, the term "antibody" refers to an immunoglobulin molecule composed of four heterologous polypeptide chains, two of which are larger and are referred to as heavy chains (H), and two of which are smaller and are referred to as light chains (L). Antibody light chains can be classified as kappa (K) and lambda (l). Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and define a different class of antibodies as IgM, IgD, IgG, IgA, and IgE, respectively. The variable regions of both light and heavy chains contain a binding domain that identifies and binds to an antigen. The N-terminal portion of each chain is highly variable, and is referred to as the variable region (V), which contains about 110 amino acids. The C-terminal portion of each chain is relatively invariant, and is referred to as the constant region (C), which contains about 330 amino acids. Accordingly, the variable region of the light chain and the variable region of the heavy chain are referred to as the VL and VH regions, respectively, and the constant region of the light chain and the constant region of the heavy chain are referred to as the CL and CH regions, respectively.

[0135] The V regions of the heavy and light chains are referred to as VH and VL, respectively. Each of VH and VL contains three regions of hypervariability, referred to as hypervariable regions (HVRs) or complementarity determining regions (CDRs), that are referred to as HVR1 (CDR1), HVR2 (CDR2), and HVR3 (CDR3), in which HVR3 (CDR3) is the most variable. Collectively, the three CDRs of the VH and VL together form the antigen-binding site of an antibody (antigen-binding site) that determines the specificity of the antibody for an antigen. Outside the CDRs, the amino acid sequences and their arrangements are relatively conserved in the V regions and are referred to as framework regions (FRs). Each of VH and VL has four FR regions, designated FR1, FR2, FR3, and FR4.

[0136] The C regions of the heavy and light chains are referred to as CH and CL, respectively. The length of CL is essentially the same for different types (kappa or lambda) of Ig, but the length of CH is different for different classes of Ig, such as IgG, IgA, and IgD, which include CH1, CH2, and CH3, and IgM and IgE, which include CH1, CH2, CH3, and CH4.

[0137] In the present application, the term "specifically binds" refers to a non-random binding reaction between two molecules, such as between an antibody and its targeted antigen. The strength or affinity of a specific binding interaction can be expressed in terms of the equilibrium dissociation constant (KD) of the interaction. In the present application, the term "KD" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen.

[0138] In the present application, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. When the vector is capable of directing the expression of a polynucleotide inserted into it, the vector is referred to as an expression vector. A vector can be introduced into a host cell by transformation, transduction or transfection, and the host cell into which the vector is introduced can then be used to produce the protein encoded by the inserted polynucleotide. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1 -derived artificial chromosomes (PAC), bacteriophages such as lambda or M13 bacteriophage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), pox viruses, baculoviruses, papillomaviruses, papova viruses (e.g., SV40). A vector can contain a variety of control elements to control expression, including but not limited to, promoter sequences, transcriptional start sequences, enhancer sequences, selection elements, and reporter genes. In addition, a vector can contain a replication origin.

[0139] In the present application, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the intended properties of the protein / polypeptide comprising the amino acid sequence. Conservative amino acid substitutions include substitutions of an amino acid residue with an amino acid residue having a similar side chain, e.g., substitutions made with residues that are physically or functionally similar (have similar size, shape, charge, chemical properties, including ability to form covalent or hydrogen bonds, etc.) to the corresponding amino acid residue. These conservative substitutions are preferably made according to the following table to generate.

[0140] Original residue Representative substitution Preferred substitution Ala (A) Val; Leu; lie Val Arg (R) Lys; Gin; Asn Lys Asn (N) Gin; His; Lys; Arg Gin Asp (D) Glu Glu Cys (C) Ser Ser Gin (Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro; Ala Ala His (H) Asn; Gin; Lys; Arg Arg lie (I) Leu; Val; Met; Ala; Phe Leu Leu (L) lie; Val; Met; Ala; Phe lie Lys (K) Arg; Gin; Asn Arg Met (M) Leu; Phe; lie Leu Phe (F) Leu; Val; lie; 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) lie; Leu; Met; Phe; Ala Leu

[0141] Once the relevant sequence is obtained, it can be obtained in large quantities using recombinant methods. This is typically done by cloning it into a vector, which is then introduced into a cell, and then isolating the relevant sequence from the propagated host cell by conventional methods.

[0142] As used herein, the term "vector," "expression system," or "expression vector" refers to a nucleic acid sequence containing a desired coding sequence and control sequences in operable linkage, such that a host transformed with the sequence produces the encoded protein. To effect transformation, the expression system can be included on a vector; however, the relevant nucleic acid molecule can subsequently also be integrated into the host chromosome.

[0143] As used herein, the term "host cell" is a cell that can support the replication or expression of a vector. Host cells can be prokaryotic cells, such as E. coli, or eukaryotic cells, such as yeast cells, insect cells, amphibian cells, or mammalian cells.

[0144] As used herein, the term "transfection," "stable transfection," or "transient transfection" means the uptake of an expression vector by a host cell, but does not necessarily mean that the encoded sequences are actually expressed. A variety of transfection techniques are known by those skilled in the art. For example, transfection is accomplished by electroporation in the presence of an expression vector and high concentrations of calcium phosphate, by insertion of the host cell with bacteriophage or viral expression vectors, by mechanical insertion of the nucleic acid, or even by culturing the host cell in the presence of unpackaged nucleic acid fragments. Transfection is generally confirmed when any indication that the vector of interest is manipulated appears in the host cell.

[0145] Anti-human interleukin 2 monoclonal antibody

[0146] The present application provides an isolated anti-human interleukin 2 monoclonal antibody, comprising three heavy chain complementarity determining regions (CDR-H1, CDR-H2, CDR-H3) and three light chain complementarity determining regions (CDR-L1, CDR-L2, CDR-L3), wherein:

[0147] the amino acid sequence of CDR-H1 is shown as SEQ ID No: 2;

[0148] the amino acid sequence of CDR-H2 is shown as SEQ ID No: 4;

[0149] the amino acid sequence of CDR-H3 is shown as SEQ ID No: 6;

[0150] the amino acid sequence of CDR-L1 is shown as SEQ ID No: 8;

[0151] the amino acid sequence of CDR-L2 is shown as SEQ ID No: 10;

[0152] the amino acid sequence of CDR-L3 is shown as SEQ ID No: 12.

[0153] In one specific embodiment, the above-mentioned monoclonal antibody comprises an antibody heavy chain variable region HCVR and an antibody light chain variable region LCVR, wherein:

[0154] the amino acid sequence of HCVR is shown as SEQ ID No: 14;

[0155] the amino acid sequence of LCVR is shown as SEQ ID No: 16.

[0156] In one specific embodiment, the anti-human interleukin 2 monoclonal antibody of the present application is anti-human interleukin 2 monoclonal antibody 2#, the heavy chain of which has an amino acid sequence shown as SEQ ID No: 18, and the light chain of which has an amino acid sequence shown as SEQ ID No: 20.

[0157] The present application also provides an antibody combination comprising a first antibody S1 and an antibody (S2) according to the first aspect of the present application. In a particular embodiment, the CDR-H1 of the first antibody has the amino acid sequence as shown in SEQ ID No: 1,

[0158] the CDR-H2 has the amino acid sequence as shown in SEQ ID No: 3,

[0159] the CDR-H3 has the amino acid sequence as shown in SEQ ID No: 5,

[0160] the CDR-L1 has the amino acid sequence as shown in SEQ ID No: 7,

[0161] the CDR-L2 has the amino acid sequence as shown in SEQ ID No: 9,

[0162] the CDR-L3 has the amino acid sequence as shown in SEQ ID No: 11.

[0163] In a particular embodiment, the antibody S1 comprises an antibody heavy chain variable region HCVR and an antibody light chain variable region LCVR, wherein:

[0164] the amino acid sequence of the HCVR is as shown in SEQ ID No: 13;

[0165] the amino acid sequence of the LCVR is as shown in SEQ ID No: 15.

[0166] In a particular embodiment, the antibody S1 is anti-human interleukin 2 monoclonal antibody 1#, the heavy chain of which has the amino acid sequence as shown in SEQ ID No: 17, and the light chain of which has the amino acid sequence as shown in SEQ ID No: 19.

[0167] In the present specification, an "isolated" antibody is one which has been separated from a component of its natural environment. In certain embodiments, an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC) methods. Methods for evaluating the purity of an antibody are well known in the art, for example, see Flatman et al., J. Chromatogr. B 848: 79-87 (2007).

[0168] In the present specification, "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind the same epitope except for possible variants that can arise during production of the monoclonal antibody, such variants being typically present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present application can be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and use of transgenic animals containing all or part of the human immunoglobulin loci, as described herein and as described generally by Harlow et al., 1999, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

[0169] In the present specification, "anti-human interleukin 2 monoclonal antibody" refers to a monoclonal antibody that is capable of binding to human interleukin 2 with sufficient affinity so that the monoclonal antibody can be used as a diagnostic and / or therapeutic agent in targeting human interleukin 2.

[0170] In the present specification, "affinity" refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, "binding affinity" as used in the present specification refers to the intrinsic binding affinity reflecting the 1 : 1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can be generally represented by the equilibrium dissociation constant (K D). Affinity can be measured by common methods known in the art.

[0171] The anti-human interleukin 2 monoclonal antibodies of the present application do not bind to proteins other than human interleukin 2, which is the target. Here, "other proteins" refers to proteins other than human interleukin 2, which is the target; here, "do not bind" means that the binding ability of the anti-human interleukin 2 monoclonal antibodies of the present application to the other proteins is less than 10%, e.g., 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0, in the case where the binding ability of the anti-human interleukin 2 monoclonal antibodies of the present application to human interleukin 2, which is the target, is taken as 100%.

[0172] The anti-human interleukin 2 monoclonal antibodies of the present application do not bind to interleukin 2 of other animal species. Here, "other animal species" means other animal species than human, such as rhesus monkey, cynomolgus monkey, rat, mouse, etc.; and here, "do not bind" means that the binding ability of the anti-human interleukin 2 monoclonal antibodies of the present application to interleukin 2 of other animal species is less than 10%, such as 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0%, when the binding ability of the anti-human interleukin 2 monoclonal antibodies of the present application to human interleukin 2, which is the target of the anti-human interleukin 2 monoclonal antibodies, is taken as 100%.

[0173] The anti-human interleukin 2 monoclonal antibodies of the present application have an equilibrium dissociation constant (KD) of < 1 μM, < 100 nM, < 50 nM, or < 40 nM for human interleukin 2.

[0174] Further, the anti-human interleukin 2 monoclonal antibodies in the present application are all murine monoclonal antibodies.

[0175] CBA

[0176] Cytometric Bead Array (CBA), i.e., cytokine microsphere detection technology. It is a multiple protein quantitative detection method based on flow cytometric detection system, which can simultaneously detect multiple indicators in a single sample. BD company uses the characteristic of flow cytometer that can amplify the fluorescence signal by several orders of magnitude, and attaches the soluble factors to be detected to some microparticles with approximate cell diameter, so that various soluble factors in the sample to be detected can be detected. The basic principle of CBA is similar to the detection of ELISA, i.e., using small and dispersed particles to capture liquid to be detected, and using flow cytometer to detect the fluorescence emitted by the "sandwich" particles-to-be-detected complex, so as to determine the amount of to-be-detected substance.

[0177] Each CBA microsphere is uniform in size, has a specific fluorescence intensity, and is coated with a specific capture antibody (Capture Antibody) suitable for a specific analysis (such as other antibodies or soluble proteins), which provides a capture surface similar to an ELISA well plate. When the microspheres and the sample solution to be detected are mixed, the specific antibodies on the microspheres bind to the corresponding antigens or proteins in the sample (serum, plasma or cell culture solution), and then the detection antibody labeled with fluorescence is added, forming a "sandwich" sandwich complex. Finally, the specific target protein is detected by flow cytometer. Each microsphere of CBA carries different intensity of red fluorescence, and the difference in fluorescence intensity of the microspheres is detected in the FL3 channel of the flow cytometer to qualitatively determine the target protein; the detection antibody is labeled with PE fluorescence, which is detected in the FL2 channel of the flow cytometer, and the intensity of the PE fluorescence of the detection antibody is detected to quantitatively determine the target protein.

[0178] In the present specification, "avidin" is a glycoprotein consisting of 4 subunits per molecule, which can bind 4 biotin molecules. More often used is streptavidin extracted from Streptomyces.

[0179] In the present specification, biotin is a small molecule growth factor widely distributed in animals and plants, also known as coenzyme R or vitamin H. Avidin is a basic glycoprotein extracted from egg white, which has very high affinity for biotin. The avidin molecule combined with the enzyme reacts with the biotin molecule combined with the specific antibody, which not only plays a multi-level amplification role, but also develops color due to the catalytic effect of the enzyme when encountering the corresponding substrate, so as to determine the amount of antibody combined. Therefore, the combination of avidin and biotin with CBA can greatly improve the sensitivity of CBA. The biotin-avidin system has various forms in CBA and can be used for indirect coating and final reaction amplification. The enzyme-labeled antibody in the conventional CBA can be replaced by biotinylated antibody, and then the avidin-enzyme conjugate is connected to amplify the reaction signal.

[0180] Detection kit

[0181] The present application provides a detection kit for detecting human interleukin 2, which comprises the antibody or antibody combination of the present application.

[0182] In one embodiment, the detection kit uses a double antibody sandwich method (sandwich method) for detection. The detection method of the present application can use CBA detection.

[0183] In a specific embodiment, the anti-human interleukin monoclonal antibody in the kit of the present application comprises a first antibody and a second antibody, wherein the first antibody is a coating antibody which has been fixed to a solid phase carrier (such as a microsphere); and the second antibody is a detection antibody which is labeled with biotin. Preferably, the microspheres have a particle size of fluorescent microspheres. Further preferably, the particle size is 5 μm. In a specific embodiment, Chinese nanometer polystyrene microspheres can be used.

[0184] In a specific embodiment, the first antibody and the second antibody are the anti-human interleukin 2 monoclonal antibodies of the present application.

[0185] In a specific embodiment, the first antibody in the CBA kit is anti-human interleukin 2 monoclonal antibody 1#, which has a heavy chain with an amino acid sequence as shown in SEQ ID No: 17 and a light chain with an amino acid sequence as shown in SEQ ID No: 19; and the second antibody is anti-human interleukin 2 monoclonal antibody 2#, which has a heavy chain with an amino acid sequence as shown in SEQ ID No: 18 and a light chain with an amino acid sequence as shown in SEQ ID No: 20.

[0186] Further, the CBA kit further comprises devices or reagents necessary for detecting interleukin 2.

[0187] Specifically, the CBA kit further comprises peroxidase or fluorescent protein-labeled streptavidin, interleukin 2 standard, substrate, coating antibody diluent, washing solution, blocking solution / sample diluent and termination solution.

[0188] In a specific embodiment, the fluorescent protein is preferably phycoerythrin (PE), and the labeled streptavidin is streptavidin-phycoerythrin conjugate (SA-PE).

[0189] In a specific embodiment, the interleukin 2 standard in the kit is human IL-2 protein; the coating antibody diluent is phosphate buffer (PBS) pH 7.4; the washing solution is phosphate buffer (PBS) containing 0.05% Tween 20; and the blocking solution / sample diluent is phosphate buffer (PBS) containing 0.5% BSA, 0.05% Tween 20 and 0.05% Proclin 300.

[0190] Preferably, the CBA kit comprises the following components:

[0191] 1) Pre-coated antibody microspheres: anti-human interleukin 2 monoclonal antibody 1# (i.e. first antibody), 2.5 mL / tube, 1 tube;

[0192] 2) Detection antibody: anti-human interleukin 2 monoclonal antibody 2# (i.e. second antibody) (labeled with biotin), 2.5 mL / tube, 1 tube;

[0193] 3) Standard: lyophilized human 2 protein, 10 ng / tube, 1 tube;

[0194] 4) Streptavidin-phycoerythrin conjugate (Streptavidin-PE, abbreviated as SA-PE), 2.5 mL / tube, 1 tube;

[0195] 5) Matrix buffer: 2.5 ml / tube, 1 tube;

[0196] 6) Washing buffer: phosphate buffer (10X PBS), pH 7.4, 5 ml / bottle, 1 bottle;

[0197] Detection method

[0198] The present application also provides a method for detecting interleukin 2 using the antibody or kit of the present application. The detection method of the present application can be a double antibody sandwich method. The detection method of the present application can use CBA detection.

[0199] The present application labels the anti-human interleukin 2 monoclonal antibody with biotin, so that an antibody-biotin-avidin system (ABAS) can be formed with the phycoerythrin-labeled streptavidin, and the high-affinity firm binding can play a multi-level amplification effect, so that the kit of the present application has higher sensitivity and wider detection range.

[0200] One particular method is to bind a known amount of a first antibody to a solid support surface. The sample to be tested is then applied to the surface so that any interleukin 2 material present in the cells and / or serum therein is captured by the immobilized first antibody. Unbound material is preferably removed by one or more washing steps. A second antibody, i.e. a detection antibody, labeled with biotin is then added and allowed to bind to any interleukin 2 material captured by the first antibody, so that each unit of interleukin 2 binds two antibodies to form a "sandwich". The amount of bound second antibody is then determined in a direct or indirect detection method. Specifically, a label or enzyme can be attached to the second antibody either directly or indirectly via a linkage such as biotin-streptavidin or biotin-avidin linkage.

[0201] In one embodiment, the detection method of the present application comprises the following steps,

[0202] The anti-human interleukin 2 monoclonal antibody 1# (i.e. the first antibody) is used to coat the fluorescent microspheres;

[0203] The anti-human interleukin 2 monoclonal antibody 2# (i.e. the second antibody) is labeled with biotin;

[0204] The microspheres coated with the first antibody, the sample to be tested, and the biotin-labeled second antibody are sequentially added to the flow tube and incubated;

[0205] The phycoerythrin-labeled streptavidin is added to the flow tube and incubated;

[0206] The MFI value of the interleukin 2 standard is used to fit a standard curve, and the MFI value of the sample to be tested is substituted into the equation to calculate the content of interleukin 2 in the sample to be tested.

[0207] In one particular embodiment, the method for quantitatively detecting the content of human interleukin 2 in a sample using the CBA kit of the present application comprises the following steps:

[0208] (1) Coating: The coating antibody anti-human interleukin 2 monoclonal antibody 1# (i.e. the first antibody) is prepared into a coating antibody working solution with a concentration of 1 g / L using a coating antibody diluent (phosphate buffer (PBS), pH 7.4), then mixed in clean EP at a ratio of 10 μg per 1E7 microspheres, and incubated at room temperature for 2 hours;

[0209] (2) Blocking: centrifugal discard coated antibody working solution in EP tube, wash twice with washing buffer (phosphate buffer (PBS) containing 0.05% Tween20), then add blocking solution (phosphate buffer (PBS) containing 0.5% BSA, 0.05% Tween20 and 0.05% Proclin300) at a dosage of 100 μL / well, and place on a shaker (120 rpm) at room temperature for 2 hours of blocking;

[0210] (3) Protein standard preparation: take 7 EP tubes and number them in turn, add 150 μL sample diluent to each of the first 6 tubes and place them on the EP tube rack; prepare the standard protein (human IL-2 protein) into a liquid with a concentration of 10 μg / L with sample diluent, take 50 μL and add to the first EP tube, then take 50 μL from the first EP tube and add to the second EP tube for four-fold dilution, and so on to the sixth EP tube, with concentrations of 10000 pg / mL, 2500 pg / mL, 625 pg / mL, 156.3 pg / mL, 39.6 pg / mL, 9.7 pg / mL, 2.4 pg / mL in turn;

[0211] (4) Sample addition: add the coated first antibody microspheres, protein standard solution, and biotin-labeled second antibody in turn at a dosage of 25 μL / tube, and incubate at room temperature for 2 hours on a shaker (500 rpm);

[0212] (5) SA-PE addition: add to the EP at a dosage of 25 μL / tube, and incubate the reaction for 0.5 hours on a shaker (500 rpm);

[0213] (6) Washing: centrifugal discard the reaction solution in the EP tube, wash twice with washing buffer, and resuspend the microspheres with sample diluent;

[0214] (7) Detection: use a flow cytometer to measure the MFI value of each EP tube at a wavelength of 585 nm, and fit a standard curve according to the MFI value of the standard.

[0215] The antibody, detection kit, and detection method of the present application can be applied to the detection of interleukin 2 in cell culture medium or human serum.

[0216] Pharmaceutical composition

[0217] The present application also provides a composition. Preferably, the composition is a pharmaceutical composition comprising the above-mentioned antibody or active fragment thereof or fusion protein thereof, 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 the pH is about 6-8, although the pH value can vary depending on the nature of the formulated substance and the condition to be treated. The prepared pharmaceutical composition can be administered by conventional routes, including but not limited to intratumoral, intraperitoneal, intravenous, or local administration.

[0218] The pharmaceutical composition of the present application can contain any anti-tumor drug (such as an anti-tumor antibody) linked to the anti-human interleukin 2 antibody of the present application, and thus can be used for treating tumors. In addition, other therapeutic agents can also be used simultaneously.

[0219] The pharmaceutical composition of the present application contains a safe and effective amount (such as 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-mentioned nanobody (or conjugate thereof) of the present application 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 be matched with the administration method. The pharmaceutical composition of the present application can be prepared in the form of a needle, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition such as the needle, solution should be manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example, about 10 μg / kg body weight to about 50 mg / kg body weight per day. In addition, the polypeptide of the present application can also be used with other therapeutic agents.

[0220] When using the pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to a mammal, wherein the safe and effective amount is generally at least about 10 μg / kg body weight, and in most cases does not exceed about 50 mg / kg body weight, preferably the dose is about 10 μg / kg body weight to about 10 mg / kg body weight. Of course, the specific dose should also take into account the administration route, the patient's health status, etc., which are within the skill of a skilled physician.

[0221] The main advantages of the present application include:

[0222] 1) The present application provides antibodies and antibody pairs that specifically target human interleukin 2, which can greatly expand the detection range and improve the detection sensitivity of the CBA kit for detecting human interleukin 2. Multiple repeated detection shows that it has high accuracy.

[0223] 2) The antibodies and combinations of the present application do not cross-react with human interleukin 4, human interleukin 7, human interleukin 9, human interleukin 15 and human interleukin 21, avoiding the interference of similar cytokines and improving the detection specificity.

[0224] The present application is further described in conjunction with the following specific examples. It should be understood that these examples are used solely to illustrate the present application and should not be construed as limiting the scope of the present application. Unless otherwise indicated, the experimental methods in the following examples were carried out according to conventional conditions, for example, the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are percentages by weight and parts by weight.

[0225] Example 1 Screening and preparation of anti-human interleukin 2 monoclonal antibodies

[0226] Human interleukin 2 was prepared and used to immunize Balb / c mice. After the immunization titer reached the standard, the B cell suspension was collected for sorting. Figure 1 The results of the human interleukin 2 immunized mice are shown in the figure, with the horizontal axis representing the dilution fold of the serum collected from the tail vein of different mice, and the vertical axis representing the OD450nm value. The results show that the immunization titer has reached the expected value, and one of the mice was selected for single B cell sorting. First, specific memory B cells were screened using cell surface markers and antigens. Then, specific plasma cells were obtained by using the DeepLight On-chip Cell Screening platform for sorting. Single B cells that bind to IL-2 were selected, and after short-term culture, positive / negative screening detection was performed by Binding ELISA. Monoclonal antibodies that bind to IL-2 and have no binding activity to IL-4, IL-7, IL-9, IL-15 and IL-21 were selected, thereby obtaining the first antibody and the second antibody. The obtained first antibody and the second antibody are murine monoclonal antibodies, and human-murine antibody chimerization was performed. The above immunization and screening process was entrusted to DeTai Bio to complete.

[0227] The heavy and light chain variable region sequences of the first antibody and the second antibody were obtained by sequencing, and the CDR sequences were defined according to the Kabat rule. The antibody sequences are shown in Table 1.

[0228] Table 1 Antibody sequences

[0229]

[0230]

[0231]

[0232] Example 2 Identification of specificity of anti-human interleukin 2 monoclonal antibodies

[0233] 1. Antibody recognition of antigen specificity experiment:

[0234] Dilute human IL-4, IL-7, IL-9, IL-15 and IL-21 to 10 μg / mL with coating solution, coat the enzyme-labeled plate at 2-8°C overnight, and block at room temperature for 2 hours the next day. After washing the plate, add 100 μL of diluted cell culture supernatant, and incubate at room temperature for 1 hour. After washing the plate, add diluted enzyme-labeled secondary antibody, and incubate at room temperature for 1 hour. Finally, after washing the plate, develop the color, and read the absorbance at OD450 nm. As shown in Table 2, antibodies 1# and 2# do not bind to human IL-4, IL-7, IL-9, IL-15 and IL-21 antigens, indicating that antibodies 1# and 2# have good specificity for IL-2 protein. Antibodies 1# and 2# can be used for pairing experiments. Figure 2

[0235] Table 2 Antibody specificity detection

[0236] OD450 IL-2 IL-4 IL-7 IL-9 IL-15 IL-21 Antibody 1# 3.0121 0.0687 0.059 0.0637 0.0549 0.0869 Antibody 2# 3.1142 0.0853 0.0744 0.0667 0.0634 0.0613 Positive control 3.2253 3.1352 2.9034 2.9113 3.0124 3.0311 Negative control 0.0782 0.0852 0.0577 0.056 0.0568 0.061

[0237] 2. Antibody pairing experiment for anti-human IL-2 antibodies

[0238] Use the Sulfo-NHS-LC-Biotin kit to biotinylate the two antibodies, and strictly follow the kit instructions for the specific steps. After labeling, use a dialysis bag to dialyze to remove free biotin, and the resulting solution is the biotinylated antibody.

[0239] Dilute the two antibodies to 10 μg / mL with coating solution, respectively, and coat the fluorescent microspheres at 2-8°C, and incubate at room temperature for 2 hours. After centrifugation, add blocking solution and incubate at room temperature for 2 hours. After washing, add the preservation solution and count for use.

[0240] Add the matrix buffer, human IL-2 protein, coated antibody microspheres, and biotinylated antibody in sequence, and incubate at room temperature for 2 hours. Then add diluted phycoerythrin-labeled streptavidin, and incubate at room temperature for 0.5 hours. Finally, after washing, use a flow cytometer to read the fluorescence value. The results are shown in Table 3 below, and antibodies 1# and 2# can be paired to assemble a human IL-2 CBA quantitative detection kit.

[0241] Table 3 Antibody pairing detection results

[0242]

[0243] Example 3 Composition of human IL-2 CBA quantitative detection kit

[0244] ​The antibody 1# coated fluorescent microspheres are used as the capture antibody, and the antibody 2# is biotin-labeled as the detection antibody to prepare the CBA kit. The reagent composition, specification, source and storage condition of the kit are shown in Table 4.

[0245] 1) Microspheres coated with pre-coated antibodies: anti-human interleukin 2 monoclonal antibody 1# (i.e. first antibody), 2.5 mL / tube, 1 tube;

[0246] 2) Detection antibody: anti-human interleukin 2 monoclonal antibody 2# (i.e. second antibody) (labeled with biotin), 2.5 mL / tube, 1 tube;

[0247] 3) Standard: lyophilized human IL-2 protein, 10 ng / tube, 1 tube;

[0248] 4) Streptavidin-phycoerythrin conjugate (Streptavidin-PE, abbreviated as SA-PE), 2.5 mL / tube, 1 tube;

[0249] 5) Matrix buffer: 2.5 mL / tube, 1 tube;

[0250] 6) Wash and replace liquid: phosphate buffer (10X PBS), pH 7.4, 5 mL / bottle, 1 bottle;

[0251] The components are shown in Table 4:

[0252] Table 4 CBA kit component composition

[0253] Component Main component Content Storage Capture antibody Antibody 1# coated fluorescent microspheres 2.5 Ml, 1 bottle 2-8°C 1 year Detection antibody Biotin labeled antibody 2# 2.5 Ml, 1 bottle 2-8°C 1 year Standard Human IL-2 protein 10 ng, 1 tube -20°C 1 year SA-PE Streptavidin-phycoerythrin conjugate 2.5 Ml, 1 bottle 2-8°C 1 year Matrix buffer BSA, protein protectant 5 Ml, 1 bottle -20°C 1 year Washing buffer 10X PBST 5 Ml, 1 bottle Room temperature 1 year

[0254] Example 4 Performance index evaluation of human IL-2 CBA kit

[0255] The human IL-2 in the quality control sample was quantitatively detected using the human IL-2 CBA kit in Example 3.

[0256] 1. Linear range:

[0257] (1) The human IL-2 protein was diluted with the sample diluent to prepare the protein standard, and the human IL-2 protein was diluted with the cell culture medium to prepare the quality control sample as the sample to be detected. The standard curve was fitted according to the MFI value of the protein standard. The MFI value of the sample to be detected was substituted into the standard curve to obtain the theoretical concentration value of human IL-2 in the sample to be detected, and then the recovery rate of the sample to be detected was calculated (recovery rate = theoretical concentration value / actual concentration value*100%), so as to analyze the quantitative detection effect of the CBA kit in Example 3 for human IL-2 in the cell culture medium.

[0258] (2) Quantitative detection operation steps:

[0259] 1) Protein standard preparation: Take 8 EP tubes and number them in order. Add 300 μL of sample diluent to each tube from the 2nd tube and place them on the EP tube rack. Prepare the standard protein with a concentration of 10 ng / mL in the 1st tube, take 100 μL and add to the 2nd EP tube, dilute by 4 times, and so on to the 7th EP tube. The concentrations are 10000 pg / mL, 2500 pg / mL, 625 pg / mL, 156.3 pg / mL, 39.6 pg / mL, 9.7 pg / mL, and 2.4 pg / mL, respectively.

[0260] 2) Quality control sample preparation: Take 6 EP tubes, and prepare quality control samples with concentrations of 6250 pg / mL, 1250 pg / mL, 250 pg / mL, 50 pg / mL, 10 pg / mL, and 2 pg / mL, respectively, from 10000 pg / mL.

[0261] 3) Sample addition: Add the matrix buffer, protein standard, coated microspheres with the first antibody, and biotin-labeled second antibody to the tubes in an amount of 25 μL per tube, and incubate at room temperature for 2 hours on a shaker (500 rpm).

[0262] 4) SA-PE addition: Add SA-PE to the EP tubes in an amount of 25 μL per tube, and incubate the reaction for 0.5 hours on a shaker (500 rpm).

[0263] 5) Washing: Centrifuge to discard the reaction solution in the EP tubes, wash twice with washing solution, and resuspend the microspheres with sample diluent.

[0264] 6) Detection: Measure the MFI values of the EP tubes at a wavelength of 585 nm using a flow cytometer. According to the MFI values of the standard, a standard curve is fitted, and the MFI values of the quality control samples are substituted into the equation to calculate the concentrations of the quality control samples.

[0265] (3) Results and discussion

[0266] 1) The linear range detection data of the human IL-2 standard are shown in Table 5:

[0267] Table 5 Detection data of human IL-2 standard

[0268]

[0269] The detection curve of human IL-2 can recover in the concentration range of 2.4-10000 pg / mL.

[0270] 2) The curve equation is shown in Table 6: Figure 3 y = 0.0799x + 0.3739x - 1.698 (y: Log of Con; x: Log of MFI), R 2 2 ​= 0.9995.

[0271] 2. Specificity

[0272] (1) Different cytokines were diluted to a concentration of 10 ng / mL, and their concentrations were detected according to the kit operation. The specificity of the kit was expressed by the cross-reactivity (%). The calculation method was: cross-reactivity (%) = detected concentration / nominal concentration x 100%.

[0273] (2) The specificity detection data of the human IL-2 kit are shown in Table 6.

[0274] Table 6

[0275]

[0276] (3) Results and discussion:

[0277] The results of Table 6 show that the cross-reactivity of the related cytokines is ≤0.005%. The above results show that when the human IL-2 in the sample is quantitatively detected by using the human IL-2 CBA kit provided in the present application, it is not interfered by the related cytokines in the sample, indicating that the detection method provided in the present application has good specificity.

[0278] 3. Accuracy:

[0279] (1) In 3 different experiments, each analysis batch contained 3 sets of samples to be tested, and each set of samples to be tested contained 6 concentrations (2, 10, 50, 250, 1250, 6250 pg / mL). The precision of the method was expressed by the coefficient of variation CV%, CV% = SD / average value x 100. The accuracy was expressed by the relative error RE%, RE% = (average detected concentration-theoretical concentration) / theoretical concentration x 100. The accuracy of the kit detection was analyzed by the above parameters. The analysis results are shown in Table 7.

[0280] Table 7 Detection data of samples to be tested

[0281]

[0282] (2) Results and discussion:

[0283] The results of Table 7 show that the within-batch variation coefficient CV of the 6 concentration levels is ≤5%. The accuracy (RE) is ≤5%. The above results show that when the human IL-2 in the sample is quantitatively detected by using the human IL-2 CBA kit provided in the present application, it can meet the within-batch variation coefficient CV ≤5% and the accuracy (RE) ≤5%, indicating that the detection method provided in the present application has good accuracy.

[0284] 4. Precision

[0285] (1) In 3 different experiments, each analysis batch contained 3 sets of samples to be tested, and each set contained 6 concentrations (2, 10, 50, 250, 1250, 6250 pg / ml). The precision of the method was expressed by the coefficient of variation CV% between batches, CV% = SD / average value x 100%. The reproducibility of the kit detection was analyzed by the above parameters. The analysis results are shown in Table 8.

[0286] Table 8 quantitative detection precision experiment data of human IL-2

[0287]

[0288] (2) Results and discussion

[0289] The results of Table 8 show that the coefficient of variation CV% between batches of 6 concentration levels (including 2 concentrations of low, medium and high) is ≤5%. The above results show that the quantitative detection of human IL-2 in samples using the human IL-2 CBA kit provided in the present application can meet the coefficient of variation between batches ≤5%, indicating that the detection method provided in the present application has good reproducibility.

[0290] 5. Sensitivity

[0291] (1) In 3 different experiments of different kits, each analysis batch detected 20 sample dilutions. The sensitivity calculation method was: the average value M of the determination results was calculated, the average value M was added to the value of 2 times the standard deviation SD, and the result calculated by the standard curve in Example 4 was the sensitivity. The analysis results are shown in Table 9.

[0292] Table 9 sensitivity experiment data of human IL-2 CBA detection kit

[0293] Detection 1 Detection 2 Detection 3 MFI mean (M) 226 240 235 Standard deviation SD 4.5 3.2 3.8 M+2SD 235 246.4 242.6 Sensitivity (pg / mL) 0.43 0.45 0.44

[0294] (2) Results and discussion

[0295] The results of Table 9 show that the sensitivity of the 3 batches of kits is ≤0.5 pg / mL. The above results show that the kit provided in the present application has good sensitivity.

[0296] All the documents mentioned in the present application are cited as references in the present application, just as each document is cited as a reference. In addition, it should be understood that those skilled in the art can make various modifications or modifications to the present application after reading the above teachings of the present application, and these equivalent forms also fall within the scope defined by the claims attached to the present application.

Claims

1. An antibody against human interleukin 2 protein, characterized in that, The antibody comprises a heavy chain and a light chain, the heavy chain comprises a heavy chain variable region HCVR, and the light chain comprises a light chain variable region LCVR, wherein the heavy chain variable region comprises the following complementarity determining regions CDRs: a CDR-H1 with an amino acid sequence as set forth in SEQ ID No: 2; a CDR-H2 with an amino acid sequence as set forth in SEQ ID No: 4; a CDR-H3 with an amino acid sequence as set forth in SEQ ID No: 6; the light chain variable region comprises the following complementarity determining regions CDRs a CDR-L1 with an amino acid sequence as set forth in SEQ ID No: 8; a CDR-L2 with an amino acid sequence as set forth in SEQ ID No: 10; a CDR-L3 with an amino acid sequence as set forth in SEQ ID No:

12.

2. The antibody of claim 1, wherein The HCVR of the antibody has an amino acid sequence as set forth in SEQ ID No: 14, and / or the LCVR has an amino acid sequence as set forth in SEQ ID No:

16.

3. The antibody of claim 1, wherein The heavy chain of the antibody has an amino acid sequence as set forth in SEQ ID No: 18, and / or the light chain has an amino acid sequence as set forth in SEQ ID No:

20.

4. A fusion protein, characterized in that, The fusion protein comprises: (1) the antibody of claim 1; and (2) a tag sequence for facilitating expression and / or purification.

5. A polynucleotide comprising a nucleic acid sequence encoding a polypeptide of any one of claims 1-4. The polynucleotide encodes the antibody of claim 1 or the fusion protein of claim 4.

6. A vector, characterized in that, The vector contains the polynucleotide of claim 5.

7. A genetically engineered host cell, characterized in that, The host cell contains the vector of claim 6, or the polynucleotide of claim 5 is integrated into the genome of the host cell.

8. An antibody conjugate, characterized in that, The antibody conjugate comprises: (a) an antibody moiety, the antibody moiety being the antibody of claim 1; and (b) a conjugating moiety conjugated to the antibody moiety, the conjugating moiety being a detectable label.

9. An antibody combination against human interleukin 2 protein, characterized in that, The antibody combination comprises: an antibody S1, the heavy chain variable region of the antibody S1 comprising the following complementarity determining regions CDRs: a CDR-H1 with an amino acid sequence as set forth in SEQ ID No: 1, a CDR-H2 with an amino acid sequence as set forth in SEQ ID No: 3, a CDR-H3 with an amino acid sequence as set forth in SEQ ID No: 5, a light chain variable region of the antibody S1 comprising the following complementarity determining regions CDRs: a CDR-L1 with an amino acid sequence as set forth in SEQ ID No: 7, a CDR-L2 with an amino acid sequence as set forth in SEQ ID No: 9, a CDR-L3 with an amino acid sequence as set forth in SEQ ID No: 11; and an antibody S2, the antibody S2 being the antibody of claim 1.

10. Use of the antibody of claim 1, the polynucleotide of claim 5, the vector of claim 6, the host cell of claim 7, the antibody conjugate of claim 8, or the antibody combination of claim 9, wherein, A reagent or a kit for detecting human interleukin 2 protein.

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