Anti-human interleukin 2 antibody and application thereof

Abstract

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

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and in particular to an anti-human interleukin 2 antibody and its application. Background Art

[0002] Interleukin-2, also known as interleukin-2 (IL-2), is a cytokine that plays an important role in the chemokine family. IL-2 consists of a bundled structure consisting of four α-helical cores and β-fragments, with a molecular weight of 15.5kD. 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 a small amount of IL-2. IL-2 is a cytokine with a wide range of biological activities. IL-2 belongs to a type of lymphocyte growth factor, which has the function of promoting the proliferation and cell transformation and differentiation of T cells after antigen stimulation. Its main mechanism of action is to enhance the secretion of cytokines to natural killer (NK) cells. NK cells are enhanced by proliferation stimulation signals, and at the same time, because they are not blocked by MHC, the killing power of NK cells is effectively improved. Lymphokine-activated LAK cells receive signal induction, and the proliferation rate of B lymphocytes is accelerated, thereby promoting antibody secretion and increasing the activity of autophagic cells. It has pharmacological effects such as antiviral, immunity enhancement, and anti-tumor in clinical practice.

[0003] Interleukin 2 (IL-2) plays an important role in the regulation of the immune system. Under normal circumstances, IL-2 levels vary with the body's immune status. The normal range of IL-2 is 5-50pg / ml, and IL-2 levels may be affected by many factors, including age, gender, weight, disease status, etc. In some diseases, IL-2 levels may change abnormally. For example, in certain cancers, autoimmune diseases, or infectious diseases, IL-2 levels may increase or decrease. Therefore, for the diagnosis and treatment of specific diseases, doctors need to understand the patient's IL-2 levels and changes. Therefore, the development of a highly sensitive interleukin 2 protein detection methodology is of great clinical significance.

[0004] Most of the detection methods on the market are ELISA, i.e., enzyme-linked immunosorbent assay. Human interleukin-2 (IL-2) ELISA KIT is based on the classic double antibody sandwich ELISA method, which uses HRP-coupled antibodies to catalyze TMB to produce color substances. Based on the standard curve, the content of human interleukin-2 in the sample is calculated. The detection principle of this method is: the double antibody sandwich method is used to determine the level of human interleukin-2 (IL-2) in the specimen. The microplate is coated with purified human interleukin-2 (IL-2) antibodies to make solid-phase antibodies. Human interleukin-2 (IL-2) is added to the microwells coated with monoclonal antibodies in turn, and then combined with HRP-labeled human interleukin-2 (IL-2) antibodies to form an antibody-antigen-enzyme-labeled antibody complex. After thorough washing, the substrate TMB is added for color development. TMB is converted into blue under the catalysis of HRP enzyme, and converted into the final yellow under the action of acid. The depth of color is positively correlated with human interleukin-2 (IL-2) in the sample. The absorbance (OD value) was measured at a wavelength of 450nm using an ELISA instrument, and the concentration of human interleukin 2 (IL-2) in the sample was calculated using a standard curve. The 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 ELISA plate, a large amount of coated antibodies or antigens is required, which conflicts with the pain point that clinical samples are difficult to obtain. In addition, the sensitivity and linearity still need to be improved. For example, SinoBiological's Human IL-2 ELISA kit (Cat: KIT11848) has a sensitivity of 4.47pg / ml and a linear range of 18.75-1200pg / mL. The linearity and sensitivity of the kit still have a lot of room for improvement.

[0005] Currently, some of the detection methods on the market are AlphaLISA methods. AlphaLISA technology mainly relies on the interaction between Alpha donor microbeads and acceptor microbeads. When the biological reaction brings the donor microbeads and acceptor microbeads close to each other, the laser excites the cascade reaction, thereby generating a greatly amplified signal. Specifically, under the irradiation of a 680nm laser, the photosensitizer on the donor microbead converts oxygen in the surrounding environment into more active monomeric oxygen. Monomeric oxygen diffuses to the acceptor microbead, producing a series of chemiluminescent reactions, and finally transfers energy to europium with an emission wavelength of 615nm. In the absence of specific interactions between biomolecules, monomeric oxygen cannot diffuse to the acceptor microbead, and no signal will be generated. One antibody is biotinylated and binds to the streptavidin-coated donor microbead, and the acceptor microbead directly binds to another antibody. In the presence of cytokines, the antibody binds to the cytokine to bring the donor and acceptor microbeads close to each other. The excitation of the donor microbead causes the singlet oxygen to diffuse to the acceptor microbead, thereby emitting a light signal with a wavelength of 615nm. The detection signal of AlphaLISA is proportional to the number of cytokines present in the sample. AlphaLISA technology also uses the sandwich method to detect cytokines, which has high sensitivity and a large detection range, such as Revvity Human Interleukin-2 (IL2) Detection Kit (Cat. No. AL221C / F) has a sensitivity of 1.8 pg / ml and a linear range of 1.8-100 000 pg / mL. However, the application of this technology requires a multifunctional microplate reader capable of AlphaLISA detection. The instrument is expensive, and the AlphaLISA detection kit is much more expensive than other cytokine detection kits on the market. The detection cost is high and it is not suitable for high-throughput sample detection.

[0006] At present, some of the detection methods on the market are CBA methods. CBA (Cytometric Bead Array), that is, micro-sample multi-index flow protein quantification technology, is a multiple protein quantification detection method based on a flow cytometric detection system, which can simultaneously detect multiple indicators in a single sample. Based on the CBA principle, the corresponding cytokine antibodies are connected to the specific fluorescent-coded microspheres. When there are specific antigens in the test sample, these capture antibodies connected to the fluorescent-coded microspheres will bind to them, and finally, by adding the specific detection antibody labeled with phycoerythrin (PE), a capture antibody-antigen-PE-labeled detection antibody complex connected to the microspheres 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 detected object in the sample, and the fluorescence signal of each calibrator and its corresponding fluorescence signal can be fitted into a concentration-fluorescence intensity standard curve, and the concentration of each antigen can be calculated by the curve equation. For example, HumanIL-2BD of BD company TM The detection limit of the Cytometric Bead Array (CBA) kit is 11.2 pg / ml and the linear range is 10-2,500 pg / mL. There is still room for improvement in sensitivity and linear range.

[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 false positives are prone to occur when detecting human IL-2 in samples. Therefore, a method for highly specific detection of human IL-2 is needed in the art.

[0008] In order to improve the specificity and sensitivity of the CBA detection method, the current consensus in the industry is to use anti-interleukin 2 monoclonal antibody pairs to develop corresponding CBA detection kits. However, the sensitivity and specificity of CBA detection kits for interleukin 2 currently on the market are generally low. In addition, the mouse monoclonal antibodies used rely on the traditional hybridoma method for development and production. The preparation process is more complicated than that of recombinant monoclonal antibodies, and there are also large batch differences. Therefore, the detection kits developed using mouse monoclonal antibodies have challenges such as low sensitivity, narrow linear range, and difficult to control batch differences.

[0009] Therefore, there is a need in the art to establish a highly sensitive, wide detection range, and highly specific IL-2 detection method. Summary of the invention

[0010] The purpose of the present invention is to provide a method for detecting IL-2 with high sensitivity, wide detection range and high specificity.

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

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

[0013] In the first aspect of the present invention, an antibody against human interleukin-2 protein is provided, wherein the antibody comprises a heavy chain and a light chain, wherein 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 complementary determining region CDR:

[0014] CDR-H1 having an amino acid sequence as shown in SEQ ID No: 2;

[0015] CDR-H2 having an amino acid sequence as shown in SEQ ID No: 4;

[0016] CDR-H3 having an amino acid sequence as shown in SEQ ID No: 6;

[0017] The light chain variable region includes the following complementarity determining regions CDR

[0018] CDR-L1 having an amino acid sequence as shown in SEQ ID No: 8;

[0019] CDR-L2 having an amino acid sequence as shown in SEQ ID No: 10;

[0020] The amino acid sequence of CDR-L3 is shown in SEQ ID No:12.

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

[0022] In another preferred embodiment, the CDR1, CDR2 and CDR3 of the light and heavy chain variable regions 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 shown in SEQ ID No: 14, or has a sequence identity of ≥85%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98% or ≥99% thereto.

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

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

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

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

[0028] In another preferred embodiment, the heavy chain amino acid sequence of the antibody is as shown 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 amino acid sequence of the antibody is such as SEQ ID No: 20 or has a sequence identity of ≥85%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98% or ≥99% thereto.

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

[0031] In another preferred embodiment, any one of the above amino acid sequences also includes a derivative sequence that is optionally subjected to addition, deletion, modification and / or substitution of at least one amino acid and can retain the binding affinity for human interleukin-2 protein.

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

[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 invention, a fusion protein is provided, the fusion protein comprising:

[0036] (1) the antibody according to the first aspect of the present invention; and

[0037] (2) Optional tag sequence to facilitate expression and / or purification.

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

[0039] In the third aspect of the present invention, a polynucleotide is provided, wherein the polynucleotide encodes the antibody as described in the first aspect of the present invention or the fusion protein as described in the second aspect of the present invention.

[0040] In the fourth aspect of the present invention, a vector is provided, wherein the vector contains the polynucleotide as described in the third aspect of the present invention.

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

[0042] In the fifth aspect of the present invention, a genetically engineered host cell is provided, wherein the host cell contains the vector as described in the fourth aspect of the present invention, or an exogenous polynucleotide as described in the third aspect of the present invention is integrated into the genome.

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

[0044] In another preferred embodiment, the host cell is selected from the following group: Escherichia coli, yeast cells, and mammalian cells.

[0045] In the sixth aspect of the present invention, an antibody conjugate is provided, the antibody conjugate comprising:

[0046] (a) an antibody portion, wherein the antibody portion is the antibody according to the first aspect of the present invention; and

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

[0048] In another preferred embodiment, the antibody portion is coupled to the detectable marker via a chemical bond or a linker.

[0049] In another preferred embodiment, the detectable marker is selected from the following group: fluorescent or luminescent markers, biotin, radioactive markers, MRI (magnetic resonance imaging) or CT (computer tomography) contrast agents, or enzymes capable of producing detectable products, gold nanoparticles / nanorods, nanomagnetic particles, or any form of nanoparticles.

[0050] In another preferred embodiment, the detectable marker 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 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.

[0054] In the seventh aspect of the present invention, an anti-human interleukin-2 protein antibody combination is provided, the antibody combination comprising:

[0055] Antibody S1, wherein the heavy chain variable region of antibody S1 includes the following complementarity determining regions CDR:

[0056] The amino acid sequence of CDR-H1 is shown in SEQ ID No: 1,

[0057] The amino acid sequence of CDR-H2 is shown in SEQ ID No: 3,

[0058] The amino acid sequence of CDR-H3 is shown in SEQ ID No: 5,

[0059] The light chain variable region of antibody S1 includes the following complementarity determining regions CDR:

[0060] The amino acid sequence of CDR-L1 is shown in SEQ ID No: 7,

[0061] The amino acid sequence of CDR-L2 is shown in SEQ ID No:9,

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

[0063] Antibody S2, wherein the antibody S2 is the antibody as described in the first aspect of the present invention.

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

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

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

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

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

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

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

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

[0072] In another preferred embodiment, any one of the above amino acid sequences also includes a derivative sequence that is optionally subjected to addition, deletion, modification and / or substitution of at least one amino acid and can retain the binding affinity for human interleukin-2 protein.

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

[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 the eighth aspect of the present invention, there is provided a use of the antibody according to the first aspect of the present invention, the polynucleotide according to the third aspect of the present invention, the vector according to the fourth aspect of the present invention, the host cell according to the fifth aspect of the present invention, the antibody conjugate according to the sixth aspect of the present invention, or the antibody combination according to the seventh aspect of the present invention, for preparing:

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

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

[0079] In another preferred embodiment, the human interleukin-2-related disease is selected from the following group: inflammation-related diseases, tumors, autoimmune diseases, or a combination thereof.

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

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

[0082] In the ninth aspect of the present invention, a human interleukin-2 protein detection kit is provided, the kit comprising the antibody as described in the first aspect of the present invention, the antibody conjugate as described in the sixth aspect of the present invention, or the antibody combination as described in the seventh aspect of the present invention.

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

[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 as described in the sixth aspect of the present invention, wherein antibody S1 is fixed to a carrier and used to capture human interleukin-2 protein, and antibody S2 is coupled to a detection marker.

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

[0088] In another preferred embodiment, the detection kit includes antibody S2 containing 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 following group: fluorescent protein-labeled streptavidin, freeze-dried interleukin-2 standard, washing solution, sample diluent, matrix buffer, or a combination thereof.

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

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

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

[0094] 1) Immobilizing antibody S1 on a carrier;

[0095] 2) labeling antibody S2 with a detectable marker;

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

[0097] 4) washing away the reagent not bound to the carrier, detecting the detectable marker, and thus 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 marker is biotin.

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

[0102] Avidin labeled with fluorescent protein is added and incubated to bind the biotin on the antibody S2, and the fluorescent protein is detected.

[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 steps of: detecting an interleukin-2 standard substance 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 following group: cell culture fluid, whole blood, serum, plasma, body fluid, or a combination thereof.

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

[0109] (i) the antibody according to the first aspect of the present invention, the polynucleotide according to the third aspect of the present invention, the vector according to the fourth aspect of the present invention, the host cell according to the fifth aspect of the present invention, the antibody conjugate according to the sixth aspect of the present invention, or the antibody combination according to the seventh aspect of the present invention, 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 also contains other drugs for treating inflammation-related diseases, tumors, or autoimmune diseases.

[0113] 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

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

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

[0116] Figure 2 Shows the binding results of anti-human interleukin monoclonal antibodies 1# and 2# with antigens human IL-4, IL-7, IL-9, IL-15, and IL-21.

[0117] Figure 3 Shows the standard curve of human IL-2 concentration detection. DETAILED DESCRIPTION

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

[0119] the term

[0120] In order to more easily understand the present invention, some technical and scientific terms are specifically defined below. Unless otherwise clearly defined in this article, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, because such methods and conditions can be changed. It should also be understood that the terms used herein are intended only to describe specific embodiments, and are not intended to be restrictive, and the scope of the present invention will be limited only by the appended claims.

[0121] As used herein, when used in reference to a specific recited numerical value, the term "about" means that the value may vary by no more than 1% from the recited value. 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 "comprise", "include", and "contain" are used interchangeably and include not only closed definitions, but also semi-closed and open definitions. In other words, the terms include "consisting of", "consisting essentially of".

[0123] As used herein, the term "pharmaceutically acceptable carrier" refers to a substance that is suitable for use in humans and / or animals without excessive adverse side effects (such as toxicity, irritation and allergic response), ie, has a reasonable benefit / risk ratio.

[0124] As used herein, the term "therapeutically effective amount" refers to an amount that produces a function or activity in humans and / or animals and is acceptable to humans and / or animals. It should be understood by those skilled in the art that the "therapeutically effective amount" may vary depending on the form of the pharmaceutical composition, the route of administration, the adjuvants of the drug used, the severity of the disease, and the combination with other drugs.

[0125] IL-2

[0126] In this specification, the term "interleukin 2 (IL-2)" is a cytokine that plays an important role in the chemokine family. IL-2 is a bundled structure consisting of four α-helical cores and β-fragments, with 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 is a type of lymphocyte growth factor that promotes the proliferation and cell transformation and differentiation of T cells after antigen stimulation. Its main mechanism of action is to enhance the secretion of cytokines to natural killer (NK) cells. NK cells are enhanced by proliferation stimulation signals, and because they are not blocked by MHC, the killing power of NK cells is effectively improved. Lymphokine-activated LAK cells receive signal induction, and the proliferation rate of B lymphocytes is accelerated, thereby promoting antibody secretion and increasing the activity of autophagic cells. It has clinical pharmacological effects such as antiviral, immunity enhancement, and anti-tumor.

[0129] The present invention provides an antibody, a detection kit and a detection method for detecting interleukin 2 (especially human interleukin 2).

[0130] Antibody

[0131] In this application, the term "antibody" should be interpreted in the broadest sense, including various antibody structures, including but not limited to Y-shaped antibodies, so-called full-length antibodies, antigen-binding portions of Y-shaped antibodies, and genetic or chemical modifications thereof. Among them, "antigen-binding portion" refers to one or more parts or fragments of a Y-shaped antibody that 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 identical antigenic determinants. That is, in the antibody population, the individual antibodies are substantially identical, except for a small amount of mutations that may occur naturally. A monoclonal antibody can show a single binding specificity and affinity for a specific epitope on an antigen. Compared to polyclonal antibodies that typically contain antibodies directed against different epitopes, each monoclonal antibody can be directed against the same or substantially identical epitope on the antigen. The modifier "monoclonal" indicates that the characteristics of the antibody are obtained from a substantially homogeneous antibody population and cannot be interpreted as an antibody that needs to be made by any specific method. The antibody can be prepared by a variety of methods, including but not limited to hybridoma method, recombinant DNA method, phage antibody library and similar methods.

[0133] In the present application, the modifier "mouse" in the term "mouse antibody" or "anti-human interleukin 2 monoclonal antibody" or similar terms indicates that the complementary determining region (CDR) of the antibody is derived from a mouse immunoglobulin sequence. In one embodiment, the anti-human interleukin 2 mouse monoclonal antibody may comprise the CDR and framework region (FR) of an antibody from a rabbit immunoglobulin sequence. In one embodiment, the anti-human interleukin 2 mouse antibody or mouse monoclonal antibody may comprise the CDR of an antibody from a mouse immunoglobulin sequence. In one embodiment, the anti-human interleukin 2 mouse monoclonal antibody may be an antibody whose CDR region is derived from a mouse immunoglobulin sequence and whose FR is derived from the germline immunoglobulin sequence of other mammals (such as mouse or human). The term "anti-human interleukin 2 mouse monoclonal antibody" may also include antibodies having amino acid residues encoded by non-rabbit immunoglobulin sequences, for example, mutations introduced by random mutations or point-specific mutations in vitro, or mutations introduced by somatic mutations in vivo. However, the term "anti-human interleukin-2 mouse monoclonal antibody" does not include antibodies whose CDR regions are derived from the germline of other mammals (eg, human).

[0134] In the present application, the term "antibody" refers to an immunoglobulin molecule composed of four heterologous polypeptide chains, wherein the two chains with larger molecular weight are called heavy chains (H), and the two chains with smaller molecular weight are called light chains (L). Antibody light chains can be classified into κ (kappa) and λ (lambda) light chains. Heavy chains can be classified into μ, δ, γ, α or ε, and the isotypes of antibodies are defined as IgM, IgD, IgG, IgA and IgE, respectively. The sequences of about 110 amino acids near the N-terminus of the heavy and light chains vary greatly, while the amino acid sequences of other parts are relatively constant. Therefore, the regions of the light and heavy chains where the amino acid sequences near the N-terminus vary greatly are called variable regions (V), accounting for 1 / 4 and 1 / 2 of the heavy and light chains, respectively; the regions where the amino acid sequences near the C-terminus are relatively stable are called constant regions (C), accounting for 3 / 4 and 1 / 2 of the heavy and light chains, respectively.

[0135] The V regions of the heavy chain and light chain are called VH and VL, respectively. VH and VL each contain three regions with highly variable amino acid composition and arrangement order, called hypervariable regions (HVR) or complementarity determining regions (CDR), including HVR1 (CDR1), HVR2 (CDR2) and HVR3 (CDR3), among which HVR3 (CDR3) has a higher degree of variation. The three CDRs of VH and VL together constitute the antigen-binding site of the antibody, which determines the specificity of the antibody and is the site where the antibody recognizes and binds to the antigen. In the V region, the amino acid composition and arrangement order of the region outside the CDR are relatively conservative, called the framework region (FR). VH or VL each has four framework regions, represented by FR1, FR2, FR3 and FR4, respectively.

[0136] The C region of the heavy chain and light chain is called CH and CL respectively. The length of CL of different types (κ or λ) of Ig is basically the same, but the length of CH of different classes of Ig is different. For example, IgG, IgA and IgD include CH1, CH2 and CH3, while IgM and IgE include CH1, CH2, CH3 and CH4.

[0137] In the present application, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen to which it is directed. The intensity or affinity of a specific binding interaction can be represented by the equilibrium dissociation constant (KD) of the interaction. In the present application, the term "KD" refers to the dissociation equilibrium constant of a specific 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 carrier into which a polynucleotide can be inserted. When the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries are expressed in the host cell. 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 artificial chromosomes (PAC) derived from P1; bacteriophages such as lambda phage or M13 phage and animal viruses, etc. Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40). A vector can contain a variety of elements that control expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication initiation site.

[0139] In this application, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the expected properties of the protein / polypeptide comprising the amino acid sequence. Conservative amino acid substitutions include substitutions that replace amino acid residues with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). These conservative substitutions are preferably generated by amino acid replacement according to the following table.

[0140] 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

[0141] 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 propagated host cells 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 a control sequence in an operable connection so that a host transformed with these sequences can produce the encoded protein. To achieve transformation, the expression system can be included on a vector; however, the relevant nucleic acid molecule can also be subsequently integrated into the host chromosome.

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

[0144] As used herein, the terms "transfection", "stable transfection" or "transient transfection" refer to the uptake of an expression vector by a host cell, regardless of whether any coding sequence is actually expressed. A variety of transfection methods are known to those skilled in the art. For example, transfection is accomplished in the presence of an expression vector and a high concentration of calcium phosphate, by electroporation, by using a bacteriophage or viral expression vector to insert into a host cell, by mechanical insertion of nucleic acids, or even by culturing host cells in the presence of unpackaged nucleic acid fragments. Successful transfection is usually confirmed when any indication of manipulation of the vector of interest 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, the monoclonal antibody comprising three heavy chain complementary determining regions (CDR-H1, CDR-H2, CDR-H3) and three light chain complementary determining regions (CDR-L1, CDR-L2, CDR-L3), wherein:

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

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

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

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

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

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

[0153] In a specific embodiment, the above 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 in SEQ ID No: 14;

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

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

[0157] The present invention also provides an antibody combination, which comprises a first antibody S1 and the antibody (S2) described in the first aspect of the present invention. In a specific embodiment, the CDR-H1 of the first antibody has an amino acid sequence as shown in SEQ ID No: 1,

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

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

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

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

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

[0163] In a specific embodiment, 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 HCVR is shown in SEQ ID No: 13;

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

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

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

[0168] In this specification, "monoclonal antibody" means an antibody derived from a colony of substantially homologous antibodies, that is, each antibody constituting the colony is identical and / or binds to the same epitope, except for possible variant antibodies (e.g., containing naturally occurring mutations or produced in the production process of monoclonal antibody products), such variants are usually present in trace amounts. Different from polyclonal antibody products that generally include different antibodies for different determinants (epitopes), each monoclonal antibody of a monoclonal antibody product is directed to a single determinant on an antigen. Thus, the modifier "monoclonal" indicates that the antibody is derived from a substantially homologous antibody colony, and should not be construed as requiring the antibody to be produced by any ad hoc method. For example, the monoclonal antibody to be used according to the present invention can be prepared by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods using all or part of transgenic animals comprising human immunoglobulin loci, and such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

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

[0170] In this specification, "affinity" means the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity" used in this specification means the intrinsic binding affinity reflecting a 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 generally be represented by an equilibrium dissociation constant (KD). Affinity can be measured by common methods known in the art.

[0171] The anti-human interleukin-2 monoclonal antibody of the present application does not bind to proteins unrelated to the target. Here, "unrelated proteins" refer to proteins other than human interleukin-2 as the target; here, "no binding" means: when the binding ability of the anti-human interleukin-2 monoclonal antibody of the present application to human interleukin-2 as its target is taken as 100%, the binding ability of the anti-human interleukin-2 monoclonal antibody of the present application to the unrelated protein is less than 10%, for example, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0.

[0172] The anti-human interleukin-2 monoclonal antibody of the present application does not bind to interleukin-2 of other animal species. Here, "other animal species" refers to animal species other than humans, such as rhesus monkeys, cynomolgus monkeys, rats, mice, etc.; here, "no binding" means: when the binding ability of the anti-human interleukin-2 monoclonal antibody of the present invention to human interleukin-2 as its target is taken as 100%, the binding ability of the anti-human interleukin-2 monoclonal antibody of the present invention to interleukin-2 of other animal species is less than 10%, such as 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0.

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

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

[0175] CBA

[0176] Cytometric Bead Array (CBA), also known as cytokine microsphere detection technology, is a multiplex protein quantitative detection method based on a flow cytometric detection system, which can simultaneously detect multiple indicators in a single sample. BD uses the characteristic of flow cytometers to amplify fluorescent signals in series, attaches the soluble factors to be tested to some microparticles with a diameter similar to that of cells, and can detect various soluble factors in the tested samples. The basic principle of CBA is similar to that of ELISA, that is, using tiny, dispersed particles to capture liquid analytes, and using flow cytometry to detect the fluorescence emitted by the "sandwich"-like particles-the analyte complex, thereby determining the amount of the analyte.

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

[0178] In this specification, "avidin" is a glycoprotein, each molecule of which is composed of 4 subunits and can bind closely to 4 biotin molecules. Streptavidin extracted from Streptomyces is more commonly used.

[0179] In this specification, biotin is a small molecule growth factor widely distributed in animals and plants, also known as coenzyme R or vitamin H. Avidin is an alkaline glycoprotein extracted from ovalbumin, which has a 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-stage amplification role, but also develops color due to the catalytic action of the enzyme when encountering the corresponding substrate, thereby achieving the effect of measuring the amount of antibody bound to it. Therefore, combining avidin and biotin with CBA can greatly improve the sensitivity of CBA. The application of biotin-avidin system in CBA has various forms, which can be used for indirect coating and can also be used for final reaction amplification. The enzyme-labeled antibody in conventional CBA can also be replaced by a biotinylated antibody, and then connected to an avidin-enzyme conjugate to amplify the reaction signal.

[0180] Detection Kits

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

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

[0183] In a specific embodiment, the anti-human interleukin monoclonal antibody in the kit of the present application includes 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); the second antibody is a detection antibody, which is labeled with biotin. Preferably, the microsphere particle size is a fluorescent microsphere. Further preferably, the particle size is 5 μm. In a specific embodiment, polystyrene microspheres of China Nano 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#, whose heavy chain has the amino acid sequence shown in SEQ ID No: 17, and the light chain has the amino acid sequence shown in SEQ ID No: 19; the second antibody is anti-human interleukin-2 monoclonal antibody 2#, whose heavy chain has the amino acid sequence shown in SEQ ID No: 18, and the light chain has the amino acid sequence shown in SEQ ID No: 20.

[0186] Furthermore, the CBA kit also includes devices or reagents necessary for detecting interleukin-2.

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

[0188] In a specific embodiment, the fluorescent protein is preferably phycoerythrin (PE), and the streptavidin labeled therewith is a 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 buffered saline (PBS) pH 7.4; the washing solution is phosphate buffered saline (PBS) containing 0.05% Tween 20; and the blocking solution / sample diluent is phosphate buffered saline (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) Microspheres pre-coated with antibodies: anti-human interleukin-2 monoclonal antibody 1# (i.e., the first antibody), 2.5 mL / tube, 1 tube;

[0192] 2) Detection antibody: anti-human interleukin-2 monoclonal antibody 2# (i.e., the 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 (SA-PE), 2.5 mL / tube, 1 tube;

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

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

[0197] Detection Methods

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

[0199] The present invention forms an antibody-biotin-avidin system (ABAS) by biotin-labeling the anti-human interleukin-2 monoclonal antibody with phycoerythrin-labeled streptavidin. The strong binding with high affinity can achieve a multi-stage amplification effect, so that the kit of the present application has higher sensitivity and a wider detection range.

[0200] A specific method is to bind a known amount of a first antibody to a solid phase carrier surface. Then the sample to be tested is applied to the surface so that any interleukin 2 substance in any cell and / or serum present therein is captured by the fixed first antibody. Unbound substances are preferably removed by one or more washing steps. Then a second antibody, i.e., a detection antibody, labeled with biotin is added, and allowed to bind to any cell and / or interleukin 2 substance in serum captured by the first antibody, so that each unit of interleukin 2 simultaneously binds to two antibodies to form a "sandwich". The amount of the second antibody bound is then determined in a direct or indirect detection method. Specifically, a label or enzyme can be directly or indirectly connected to the second antibody via a connection such as a biotin-streptavidin or a biotin-avidin connection.

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

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

[0203] Labeling biotin onto anti-human interleukin-2 monoclonal antibody 2# (i.e., the second antibody);

[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] Add phycoerythrin-labeled streptavidin to the flow tube and incubate;

[0206] The standard curve was fitted using the MFI value of the interleukin-2 standard, and the MFI value of the sample to be tested was substituted into the equation to calculate the content of interleukin-2 in the sample to be tested.

[0207] In a specific 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) was prepared into a coating antibody working solution with a concentration of 1 g / L using a coating antibody diluent (phosphate buffered saline (PBS), pH 7.4), and then mixed in clean EP at a ratio of 10 μg / 1E7 microspheres and incubated at room temperature for 2 hours;

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

[0210] (3) Preparation of protein standards: Take 7 EP tubes and number them in sequence. Add 150 μL of sample diluent to each tube 1-6 and place them on the EP tube rack. Use the sample diluent to prepare the standard protein (human IL-2 protein) into a liquid with a concentration of 10 μg / L. Pipet 50 μL of the liquid and add it to the first EP tube. Then, pipette 50 μL of the liquid from the first EP tube and add it to the second EP tube for four-fold dilution. Repeat this process until the sixth 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.

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

[0212] (5) Adding SA-PE: Add 25 μL / tube to EP and incubate on a shaker (500 rpm) for 0.5 h;

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

[0214] (7) Detection: The MFI value of each EP tube was measured at a wavelength of 585 nm using a flow cytometer, and a standard curve was fitted based on the MFI value of the standard.

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

[0216] Pharmaceutical composition

[0217] The present invention also provides a composition. Preferably, 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): intratumoral, intraperitoneal, intravenous, or local administration.

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

[0219] 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 nanoantibody (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 10 micrograms / kg body weight to about 50 mg / kg body weight per day. In addition, the polypeptide of the present invention can also be used with other therapeutic agents.

[0220] 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 50 milligrams / kg body weight, preferably the dosage is about 10 micrograms / kg body weight to about 10 milligrams / 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.

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

[0222] 1) The present invention provides antibodies and antibody pairs that specifically target human interleukin 2, which can be used in CBA kits to detect human interleukin 2 and can greatly expand the detection range and improve the detection sensitivity. Repeated tests have shown that they have high accuracy.

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

[0224] 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.

[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 immune titer reached the standard, B cell suspension was collected and prepared for sorting. Figure 1 The results of immunizing mice with human interleukin 2 are shown. The horizontal axis is the dilution multiple after collecting the tail vein serum of different mice, and the vertical axis is the OD450nm value. The results show that the immune titer has reached the expectation, and one of the mice is selected for single B cell sorting. First, specific memory B cells are screened out using cell surface markers and antigens. Subsequently, the DeepLight On-chip Cell Screening platform is used for sorting to obtain specific plasma cells, and single B cells bound to IL-2 are selected. After a short period of culture, Binding ELISA is used for positive / reverse screening detection to select monoclonal clones that bind to IL-2 and have no binding activity to IL-4, IL-7, IL-9, IL-15 and IL-21, thereby screening and obtaining the first antibody and the second antibody. The obtained first and second antibodies are mouse monoclonal antibodies, and human-mouse antibody chimerism is performed. The above immunization and screening processes were entrusted to Detai Bio.

[0227] The heavy and light chain variable region sequences of the first antibody and the second antibody were obtained by sequencing, and their 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 Specificity Identification of Anti-Human Interleukin-2 Monoclonal Antibodies

[0233] 1. Antibody recognition 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 ELISA plate at 2-8℃ 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, wash the plate for color development and read the absorbance at OD450nm. The results are as follows: Figure 2 As shown, antibodies 1# and 2# did 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. These antibodies can be paired for experiments. The test results of antibodies 1# and 2# are shown in Table 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. Anti-human IL-2 antibody pairing experiment

[0238] The two antibodies were biotin-labeled using the Sulfo-NHS-LC-Biotin kit, and the specific steps were strictly followed in accordance with the kit instructions. After labeling, the free biotin was dialyzed using a dialysis bag, and the resulting solution was the biotin-labeled antibody.

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

[0240] Matrix buffer, human IL-2 protein, antibody-coated microspheres, and biotin-labeled antibodies were added in sequence and incubated at room temperature for 2 hours. Diluted phycoerythrin-labeled streptavidin was then added and incubated at room temperature for 0.5 hours. After the final wash, the fluorescence value was read using a flow cytometer. The results are shown in Table 3 below. Antibody 1# and antibody 2# can be paired for assembly and detection of human IL-2CBA quantitative detection kit.

[0241] Table 3 Antibody pairing test results

[0242]

[0243] Example 3 Composition of Human IL-2CBA Quantitative Detection Kit

[0244] The antibody 1# was coated with fluorescent microspheres as the capture antibody, and the antibody 2# was biotin-labeled as the detection antibody to prepare a CBA kit. The reagent composition, specifications, sources, storage conditions, etc. in the kit are shown in Table 4.

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

[0246] 2) Detection antibody: anti-human interleukin-2 monoclonal antibody 2# (i.e., the 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 (SA-PE), 2.5 mL / tube, 1 tube;

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

[0250] 6) Washing solution: phosphate buffered saline (10XPBST), pH 7.4, 5 mL / bottle, 1 bottle;

[0251] The components are shown in Table 4 below:

[0252] Table 4 Composition of CBA kit

[0253] Components Main Ingredients content Storage Capture Antibody Fluorescent microspheres coated with antibody 1# 2.5Ml, 1 bottle 2-8℃ 1 year Detection antibodies Biotinylated Antibody 2# 2.5Ml, 1 bottle 2-8℃ 1 year Standards Human IL-2 protein 10ng, 1 tube -20℃ 1 year SA-PE Streptavidin-phycoerythrin conjugate 2.5Ml, 1 bottle 2-8℃ 1 year Matrix buffer BSA, protein protective agent 5Ml, 1 bottle -20℃ 1 year Wash buffer 10XPBST 5Ml, 1 bottle Room temperature 1 year

[0254] Example 4 Evaluation of performance indicators of human IL-2 CBA kit

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

[0256] 1. Linear range:

[0257] (1) Dilute human IL-2 protein with sample diluent to prepare protein standard, and dilute human IL-2 protein with cell culture medium to prepare quality control sample as the sample to be tested. Fit the standard curve according to the MFI value of the protein standard. Substitute the MFI value of the sample to be tested into the standard curve to obtain the theoretical concentration value of human IL-2 in the sample to be tested, and then calculate the recovery rate of the sample to be tested (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 cell culture medium.

[0258] (2) Quantitative detection steps:

[0259] 1) Preparation of protein standards: Take 8 EP tubes and number them in sequence. Add 300μL of sample diluent to each tube starting from the second tube and place them on the EP tube rack. Use sample diluent to prepare the standard protein to a concentration of 10ng / mL in the first tube, draw 100μL and add it to the second EP tube for four-fold dilution, and so on to the seventh EP tube. The concentrations are 10000pg / mL, 2500pg / mL, 625pg / mL, 156.3pg / mL, 39.6pg / mL, 9.7pg / mL, and 2.4pg / mL, respectively.

[0260] 2) Preparation of quality control samples: Take 6 EP tubes, take samples from 10000 pg / mL respectively, and prepare quality control samples of 6250 pg / mL, 1250 pg / mL, 250 pg / mL, 50 pg / mL, 10 pg / mL, and 2 pg / mL;

[0261] 3) Sample addition: Add matrix buffer, protein standard solution, microspheres coated with the first antibody, and biotin-labeled second antibody in order at a volume of 25 μL / tube, and incubate at room temperature for 2 hours on a shaker (500 rpm);

[0262] 4) Add SA-PE: Add 25 μL / tube to EP and incubate on a shaker (500 rpm) for 0.5 h;

[0263] 5) Washing: Discard the reaction solution in the EP tube by centrifugation, wash twice with washing solution, and resuspend the microspheres with sample diluent;

[0264] 6) Detection: Use flow cytometry to measure the MFI value of each EP tube at a wavelength of 585 nm, fit a standard curve based on the MFI value of the standard, and substitute the MFI value of the quality control sample into the equation to calculate the concentration of the quality control sample.

[0265] (3) Results and discussion

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

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

[0268]

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

[0270] 2) The curve equation is as follows Figure 3 As shown: y = 0.0799x 2 +0.3739x-1.698(y:Log of Con;x:Log ofMFI),R 2=0.9995.

[0271] 2. Specificity

[0272] (1) Take different cytokines and dilute them to a concentration of 10 ng / mL, and detect their concentrations according to the kit operation. The specificity of the kit is expressed by the cross-reaction rate (%). The calculation method is: cross-reaction rate (%) = detection concentration / nominal concentration × 100%.

[0273] (2) The specific 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 in Table 6 show that the cross-reaction rate of related cytokines is ≤0.005%. The above results show that when the human IL-2CBA kit provided by the present application is used to quantitatively detect human IL-2 in the sample, it is not interfered by related cytokines in the sample, indicating that the detection method provided by the present application has good specificity.

[0278] 3. Accuracy:

[0279] (1) In 3 different batches of 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 is expressed by the coefficient of variation CV%, CV% = SD / mean value × 100. The accuracy is expressed by the relative error RE%, RE% = (mean detection concentration - theoretical concentration) / theoretical concentration × 100. The accuracy of the test kit was analyzed by the above parameters. The analysis results are shown in Table 7.

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

[0281]

[0282] (2) Results and Discussion

[0283] The results in Table 7 show that the intra-batch coefficient of variation CV≤5% at 6 concentration levels and the accuracy (RE)≤5%. The above results show that the quantitative detection of human IL-2 in samples using the human IL-2 CBA kit provided by the application can meet the intra-batch coefficient of variation CV≤5% and the accuracy (RE)≤5%, indicating that the detection method provided by the application has good accuracy.

[0284] 4. Precision

[0285] (1) In 3 different batches of 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 inter-batch coefficient of variation CV%, CV% = SD / mean value × 100%. The reproducibility of the test kit was analyzed by the above parameters. The analysis results are shown in Table 8.

[0286] Table 8 Experimental data on the precision of quantitative detection of human IL-2

[0287]

[0288] (2) Results and discussion

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

[0290] 5. Sensitivity

[0291] (1) In the experiment of 3 batches of different kits, each analysis batch was tested 20 times of sample dilution. The sensitivity calculation method is: calculate the mean value M and standard deviation SD of the measurement results, and bring the value of the mean value M plus 2 times the standard deviation SD into the standard curve in Example 4. The calculated result is the sensitivity. The analysis results are shown in Table 9.

[0292] Table 9 Sensitivity experimental 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 in Table 9 show that the sensitivity of the three batches of kits is ≤0.5 pg / mL. The above results show that the kit provided by the present application has good sensitivity.

[0296] 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. An antibody against human interleukin-2 protein, characterized in that The antibody comprises a heavy chain and a light chain, wherein 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 region CDR: CDR-H1 having an amino acid sequence as shown in SEQ ID No: 2; CDR-H2 having an amino acid sequence as shown in SEQ ID No: 4; CDR-H3 having an amino acid sequence as shown in SEQ ID No: 6; The light chain variable region includes the following complementarity determining regions CDR CDR-L1 having an amino acid sequence as shown in SEQ ID No: 8; CDR-L2 having an amino acid sequence as shown in SEQ ID No: 10; The amino acid sequence of CDR-L3 is shown in SEQ ID No:

12.

2. The antibody according to claim 1, characterized in that The HCVR of the antibody has the amino acid sequence shown in SEQ ID No:14, and / or the LCVR has the amino acid sequence shown in SEQ ID No:

16.

3. The antibody according to claim 1, characterized in that The heavy chain of the antibody has the amino acid sequence shown in SEQ ID No: 18, and / or the light chain has the amino acid sequence shown in SEQ ID No:

20.

4. A fusion protein, characterized in that The fusion protein comprises: (1) the antibody according to claim 1; and (2) Optional tag sequence to facilitate expression and / or purification.

5. A polynucleotide, characterized in that The polynucleotide encodes the antibody according to claim 1 or the fusion protein according to claim 4.

6. A carrier, characterized in that The vector contains the polynucleotide according to claim 5.

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

8. An antibody conjugate, characterized in that: This antibody conjugate contains: (a) an antibody portion, wherein the antibody portion is the antibody according to claim 1; and (b) a conjugated moiety conjugated to the antibody portion, wherein the conjugated moiety is selected from the group consisting of a detectable label, a drug, or a combination thereof.

9. An antibody combination against human interleukin-2 protein, characterized in that: The antibody combination includes: Antibody S1, wherein the heavy chain variable region of antibody S1 includes the following complementarity determining regions CDR: The amino acid sequence of CDR-H1 is shown in SEQ ID No: 1, The amino acid sequence of CDR-H2 is shown in SEQ ID No: 3, The amino acid sequence of CDR-H3 is shown in SEQ ID No: 5, The light chain variable region of antibody S1 includes the following complementarity determining regions CDR: The amino acid sequence of CDR-L1 is shown in SEQ ID No: 7, The amino acid sequence of CDR-L2 is shown in SEQ ID No:9, CDR-L3 having an amino acid sequence as shown in SEQ ID No: 11; and Antibody S2, wherein the antibody S2 is the antibody according to claim 1.

10. Use of the antibody according to claim 1, the polynucleotide according to claim 5, the vector according to claim 6, the host cell according to claim 7, the antibody conjugate according to claim 8, or the antibody combination according to claim 9, characterized in that: For preparation: i) a reagent or kit for detecting human interleukin-2 protein; ii) a preparation or a drug for treating human interleukin-2 related diseases.

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